evaluation of gold nanoparticle based lateral flow assays...
TRANSCRIPT
Food Chemistry 170 (2015) 470–483
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier .com/locate / foodchem
Review
Evaluation of gold nanoparticle based lateral flow assays for diagnosisof enterobacteriaceae members in food and water
http://dx.doi.org/10.1016/j.foodchem.2014.08.0920308-8146/� 2014 Elsevier Ltd. All rights reserved.
⇑ Corresponding author. Tel.: +91 0532 2271245.E-mail addresses: [email protected], [email protected] (S. Nara).
Jyoti Singh, Shivesh Sharma, Seema Nara ⇑Department of Biotechnology, Motilal Nehru National Institute of Technology, Allahabad 211004, U.P., India
a r t i c l e i n f o
Article history:Received 10 February 2014Received in revised form 15 August 2014Accepted 21 August 2014Available online 29 August 2014
Keywords:Lateral flow immunoassayGold nanoparticleEnterobacteriaceaeSalmonellaE. coli
a b s t r a c t
Lateral flow immunoassays (LFIAs) are advantageous over conventional detection methods in terms oftheir simplicity and rapidity. These assays have been reported using various types of labels but colloidalgold nanoparticles are still the preferred choice as a label because of their easy synthesis, visual detectionand stability. Bacterial contamination of food and drinking water is a major threat and hindrance towardsensuring food and water safety. Enterobacteriaceae family members are mainly transmitted by the con-sumption of contaminated water and food and implicated in various food or water borne infections. TheLFIAs have been popularly used for detection of bacterial cells in different matrices. Therefore, this reviewintends to provide an analysis of the gold nanoparticle based lateral flow assays developed for detectingenterobacteriaceae family members in food and water samples. The review includes detailed data anddiscusses the factors that influence the performance of LFIAs and their shortcomings.
� 2014 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4702. Structure of lateral flow immunoassay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
2.1. Antibody based LFIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
2.1.1. Sandwich assay format. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4722.1.2. Competitive assay format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4722.2. Nucleic acid based lateral flow assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
2.2.1. Target analyte (PCR amplicon). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4722.2.2. Target analyte (whole bacteria or its surface antigen). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4723. LFIA for enterobacteriaceae members in food/water samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
3.1. Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4773.2. E. coli O157. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4784. Factors affecting the performance of LFIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4805. LFIA-a potential tool in HACCP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4806. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
1. Introduction
Enterobacteriaceae is a large family of gram negative, non-sporeforming rods, which are facultative anaerobes and capable of
fermenting sugars to various end products. Several members ofthis group such as some species of Escherichia coli, Salmonella,Shigella, Yersinia entercolitica, Enterobacter, etc. are capable of caus-ing primary infections of the human gastrointestinal tract and thus,they are referred to as ‘‘enterics’’. Some members act as opportu-nists and cause nosocomial infections. Their natural habitat is theintestinal tract of humans and animals and they get transmitted
J. Singh et al. / Food Chemistry 170 (2015) 470–483 471
by the consumption of contaminated water and food. Members ofthis family are known to be fastidious in nature and the pathoge-nicity is mainly due to various endotoxins or exotoxins producedin the stomach. Toxins interact with the digestive juices resultingin huge water loss from the body which may lead to death(Paton & Paton, 1998). They are also the leading cause of gastroin-testinal disorders especially in the developing world population.Some may cause systemic diseases with chronic implications suchas hemolytic uremic syndrome (E. coli O157:H7), Guillain-Barresyndrome (Campylobacter spp.) and disease with metastaticinfections (non typhoidal Salmonella spp.). The disease symptomsoriginate due to favourable high temperatures. The Indian subcon-tinent is considered to be a hub of food and water borne tropicaldiseases, specially caused by the members of the enterobacteria-ceae family, owing to the fluctuations in temperature and humid-ity. The food and water borne diseases are less prevalent intemperate climates, due to the occurrence of a cold season, whichcontrols the rapid multiplication of potentially pathogenic bacteria(Lafferty, 2009).
The biological contamination in drinking water is a major prob-lem of public health in developing world. WHO estimates thatabout 1.1 billion people globally drink unsafe water and the vastmajority of diarrhoea disease in the world (88%) is attributable tounsafe water, sanitation and hygiene. A diversity of enteric bacteriaand viruses has been associated with outbreaks of waterbornegastroenteritis (Espinosa, Arias, Sánchez-Colón, & Mazari-Hiriart,2009). In 2008, a large Salmonella outbreak caused by contamina-tion of the municipal drinking water supply occurred in Alamosa,Colorado (Ailes et al., 2013). .Ganges river is the lifeline of majorIndian population and an estimated 200 million litres sewagewaste is discharged daily in it. The enteric disease incidence inthe population residing along the banks of river was estimated tobe 66% annually (Abraham, 2011). Food-borne diseases also con-tribute to a significant disease burden particularly in developingcountries. World Health Organization has created an initiative toestimate the burden of disease related to food (WHO, 2008). TheWorld Health Organization estimates that in 2005, 1.5 millionpeople died, worldwide, from diarrheal diseases (Buzby &Roberts, 2009). In the United States, food-borne illnesses affectup to 80 million people and cost an estimated $5 billion US onan annual basis (Altekruse, Cohen, & Swerdlow, 1997). The costmay be even higher for developing countries. Several factorscurrently contribute to the emergence of food and water-borneenteric diseases. These include changes in human demographicsand behaviour, international travel, microbial factors, lack of sew-erage and toilets at residence, children defecating outdoors, poorsanitation, low income and low education levels, percolation ofreservoir water in drinking water and breakdown of public healthmeasures. These factors pose a hurdle in ensuring food and watersecurity to the masses. Therefore, rapid food and water qualitymonitoring is indispensable to avoid the outbreak of food/waterborne illnesses.
Preventing the outbreaks of infection due to consumption ofcontaminated food/water is a challenging task particularly in thelarge populations of developing world. The detection of thesepathogens or their preformed toxins in food and water is thus agrowing concern. Several methods have been developed for detec-tion of pathogenic bacteria in food and water which include phys-iological and biochemical methods like most probable number(MPN) analysis, differential media culturing, enzyme assays (Li &Gu, 2011; Rompre, Servais, Baudart, de-Roubin, & Laurent, 2002),PCR (Navas et al., 2006; Wang, 2002), loop-mediated isothermalamplification (Notomi et al., 2000; Saleh, Soliman, & El-Matbouli,2008; Tao et al., 2011) and immunological techniques like ELISA(Crowther, 1995). The culture methods although considered thegold standard for bacterial detection are often time taking giving
results in 48 h. Other methods like enzyme assays or PCR orimmunoassays are restricted to the laboratory set up. Rapid meth-ods which can be used outside the laboratory by unskilled personscan prevent the occurrence of these infections and thus, is the needof hour. Therefore the recent focus is on the development of rapiddetection techniques, which are user friendly, require less techni-cal expertise and time efficient. In this regard, techniques basedon immunological principles are considered to be most suitableto make on-site decisions due to their rapid, precise, and sensitiveresult producing ability.
Lateral flow immunoassays represent a well established andvery appropriate technology in the category of rapid assays. Theyhave been used to detect levels of various hormones in blood andserum (Eisinger, Khalil, Katz, & Sargeant, 1990; Tripathi, Nara,Singh, Singh, & Shrivastav, 2012) heavy metals like Hg, Ar, Pb fromenvironment (Chai, Wang, Wang, Li, & Su, 2010; Knecht & Sethi,2009), on site rapid plant pathogen detection (Zhao et al., 2011),toxicity and adulterant detection in both cooked as well as raw foodsubstances (Blazkova et al., 2009), industrial water toxicity for dif-ferent contaminants, on site detection of various plant viruses(Cambra et al., 2000; Danks & Barker 2000), many human infectiousdiseases like syphilis (Yang et al., 2013) malaria (Gillet et al., 2010;Piper, Buchanan, Choi, & Makler, 2011), and tuberculosis (Werneryet al., 2007). There are variants of LFIA depending upon the labelsused for the signal generation and final read out. An ideal labelshould possess some features such as amenable to detection bymultiple methods over useful range, easy to conjugate withbiological or chemicals without losing its integrity, possess low ornegligible non specific binding over a range of assay conditions,stable under different assay conditions and commercially availableat low cost. However, it is difficult for one label to satisfy all thesecriteria; therefore, the choice of label is greatly determined bythe assay to be developed. Labels like carbon nanoparticles(Posthuma-Trumpie, Korf, & Amerongen, 2009), latex beads(Doumanas, Bonenberger, & Michael, 2006; Linares, Kubota,Michaelis, & Thalhammer, 2012), selenium (Lou, Patel, Ching, &Gordon, 1993), colloidal carbon (Lonnberg & Carlsson, 2001; VanAmerongen et al., 1993), dye-loaded liposomes (Baeumner, Jones,Wong, & Price, 2004; Ho & Huang, 2005; Zaytseva, Montagna, Lee,& Baeumner, 2004), Europium Chelate–Loaded Silica Nanoparticles(Xiaohu, Ye, Xilin, & Qingge, 2009), fluorescent europium (III) chea-ted nanoparticles, have been shown to be applicable in various for-mats of LFIAs (Huhtinen, Pelkkikangas, Jaakohuhta, Lövgren, &Härmä, 2004). The newest labels include quantum dots (Goldmanet al., 2004) and upconverting phosphors (Corstjens et al., 2001;Zuiderwijk, Tanke, Niedbala, & Corstjens, 2003). However, use ofgold nanoparticles as label in LFIA is still popular and most widelyused. The most sought after property of the gold label is its ability toproduce colour while allowing an unobstructed flow of the testsolution through the nitrocellulose membrane. Gold being inertpossess optical properties and is easy to visualise, with simpleand inexpensive laboratory preparation, stable in liquid or driedform, easy to conjugate with biological material, due to which itis the most preferred label for the LFIAs.
Number of gold nanoparticle based lateral flow immunoassayshave been reported for the detection of bacterial pathogens butmost of these reported assays are restricted to the publicationsand are not in actual use. Therefore, this paper intends to reviewthe recent status of gold nanoparticle based LFIAs developed/reported so far for detection of enterobacteriaceae family membersin food and water. It discusses their advantages and shortcomingsin order to understand the lacunae which need to be appropriatelyaddressed through future research. In addition, this paper alsohighlights the factors which can affect the performance of LFIAand how LFIA can be used as a tool for HACCP (Hazard AnalysisCritical Control Points) analysis.
472 J. Singh et al. / Food Chemistry 170 (2015) 470–483
2. Structure of lateral flow immunoassay
Lateral flow immunoassay is also commonly known as immu-nochromatographic assay as these assays utilise immunologicalentity (antibody/antigen) which moves across the membrane sur-face via capillary action. The standard LFIA strip includes a backingcard, membrane, sample pad, absorbent pad, conjugate pad, andsometimes a wick. The various components of LFIA strip arearranged in a way that they overlap each other as shown inFig. 1. All possible variations of immunochromatographic stripshave one thing in common i.e. they employ the formation of com-plex between a detection reagent coupled with a colour label(which moves along with the sample on the membrane) and thecapture reagent immobilised on the membrane. The variations inthe set up of LFIA arise by virtue of the labels and the detection/capture regents employed. On the basis of the detection/captureregents used in the LFIA, following variants have been reportedfor bacterial detection.
2.1. Antibody based LFIA
The LFIA employing antibodies as both capture and detectorreagents have been developed in two basic formats namely sand-wich and competitive.
2.1.1. Sandwich assay formatPrimary antibody against the antigen is immobilised on the
membrane and another antibody against a different epitope ofthe same antigen is labelled with gold NPs. When the gold labelledantibody flows along with the test sample (which is expected tohave the antigen), it interacts with the antigen in the sample. Thiscomplex interacts with the antibody coated at the test line on themembrane and forms a sandwich (Fig. 2a). A control line willalways form. Thus in sandwich assay format, a positive test sampleis indicated by the appearance of two red lines and a negative testsample is indicated by the appearance of one red line i.e. only con-trol line.
2.1.2. Competitive assay formatIs typically used for determining small molecules with single
antigenic determinants, which cannot bind to two antibodiessimultaneously. There are variations in competitive assay format.In format 1 a positive result is indicated by the absence of a testline on the reaction matrix. A control line should still form, irre-spective of the result on the test line (Posthuma-Trumpie, Korf, &Amerongen, 2008; Qian & Bau, 2004). In this format, standard anti-gen (analyte) is labelled with gold NPs, and coated on reservoirmatrix. For negative sample, this labelled antigen flows and bindswith antibody at test line, resulting in the red colour at test line
Fig. 1. Basic structure of late
(Fig. 2-b1). For positive sample, the unlabelled antigen present inthe test sample will prevent the binding of labelled analyte at testline due to which red line will not form at test (Fig. 2-b2). Anotherversion of the competitive format is either quantitative or semi-quantitative where colour intensity at the test line changes withconcentration of analyte in the sample. The signal at the capturezone can be directly or indirectly proportional to the analyte con-centration (Fig. 2-c1 and c2).
2.2. Nucleic acid based lateral flow assays
Nucleic acid lateral flow (NALF) uses nucleic acid hybridizationto capture and detect nucleic acid amplification products in a man-ner akin to lateral-flow immunoassays. On the basis of the targetanalyte to be detected they can categorised in two groups.
2.2.1. Target analyte (PCR amplicon)Here, the target analyte to be detected in the sample can be any
characteristic gene of the bacterial pathogen in question. Suchassays depend on isolation and PCR amplification of the desiredgene of interest from the bacterial pathogen in sample. The pres-ence of specific amplicon can then be detected by developing NALFassays. These assays can be developed in two formats: antibodydependent and antibody independent. In the antibody dependentformat (Fig. 3a), PCR amplicon (Target analyte) is mixed withtwo specific primers – one labelled with biotin and other labelledwith FITC. After incubation, this solution is put on the samplepad and allowed to run along the IC strip. The colloidal goldlabelled avidin coated at reservoir matrix will bind to the ampli-con-primer complex through biotin–avidin interaction. The anti-FITC antibody immobilised at the test line will capture the goldlabelled amplicon complex by interacting with FITC. The controlline has anti-avidin antibody. The presence/absence of ampliconin the test sample is indicated by the presence/absence of red col-our at the test line (Wang et al., 2012). In antibody independentformat (Fig. 3b), one of the specific primers to amplicon is labelledwith colloidal gold which will hybridise with the amplicon put insample pad. The complex will then interact with another primerto amplicon which is immobilised at the test line either directly(passive adsorption) or through BSA or through biotin–avidininteraction and gets captured at the test line producing a red col-our (Blazkova et al., 2009).
2.2.2. Target analyte (whole bacteria or its surface antigen)Besides, determining the presence/absence of PCR amplified
products by lateral flow assays, aptamers (DNA/RNA sequencesthat specifically binds to non-oligonucleotide target) have alsobeen used in developing lateral flow assays for directly detectingthe bacterial pathogen or its surface antigen. Such aptamers are
ral flow immunoassay.
Fig. 2. (A–C) Antibody based LFIAs (A) sandwich assay format, (B1 and B2) competitive assay format where presence of analyte is indicated by absence of test line, (C1 and C2)competitive assay format where the colour intensity at the test line is inversely proportional to the analyte in sample.
J. Singh et al. / Food Chemistry 170 (2015) 470–483 473
developed using SELEX (Systematic Evolution of Ligands by Expo-nential Enrichment) technique. In this approach, one of the apt-amer specific to the analyte is immobilised on the NC membraneas test line directly or through biotin–avidin interaction. Anotheraptamer is conjugated to colloidal nanoparticles. The bacterialpathogen (analyte) is put on the sample pad which interacts withthe nanoparticle conjugated aptamer. As the complex moves alongthe strip, the aptamer immobilised at test line will also interactwith the bacterial pathogen to form a sandwich and the gold nano-particles get captured at the test line producing a red colour(Fig. 4).
3. LFIA for enterobacteriaceae members in food/water samples
The classical practise of bacterial detection comprises of culturebased assays. With the emergence of LFIAs strips, the detection ofpathogenic bacteria has become easier and rapid. LFIAs have been
reported for detection of bacterial pathogens in various samplematrices like plasma/serum, faeces/stool, food samples and drink-ing water. The detection of bacterial pathogens in plasma, serum orstool samples indicates the entry/presence or infection of bacterialpathogen in the body and positive confirmation of results is fol-lowed by some line treatment. However, it is usually said that‘‘prevention is better than cure’’ and it would be beneficial if theoccurrence of infection can be timely prevented. The most com-mon vehicles of these bacterial pathogens are food and water sam-ples and in order to avoid/prevent the infections to occur, thesevehicles should be contamination free. In this regard, it is requiredto test food/water samples for bacterial contamination before theirconsumption. At this point, rapid and easy to use detection meth-ods which can quickly screen the food/water samples for any kindof bacterial contamination play very crucial role. Keeping in viewthe significance of such assays for screening water/food samples,this section reviews the gold nanoparticle based LFIAs developedfor detecting enterobacteriaceae members in food and water. After
Fig. 3. (a and b) Nucleic acid based LFIAs where target analyte is a PCR amplicon (a) antibody dependant format where the amplicon in analyte is recognised though antibodyimmobilised at the test line (b) antibody independent format where the amplicon in the sample is recognised through an oligonucleotide immobilised at test line.
Fig. 4. Nucleic acid based LFIAs where target analyte is a bacterial cell or its antigen which is recognised through a pair of aptamers.
474 J. Singh et al. / Food Chemistry 170 (2015) 470–483
extensive literature search, it was observed that out of variousenterobacteriaceae members that could be present in contami-nated water and food like Aeromonas spp., Campylobacter spp.,Clostridium spp., E. coli (including VTEC types such as O157),
Legionella spp., Leptospira spp., Pseudomonas aeruginosa, Salmonellaenterica, Shigella spp., Vibrio spp. and Yersinia spp., most of the goldnanoparticle based LFIAs have been developed for Salmonella and E.coli as the later two are the most common pathogens found to
Table 1LFIA developed for detection of various E. coli serovars in different food/water samples.
Entero-bacteriaceaemember (targetantigen)
Assayformat
Sample usedfor detection
Sampleprocessing/enrichment
Sensitivity Specificity Remarks References
E. coli O157 LFIA Raw beef,pork, bovineand swinefeces
Traditionalculture media forE. coli
1.8 � 10(5) CFU/ml withoutenrichment and1.8 CFU/ml afterenrichment
With pork (98.8%) – Jung et al. (2005)
E. coli O157 LFIA Milkpowder,flour, starch,coffee,biscuit, cake,jelly andjuice
– 1 � 10(5) cfu/ml No cross-reactionwith 30 strains of 24species inEnterobacteriaceae(including non-O157 E. coli,Salmonella, Shigella,Proteus, Citrobacter,Enterobacter,Serratia andYersinia),Staphylococus,Listeria, Aeromonasand Vibrios
– Wang et al. (2006)
E. coli O157:H7 LFIA Spikedsamples ofbeef, milk,cakes andwater
Spiked 5 g ofground beefsample in 45 mlPBS washomogenised in astomacher for1 min andsupernatant wasused for analysis.Spiked milk andwater sampleswere 10-foldserially dilutedEnrichment ofculture was donein tryptic soybroth for 18 h at37 �C
2.3 � 103 cfu/mlwithoutenrichment and2.3 cfu/ml afterenrichment
For food samples94.9–99.2%For pure cultures98.5%
Use of biotin–streptavidin systemhas improved theassay sensitivitynearly 100 times
Zhao et al. (2010)
E. coli O157:H7 LFIA Spiked milk,purifiedwater andbeef samples
Enrichment wasdone usingimmunomagneticnanoparticlescoupled with E.coli O157:H7polyclonalantibodies
P105 cfu/ml(withoutenrichment)P103 cfu/ml(IMPenrichment)
Without enrichment(93.3%)After enrichment(100%)
IMP enrichedbacteria weredetached by heatingso that their largersize do not interferewith assay flow andthe epitopes on theantigen surface arealso available forICA
Qi et al. (2011)
Shiga toxin producingE. coli [O157, O26,O111]
MultiplexLFA
Spikedground beef
Spiked groundbeef washomogenised in astomacher for 30 sfollowed byincubation at42 �C for 18 h
104 cfu/ml afterenrichment
Specificallydetermines thethree serovars
The use of AMPs asprobe fordeveloping LFIA forthe first time
Yonekita et al.(2013a),Yonekitaet al. (2013b)
E. coli O111 LFIA Meatsamples
Enrichment in themodified E. colibroth withnovobiocin for22 h at 42 �C
8 � 10(3)–5.6 � 10(5) CFU/ml of cellsuspension
100% when testedwith cellsuspensions of 8E. coli O111 strainsand 77 non-E. coliO111 strains
– Terao et al. (2013)
Shiga toxin-producingEscherichia coli(STEC) O26
LFIA Ground beef,beef liver,groundchicken,alfalfasprout,radishsprout,spinach,naturalcheese, andapple juice
Enrichment for18 h
2.2 � 10(3) –1.0 � 10(5) cfu/ml1 cfu/25 g offood samplesafter 18-henrichment
100% with purecultures of 67 E. coliand 22 non-E. colistrains
IC strip results werein accordance withthe PCR resultsusing 115 meatsamples from themarket
Yonekita et al.(2013a,b)
(continued on next page)
J. Singh et al. / Food Chemistry 170 (2015) 470–483 475
Table 1 (continued)
Entero-bacteriaceaemember (targetantigen)
Assayformat
Sample usedfor detection
Sampleprocessing/enrichment
Sensitivity Specificity Remarks References
E. coli O157:H7 Evaluationof Quix™
E. coli O157sprout ICassay
Alfalfa spentsproutirrigationwater
In brain heartinfusion broth at37 �C for 20 h
Able to detectbacteria inwater collectedafter 30 h(withoutenrichment)and 8 h (afterenrichment)from the start ofsproutingprocess
16 of 32 E. coli O157were detected
LFIA was lesssensitive than PCRassay
Fratamico and Bagi(2001)
E. coli O157:H7 Comparisonof Quix™ ICassay with aPCR assay
Minced beef 8 h incubation at37 �C or 42 �C inbuffered peptonewater
Minced beefinoculated with1–100 cells ofbacteria couldbe detected onlyafterenrichment
Extraction reagent isused to extract theantigen from intactorganismsThe assay time wasalmost double(15 min) than thatrecommended bythe manufacturer(7 min)
Gryko et al. (2002)
E. coli O157 SMART™ IIrapid E. coliO157 striptest
Foodsamples
An selectivegrowth mediumfor E. coli O157
Analyticalsensitivity was3.3 � 104 cfu/ml. Afterenrichment, thekit was able todetect 1 cfu/25 g of thesample
– – http://lifesci.com/ecoli/
E. coli O157 DuPont™lateral flowsystems
Raw groundbeef andbonelessbeef trim
8 h and 10–15 hfor 25 and 375 gof sample inselective E. coliO157 medium
1 cfu/25 g ofsample
– – http://www2.dupont.com/Qualicon/en_US/products/Lateral_Flow_System/lfs_ecoli.html
Table 2LFIA developed for detection of various Salmonella serovars in different food/water samples.
Entero-bacteriaceaemember (targetantigen)
Assay format Sample usedfor detection
Sample processing/enrichment
Sensitivity Specificity Remarks References
Salmonella enteritidis LFIA Raw eggsSpikingconcentration:100 ll of SE cellsuspension tomake a finalconcentrationof 10 cells/ml
10 eggs were polledand homogenisedfor 30 s in astomacher.Enrichment wasdone in bufferedpeptone broth at37 �C for 24 hAntigen extractionwas carried outfrom the eggcontents using amixture of oleic acidand caprylic acid(2:1)
106–105 cells/ml No cross-reaction wasreported withtwo otherserovars STad SK
Extractionprocedure hasadded advantage ofproviding effectivewicking of thesample
Seo et al. (2003)
S. Typhimurium andS. enteritidis
MultiplexLFIA
Chickensamples
Enrichment wasdone in bufferedpeptone broth at37 �C for 24 h
104 and 106 cfu/mlof S. typhimuriumand S. enteritidisfrom culturesamples sensitivityin the spikedsamples wasobserved to be98.89% and 87.50%for of S.typhimurium and S.enteritidis
100% – Moongkarndi et al.(2011)
476 J. Singh et al. / Food Chemistry 170 (2015) 470–483
Table 2 (continued)
Entero-bacteriaceaemember (targetantigen)
Assay format Sample usedfor detection
Sample processing/enrichment
Sensitivity Specificity Remarks References
Salmonella enteritidis LFIA Shell eggs andenvironmentalsamples
Enriched accordingto the procedureprescribed by theU.S. Food and DrugAdministration
100% – – Jagadeesan et al.(2011)
Salmonella entericaserovar Typhi
Dot boltimmuno-bioprobe
Capsular Vipolysaccharideof Salmonellaenterica serovarTyphi (surfaceantigen) inclinical sampleof blood
LB brothsupplemented with200 mMconcentration andat pH 7 formaximumexpression of viantigen
102 cells/ml inclinical samples
No crossreaction wasshown withserovarTyphimurium
Vi positive serovarstyphi strains wereidentified based onthe colourdevelopment on themembranecontaining strips
Pandey et al. (2012)
Salmonella Lateral flowimmunoassay
Food samples Cultivatedovernight at 37 �C inLuria–Bertani (LB)broth
104 cells 100% DNA is used forconjugation withgold for LFIA format
Liu et al. (2013)
S. typhimurium Evaluation ofan LFIA
Food samples Differentenrichment media
104–105 cfu/ml withpure cultures
Successfullydetected 19of 22 strainsof Salmonellaspp. and didnot detect 27differententericbacteria
absorbent pad wascoated with driedextraction bufferthat after coming incontact with testsample releases theantigen from intactsalmonella cells
Bautista et al. (2002)
Salmonella DuPont™lateral flowsystem test
Food includingraw meat,poultry, dairyproducts,processed meatand freshproduce
Salmonella media – – http://www2.dupont.com/Qualicon/en_US/products/Lateral_Flow_System/lfs_ecoli.html
Salmonella RapidChekSelect
Food samples Phage basednegative selection ofany cross reactingbacteria from thetest sample withtwo stepenrichment for�32–48 h
– – – http://www.romerlabs.com/en/products/food-pathogen-testing/salmonella/
Salmonella LFIA Meat and meatproducts
RapidChek Selectpre-enrichmentbroth pre-warmedat 41.5 �C
LOD50 of RapidChekSelect and ISOmethod were2.00 cfu/25 g
No crossreactivity for30salmonellastrains
High contaminationrates in this studyverified that meatand meat productsshould bemonitored regularlyfor the Salmonellacontamination
Torlak et al. (2012)
Salmonella spp. LFIA Raw groundchicken,chicken carcassrinse, slicedcooked turkey,and liquid eggs
The method usestwo proprietaryenrichment broths(primary (16–22 h,42 �C)) andsecondary (6–8 h(42 �C).))
0% false-negativerate and 0% false-positive
100% The RapidChekSelect Salmonellamethod was foundto be capable ofdetecting very lowlevels (0.1 MPN/25 g) of Salmonellain peanut butter;moreover, adversematrix effects onthe lateral flowdevice were notobserved whenpeanut butter wastested
Muldoon et al. (2009)
J. Singh et al. / Food Chemistry 170 (2015) 470–483 477
involve in major food/waterborne outbreaks. The LFIA discussed inthe following section have also been summarised in Tables 1 and 2.These assays can be classified in three groups- (1) commercialassays available in the market, (2) the reports that evaluated thesecommercial assay performances in author’s laboratories and (3)those actually developed and reported by the author but havenot been commercialised yet.
3.1. Salmonella
Salmonella is genus within enterobacteriaceae family andknown to be a leading cause of intestinal illnesses. Contaminateddrinking water is reported to be a common vehicle of typhoidalSalmonella serovars while non typhoidal serovars are common foodborne pathogens. The species has a widespread occurrence in
478 J. Singh et al. / Food Chemistry 170 (2015) 470–483
animals, especially in poultry and swine. Environmental sources ofthe organism include water, soil, insects, factory surfaces, kitchensurfaces, animal faeces, raw meats, raw poultry, and raw seafoodsetc. Food borne illnesses caused by Salmonella are a significantcause of health problems. The most common serovar of Salmonellabehind food borne illnesses are Salmonella typhimurium andS. enteritidis (Vugia et al., 2004). Salmonella typhi and theparatyphoid bacteria normally cause septicaemia and producetyphoid or typhoid-like fever in humans. Rapid, specific andinexpensive detection methods can help in preventing the occur-rence of infections to some extent.
A lateral flow test was reported for the detection of onlySalmonella enteritidis (SE) from egg which was a major cause ofhuman salmonellosis (82%) in U.S. between 1985 and 1998 (Seo,Holt, Stone, & Gast, 2003). Homogenised egg samples were spikedwith 100 ll of SE cell suspension to make a final concentration of10 cells/ml. Enrichment was done in buffered peptone broth at37 �C for 24 h. This study employed the use of a new method forantigen extraction from the egg contents using a mixture of oleicacid and caprylic acid (2:1). This extraction method improved thedetection limit of the assay by 10-fold (106–105 cells/ml) comparedto the conventional PBS dilution method. The extraction procedurehas added advantage of providing effective wicking of the sample.It is an easy alternative to the conventional pre-enrichment orselective enrichment steps required before final detection infood/water samples. A multiplex immunochromatographic teststrip for detection of S. typhimurium and S. enteritidis in chickensamples was reported (Moongkarndi, Rodpai, & Kanarat, 2011). Aspecific polyclonal rabbit antibody was conjugated to goldnanoparticles and monoclonal anti S. typhimurium and S. enteritidisantibodies were immobilised on nitrocellulose membrane. The teststrips detected 104 and 106 cfu/ml of S. typhimurium and S. enteri-tidis from culture samples. The sensitivity in the spiked sampleswas observed to be 98.89% and 87.50% for of S. typhimurium andS. enteritidis, respectively. Reveal S. enteritidis (SE) is a lateralflow-based immunodiagnostic assay used for rapid detection ofS. enterica serovar Enteritidis from pooled shell eggs and environ-mental samples. This assay uses highly specific antibodies to accu-rately detect S. enteritidis. Jagadeesan et al. (2011), compared theperformance of this test against reference procedures for detectionof S. enteritidis from both pooled shell eggs (after spiking andenrichment) and environmental samples. Reveal SE exhibited100% sensitivity and 100% specificity in comparison to the refer-ence method with pooled shell eggs and 245 natural drag swabs.147 poultry house environmental samples were screened by anexternal laboratory, giving Reveal SE sensitivity of 100% and spec-ificity of 90%. Inoculation trials with drag swabs resulted in 96%sensitivity and 100% specificity. An enrichment procedure was alsodeveloped and validated for detection of S. enteritidis from pooledshell egg samples. This enrichment procedure can be used in con-junction with the Reveal SE test and offer a significant enrichmenttime savings of 96 h. A gold nanoparticle based strip immunoassayhas been reported for detection and diagnosis of typhoid with itspotential application in clinic. The assay specifically detectsS. enterica serovar typhi by targeting its capsular polysaccharideVi antigen, an important virulence factor. The Vi antigen is alsopresent in serovar Dublin and Paratyphi C, however the specificitystudies were not carried out with these serovars due to lessfrequency of incidence of these serovars than serovar typhi. Theassay reportedly did not detect S. enterica serovar typhimurium asthis serovar lacks Vi antigen. 30 nm Gold nanoparticles werenon-covalently coupled with anti-Vi antibody and used to developa sandwich type immunostrip assay by coating anti-salmonellapolyclonal IgG on Nitrocellulose membrane. The detection limitin the standard and clinical strains was reported to be 104 cells/mL and 102 cells/mL. This difference in detection limits was
attributed to the variation in expression of Vi antigen in differentstrains (Pandey, Suri, Chaudhry, Tiwari, & Rishi, 2012). Liu,Yeung, Chen, Yeh, and Hou (2013), make use of a 16S rRNA/DNAfor developing a gold nanoparticle based lateral flow assay forsalmonella detection. The probe DNA is complementary to 16SrRNA/DNA of salmonella and conjugated to gold nanoparticlesand biotin. The antiavidin antibody is immobilised on the nitrocel-lulose membrane. The probe is allowed to hybridise with DNA/RNAsamples isolated from salmonella cultures followed by incubationwith avidin. Test strip was dipped in this solution to get the results.Nucleic acids from 107 cultured salmonella cells were detected in30 min. The assay was specific but requires additional time forDNA/RNA isolation and hybridization with probe.
Besides developing LFIA, some laboratories have also evaluatedthe LFIAs developed by others in their own laboratories. In thiscontext, an immunochromatographic strip for salmonella detectionwas evaluated (Bautista, Elankumaran, Arking, & Heckert, 2002).The sample pad was coated with a dried extraction buffer that aftercoming in contact with test sample releases the antigen from intactsalmonella cells. Antigen binds with gold antibody conjugate andfurther follows the principle of lateral flow assay. The analyticalsensitivity of the test with pure colonies of different salmonellaspp. was in the range of 104–105 cfu/ml. The strip had a sensitivityof 12.3% when directly tested for S. typhimurium in chicken faeces.The sensitivity was further improved to more than 90% by pre-enrichment in different media. The assay successfully detected19 of 22 strains of Salmonella spp. and did not detect 27 differententeric bacteria. DuPont™ Lateral flow System Test is tested andcertified by AOAC for Salmonella detection in variety of foods(http://www2.dupont.com/Qualicon/en_US/products/Lateral_Flow_System/lfs_ecoli.html). This test requires pre-enrichment for 22 hin case of liquid eggs and processed foods and 32 h for raw meatsand carcass rinses. Another such assay is marketed by RapdChekSelect for Salmonella detection in food samples (http://www.romerlabs.com/en/products/food-pathogen-testing/salmonella/).It includes a phage based negative selection of any cross reactingbacteria from the test sample with two step enrichment for�32–48 h. The test strip is directly added into the sample tubeand results are available within 10 min in both the test systemssingle red line indicates a negative result and two red lines indicatepositive results. Rapidchek select has been validated by somegroups for detecting Salmonella in meat and Meat products(Torlak, Akan, & _Inal, 2012) and poultry house drag swabs, shellegg pools, and chicken carcass rinsates (Muldoon et al., 2009). Bothvalidation studies showed acceptable degree of correlationbetween the results of Rapidchek select and reference method usedin respective studies.
3.2. E. coli O157
E. coli are gram negative bacteria and the natural inhabitants ofhuman and animal gut. The major serogroup of shiga toxinproducing E. coli (O157) have been implicated in various food borneinfections and is a major cause of hemorrhagic colitis and haemo-lytic-uremic syndrome (Gyles, 2007). Based on serotyping, E. coliO157:H7 is the predominant disease causing strain. Major sourcesof E. coli O157 are ground beef, unpasteurised milk, juice, sprouts,lettuce and salami. Cattle are the principal reservoir of entero-haemorrhagic E. coli (EHEC) and the majority of large outbreakshave been foodborne (Welinder-Olsson et al., 2004). Rapid andsensitive tests which can preliminary characterise these EHEC infood samples can effectively control the risk presented by them.
Jung, Jung, and Kweon (2005), also developed an immunochro-matographic assay for E. coli O157 detection in various samples(raw beef, pork, bovine and swine feces). The assay sensitivitywas 1.8 � 10(5) cfu/ml without enrichment and 1.8 cfu/ml after
J. Singh et al. / Food Chemistry 170 (2015) 470–483 479
enrichment. The specificity of the IC strip was tested with 48pure-cultured bacteria, including 32 E. coli strains and 16 non-E.coli strains. Amongst 16 non-E. coli strains, only Citrobacter amalo-naticus yielded a positive signal. The specificity of the strip washigher with pork (98.8%) than with bovine feces (87.9%) and swinefeces (93.4%). Wang, Chen, Hu, and Li (2006) reported and testedthe feasibility of a sandwich immunochromatographic strip withvarious food samples (milk powder, flour, starch, coffee, biscuit,cake, jelly and juice) for detecting E. coli O157. The sensitivity ofthe test was 1 � 105 cfu/ml. No cross-reaction with 30 strains of24 species in enterobacteriaceae (including non-O157 E. coli,Salmonella, Shigella, Proteus, Citrobacter, Enterobacter, Serratia andYersinia), Staphylococus, Listeria, Aeromonas and Vibrios was found.Zhao et al. (2010), developed an immunochromatographic stripusing biotin-streptavidin binding for E. coli O157:H7 detection.The anti E. coli polyclonal antibody was biotinylated and jettedon the sample pad so that the sample containing E. coli will interactwith it to form an antigen–antibody complex. Colloidal gold cou-pled with anti E. coli MAb was coated on the reservoir matrixand it interacts with the biotinylated antigen–antibody complexto form a sandwich. As this sandwich moves further along themembrane, it interacts with the streptavidin immobilised at thetest line of membrane to produce a visual colour. The assay wasable to detect 2.3 cfu/ml after enrichment and 2.3 � 103 cfu/mlwithout enrichment, which was reportedly 100 times higher thanthe immunochromatographic strip assay of Jung et al. The stripwas tested with spiked samples of beef, milk, cakes and waterand showed a specificity ranging from 94.9 to 99.2 in various foodsamples. The specificity of the strip with pure cultures was 98.5%.Qi et al. (2011) demonstrated the use of immunomagneticnanoparticles (IMPs) as a tool for enrichment of E. coli O157:H7 fol-lowed by its detection using gold nanoparticle based LFIA. TheIMPS (50 nm) were coupled with anti E. coli O157:H7 polyclonalantibodies. These antibody coupled IMPs were added into pure/contaminated food samples to gather or collect the bacteria. After10 min, the IMPs were separated using magnetic separator fol-lowed by their washing. IMPs enriched with E. coli O157:H7 wereplaced in 60 �C water bath for 15 min followed by magnetic sepa-ration. This supernatant was then evaluated using immunochro-matographic assay. The enrichment step was shown to increasethe assay sensitivity from 105 cfu/ml (without enrichment) to103 cfu/ml (IMP enrichment). When IMP enriched bacteria wasdetected in spiked milk, purified water and beef samples, positivityrate was found to be 80% and the sensitivity and specificity wasincreased to 95.5% and 100% respectively compared to the normalLFIA method i.e. without enrichment (sensitivity 29.6% and 93.3%).However, the use of IMPs associated bacteria for testing with thestrip may delay the assay time and interfere with assay flow dueto their larger size. Secondly, number of binding sites of antigenis occupied by the IMP-bound anti E. coli antibody which can affectthe colour intensity of colloidal gold particles. To alleviate theseproblems the IMP enriched bacteria were detached by heatingand then used for analysis by the strip assay. This could furtherincrease the assay time. Yonekita et al. (2013a), has reported theuse of antimicrobial peptide (AMP) for developing a multiplex LFIAto detect three different serogroups (O157, O26, O111) of shigatoxin producing E. coli. The assay specifically detected the targetserogroups in a single strip using cultured as well as enrichedspiked ground beef samples. However, the sensitivity was low inbeef samples even after 18 h enrichment. But the assay demon-strated the use of AMPs as probe for developing LFIA for the firsttime. Due to the broad binding capacity of AMPs, they can bepotentially used for detecting any set of microbes and multipletargets simultaneously. Further, the binding capacity of theseAMPs is not necessarily related with their antimicrobial activity,hence mutant AMP probes can be produced to increase the assay
sensitivity. AMPs like CP1 have antimicrobial activity against num-ber of gram positive and gram negative bacteria. Such AMPs can beidentified and used for developing multiplex assays. Terao et al.(2013) developed and evaluated an immunochromatographicassay for direct detection of E. coli O111 in food after enrichment.The assay yielded positive results with meat samples (in whichthe original load of the bacteria was low 1.6 � 100 to 1.6 �101 cfu/25 g of food) only after enrichment in the modified E. colibroth with novobiocin for 22 h at 42 �C. The results correspondedwell with the PCR results. The assay specificity was found to be100% when tested with cell suspensions of 8 E. coli O111 strainsand 77 non-E. coli O111 strains. The minimum detection limitsfor the E. coli O111 strains were 1.8 � 103–5.6 � 105 cfu/ml of cellsuspension. Moreover, the LFIA was able to detect live cultures orthose killed by autoclaving at nearly the same level of sensitivity.Yonekita, Fujimura, Morishita, Matsumoto, and Morimatsu(2013b) reported an immunochromatographic strip for detectingShiga toxin-producing Escherichia coli (STEC) O26 in food samples(ground beef, beef liver, ground chicken, alfalfa sprout, radishsprout, spinach, natural cheese, and apple juice). The IC strip wasable to detect E. coli O26 at a level of approximately 1 cfu/25 g offood samples after 18-h enrichment. The IC strip results were in100% agreement with the results of the culture method and PCRassay. The minimum detection limits for E. coli O26 were2.2 � 103–1.0 � 105 cfu/ml. The specificity of the IC strip as testedwith pure cultures of 67 E. coli and 22 non-E. coli strains was 100%.The IC strip results were in accordance with the PCR results using115 meat samples from the market.
E. coli and Salmonella contaminated raw sprouts have resultedinto food borne diseases in recent years. To determine the safetyof individual batches of sprouts, FDA has proposed testing of spentirrigation water during sprout production. Fratamico and Bagi(2001) compared a commercial immunochromatographic assay(Quix™ E. coli O157 sprout assay) with TaqMan
�assay for detecting
E. coli O157:H7 in alfalfa spent sprout irrigation water. The LFIAwas able to detect the bacteria in water collected after 30 h(without enrichment) and 8 h (after enrichment) from the startof sprouting process. Although the LFIA was less sensitive thanPCR assay but was more rapid. Likewise, Gryko, Sobieszczanska,Stopa, and Bartoszcze (2002) compared the sensitivities of a multi-plex PCR assay and an immunochromatographic method (Quix™)marketed by New horizons Diagnostics, Columbia, MD, to detectE. coli O157:H7 in minced beef. Both these methods had compara-ble sensitivities and were able to detect 1–100 bacteria in the beefsample but after enrichment for 8 h in a non-selective medium.The kit also employed an extraction reagent to release antigenfrom the intact organisms which is then subsequently used fordetection. However, the assay time was almost double (15 min)than that recommended by the manufacturer (7 min).
Another rapid lateral flow assay kit ‘‘SMART™ II Rapid E. coliO157 strip test’’ was developed to detect E. coli O157 in enrichedfood samples (http://lifesci.com/ecoli/). The analytical sensitivityof the kit was claimed to be 3.3 � 104 cfu/ml. After enrichment,the kit was able to detect 1 cfu/25 g of the sample. The DuPont™lateral flow systems is also designed and marketed to detect E. coliO157 in 25 or 375 g of raw ground beef and boneless beeftrim (http://www2.dupont.com/Qualicon/en_US/products/Lateral_Flow_System/lfs_ecoli.html). Enrichment is required for 8 h and10–15 h for 25 and 375 g of sample respectively. The detectionlimit of the test is reported to be 1 cfu/25 g of sample. Infectionswith shiga toxin producing E. coli (STEC) have also been implicatedthrough various food types such as beef, cheese, lettuce andspinach. Arakawa, Sawada, Takatori, Lee, and Hara-Kudo (2011)compared various types of enrichment broth for detecting STECusing an immunochromatographic strip. They recommended theuse of mEC with novobiocin for enrichment at 42 �C, 8hr along
480 J. Singh et al. / Food Chemistry 170 (2015) 470–483
with polymyxin B treatment for rapid detection using the immu-nochromatography kit. Although the details of the kit which wasused for this detection were not given.
4. Factors affecting the performance of LFIA
The performance of any lateral flow assay for bacterial detectioncan be assessed in terms of its lower detection limit, specificity(false negatives/positives) and the total time taken for the assay.There are number of factors that can influence these parametersand hence the assay performance. Such factors can be broadly cat-egorised as (a) factors inherent to matrix/pathogen (test sample)(b) factors inherent to the LFIA system (c) capture reagent used.
(a) Factors inherent to matrix/pathogen (test sample): there arenumber of possible factors owing to characteristic nature ofthe test sample that can exert effect on the LFIA perfor-mance. Firstly, the complexity of the sample matrix: theease, with which the bacterial pathogen suspected to bepresent in the food/water sample, interacts with the capturereagent immobilised at test line is governed by the nature oftest sample. For example, samples like raw meat, beef, egg,are more complex in their constitution as compared to milk,juices or water samples. In these complex sample matrices,pathogens can be present in small numbers along with largenumbers of background microflora in a complex samplematrix (Kelly, Stevens, & Lee-Ann, 2004). Additionally, thetype of food being analysed, e.g., fruits vs. vegetables orsmooth surface vs. an irregular surface, and pH of foodmatrix in the extraction solution can affect the attachment,release and recovery of pathogenic organisms (Burnett &Beuchat, 2001). Sample complexity can also interfere withthe chemistries and signal transduction associated withemerging rapid technologies slowing down the detectionprocess. To compensate for these negative effects, complexmatrices like raw meat, beef, egg samples, need to be pro-cessed (homogenisation, separation from interfering mole-cules like proteins or lipids) for antigen extraction and lesscomplex matrices like milk, juices or water samples needto be diluted with appropriate buffer or diluents to minimiseinterfering substances that are inherent to them. The lowerdetection limit of LFIA is affected by the concentration ofbacteria in the processed sample (which is often low afterprocessing leading to less sensitive assays). Secondly, inorder to increase the concentration of the bacterial antigenin processed sample, enrichment is done in an appropriatemedium for 8–72 h (depending upon the sample and proto-col). With processed and enriched samples, it could beexpected to have a sensitive assay. All LFIAs developed forSalmonella/E. coli detection in food/water samples (Tables 1and 2) have employed the sample processing and enrich-ment steps. These steps although improves the sensitivityof the assay, but increases the total time taken for the anal-ysis, depends upon a laboratory set up and hence defeats oneof the advantages of LFIA i.e. on-site detection by the enduser.
(b) Factors inherent to the LFIA system: Besides, nature of testsample, the various components of LFIA assembly also caninfluence the assay performance and therefore need to becarefully chosen. The nature of membrane (chemical compo-sition, pore size, flow rate, etc.) used in the LFIA is most det-rimental in influencing the assay performance. Industries likeWhatman and Millipore are the leading producers of lateralflow assay membranes of different grades. Lee et al. (2012),has discussed the importance of membrane selection indeveloping immunochromatographic assays and compared
various membranes like nitrocellulose 8.0, Immunopore RP,FP, and SP from Whatman, UK. It was concluded that relativemigration speed of analyte and antibody-gold conjugate onthe test strip is important for membrane selection. The mem-brane on which the migration speed of the analyte is muchhigher than gold conjugate is most appropriate for testingsmall analytes. For effective immobilization of capturereagent on the membrane pad, incubation temperature(37 �C, RT), time, volume of antibody immobilised on the testline shall be optimised for every assay. Factors like the dryingtemperature was found to be an important parameter insynthesising membranes as it affects pore structures. A highdrying temperature causes agglomeration of the polymermatrix, and thus smaller pores were observed on the mem-brane surface. This further decreases the membrane lateralliquid migration rate, besides reducing the membrane bind-ing ability for bacteria detection. Results show that bydecreasing polymer concentration, membrane surface poresbecame apparently larger, thus creating a faster lateralmigration speed of the water solution. A larger pore sizeincreases the chance for the bacteria detecting agent to bindonto the pore layers, which ultimately enhances the bacteriadetection ability of the device (Ahmada, Lowa, Shukora,Ismailb, & Sunarti, 2009). To increase the wetability of themembrane, methanol can also be added in the capturereagent which would result in effective immobilization.Although the membranes which are commercially availablethese days are already treated with blocking agents butdepending upon the assay, various blocking agents like BSA,milk powder can be used to reduce the non specific binding.Additionally, the colloidal gold-antibody conjugate (label)prepared shall also be stable and able to effectively competewith the analyte. The material of which the conjugate pad ismade of, should facilitate the release of label. For effectiverelease of label from the conjugate pad the use of detergents(tween-20) and alcohols (methanol) is recommended in therunning buffer. Thus, while developing lateral flow assays,the membrane, sample pad, absorbent pad and conjugatepad should be very carefully chosen. For more sensitiveassays, membranes with slow migration rate can serve thepurpose whereas, for those assays where sensitivity is notan issue and time of assay run is important, membranes withfast migration rates should be the preferred choice.
(c) Capture reagent: the capture reagent employed in develop-ing an LFIA influences the specificity and accuracy of theassay. Antibodies/oligonucleotides/aptamers have been usedfor developing any LFIA. The specificity with which thesecapture reagents detects the analyte governs the numberof false positive or false negatives. Therefore, the captureregents should be developed and so chosen to minimisethe degree of cross-reaction. Although, antibodies/aptamerswhich recognise more than one serotype of a bacteria or dif-ferent bacteria can also prove to be advantageous as theycan be employed for developing multiplex assays.
5. LFIA-a potential tool in HACCP
Although the current generation technologies are quite advancebut in food and beverage industries where mass production is donewe need lower analytical costs, higher automation and integrationwith existing workflow, need results in few hours and not in days,no compromise on performance, high throughput with multiplexcapability etc. To fulfil the norms of Hazard Analysis CriticalControl Points (HACCP), LFIA could act as a boon for the food andbeverage industries as it dramatically will reduce the cost of labtesting without compromising the production quality. Considering
J. Singh et al. / Food Chemistry 170 (2015) 470–483 481
this LFIA have been projected to be used in food industry as per theHACCP norms for pathogen detection by many authors. Aldus et al.(2003) has detected the verotoxigenic E. coli (VTEC) serotype fromraw milk, minced beef, apple juice and salami, with the use of rapiddipstick test device and projected it as useful tool in the develop-ment and/or verification of hazard analysis critical control point(HACCP) plans and other control measures. Liu, Zanardi, Powersand Suman (2012) detected mycotoxin deoxynivalenol (DON), acommon contaminant of cereals such as wheat, barley and cornproduced by a number of species of Fusarium and some otherfungi. They have shown the LIFA with a detection limit of about0.30 mg kg�1 in wheat spiked samples and the results were similarto the results of LC–MS. They reported a LFIA-based DON test,using confocal optical reader with built-in calibration, to be poten-tially useful as a tool in HACCP type approaches to mycotoxin con-trol in the cereal food industry. The implementation of suchtechnologies has potential to provide the consumer with higherquality and healthier products. Lattanzio et al. (2012) also plannedLFIA suitable for rapid mycotoxin monitoring in HACCP plans atdifferent points of the food chain, from harvesting to storage anddistribution of raw cereals. Odumeru and León-Velarde (2012)has proposed the application of rapid methods for the detection,identification, and characterisation of Salmonella for assessmentof the safety of food products like raw materials and food ingredi-ents used in food for food safety programs such as the Hazard Anal-ysis Critical Control Point (HACCP). Rapid check for E. coli and Rapidcheck for Salmonella, clear view and REVEAL are some of the kitsavailable in the market for the rapid detection of the most commonpathogenic strains of E. coli and Salmonella (Tothill & Magan,2003).
6. Conclusions and outlook
Numbers of analytical methods are available to detect bacterialpathogens in different matrices like serum, plasma, stool, food orwater but they suffer from some limitations such as dependencyon costly equipments, trained personnel’s, time consuming, expen-sive and complicated to use. This review paper was intended toprovide an overview of the gold nanoparticle based lateral flowassays that have been either commercialised or evaluated in differ-ent laboratories or just reported in the literature for detection ofenterobacteriaceae family members in food/water samples. Outof various enterobacteriaceae family members, different serotypesof E. coli and Salmonella have been found to be implicated in food/water borne infections. Therefore, the LFIAs have been developedagainst these two members only. The literature also shows thatmost of the developed LFIAs detect these bacterial species in foodsamples. Only two reports were found that evaluated the assayswith water samples. With the deteriorating water quality in thedeveloping countries and looking at the occurrence of water borneinfections, it becomes indispensable to have some point of carediagnostic system for water quality assessment. Since E. coli is amajor indicator organism for water quality monitoring, there existsa scope for developing such rapid LFIAs in this direction.
The sensitivity is a major concern when developing any LFIA,since the infectious dose necessary to cause the disease is verylow for these pathogens for example, for E. coli, it is 2–2000ingested cells (Buchanan & Doyle, 1997). The literature showedthat specimen enrichment in selective broth markedly improvesthe detection limits of the reported LFIAs. The lower detection limitfor E. coli in the reviewed literature lies in the range of 105 cfu/ml(without enrichment) to 1 cfu (after enrichment) but for Salmonellathese are comparatively higher (107 cfu/ml–104 cfu/ml). Themethod and medium used for enrichment significantly influencesassay sensitivity and also enhances the overall assay time. In
addition, it defeats the purpose of using LFIAs outside the labora-tory settings by unskilled persons. Therefore, efforts shall be direc-ted towards simplifying the enrichment/antigen extraction steps inorder to provide rapid and accurate pathogen detection tests withmaximum sensitivity. To improve the specificity of the assay,newer capture reagents like antimicrobial peptides and immuno-magnetic particles may also be evaluated further for their efficacyin LFIAs. In the development of a highly sensitive LFIA, the chal-lenge is also to obtain lateral flow membranes with the desiredmorphologies. The review also concludes that the multiplex assaysfor pathogen detection are still lacking and the future researchshould focus towards multiplexing of the assays as it wouldminimise the time, efforts and overall cost of the assay run.Multiplexing seems to be the future of point of care diagnosticsand shall be taken up further. Thus the present situation demandstranslation of new research findings into safe, stable, and effectivetechnologies that can be applied worldwide, for the benefit of thepoor people living in the developing countries.
References
Abraham, W. (2011). Megacities as sources for pathogenic bacteria in rivers andtheir fate downstream. International Journal of Microbiology, 798292. http://dx.doi.org/10.1155/2011/798292.
Ahmada, A. L., Lowa, S. C., Shukora, S. R. A., Ismailb, A., & Sunarti, A. R. (2009).Development of lateral flow membranes for immunoassay separation.Desalination and Water Treatment, 5, 99–105.
Ailes, E., Budge, P., Shankar, M., Collier, S., Brinton, W., Cronquist, A., et al. (2013).Economic and health impacts associated with a Salmonella typhimuriumdrinking water outbreak � Alamosa, CO, 2008. PLoS ONE, 8, e57439. http://dx.doi.org/10.1371/journal.pone.0057439.
Aldus, C. F., Van Amerongen, A., Ariens, R. M. C., Peck, M. W., Wichers, J. H., & Wyatt,G. M. (2003). Principles of some novel rapid dipstick methods for detection andcharacterization of verotoxigenic Escherichia coli. Journal of Applied Microbiology,95, 380–389.
Altekruse, S. F., Cohen, M. L., & Swerdlow, D. L. (1997). Emerging foodborne diseases.Emerging Infectious Diseases, 3, 85–93.
Arakawa, Y., Sawada, T., Takatori, K., Lee, K. I., & Hara-Kudo, Y. (2011). Rapiddetection of shiga toxin-producing Escherichia coli in ground beef by animmunochromatography kit in combination with short-term enrichment andtreatment for shiga toxin release. Biocontrol Science, 16, 159–164.
Baeumner, A. J., Jones, C., Wong, C., & Price, A. (2004). Development of a microfluidicbiosensor module for pathogen detection. Analytical and Bioanalytical Chemistry,378, 1587–1593.
Bautista, D. A., Elankumaran, S., Arking, J. A., & Heckert, R. A. (2002). Evaluation of animmunochromatography strip assay for the detection of Salmonella sp. frompoultry. Journal of Veterinary Diagnostic Investigation, 14, 427–430.
Blazkova, M., Koet, S., Wicher, J. H. S., Merongen, V., Fukal, Rauch., et al. (2009).Nucleic acid lateral flow immunoassay for the detection of pathogenic bacteriafrom food. Czech Journal of Food Sciences, 27, S350–S353.
Buchanan, R. L., & Doyle, M. P. (1997). Foodborne disease significance of Escherichiacoli O157:H7 and other enterohemorrhagic E. coli. Food Technology, 51(10), 69–76.
Burnett, A. B., & Beuchat, L. R. (2001). Comparison of sample preparation methodsfor recovering Salmonella from raw fruits, vegetables, and herbs. Journal of FoodProtection, 64, 1459–1465.
Buzby, J. C., & Roberts, T. (2009). The economics of enteric infections: Humanfoodborne disease costs. Gastroenterology, 136, 1851–1862.
Cambra, M., Gorris, M. T., Román, M. P., Terrada, E., Garnsey, S. M., Camarasa, E.,et al. (2000). Routine detection of Citrus tristeza virus by directimmunoprinting-ELISA method using specific monoclonal and recombinantantibodies. In J. V. da Graça, R. F. Lee, & R. K. Yokomi (Eds.), Proceedings of the14th IOVC Conference (pp. 34–41). Calif.: IOCV, Riverside.
Chai, F., Wang, C., Wang, T., Li, L., & Su, Z. (2010). Colorimetric detection of Pb2+
using glutathione functionalized gold nanoparticles. ACS Applied Materials &Interfaces, 2, 1466–1470.
Corstjens, P., Zuiderwijk, M., Brink, A., Li, S., Feindt, H., Niedbala, R. S., et al. (2001).Use of up-converting phosphor reporters in lateral-flow assays to detect specificnucleic acid sequences: A rapid, sensitive DNA test to identify humanpapillomavirus type 16 infection. Clinical Chemistry, 47, 1885–1893.
Crowther, J. R. (1995). ELISA theory and practice. 0-89603-279-5. USA: Humana PressInc.
Danks, C., & Barker, I. (2000). On-site detection of plant pathogens using lateral-flowdevices. EPPO Bulletin, 30421–30426.
Doumanas, Bonenberger, J., Michael (2006). Overcoming sensitivity limitations oflateral-flow immunoassays with a novel labeling technique. IVD Technology,2006-05-01 03:00.
Eisinger, R., WKhalil, M.H., Katz, D.H., Sargeant, R.B. (1990). U.S. Patent No.4,943,522. Washington, DC: U.S. Patent and Trademark Office.
Espinosa, A. C., Arias, C. F., Sánchez-Colón, S., & Mazari-Hiriart, M. (2009).Comparative study of enteric viruses, coliphages and indicator bacteria for
482 J. Singh et al. / Food Chemistry 170 (2015) 470–483
evaluating water quality in a tropical high-altitude system. EnvironmentalHealth, 8–49.
Fratamico, P. M., & Bagi, L. K. (2001). Food-borne pathogens comparison of animmunochromatographic method and the TaqMan1 E. coli O157:H7 assay fordetection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water andin sprouts after blanching. Journal of Industrial Microbiology & Biotechnology, 27,129–134.
Gillet, P., Mukadi, P., Vernelen, K., Van Esbroeck, M., Muyembe, J., Bruggeman, C.,et al. (2010). External quality assessment on the use of malaria rapid diagnostictests in a non-endemic setting. Malaria Journal, 9–359.
Goldman, E. R., Clapp, A. R., Anderson, G. P., Uyeda, H. T., Mauro, J. M., Medintz, I. L.,et al. (2004). Multiplexed toxin analysis using four colors of quantum dotfluororeagents. Analytical Chemistry, 76, 684–688.
Gryko, R., Sobieszczanska, M. B., Stopa, P. J., & Bartoszcze, M. A. (2002). Comparisonof multiplex PCR, and an immunochromatographic method sensitivity for thedetection of Escherichia coli O157: H7 in minced beef. Acta MicrobiologicaPolonica, 51, 121–129.
Gyles, C. L. (2007). Shiga toxin-producing Escherichia coli: An overview. Journal ofAnimal Science, 85, E45–E62.
Ho, J. A., & Huang, M. (2005). Application of a liposomal bioluminescent label in thedevelopment of a flow injection immunoanalytical system. Analytical Chemistry,77, 3431–3436.
Huhtinen, P., Pelkkikangas, A. M., Jaakohuhta, S., Lövgren, T., & Härmä, H. (2004).Quantitative, rapid europium (III) nanoparticle-label- based all-in-one dry-reagent immunoassay for thyroid- stimulating hormone. Clinical Chemistry, 50,1935–1936.
Jagadeesan, B., Curry, S., Foti, D., Peterson, L., Wilson, R., & Mozola, M. (2011). RevealSalmonella enteritidis test for detection of Salmonella enteritidis in shell eggs andenvironmental samples. Journal of AOAC International, 94, 1125–1137.
Jung, B. Y., Jung, S. C., & Kweon, C. H. (2005). Development of a rapidimmunochromatographic strip for detection of Escherichia coli O157. Journal ofFood Protection�, 10, 2012–2241.
Kelly, A., Stevens, J., & Lee-Ann (2004). Bacterial separation and concentrationfrom complex sample matrices: A review. Critical Reviews in Microbiology, 30,7–24.
Knecht, M. R., & Sethi, M. (2009). Bio-inspired colorimetric detection of Hg2+ andPb2+ heavy metal ions using Au nanoparticles. Analytical and BioanalyticalChemistry, 394, 33–46.
Lafferty, K. D. (2009). The ecology of climate change and infectious diseases. Ecology,90, 888–900.
Lee, J. Y., Kim, Y. A., Kim, M. Y., Lee, Y. T., Hammock, B. D., & Lee, H. S. (2012).Importance of membrane selection in the development ofimmunochromatographic assays for low-molecular weight compounds.Analytica Chimica Acta, 757, 69–74.
Li, M., & Gu, J.-D. (2011). Advances in methods for detection of anaerobicammonium oxidizing (anammox) bacteria. Applied Microbiology andBiotechnology, 90, 1241–1252.
Linares, E. M., Kubota, L. T., Michaelis, J., & Thalhammer, S. (2012). Enhancement ofthe detection limit for lateral flow immunoassays: Evaluation and comparisonof bioconjugates. Journal of Immunological Methods, 375, 264–270.
Liu, C. C., Yeung, C. Y., Chen, P. H., Yeh, M. K., & Hou, S. Y. (2013). Salmonelladetection using 16S ribosomal DNA/RNA probe gold nanoparticles and lateralflow immunoassay. Food Chemistry, 141, 2526–2532.
Liu, J., Zanardi, S., Powers, S., & Suman, M. (2012). Development and practicalapplication in the cereal food industry of a rapid and quantitative lateral flowimmunoassay for deoxynivalenol. Food Control, 26, 88–91.
Lattanzio, V. M., Nivarlet, N., Lippolis, V., Gatta, S. D., Huet, A. C., Delahaut, P., et al.(2012). Multiplex dipstick immunoassay for semi-quantitative determinationof Fusarium mycotoxins in cereals. Analytica Chimica Acta, 718, 99–108.
Lonnberg, M., & Carlsson, J. (2001). Quantitative detection in the attomole range forimmunochromatographic tests by means of a flatbed scanner. AnalyticalBiochemistry, 293, 224–231.
Lou, S. C., Patel, C., Ching, S., & Gordon, J. (1993). One-step competitiveimmunochromatographic assay for semiquantitative determination oflipoprotein(a) in plasma. Clinical Chemistry, 39, 619–624.
Moongkarndi, P., Rodpai, E., & Kanarat, S. (2011). Evaluation of animmunochromatographic assay for rapid detection of Salmonella entericaserovars Typhimurium and Enteritidis. Journal of Veterinary DiagnosticInvestigation, 23, 4797–4801.
Muldoon, M. T., Sutzko, J., Li, M., Olsson-Allen, A. C., Teaney, G., & Gonzalez, V.(2009). RapidChek SELECT Salmonella performance tested method 080601.Journal of AOAC International, 92, 1890–1894.
Navas, J., Ortiz, S., Lopez, P., Jantzen, M. M., Lopez, V., & Martinez-Suarez, J. V. (2006).Evaluation of effects of primary and secondary enrichment for the detection oflisteria monocytogenes by real-time PCR in retail ground chicken meat.Foodborne Pathogens and Disease, 3, 347–354.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N.,et al. (2000). Loop-mediated isothermal amplification of DNA. Nucleic AcidsResearch. 12Pp. e63.
Odumeru, J. A., & León-Velarde, C. G. (2012). Salmonella detection methods for foodand food ingredients. In B. S. M. Mahmoud (Ed.), Salmonella – A dangerousfoodborne pathogen (pp. 373–392). InTech.
Pandey, S. K., Suri, C. R., Chaudhry, M., Tiwari, R. P., & Rishi, P. (2012). A goldnanoparticles based immuno-bioprobe for detection of Vi capsularpolysaccharide of Salmonella enterica serovar Typhi. Molecular BioSystems, 8,1853–1860.
Paton, J. C., & Paton, A. W. (1998). Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clinical Microbiology Reviews, 11, 450–479.
Piper, R. C., Buchanan, I., Choi, Y. H., & Makler, M. T. (2011). Opportunities forimproving pLDH-based malaria diagnostic tests. Malaria Journal, 10–213. http://dx.doi.org/10.1186/1475-2875-10-213.
Posthuma-Trumpie, G. A., Korf, J., & Amerongen, A. V. (2008). Development of acompetitive lateral flow immunoassay for progesterone: Influence of coatingconjugates and buffer components. Analytical and Bioanalytical Chemistry, 392,1215–1223.
Posthuma-Trumpie, G. A., Korf, J., & Amerongen, A. V. (2009). Lateral flow (immuno)assay: Its strengths, weaknesses, opportunities and threats. A literature survey.Analytical and Bioanalytical Chemistry, 393, 569–582.
Qi, H., Zhong, Z., Zhou, H., Deng, C., Zhu, H., Li, J., et al. (2011). A rapid and highlysensitive protocol for the detection of Escherichia coli O157:H7 based onimmunochromatography assay combined with the enrichment technique ofimmunomagnetic nanoparticles. International Journal of Nanomedicine, 6,3033–3039.
Qian, S., & Bau, H. H. (2004). Analysis of lateral flow biodetectors: Competitiveformat. Analytical Biochemistry, 326, 211–224.
Rompre, A., Servais, P., Baudart, J., de-Roubin, M.-R., & Laurent, P. (2002). Detectionand enumeration of coliforms in drinking water: Current methods andemerging approaches. Journal of Microbiological Methods, 49, 31–54.
Saleh, M., Soliman, H., & El-Matbouli, M. (2008). Loop-mediated isothermalamplification as an emerging technology for detection of Yersinia ruckeri thecausative agent of enteric red mouth disease in fish. BMC Veterinary Research.http://dx.doi.org/10.1186/1746-6148-4-31.
Seo, K. H., Holt, P. S., Stone, H. D., & Gast, R. K. (2003). Simple and rapid methods fordetecting Salmonella enteritidis in raw eggs. International Journal of FoodMicrobiology, 87, 139–144.
Tao, Z. Y., Zhou, H. Y., Xia, H., Xu, S., Zhu, H. W., Culleton, R. L., et al. (2011).Adaptation of a visualized loop-mediated isothermal amplification techniquefor field detection of Plasmodium vivax infection. Parasites & Vectors, 21, 4–115.
Terao, Y., Yonekita, T., Morishita, N., Fujimura, T., Matsumoto, T., & Morimatsu, F.(2013). Potential rapid and simple lateral flow assay for Escherichia coli O111.Journal of Food Protection, 76, 755–761.
Torlak, E., Akan, I. M., & _Inal, M. (2012). Evaluation of RapidChek select for thescreening of Salmonella in meat and meat products. Journal of MicrobiologicalMethods, 90, 217–219.
Tothill, I. E., & Magan, N. (2003). Rapid detection methods for microbialcontamination. Rapid and On-Line Instrumentation for Food Quality Assurance,89, 36.
Tripathi, V., Nara, S., Singh, K., Singh, H., & Shrivastav, T. G. (2012). A competitiveimmunochromatographic strip assay for 17-[alpha]-hydroxy progesteroneusing colloidal gold nanoparticles. Clinica Chimica Acta, 413, 262–268.
Van Amerongen, A., Wichers, J. H., Berendsen, L. B. J. M., Timmermans, A. J. M.,Keizer, G. D., van Doorn, A. W. J., et al. (1993). Colloidal carbon particles as a newlabel for rapid immunochemical test methods: Quantitative computer imageanalysis of results. Journal of Biotechnology, 30, 185–195.
Vugia, D. J., Samuel, M., Farley, M. M., Marcus, R., Shiferaw, B., Shallow, S., et al.(2004). Invasive Salmonella infections in United States, FoodNet, 1996–1999:Incidence, serotype distribution and outcome. Clinical Infectious Diseases, 38,S149–S156.
Wang, H. (2002). Rapid method for detection and enumeration of Campylobacterspp. in food. Journal of AOAC International, 85, 996–999.
Wang, J., Chen, W. N., Hu, K. X., & Li, W. (2006). Development of a gold-immunochromatography test for rapid detecting E. coli O157. Wei Sheng Yan Jiu,35, 439–441.
Wang, J., Wang, X., Li, Y., Yan, S., Zhou, Q., Gao, B., et al. (2012). A novel, universaland sensitive lateral-flow based method for the detection of multiple bacterialcontamination in platelet concentrations. Analytical Sciences, 28, 237–241.
Welinder-Olsson, C., Stenqvist, K., Badenfors, M., Brandberg, A., Floren, K., Holm, M.,et al. (2004). EHEC outbreak among staff at a children’s hospital – Use of PCR forverocytotoxin detection and PFGE for epidemiological investigation.Epidemiology and Infection, 13, 243–249.
Wernery, U., Kinne, J., Jahans, K. L., Vordermeier, H. M., Esfandiari, J., Greenwald, R.,et al. (2007). Tuberculosis outbreak in a dromedary racing herd and rapidserological detection of infected camels. Veterinary Microbiology, 122, 108–115.
WHO (2008). World Health Organization initiative to estimate the global burden offoodborne diseases – A summary document. Geneva: World Health Organization.
Xiaohu, X., Ye, X., Xilin, Z., & Qingge, L. (2009). Lateral flow immunoassay usingeuropium chelate-loaded silica nanoparticles as labels. Clinical Chemistry,55–61.
Yang, D., Ma, J., Zhang, Q., Yang, N., Li, J., Raju, P. A., et al. (2013). Polyelectrolyte-coated gold magnetic nanoparticles for immunoassay development: Towardpoint of care diagnostics for syphilis screening. Analytical Chemistry, 85,6688–6695.
Yonekita, T., Fujimura, T., Morishita, N., Matsumoto, T., & Morimatsu, F. (2013b).Simple, rapid, and reliable detection of Escherichia coli O26 usingimmunochromatography. Journal of Food Protection, 76(5), 748–754.
Yonekita, T., Ohtsuki, R., Hojo, E., Morishita, N., Matsumoto, T., Aizawa, T., et al.(2013a). Development of a novel multiplex lateral flow assay using anantimicrobial peptide for the detection of Shiga toxin-producing Escherichiacoli. Journal of Microbiological Methods, 93, 251–256.
Zaytseva, N. V., Montagna, R. A., Lee, E. M., & Baeumner, A. J. (2004). Multi-analytesingle-membrane biosensor for the serotype-specific detection of Dengue virus.Analytical and Bioanalytical Chemistry, 380, 46–53.
J. Singh et al. / Food Chemistry 170 (2015) 470–483 483
Zhao, X., He, X., Li, W., Liu, Y., Yang, L., & Wang, J. (2010). Development andevaluation of colloidal gold immunochromatographic strip for detection ofEscherichia coli O157. African Journal of Microbiology Research, 4, 663–670.
Zhao, W., Lu, J., Ma, W., Xu, C., Kuang, H., & Zhu, S. (2011). Rapid on-site detection ofAcidovorax avenae subsp. citrulli by gold-labeled DNA strip sensor. Biosensorsand Bioelectronics, 26, 4241–4244.
Zuiderwijk, M., Tanke, H. J., Niedbala, R. S., & Corstjens, P. L. A. M. (2003). Anamplification-free hybridization-based DNA assay to detect Streptococcuspneumoniae utilizing the up-converting phosphor technology. ClinicalBiochemistry, 36, 401–403.