REVIEW

Approaches for the detection of harmful algal bloomsusing oligonucleotide interactions

Karen L. Bruce & Sophie C. Leterme & Amanda V. Ellis & Claire E. Lenehan

Received: 27 June 2014 /Revised: 2 September 2014 /Accepted: 15 September 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Blooms of microscopic algae in our waterways arebecoming an increasingly important environmental concern.Many are sources of harmful biotoxins that can lead to deathin humans, marine life and birds. Additionally, their biomasscan cause damage to ecosystems such as oxygen depletion,displacement of species and habitat alteration. Globally, thenumber and frequency of harmful algal blooms has increasedover the last few decades, and monitoring and detection strat-egies have become essential for managing these events. Thisreview discusses developments in the use of oligonucleotide-based ‘molecular probes’ for the selective monitoring ofalgal cell numbers. Specifically, hybridisation techniques willbe a focus.

Keywords Harmful algal bloom . Nucleic acids . Bioassays

Introduction

The term ‘harmful algal bloom’ (HAB) refers to a rapidproliferation of algal cells in an aquatic environment withharmful consequences. This may be due to the production ofbiotoxins or the accumulated biomass altering the ecosystemin a harmful manner [1]. In some cases, the number of cellsmay increase to such an extent that they discolour the water,

such as in those of ‘red tides’. Globally, HABs have becomemore prevalent over the last few decades and the number oftoxins and toxic species has increased [2]. Most coastal coun-tries are now threatened by HAB formations which wereunheard of several decades ago. The cause of the expansionis still not thoroughly understood however there are claimsthat environmental impacts as well as human impacts may beto blame [1]. This increased prevalence has in-turn impactedon local economies due to sea life mortality and cessation ofseafood trade as many of the organisms are unfit for consump-tion. Bioaccumulation of biotoxins in shellfish and filter feed-ing bivalves can result in poisoning of higher organismsthrough the consumption of contaminated shellfish [3].These can have a range of side effects and are classified asparalytic (PSP), amnesic (ASP), diarrhetic (DSP), neurotoxic(NSP) and azaspiracid (AZP) shellfish poisoning, and arepotentially lethal [4–6]. With the exception of ASP (wherediatoms are the biotoxin source), all of the poisonousbiotoxins result from a type of algae known as dinoflagellates.The most commonly encountered of these include organismsfrom the Karenia, Alexandrium and Pseudo-nitzschia genus[7].

The average duration of HABs and their geographic spreadhas increased over the past few decades. A number of factorshave been hypothesised to be linked to this increase in prolif-eration, but the underlying causes are not well understood.This could be due to improved detection strategies, howevercurrent consensus is that the increase in global population hasled to increased nutrient pollution resulting in excessive algalgrowth [8, 9]. The increased geographic spread of species hasbeen linked to the release of ballast water from ships andthrough natural water movement such as currents and storms[1, 10]. Alternately, it has been suggested that increasedfishing could be affecting the food chain leading to an unbal-anced relationship between organisms that feed on the algae tocontrol growth [8].

K. L. Bruce :C. E. Lenehan (*)School of Chemical and Physical Sciences, Flinders University,Sturt Road, Bedford Park, Adelaide, SA 5042, Australiae-mail: claire.lenehan@flinders.edu.au

S. C. LetermeSchool of Biological Sciences, Flinders University, Sturt Road,Bedford Park, Adelaide, SA 5042, Australia

A. V. EllisFlinders Centre for Nanoscale Science and Technology, FlindersUniversity, Sturt Road, Bedford Park, Adelaide, SA 5042, Australia

Anal Bioanal ChemDOI 10.1007/s00216-014-8193-x

Given the increase in the number and frequency of HABsover the last few decades, there has been a significant researcheffort towards methods for monitoring the levels of suchspecies in the environment. The development of improveddetection methods and will allow greater understanding ofHAB lifecycles, and could lead to improved strategies forHAB prevention and/or control. Currently, monitoring strate-gies include (1) satellite monitoring of ocean colour [11, 12],(2) analysis for the presence of exuded biotoxins [2, 13, 14]and (3) localised monitoring of cell densities of the causativeorganisms [15, 16].

Remote sensing using satellite monitoring typically con-centrates on the detection of ocean spectral changes resultingfrom a bloom. This approach can be complicated by atmo-spheric conditions and the presence of coloured dissolvedorganic matter and suspended solids. For further informationon this approach the reader is referred to extensive reviews byBlondeau-Patissier et al. [17] and Moisan et al. [18]. Biotoxinanalysis generally focuses on either the determination of thetoxin concentration in bivalves such as mussels, or the con-centration in the waterway [2, 13]. Detection is typicallyachieved through liquid chromatography with either massspectrometry [19–21] or fluorescence detection [22–25].Generally, these methods require time consuming extractionand pre-concentration sample preparation steps [26].Examples include passive sampling devices and solid phaseadsorption toxin tracking for water sampling [14, 27]. Whilstimportant for monitoring the potential toxic effects during andpost bloom, this approach can be expensive and the time delaybetween sampling and analysis can limit monitoring in theinitial stages of a bloom [28, 29].

Monitoring of cell densities can be achieved manuallyusing light microscopy (LM). This approach has the advan-tage of being able to determine cell density during a bloomwhile also identifying the causative species. However, in somecases, species identification is difficult because of the similarmorphologies of toxic and non-toxic species of the samegenus [30]. Additionally, as many species are motile, fixationof the cells must occur prior to LM. This process can com-promise the structural integrity of the cell, further complicat-ing species identification. Although useful for identifyingcausative species, LM is labour-intensive and time-consum-ing, and is unsuitable for rapid at-site monitoring of bloomevents [31].

An alternative method for the detection of HAB specieswas demonstrated by Scholin et al. [32], who were able to useand target the cellular genome of the species. Here, the re-search reported a variable region within the large subunit(LSU) ribosomal RNA (rRNA) of the Pseudo-nitzschia dia-tom toxic and non-toxic species which could be targeted fordetection. Further work found that variable regions in the LSUrRNA were present in other HAB-causing species [33–35].These offer an ideal alternative target for a species-specific

detection approach that can be rapid, automated and indicativeof cell numbers [32]. As a result, research using specificallychosen single-strand oligonucleotide sequences that are ableto hybridise with variable regions within the HAB geneticmaterial has received much attention. Developed methodsbased on this technique include fluorescence in situhybridisation (FISH) assays, sandwich hybridisation assays(SHA), nucleic acid amplification techniques such as real-timePCR and nucleic acid sequence based amplification(NASBA), and the use of oligonucleotide alternates such aspeptide nucleic acids. These are reviewed herein.

Fluorescence in situ hybridisation

FISH, also referred to as whole cell hybridisation, was firstreported in the 1970s; however, it was almost 25 years beforethis technique was first applied to HAB detection [36–38]. Aschematic diagram illustrating the FISH process is shown inFig. 1. FISH uses a fluorescently labelled oligonucleotidecapture probe that is complementary to the variable region incellular RNA or DNA of the target organism. RNA is the mostcommonly targeted substrate, with only one report of DNAbeing used as the target [39]. Typically, cells are immobilisedwith a fixative, followed by hybridisation with oligonucleo-tide probes. In this process, the oligonucleotide probe crossesthe cell membrane into the cell, where it hybridises with theintracellular rRNA. The sample is subsequently washed toremove unbound oligonucleotide strands, and the resultingfluorescence intensity is monitored using epifluorescence mi-croscopy or flow cytometry [30, 40, 41]. Table 1 outlines thereports of the use of FISH for HAB detection, some of whichare highlighted below.

After identifying variable regions in the LSU rRNA ofPseudo-nitzschia diatoms [32], Scholin et al. [30] developeda fluorescein-labelled oligonucleotide probe specific toP. australis (aus-D1; Table 1) and tested its sensitivity andcross reactivity using FISH. This aus-D1 capture probe dem-onstrated high selectivity with no observable cross-reactivityfor other Pseudo-nitzschia species such as P. pungens,P. multiseries, P. delicatissima, P. pseudodelicatissima, Pfraudulenta, P. heimii and P. americana. They observed animproved and more uniform fluorescence response when anethanol–saline fixative was used instead of the traditionalformalin and aldehyde-type fixatives. This milder fixativereduced the exudation of cellular contents on fixation, thusreducing background fluorescence [30]. They continued this

Fig. 1 The steps required for fluorescence in situ hybridisation analysis

K.L. Bruce et al.

work and developed fluorescently labelled oligonucleotideprobes for an additional eight cultured Pseudo-nitzschia spe-cies (Table 1). Of the 15 oligonucleotide sequences tested,eight provided species-specific identification; however, thedetection limits and cross-reactivity were comparable to onlythose of the positive and negative control probes. Quantitativeassessment of the method performance was not undertaken[34]. Subsequent work by this group focused on improvingthe assays via the incorporation of a vacuummanifold in orderto increase the overall quantitative cell recovery and allow forpotential automation using robotic processing [40, 41].Collected samples underwent initial ethanol–saline fixationprior to vacuum filtration on a 13-mm Isopore membrane(Millipore). Hybridisation with fluorescein-labelled oligonu-cleotide capture probes at 45 °C followed. Washing withhybridisation buffer removed all non-hybridised oligo-nucleotides. The authors also noted that antifade treat-ment with SlowFade® prior to fluorescence microscopyextended the fluorescence signal lifetime by preventingphotobleaching [49].

In 2000 Miller and Scholin [43] extended this work, dem-onstrating simultaneous detection of two different HAB spe-cies, P. multiseries and P. pungens, by using a fluorescein-labelled oligonucleotide probe for P. pungens and a Texas Redlabelled oligonucleotide probe for P. multiseries. Stabilitytrials undertaken during this study also showed that the integ-rity of ethanol–saline-fixed cells was not compromised after6 weeks for cultured cells and after 4–6 weeks for naturalsamples. After this time there was a rapid reduction in thereactivity of intracellular material and resulting fluorescenceresponse.

A vacuum manifold sample collection system previouslydeveloped by Miller and Scholin [41] allowed the preparationof both FISH and scanning electronmicroscopy (SEM) sampleson the same apparatus. Initially, FISH was used to identify thespecies present on the basis of the unique rRNA region withinthe target cell. Subsequent SEM confirmed the results on thebasis of morphological features of the same cells [41]. The celldensity that can be effectively analysed via this FISH method isin the range of 1,250–2,500 cells per litre, which is well belowthe minimum cell concentration present in a bloom [41].

Other Pseudo-nitzschia species such as P. australis,P. multiseries, P. pseudodelicatissima and P. pungens werecompared with those found using the SHA technique(reviewed in next section). A good correlation betweenFISH and SHA results was observed, making both methodsan effective tool for tracking bloom formations [42]. Noobservable variations between the cell growth stage and signalintensity were observed during the different stages of Pseudo-nitzschia species cell growth, suggesting that the amount ofgenetic material in the cell is independent of cell growth [42].

Species-specific identification of the Alexandrium dinofla-gellates A. tamarense, A. catenella and A. affine using

oligonucleotide capture probes Atm1, Act1 and Aaf1(Table 1), respectively, was achieved using FISH in culturedsamples [44, 45, 47]. No cross-reactivity between the oligonu-cleotide probes and non-target species was reported. In contrastto Pseudo-nitzschia diatom species [30], the use of a parafor-maldehyde fixative in place of ethanol–saline provided thegreatest level of uniform fluorescence across the cells [44].Furthermore, the hybridisation time for the Alexandrium assayswas typically 5 min, in contrast to the 1–4 h needed for thePseudo-nitzschia diatom species reported previously [44].

Application of FISH to natural samples from Japan con-firmed the presence of A. tamarense and A. catenella. Theresults obtained allowed quantitative, rapid and precise detec-tion of the two species [44]. Multiple organic solvents havealso been tested on A. tamarense and A. catenella species withthe aim of decolourising fixed natural samples. Treatmentwith 80 % acetone prior to hybridisation was found to signif-icantly reduce background autofluorescence [45]. A quantita-tive spike study showed 92.5±2.1% recovery ofA. tamarenseusing the Atm1 probe and 94.1±1.0 % recovery ofA. catenella using the Act1 probe [47]. Two oligonucleotideprobes with different fluorescent labels, previously shown byMiller and Scholin [43], were trialled for the Alexandriumspecies, with a rhodamine-labelled Atm1 probe selective forA. tamarense and a fluorescein isothiocyanate labelled Act1probe selective for A. catenella. The two fluorochromes wereable to identify the two species; however, high levels ofautofluorescence interfered with the overall identification ofA. tamarense [47].

The use of FISH to detect Karenia breviswas first reportedin 2005 [46]. Initial PCR sequencing of the species allowed aseries of test oligonucleotide capture probes to be developedand tested. Kprobe-7 (Table 1) provided the highest level offluorescent labelling for formalin-fixed cells when comparedwith the other sequences tested. This oligonucleotide probedemonstrated high selectivity for K. breviswhen mixed with arange of dinoflagellate, diatom and raphidophyte taxa.Kprobe-7 was mixed with stored samples from a 2001 bloomto determine if K. breviswas the causative species. Successfulidentification of this species followed, giving rise to a poten-tial method for tracking bloom formation [34, 46].

PCR sequencing resulted in the development of specificoligonucleotide probes for Cochlodinium polykrikoides(Table 1). These were tested on cultured samples ofC. polykrikoides along with isolates from Korean and HongKong waters [48]. Of the probes tested, Cp-C4 provided thegreatest probe reactivity towards C. polykrikoides. No cross-reactivity with other dinoflagellate, diatom or raphidophytespecies was observed for the positively labelled isolates [48].However, isolates of the same species from North Americashowed no hybridisation with the any of the probes devel-oped. This is most likely due to the slight difference in thegenetic sequence for this isolate [48].

Detection of harmful algal blooms using oligonucleotides

Tab

le1

Overviewof

methods

used

previously

forthefluorescence

insitu

hybridisationbasedanalysisof

harm

fulalgalbloom

(HAB)species

Speciesidentified

Probe

name

Oligonucleotidecapture

probesequence

(5′–3′)

Sample

Hybridisatio

nconditions

Fixative

Detectio

nReference

Pseudo-nitzschiaaustralis

aus-D1

AAATGACTCACTCCACCAGG

Cultured.Maintained

inf/2-enriched

seaw

ater

medium,

15°C

,12h–12

hlight–darkcycle

3-4hat42

°CEthanol–saline

Epifluorescence

microscopy

[30]

P.australis

P.australis

Pseudo-nitzschiapungens

P.pungens

P.pungens

Pseudo-nitzschiamultiseries

P.multiseries/Pseudo-nitzschia

pseudodelicatissima

P.multiseries/P.pseudodelicatissima

Pseudo-nitzschiaheimii

P.heimii

P.heimii

P.heimii/Pseudo-nitzschia

fraudulenta/Pseudo-nitzschia

delicatissima

P.delicatissima

Pseudo-nitzschiaam

ericana

P.fraudulenta/P.delicatissima

auD1

auD1a

puD1

puD2

puD2a

muD

1muD

2

muD

2aheD1

heD2-1

heD2-2

frD1

deD1

amD1

amD3

AAATGACTCACTCCACCAGG

ATGACTCACTCCACCA

ATGACTCACTTTA

CCA

AAGTCCACAGCGCCCAGGCC

TCCACAGCGCCCAGG

ATGACTCACTCTGCCA

AAGCCCACAGCGCCCAAGCC

CCCACACGCCCAAG

CCATGACTCATTCTA

ACCAGA

CAAGGGAAATA

TGAACATA

TATCCACAGCGCCCACA

AAAGACTCATTCTA

ACCAGG

AGACTCACTCTA

CCA

ATGACTCATTCAGCCA

ATA

TCCAACCACTGTTA

Cultured.Maintainedin

f/2-enriched

seaw

ater

medium,13°C

,10h–14

hlight–darkcycle

3hat45–55°C

Ethanol–saline

SEM

andepifluorescence

microscopy

[34]

P.australis

aus-D1

AAATGACTCACTCCACCAGG

Natural.M

ontereyHarbor,

California

1hat45

°CEthanol–saline

Epifluorescence

microscopy

[40]

Alexandrium

tamarense

P.australis

P.pungens

P.multiseries

P.multiseries/P.pseudodelicatissima

P.heimii

P.fraudulenta

P.delicatissima

Nitzschiaam

ericana

NA1

auD1

puD1

muD

1muD

2heD2-2

frD1

deD1

amD1

AGTGCAACACTCCCACCA

AAATGACTCACTCCACCAGG

ATGACTCACTTTA

CCA

ATGACTCACTCTGCCA

AAGCCCACAGCGCCCAAGCC

TATCCACAGCGCCCACA

AAAGACTCATTCTA

CCAGG

AGACTCACTCTA

CCA

ATGACTCATTCAGCCA

Natural.S

antaCruz,California

1–2hat45

°CEthanol–saline

SEM

andepifluorescence

microscopy

[41]

P.australis

P.pungens

P.multiseries

P.multiseries/P.pseudodelicatissima

auD1S

puD1S

muD

1SmuD

2S

AAATGACTCACTCCACCAGGCGG

TGACTCACTTTA

CCAGGCGG

AAATGACTCACTCTGCCAGG

AGCCCACAGCGCCCAAGCCA

Natural.M

ontereyBay

and

SantaCruz,California

1–2hat45

°CEthanol–saline

SEM

andepifluorescence

microscopy

[42]

P.australis

P.pungens

P.multiseries

auD1

puD1

muD

1

AAATGACTCACTCCACCAGG

ATGACTCACTTTA

CCA

ATGACTCACTCTGCCA

Natural.M

ontereyBay

and

SantaCruz,California

1hat45

°CMultip

letrialled:

(1)ethanol–salin

e,(2)modifiedethanol–salin

e,(3)Carnoy’sfixativ

e,(4)

form

alin

then

70%

methanol,

(5)form

alin

then

90%

methanol,

(6)form

alin

then

ethanol–salin

e,(7)form

alin

then

themodified

ethanol–salin

e

Epifluorescence

microscopy

[43]

K.L. Bruce et al.

Tab

le1

(contin

ued)

Speciesidentified

Probename

Oligonucleotidecapture

probesequence

(5′–3′)

Sample

Hybridisatio

nconditions

Fixativ

eDetectio

nReference

A.tam

arense

Alexandrium

catenella

Alexandrium

affin

e

Atm

1Act1

Aaf1

ACACCCACAGCCCAAAGCTC

GCACTTGCAGCCAAAACCCA

GCACCCACAAATCAAAACCTA

Cultured.Maintainedin

SWIIm

medium,

15°C

,14–h–10

hlight–darkcycle

5min

at40

°CParaform

aldehyde

Epifluorescence

microscopyand

flow

cytometry

[44]

P.australis

auD1

AAATGACTCACTCCACCAGG

Cultured.ND

ND

Ethanol–saline

Epifluorescence

microscopy

[31]

A.tam

arense

A.catenella

Atm

1Act1

ACACCCACAGCCCAAAGCTC

GCACTTGCAGCCAAAACCCA

Natural.K

ureBay,

Tokuyama

Bay

andKitsuki

Bay,Japan

5min

at40

°C5×

PBSwith

paraform

aldehyde

Epifluorescence

microscopy

[45]

Karenia

brevis/Karenia

mikimotoi

K.brevis

Kbprobe-4

Kbprobe-5

Kbprobe-7

AAGATTGCAAGCAAGCAC

TCCTGGCACTA

GCAACCT

GCTGGTGCAGATA

TCCCAG

Natural.G

ulfof

Mexico,

USA

1.5hat45

°CEthanol–salinewith

form

alin

Epifluorescence

microscopyand

flow

cytometry

[46]

A.tam

arense

A.catenella

Atm

1Act1

CACCCACAGCCCAAAGCTC

GCACTTGCAGCCAAAACCCA

Natural.T

okuyam

aBay

andKitsukiB

ay,Japan

5min

at40

°CEthanol–paraformaldehyde

Epifluorescence

microscopyand

flow

cytometry

[47]

Heterosigmaakashiwo

H.akashiwo

HS-lsu

HS-its

CAGCATA

CCCGAGAGAGGAAC

CAGATTGGAGGTTGGTTTGGG

Cultured.Maintainedin

f/2medium

orf/2and

Simedium,2

0-22

°C,

12h–12

hlight–darkcycle

30min

at45

°CEthanol–saline

Epifluorescence

microscopy

[39]

Cochlodiniumpolykrikoides

Cp-C1

Cp-C2

Cp-C4

Cp-C5

GCCCAAGCAACTCGCACAT(+)

GCGCATGGGTTTGCAGCCC(++)

CTCGCAATTGATCAGTCGGT(+++)

GGTCTCAAACACGTA

TTTA

(+)

Cultured.Maintainedin

f/2medium/L1medium,

15°C

/25°C

/20°C

,16h–8h

light–darkcyclebased

onstrain

(see

[8])

1.5hat45

°CSalin

e–ethanolfollowed

byform

aldehyde

Epifluorescence

microscopy

[48]

NDNodata,P

BSphosphate-buffered

salin

e,SE

Mscanning

electron

microscopy

The

symbols+,+

+and+++referto

thelevelo

ffluorescence

observed

towards

C.polykrikoides

usingtherespectiv

eprobes.

Detection of harmful algal blooms using oligonucleotides

A FISH detection method was also developed forHeterosigma akashiwo, another HAB-causative species.Both rRNA and ribosomal DNAwere targeted using differentprobes specific to each. The rRNA probe successfullylabelled the cytoplasmic rRNA, with 80 % of the targetbeing detected, and the ribosomal DNA probe success-fully labelled the nucleic material only in the nucleus,with 70 % of the target being identified. When H. akashiwowas tested with eight other species, no cross-reactivity wasobserved [39].

Overall, FISH provides a quick and easy method for thedetection of HAB cells in cultured and natural samples. AsRNA can be targeted with oligonucleotide probes in situ,without the need for complex extraction and sample prepara-tion, the method is non-destructive with regard to the sample.This allows the analyst to confirm species identity whilstkeeping the cell intact for cell morphology studies.Unfortunately, FISH is typically complicated by high back-ground fluorescence from material present within a naturalsample. This can obscure labelled cells, making quantificationdifficult. Furthermore, cellular integrity must also be closelyconsidered during the fixation step as cell rupture can lead to aloss of target RNA and result in difficulties in both identifica-tion and cell studies. Counting via fluorescence microscopy isalso time-consuming, but can be overcome by using automat-ed methods such as flow cytometry.

Sandwich hybridisation assay

The SHA is another common method for the detection ofHABs. Unlike FISH, which targets whole cells, the SHAcomprises a two-step hybridisation of lysed cellular material.Two specific oligonucleotide probes, one for capture and theother to provide a signal, are used as outlined in Fig. 2 [30].The lysed sample is introduced to biotinylated capture probes,usually bound to a coated streptavidin surface on the base of amicrowell or bead surface [30, 42]. Following the tethering ofthe target sequence to the capture probe, a secondaryhybridisation event follows with a specifically labelled signalprobe. This probe can be labelled with fluorescein, biotin ordigoxigenin (DIG). Once hybridisation has occurred, a reac-tion with the corresponding antibody conjugated to horserad-ish peroxidase (HRP) follows. Depending on the substrateused in the next step, a colorimetric or electrochemical re-sponse can be detected and used in subsequent cell quantita-tion [40, 50, 51].

Fluorescein signal probes are most commonly used in SHAdetection of HAB species [35, 42, 48, 50, 52–56]. This re-quires a reaction after hybridisation with anti-fluorescein–HRP conjugates followed by a reaction with a commerciallyavailable 3,3′,5,5′-tetramethylbenzidine or 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) HRP substrate solution.

A detectable colorimetric response follows that is proportionalto the level of HRP reactivity in the system. Biotin-labelledsignal probes also provide a colorimetric response whenreacted with anti-biotin–HRP conjugates followed by reactionwith the corresponding commercially available HRP conju-gate (details not specified) [30, 40]. Electrochemical re-sponses can be obtained by using DIG-labelled signal probesthat can react with anti-DIG–HRP conjugates and a hydrogenperoxide substrate. The resulting reduction of the hydrogenperoxide with HRP leads to a readable signal [51]. A simpli-fied detection using a Cy3-labelled signal probe and recordingthe subsequent fluorescence response has also been reportedin literature [57].

This approach has the advantages of no purification stepsprior to exposing the lysate to the capture probe, and that theamplification of the genetic RNA occurs naturally within thecell, eliminating the need for PCR, prior to detection [50, 55].Caution must be exercised though, as lysed RNA isincredibly sensitive to degradation when in contact with ribo-nucleases (RNase) [55]. Table 2 outlines the reports of the useof the SHA for HAB detection, some of which are highlightedbelow.

The use of the SHA for the detection of HABs was firstreported in 1996 for the detection of P. australis in culturedsamples [30]. Here, oligonucleotide capture probes wereadsorbed onto a nylon bead which was directly added into acrude lysate. As the SHA involves two hybridisation eventswith two specific oligonucleotide probes, the selectivity isfurther enhanced when compared with the alternative FISHdetection discussed previously. Oligonucleotide sequencesspecific for P. australis showed a high level of selectivitywhen compared with other members of the genus Pseudo-nitzschia such as P. pungens, P. multiseries, P. fraudulenta,P. heimii and P. delicatissima.

Semiautomation of the SHA procedure followed, and theresults were compared with the results from LM and FISH forthe detection of P. australis [40]. The authors commented thatthe SHA method was severalfold faster that the previouslydiscussed FISH method, with quantifiable results being ob-tained with the aid of a standard curve [40]. Field samplesfrom Monterey Harbour, California, were tested and the re-sults suggested that naturally occurring particulate organicmatter and organisms, such as other diatoms and zooplankton,present in the lysate did not affect the hybridisation efficiency[40]. To determine if the growth phase affected the levels ofnucleic cellular material, different growth stages were collect-ed and analysed using the semiautomated SHA method [42].Cellular rRNA concentrations were found to remain stablethroughout the different growth stages. This is an importantfactor as some other species, such as A. catenella [56], havedisplayed differing concentrations of rRNA at differentgrowth stages, skewing cell concentrations based on thatSHA analysis [42].

K.L. Bruce et al.

Quantitative detection of Fibrocapsa japonica andH. akashiwo by the SHA was made possible with the aid ofa standard curve. Initially, FISH (discussed earlier) was usedto test the specificity of the designed oligonucleotide probes.Once developed, the probes (Table 2) were introduced to theSHA, providing the highest signal intensity and thus greatestsensitivity. Overall the method was rapid, easy and moreaffordable when compared with FISH and LM [35]. Thisapproach was applied to the detection of H. akashiwo innatural samples from Puget Sound and Hood Canal, USA[52]. Here, the fixation step was also omitted as the cells werefound to rupture on treatment with Lugol. Instead, cells werelysed immediately with the lysate and stored at -70 °C untilanalysis. Higher cell densities resulted in difficulties in quan-titation, with this being overcome by serial dilution of thecellular samples [52]. Similarly, natural samples from theCalifornian coast between 2001 and 2002 were examined bythe SHA for the presence ofH. akashiwo [53]. In this case, theSHA results showed good agreement with the end-point PCRassay on samples collected during a bloom event alongMonterey Bay and San Francisco Bay, California. With useof LM, a method used in phytoplankton monitoring programs,positive identification of H. akashiwo was difficult owing tocell rupture on fixation. The SHA and PCR methods, howev-er, were able to positively identify the species, making theSHA an alternative approach for the rapid screening of fragileHAB organisms [53].

In 2005, an SHA was developed for the validation ofF. japonica and H. akashiwo in New Zealand. This methodwas found to underestimate cell densities for F. japonica, butfor H. akashiwo counts comparable to those found from LMwere obtained. It was suggested that the samples may havebeen collected during late stationary phase and, in turn, thelevel of target rRNA may have been limited. During bloomcollapse, it was also noted that there was an increase in rRNAlevels most likely due to cell rupture on death, leading tooverestimation of cell concentration when using the SHA.On the basis of the results of this study, international accred-itation was awarded by International Accreditation NewZealand for the SHA method developed (ISO 17025) [31].

Natural samples of C. polykrikoides were collected fromKorean coastal waterways and applied to an automated SHA.The detection limit was also recorded: 115±53 cells permillilitre. Cell concentrations below this level could still bedetected however, but were not accurately quantifiable.Mikulski et al. [48] reported that in Korea, HAB warningsystems recognise 1,000 cells per millilitre as a “red tide alert”cell concentration. The detection limit resulting from this SHAmethod was found to lie below these warning cell concentra-tions, making the detection limit acceptable.

Alexandrium ostenfeldii, an HAB-causative dinoflagellate,was also detected using an SHAwith an electrochemical modeof detection as an alternative to the colorimetric approach. As

stated previously, the electrochemical response was possibleby using a DIG-labelled signal probe which can react with ananti-DIG–HRP conjugate. Finally, hydrogen peroxide isadded as the HRP substrate. All the hybridisation steps occuron a streptavidin-coated working electrode, allowing the sub-sequent detection of the electrochemical signal [51].Fragmentation of the RNAwas introduced in this research toincrease the sensitivity of the capture probe to the requiredsequence during the first hybridisation event. A “helper”oligonucleotide was also introduced which was able to suc-cessfully reduce the formation of secondary rRNA structuresin solution. Both of these changes to the SHA method wereable to increase the measured signal 6.3 times, which corre-lated to an estimated detection limit of approximately 16 ng/μL for the optimised conditions, which was previously esti-mated at 100 ng/μL for the standard conditions [51].

More recently, SHA detection has been moved to an alter-native fibre-optic microarray as opposed to the normal micro-plate format. P. australis, Alexandrium fundyense andA. ostenfeldii were detected using the method. Microspheres,the platform for oligonucleotide attachment, were introducedinto fibre-optic wells, where they were left to dry. The methodof attachment of the capture probe to the microspheres was notspecified. The lysate was then introduced, left to hybridise andthen underwent the second hybridisation with the signal oli-gonucleotide probe. The resulting epifluorescence signal wasmeasured using the fibre optic as well as direct image analysis.Samples were prepared by the serial dilution of known cellconcentrations ranging from five to 5,000 cells per millilitrefor the three species. As such, cell concentrations were notrecorded as the volume prior to filtration was dependent on thedilution of the initial culture. Detection limits of five to tencells per sample were obtained for A. fundyense andP. australis; however, the detection limits for A. ostenfeldiiwere higher at 50 cells per sample. Non-target species did notinterfere with the signal produced, making it possible toperform an SHAwith no prior RNA purification. The platformalso displayed high stability of the tethered capture probe,making is possible to use the capture probe multiple timeswith no loss in reactivity [58].

The reaction conditions of the fibre-optic microarrays wereoptimised further in 2006 by the same research group. Here, theattachment of the oligonucleotides was achieved by firstlyactivating them with cyanuric chloride prior to exposing themto amine-modified microspheres. Surface characterisation andthe density of attachment of the oligonucleotide probes to themicrospheres were not reported. The method optimisation ledto a detection limit of five cells per sample for all three speciestested [57]. This is a significant improvement for A. ostenfeldii,which previously demonstrated a detection limit of only 50cells per sample [58]. Multiplexed arrays were also trialled andwere found to be successful and highly specific for speciesdetection of HABs independent of the sample matrix [57].

Detection of harmful algal blooms using oligonucleotides

The SHAmethod was also simplified with the aid of a PCRELISA DIG detection kit for Alexandrium minutum.Hybridisation buffer, conjugate dilution buffer, substrate buff-er, anti-DIG–peroxidase, washing tablets and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) tablets were used in thereaction. The total method was simplified considerably for theassessment of the species. A standard curve was alsoproduced taking into account the RNA content permicrolitre instead of the cell density (cells per millilitreor cells per litre). This allowed the amount of RNA per cell tobe calculated as 0.028±0.003 ng. Both spiked culture samplesand field samples were tested, with the positive identificationof A. minutum using the specific oligonucleotide capture andsignal probe [54].

By targeting nucleic material in crude cell lysates, the SHAprovides, in comparison with FISH, improved quantitation ofHAB cells. This is due to the ability to reduce or removebackground fluorescence as well as the removal of the re-quirement for cell counting. Importantly, the stepwise ap-proach of detection makes the automation of the procedurepossible. The use of two probes targeting one sequence is alsoable to increase the selectivity of the process with carefuloligonucleotide sequence design. Natural amplification ofRNA within the cell means that use of potentially expensiveamplification techniques can be eliminated. RNA stability,however, is a cause for concern owing to the sensitivity ofRNA to degradation by RNase. To prevent any loss of mate-rial, samples should be analysed or stored in stability buffersto maintain integrity. Analysis is further complicated as duringdifferent cell growth phases the RNA content within the cellsof some species has also been shown to fluctuate. This couldpotentially be overcome by analysing whole-cell samples toobserve the potential growth phase of the sample collected.

Nuclease protection assay with sandwich hybridisation

More recently, a nuclease protection assay (NPA) prior tosandwich hybridisation was introduced to increase sensitivityand selectivity. For this, specific NPA probes are developedthat match the exact target sequence in the algal rRNA. Afterhybridisation with the NPA probes, a treatment with S1 nu-clease follows that removes any non-target single-strandedRNA or mismatched probes, leaving only perfectly matchedprobes stoichiometrically (see Fig. 3). Here, the remainingNPA oligonucleotide probe is used for the subsequent detec-tion, not the RNA usually targeted, making it more stable thanpreviously completing the SHAwith less stable RNA [50].

The NPAwith sandwich hybridisation (NPA-SH) was firstapplied in 2006 for the detection of Prorocentrum minimumand Prorocentrum micans in both cultured and natural sam-ples. The NPA was able to provide a much higher level ofsensitivity compared with previous methods. When the cell

counts were compared with those obtained by LM, a goodlevel of agreement was found between both results. Thesensitivity of the NPA-SH was found to be 15 cells permillilitre for P. minimum and only one cell per millilitre forP. micans. This proved that the NPA-SH could successfullydetect low cell numbers of these two HAB species [50].

Another HAB species optimised for NPA-SH analysis isPhaeocystis globosa. Both cultured and natural samples wereanalysed, with the lowest detectable limit being recorded at 18cells per millilitre. Good agreement was seen between LMresults as with previous methods, and field samples displayedno interferences when in contact with biotic and abiotic ma-terial in the sample matrix [55].

The NPA-SH has also been applied to A. catenella andA. tamarense. Comparison with an alternative testing method,competitive ELISA (cELISA), was conducted, each with theiradvantages and disadvantages. NPA-SH analysis was unableto discriminate between the two closely related Alexandriumspecies. However, cELISA successfully identified the two.The NPA-SH overestimated cell concentrations in some caseswhen compared with LM and cELISA, most likely owing tothe differing genetic concentrations at different stages of cel-lular growth. LM and cELISA were unable to track thesechanges as they were not focused on the concentrations ofgenetic material. The lowest obtainable detection for the NPA-SH method was determined to be 0.47 cells per millilitre [56].

Another modification made to increase the sensitivity ofthe NPA-SH method was developed in 2012 by Zhu et al.[ 59 ] f o r t h e de t e c t i on o f P. m in imum . He r e ,electrochemiluminescence detection is used to quantitate celldensity. Extracted lysates are introduced to the NPA probe asbefore; however, the NPA probe is labelled with biotin at oneend and Ru(bpy)3

2+ (where bpy is bipyridine) at the other.Then, rRNA hybridises with the probe and undergoes S1nuclease digestion (Fig. 4). The strands are then denatured,leaving the NPA oligonucleotide probes, which can then bindvia a streptavidin–biotin interaction onto magnetic beads. Theprobes can then be drawn to a working electrode using amagnet, allowing a subsequent electron transfer reaction atRu(bpy)3

2+ in the presence of tripropylamine and detectablephoton release after relaxation of the excited Ru(bpy)3

2+. Thisphoton is measured at 610 nm after being passed through aphotomultiplier tube. This method was able to increase sensi-tivity and simplify detection, with a detection range of 0.4pmol to 4 nmol of rRNA.

The NPH-SH offers significant improvements over theSHA. The use of an NPA probe for detection (DNA oligonu-cleotide) minimises any effects of degradation of RNA byRNase and maintains the advantage of the possibility ofautomation. The sensitivity of this method is also acceptablefor a bloom event, with the lowest obtainable detection limitsbeing well below the cell concentrations expected during abloom. Despite this advantage, variation in RNA

K.L. Bruce et al.

concentrations during the different growth phases of the cellstill needs to be taken into account. Optimisation of probesequences as well as experimental conditions is also essentialto obtain the best sensitivity and selectivity for the targetorganism.

Nucleic acid amplification based detection

An alternative to targeting rRNA is to target DNAwith the aidof amplification-based techniques. Typically, this has beendone using real-time PCR, NASBA and loop-mediated iso-thermal amplification (LAMP).

Real-time PCR applications

Real-time PCR (also called quantitative PCR) is a modifiedPCR technique whereby amplified DNA is detected in realtime as the amplification reaction progresses. Detection ofamplification is achieved through the use of fluorescent ma-terials such as a TaqMan probe, a molecular beacon and anintercalating dye such as SYBR Green (see Fig. 5). Thefluorescence intensity of the signal increases with increasingcycle of the PCR until a cycle detection threshold (Ct) isreached. Ct can then be used to calculate the concentrationof genetic material in a sample based on the inverse relation-ship between Ct and initial concentration of genetic material[60]. Production of a standard curve allows the quantificationof genetic material present [61].

Although real-time PCR has been applied to clinical andenvironmental diagnostics for some time, application of thismethod to HAB detection is relatively recent. This reviewfocuses on recent developments (2013 onwards) in real-timePCR for HAB detection; for a comprehensive review of real-time PCR applications for the detection of species that causeHABs prior to 2013, the reader is referred to Antonella andLuca [61]. The expanse of this real-time PCR research hasbeen significant, with a number of methods being reported fordinoflagellates, diatoms and members of Raphidophyceae,Haptophyceae, Pelagophyceae and Cyanophyceae [61].Applications directed at the detection of Alexandrium speciessuch as A. catenella [62–66], A. minutum [26, 67],A. fundyense [68, 69], A. taylori [64] and A. tamarense [62,

63, 70] have been thoroughly reported. Similarly, real-timePCR methods were developed for the dinoflagellate speciesDinophysis acuta andD. acuminata [71]. Recent outbreaks ofPseudo-nitzschia in the USA have propelled the developmentof real-time PCR methods for P. australis, P. pungens,P. delicatissima, P. calliantha, P. multistriata [72, 73] andP. multiseries [74]. Members of Raphidophyceae such as inthe genus Chatonella have also been applied to real-time PCR[75]. These include C. verrucolosa [76, 77], C. antiqua [76],C. ovata [76] and C. subsala [76, 77].

A method for the Raphidophyceae spec iesF. japonica was also developed [76]. Members ofHaptophyceae have been less researched; however, a numberof methods have been developed for Prymnesium parvumusing both SYBR Green intercalating dyes (Fig. 5c) [78, 79]and molecular probes (Fig. 5b) [80]. Cyanobacterial HABspecies have also been researched, with a detailed reviewbeing published recently looking into the real-time PCR ap-plication for species monitoring [81].

Detection of C. polykrikoides has also been achieved usingreal-time PCR methods on both natural and cultured samples[82]. This study demonstrated how the application of real-timePCR can be used to look at the variation on a subspecies level,most likely caused by ecological factors and cellular adapta-tions. Subspecies variable regions in the LSU rRNA wereidentified for C. polykrikoides, with no observable cross-reactivity with other HAB species. Reaction efficiencies of91 % were reported, and melting point analysis found onlythe target region was successfully amplified. A decrease inreaction efficiency was observed however when natural sam-ples were analysed. This was most likely due to the presence ofinhibitors such as mucopolysaccharides, phenolic compounds,humic acids and heavymetals. The inhibition was overcome bya simple tenfold sample dilution prior to analysis [82].

Real-time PCR method development for eight species inthe genera Karenia, Gymnodinium, Karlodinium andTakayama has also been reported recently [83]. Theoptimised assays were highly specific, and sensitivity wasreported to be below one cell in all cases. The method wassuccessfully applied to a bloom event in New Zealand, withGymnodinium catenatum being recorded at three cells perlitre, well below the detection limit of LM, reported as 100cells per litre in this case.

Fig. 2 The sandwich hybridisation assay (SHA) process. HRP horseradish peroxidases, rRNA ribosomal RNA

Detection of harmful algal blooms using oligonucleotides

Tab

le2

Overviewof

methods

used

previously

forthesandwichhybridisationassays

ofHABspecies

Speciesidentified

Capture

oligonucleotide

probesequence

(5′–3′)

Signaloligonucleotide

probesequence

(5′–3′)

Sample

Nucleicacid

extractio

nHybridisatio

nconditions

Detectio

nReferences

P.australis

AAATGACTCACTCC

ACCAGGCGG

CTCTTTA

ACTCTCTTT

TCAAAGTTCTTTGC

ATC

Cultured.Maintainedin

f/2-enriched

seaw

ater

medium,1

5°C

,12h–12

hlig

ht–darkcycle

Lysissolutio

n30

min

at23-25°C

Photographed

beadsafter

colour

developm

ent

[30]

P.australis

AAATGACTCACTCC

ACCAGGCGG

CTCTTTA

ACTCTCTTT

TCAAAGTTCTTTGC

ATC

Cultured.Maintainedin

f/2medium,13°C

,10

h–14

hlight–darkcycle.

Natural.M

ontereyHarbour,

California,USA

Lysissolutio

n10

min,tem

perature

notspecified

Photographed

beadsafter

colour

developm

ent

[40]

P.australis

AAATGACTCACTCC

ACCAGGCGG

CTCTTTA

ACTCTCTTT

TCAAAGTTCTTTGC

ATC

Cultured.Maintainedin

f/2medium,13°C

,10

h–14

hlight–darkcycle

Lysissolutio

nND

Platereader.6

55nm

then

450nm

after10

%H2SO

4

treatm

ent

[42]

P.pumgens

TGACTCACTTTA

CC

AGGCGG

P.multiseries

AAATGACTCACTCT

GCCAGG

P.pseudo-delicatissima

andP.multiseries

AGCCCACAGCGC

CCAAGCCA

H.a

kashiwo

ACCACGACTGAGCA

CGCACCTTT

CCGCTTCACTCGCCGT

TACTA

GNatural.P

ugetSo

undand

HoodCanal,U

SALy

sissolutio

nND

96-w

ellp

latespectrophotometer

[52]

H.a

kashiwo

ACCACGACTGAGCA

CGCACCTTT

CCGCTTCACTCGCCGT

TACTA

GCultured.Maintainedin

f/2medium,15°C

,12

h–12

hlight–darkcycle.

Natural.M

ontereyBay

andBerkeleyPier,

California

Lysissolutio

nND

Platereader.6

55nm

then

450nm

after10

%H2SO

4

treatm

ent

[53]

H.a

kashiwo

ACCACGACTGAGCA

CGCACCTTT

CCGCTTCACTCGCC

GTTA

CTA

GCultured.Maintainedin

f/2medium,20°C

,12

h–12

hlight–dark

cycle

Lysissolutio

nND

Platereader.6

55nm

then

450nm

after10

%H2SO

4

treatm

ent

[35]

Fibrocapsajaponica

CGGCTGGACACGCT

TCTGTA

G

H.a

kashiwo

F.japonica

P.australis

ND

ND

AAATGACTCACTCC

ACCAGGCGG

ND

ND

CTCTTTA

ACTCTCTTTTC

AAAGTTCTTTGCATC

Cultured.ND.

Natural.B

ayof

Plenty

andMarlborough

Sounds,N

ewZealand

Purchased

lysisbuffer

ND

Platereader.6

55nm

then

450nm

after10

%H2SO

4

treatm

ent

[31]

Alexandrium

fundyense

GCAAGTGCAACACT

CCCACCA

TTCAAAGTCCTTTTCATA

TTTCCC

Cultured.Maintainedin

f/2medium,15°C

,14

h–10

hlight–dark

cycle

Purchased

lysisbuffer

ND

Fluorescence

measured

aslig

htintensity

perbead

[57,58]

Alexandrium

ostenfeldii

GTGGACGCAACAAT

CTCACCA

TTCAAAGTCCTTTTCATA

TTTCCC

P.australis

AAATGACTCACTCCAC

CAGGCGG

CTCTTTA

ACTCTCTTTTC

AAAGTTCTTTGCATC

K.L. Bruce et al.

Tab

le2

(contin

ued)

Speciesidentified

Captureoligonucleotide

probesequence

(5′–3′)

Signaloligonucleotide

probesequence

(5′–3′)

Sample

Nucleicacid

extractio

nHybridisatio

nconditions

Detectio

nReferences

Alexandrium

minutum

GAAGTCAGGTTTGGATGC

TAATGACCACAACCCTTCC

Cultured.Maintainedin

seaw

ater-based

Kmedium/IMRmedium/F2

medium,1

5-22

°C,

14h–10

hlig

ht–darkcycle

basedon

strain

(see

[110])

RNeasy

Plant

MiniK

it1hat46

°CVarianCary4000

UV–vis

spectrom

eter

[54]

A.ostenfeldii

CAACCCTTCCCAATA

GTCAGGT

GAATCACCAAGGTTCC

AAGCAG

Cultured.Maintainedin

seaw

ater-based

Kmedium,

15°C

,14h–10

hlight–dark

cycle

RNeasy

Plant

MiniK

it30

min

at46

°CElectrochem

icalsignal

recorded

for10

sat-150

mV

[51]

a

A.catenella

ATTTGGCACAGCCTGA

GCATTTA

TC

ACAACTGCACTTGACT

GTGTGGTGTG

Cultured.Maintainedin

f/2

medium,2

2-25

°C,

12h–12

hlig

ht–darkcycle.

Natural.O

ldStonem

anBeach,Q

ingdao,C

hina

Freeze–thawcycle

1hat50

°CPlatereader.450

nm/630

nmratio

recorded

after2M

H2SO4treatm

ent

[56]

b

Phaeocystisglobosa

GGATTGGAGGGT

GTTCGTTTCG

GCACCTTA

CGGGAAAC

TAAAGTCTTTG

Cultured.Maintainedin

f/2medium,22-25

°C,

12h–12

hlig

ht–darkcycle.

Natural.L

uxun

Park,Q

ingdao,

China

RNAextractio

nbuffer

1hat50

°CPlatereader.450

nm/630

nmratio

recorded

after

2M

H2SO

4treatm

ent

[55]

c

Prorocentrum

minimum

Prorocentrummicans

TCATGGTA

GCTCGT

CTA

CGGGTGA

ATCGAGGAAAACTC

CAGGGACATG

GGCAGAACTCATTTGC

GGACTGT

GGGTGGGTGAATGT

GCCTGGT

Cultured.Maintainedin

f/2medium,22-25

°C,

12h–12

hlig

ht–darkcycle

Lysisbuffer

1hat50

°CPlatereader.450

nm/630

nmratio

recorded

after2

MH2SO

4treatm

ent

[50]

d

P.minimum

ACAGTCCGCAAATG

AGTTCTGCCA

AGGCTA

TTCACTCA

CCCGTA

GACGAG

CTA

CCATGA

ND

Cultured.Maintainedin

f/2medium,22°C

,12

h–12

hlig

ht–darkcycle.

Natural.E

astC

hina

Sea,China

Lysisbuffer

2hat42

°CElectrochem

iluminescence

detectionat1.0V,1.0mA

measuredin

countsper

second

[59]

e

aAhelper

probewas

also

used

inthisstudy(5′-G

CATA

TGACTA

CTGGCAGGATC-3′)

bAnuclease

protectio

nprobewas

also

used

forthenuclease

protectio

nassaywith

sandwichhybridisation(N

PA-SH)forP.minimum

(5′-A

CAGTCCGCAAATGAGTTCTGCCAAGGCTA

TTCACTCA

CCCGTA

GACGAGCTA

CCATGA-3′)andP.micans(5′-A

CCAGGCACATTCACCCACCCAGGGGTA

GGCTA

CCATGTCCCTGGAGTTTTCCTCGAT-3′)

cAnuclease

protectio

nprobewas

also

used

forNPA

-SH(5′-C

AAAGACTTTA

GTTTCCCGTA

AGGTGCTGAAGAGGTCGAAACGAACACCCTCCAATCC-3′)

dAnuclease

protectio

nprobewas

also

used

forNPA

-SH(5′-C

ACACCACACAGTCAAGTGCAGTTGTGCTTTCAAGATA

AATGCTCAGGCTGTGCCAAAT-3′)

eAnuclease

protectio

nprobewas

also

used

forNPA

-SH(5′-A

CAGTCCGCAAATGAGTTCTGCCAAGGCTA

TTCACTCACCCGTA

GACGAGCTA

CCATGA-3′)

NDNodata

Detection of harmful algal blooms using oligonucleotides

Real-time PCR is one of the most widely used nucleic acidmethods for HAB detection. The use of specifically designedprimers facilitates selective amplification of target sequencesand allows the detection of very low concentrations of targetDNA. Quantitation of HAB-causative species in natural sam-ples has been subject to some problems. Importantly, PCRinhibitors such as polyphenols and plant materials can reducethe efficiency of the real-time PCR process and reduce or evenprevent the overall amplification of target sequences. This canmake detection almost impossible in some cases [84].Consequently, elimination of PCR inhibitors is essential inorder to obtain the highest-accuracy results and can be over-come by modifications to nucleic acid extraction and purifi-cation. Digital real-time PCR, which uses dilute samples, mayovercome the inhibition of molecules, making it a good alter-native [85]. Additionally, the requirement for amplificationtypically limits the analysis of samples to a laboratory envi-ronment. Optimising the conditions for a handheld devicewould potentially allow direct bloom tracking in the field.Direct correlations to cell numbers from the RNA concentra-tion will also allow direct tracking of bloom formation.Recently there has been an increase in popularity of isother-mal nucleic acid methods such as NASBA and LAMP. Thesedo not require the use of thermocyclers for amplification andsubsequent detection and have demonstrated an increase in thesensitivity of the analysis.

Nucleic acid sequence based amplification assays

NASBA is an isothermal reaction used to amplify specificvariable regions of target messenger RNA in the lysate of acell with the aid of specific primers. There are two phasesinvolved in NASBA amplifications: a linear phase followedby an amplification phase (Fig. 6). In the linear phase, a specificforward primer (primer 1) anneals to the complementary se-quence present in the RNA sample. Avian myeloblastosis virusreverse transcriptase is then added to the reaction mixture andextends the 3′ end of the primer. This, in turn, produces a DNA

strand complementary to the RNA. Treatment with RNase Hthen removes the RNA from the duplex, leaving single-stranded complementary DNA. The reverse primer (primer 2)then anneals to the complementary 5′ region of complementaryDNA and produces the complementary DNA strand with thehelp of reverse transcriptase. Treatment with T7 RNA polymer-ase then produces the complementary antisense RNA strand(complementary to the original target RNA) from these tem-plates [86].

The amplification phase follows the linear phase, with thesecond primer once again annealing to the complementaryantisense RNA strand. Reverse transcriptase then producesthe complementary DNA strand in the form of another RNA–DNA duplex. RNase H then removes the antisense RNA,leaving the template DNA. The first primer is then reactedwith the DNA, producing a DNA–DNA duplex. RNA poly-merase is then reacted to produce the complementary RNA.The cycle then continues, leading to the overall amplificationof target RNA (Fig. 6) [86].

Real-time detection with a molecular beacon labelled with 6-c a r b o x y f l u o r e s c e i n a t t h e 5 ′ e n d a n d a4-(dimethylaminoazo)benzene-4-carboxylic acid quencher atthe 3′ end allows quantitation of initial target RNA. When notarget RNA is present, the molecular beacon remains quenchedby the 4-(dimethylaminoazo)benzene-4-carboxylic acid in ahairpin formation (see Fig. 5b). However, in the presence ofcomplementary target RNA, the molecular beacon hybridises tothe RNA, separating the quencher and 6-carboxyfluorescein,leading to a detectable fluorescent signal. The time to positivity(TTP), or the time taken for the fluorescent signal to reachexponential growth, can then be measured and used to quantifythe initial concentration of target RNA in the sample. This isdone by producing standard curves generated using known cellnumbers and their subsequent TTP values. Table 3 outlines thereports of the use of NASBA for HAB detection, some of whichare highlighted below.

In 2004, Casper et al. [87] reported the first use of NASBAfor the detection of K. brevis. They recorded an estimated

Fig. 3 Process of the nucleaseprotection assay (NPA) coupledwith the SHA

K.L. Bruce et al.

sensitivity to one cell on the basis of the detectable 1.0 fg ofin vitro transcript. The method was highly selective whenanalysing closely related organisms as no non-target organ-isms were positively identified. A tenfold excess of closelyrelated cellular rbcLmessenger RNA did not inhibit the assayresults. Ten K. brevis strains were all positively identifiedusing the designed primers. Other Karenia species, however,were not tested as there were limited sequencing data at thetime of publication. Environmental samples were alsoreanalysed with the NASBA assay and the results were com-pared with results previously obtained by the Florida Fish and

Wildlife Conservation Commission, with 72 % of eventsshowing a good level of agreement between the two methods[87]. The calculated concentration using this method wasstated as being limited by the accuracy of the standard curve.Use of the TTP for the standard curve data can result invariations owing to the three different enzymes displayingdifferent kinetics and hence reacting at different rates [87].

To overcome the issues surrounding the use of the TTPstandard curve, internal control RNA (IC-RNA) was intro-duced by the same research group in 2005. This aided in thequantification of the genetic material. This IC-RNA has a

Fig. 4 Electrochemiluminescentdetection using NPAwithsandwich hybridisation.B biotin, M magnetic beadplatform, Rtris(bipyridine)ruthenium(II)complex, TPrA tripropylamine

Fig. 5 Detection methods usedfor real-time PCR: a TaqManprobes with Taq polymerase; bmolecular beacon detection; theuse of an intercalating dye such asSYBR Green. FAMcarboxyfluorescein, DABCYL4-(dimethylaminoazo)benzene-4-carboxylic acid, TAMRAcarboxytetramethylrhodamine

Detection of harmful algal blooms using oligonucleotides

sequence that is the same as that of the target RNA but with adifferent attached fluorophore. The ratio of IC-RNA TTPvalues and the target RNA TTP values can then be taken tominimise the errors associated with the three enzymes andstandard curve. As such, both accuracy and precision of theNASBA assay were improved for the quantification of cellnumbers in a sample. Internal control NASBA showed amagnitude of predictions ranging from 59 to 740 %, whereasfor the TTP standard curve, the range was from 6 to 3,820 %.It was noted that the main associated errors were at low cellconcentrations. Furthermore, the variance with the IC-RNAwas much lower. For example, for a cell number of 1,000, theTTP standard curve predicted 677±209 cells and the IC-RNAmethod predicted 920±118 cells. At a lower cell number of 1,the TTP standard curve predicted 58±143 cells, whereas theIC-RNA method predicted 3±2.5 cells [88].

The same research group aimed to simplify the internalcontrol NASBA assay for in-field analysis in 2007. Here anextraction protocol and a portable handheld NASBA devicewere developed for the detection of K. brevis. The protocoldeveloped for the RNeasyMini Kit was altered so that insteadof centrifugation, the solutions could be pushed through thespin columns with a flow of air. A t test of the linear plot of thecounts obtained from the modified in-field RNeasy Mini Kitversus the direct extraction showed no significant differencefrom 1.0 (p>0.05). The handheld NASBA device cell

predictions were compared with LM cell counts, and a goodlevel of agreement was found. In most cases the bloom clas-sifications were similar, with very little variance, and a paired-sample t test suggested that there was no significant differencebetween the NASBA assay and LM cell counts (p>0.05) [89].

The research was extended in 2010 by the same researchgroup to include Karenia mikimotoi but not using the hand-held device discussed previously. With use of internal controlNASBA, a theoretical detection limit of 1,250 cells per litrewas calculated on the basis of a 250-mL sample size, which isreasonable when monitoring high cell concentrations in abloom. When the method tested with closely relatedK. brevis, no variation in cell number was observed, suggest-ing the method is also highly selective. Cell count compari-sons showed a linear relationship between values obtainedusing the same NASBA method and LM cell counts [90].

With the aid of an internal control oligonucleotide, quanti-fication of HAB-causative organisms using NASBA has im-proved significantly. Unlike PCR-based techniques, NASBAis an isothermal process by which the reaction can occurwithout the need for expensive equipment. The method offershigh sensitivity and selectivity with the use of two specificallydesigned primers. The method has also been successfullyautomated using a handheld NASBA device, which will allowresearch and analysis in the field. As RNA is the targetmaterial, potential degradation of the sample needs to be taken

Fig. 6 The nucleic acid sequencebased amplification processincluding the non-cyclic andcyclic phases

K.L. Bruce et al.

into account as discussed previously. Any degradation of thesample prior to analysis can severely alter the final bloomresults, potentially underestimating the concentrations pres-ent. The use of a handheld NASBA device and direct in-fieldanalysis could potentially overcome this issue. As differentenzymes are also used in this analysis, the kinetics of eachenzyme need to be thoroughly understood as each may bedifferent and subsequently affect the standard curve. This hasbeen shown to be significantly improved however with the useof internal control NASBA, making it a good alternative.

Loop-mediated isothermal amplification

LAMP has been introduced more recently as a method ofHAB detection. This rapid procedure has been reported toincrease the sensitivity of regular PCR by up to 100-fold [91].Nucleic acid amplification occurs in three stages: an initialstep, a cycling amplification step and an elongation step(Fig. 7) [92]. Unlike NASBA and real-time PCR, LAMP usesfour specifically designed primers in the amplification pro-cess. Two inner and two outer primers are able to recognise sixregions of the target DNA strand. As amplification of thetarget DNA occurs only when all six regions are present, theprocess is highly specific. Other advantages of LAMP includethe low costs involved in amplification as it occurs at isother-mal temperatures [92]. The exact method has been describedin detail elsewhere [93]. Briefly, during the non-cyclic steps, aDNA strandwith two loops at the 5′ and 3′ ends is formedwiththe aid of a forward inner primer, a forward outer primer, abackward inner primer and a backward outer primer [94]. Inthe cyclic amplification, the loops allow a strand displacementDNA synthesis in the form of inverted repeats of the targetsequence within a single strand during elongation [92].

The first example of LAMP being used for the detection ofsix HAB species in the genus Alexandriumwas not until 2008,8 years after its initial development [95]. In this study, acomparison between LAMP and PCR sensitivity was con-ducted. It was found that the sensitivity of LAMP was tenfoldhigher than that of PCR. A detection limit of five cells per tube(1 pg, total reaction mixture 25 μL) was obtained whentargeting A. minutum.

Identification of A. catenella and A. tamarense usingLAMP was possible within an amplification time of 25 minusing both cultured and natural samples [96]. The detectionlimits were not stated, and instead a five-cell and single-cell(approximately 5 ng) amplification was performed in thepresence of more than 10 ng of non-target Alexandrium spe-cies DNA. The fluorescence intensity seemed unaffected bythe non-target DNA, with the fluorescence exhibited being thesame as that expected for 5 ng of target DNA. In a continua-tion of the study, DNA extraction methods that were alterna-tives to boiling of tris(hydroxymethyl)aminomethane–EDTAbuffer, which was found to not extract DNA of the closelyT

able3

Overviewof

nucleicacid

sequence

basedam

plificationused

inthedetectionof

HABspecies

Species

identified

Sam

ple

Extractionprotocol

Forward(1)prim

ersequence

(5′–3′)

Reverse

(2)Prim

erSequence

(5′–3′)

Molecular

BeaconSequence(5′–3′)

References

K.brevis

Cultured.Medium

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ht–darkcycle

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ACGTTA

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TAGGGAGAAGGTA

CACACTT

TCGTA

AACTA

[6-FAM]-CGATCGCTTA

GT

CTCGGGTTA

TTTTTTC

GATCG-[DABCYL]

[87]

K.brevis

Cultured.Medium

notspecified,

24°C

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notspecified

RLT

lysisbuffer

(Qiagen)

follo

wed

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spin

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(Qiagen)

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n

ACGTTA

TTGGGT

CTGTGTA

AATTCTA

ATA

CGACTCACTA

TAGGGAGAAGGTA

CACACTT

TCGTA

AACTA

[6-FAM]-CGATCGCTTA

GT

CTCGGGTTA

TTTTTTC

GATCG-[DABCYL]

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a

K.brevis

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24°C

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hlig

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ACGTTA

TTGGGT

CTGTGTA

AATTCTA

ATA

CGACTCACTA

TAGGGAGAAGGTA

CACACTT

TCGTA

AACTA

[6-FAM]-CGATCGCTTA

GT

CTCGGGTTA

TTTTTTC

GATCG-[DABCYL]

[89,101]

a

K.m

ikimotoi

Cultured.L1medium,22°C

,12

h–12

hlig

ht–darkcycle.

Natural.C

oastalFlorida,U

SA

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it(Q

iagen)

AACCTA

AAAT

GATTA

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AATTCTA

ATA

CGACTCACTA

TAGGGAGGAGACCCATTCTT

GCGAAAAATA

A

[6-FAM]-CGATCGAACAAC

TAAACATGATTTTGCGAT

CG-[DABCYL]

[90]

b

DABCYL4-(dim

ethylaminoazo)benzene-4-carboxylic

acid,6

-FAM

6-carboxyfluorescein

aThe

internalcontrolR

NAmolecular

beacon

used

was

[6-ROX]-CGATCGTGGCTGCTTA

TGGTGACAATCGATCG-[DABCYL]forK.brevis

bThe

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used

was

[6-ROX]-CATGCGTGGCTGCTTA

TGGTGACAATCGCATG-[DABCYL]forK.m

ikimotoi

Detection of harmful algal blooms using oligonucleotides

K.L. Bruce et al.

related Alexandrium species [96], were trialled [97].Extraction using a tris(hydroxymethyl)aminomethane–EDTA buffer, a cetyltrimethylammonium bromide buffer,0.5 % Chelex buffer and 5 % Chelex buffer demonstrated thatthe two Chelex buffer systems were able to provide thehighest amplification success (100 %) in both species. Thedetection limits in this case were not reported, as with theprevious research.

LAMP has also been used to track A. tamarense restingcysts in sediment core samples in Funka Bay, Japan [98].Primers specific to A. tamarense and A. catenella were usedto determine if either species was present in the sample. Of theisolated cysts, 91.9 % were found to be A. tamarense, with noA. catenella being detected, suggesting there were few to nocysts of this species in Funka Bay.

A LAMPmethod for the identification ofK. mikimotoiwasdeveloped and compared with previously used PCR [91].Four specific primers were developed as before, and on com-parison with PCR, a 100-fold increase in sensitivity wasobserved for LAMP. The detection limit reported for thisspecies was approximately 6 pg of DNA (cell number notspecified), lower than that previously limits for A. minutum.High selectivity was also observed, with no amplificationoccurring with other HAB species of different genera usingthe same four primers.

Another comparison between the use of LAMP and real-time fluorescence quantitative PCR (FQ-PCR) was conductedfor the detection of P. minimum [99]. The sensitivity of LAMPwas found, again, to be tenfold higher in LAMP than inconventional PCR. The detection limit for LAMP, however,was higher at 36 pg of DNA compared with real-time FQ-PCR, which had a recorded detection limit of 10 pg of DNA.These detection limits were also higher than those of somepreviously discussed methods for other species, suggestingsome optimisation is still necessary. LAMPwas determined tobe a more applicable method for detecting samples with highthroughput in the field. Real-time FQ-PCR was also found toprovide cell counts with a high level of precision; however,analysis was restricted to laboratory environments.

Reverse-transcription-coupled LAMP has also been intro-duced for the detection of Prorocentrum donghaiense [100].Compared with the previously discussed LAMP, reverse-transcription-coupled LAMP allows RNA to be targeted in asample with the initial production of complementary DNAbefore amplification. A detection limit of 0.6 cells permillilitre (0.4 ng) was possible with the specifically designedprimers developed in this study. As was shown for LAMP,

reverse-transcription-coupled LAMP was able to provide vis-ible detection of amplification, making it a practical option forthe detection of this HAB species.

Overall, LAMP has a lot of potential for the detection ofbloom formations. The use of four designed primers makesthe procedure extremely selective. The reaction also occurs ina single tube, with no need for expensive equipment, such asthe thermal cycler required for PCR [95]. The detection limitswhen compared with those of PCR are also much lower,making the technique suitable for low cell number analysis.The issues with DNA extraction need to be thoroughlyassessed prior to using this method for detection however, assome cells require more treatment than others. The primersequences must also be very carefully considered to providethe best selectivity possible for the targeted species.

Hybridisation using synthetic oligonucleotide analogues

Synthetic oligonucleotide substitutes such as peptide nucleicacids (PNA) and locked nucleic acids (LNA) (Fig. 8) havebeen reported in a number of molecular biology applications.The PNA analogue mimics the structure of DNA, but insteadof a phosphate backbone it consists of an uncharged peptidebackbone [102, 103]. LNA, however, still contains the chargedbackbone of DNA but contains a methylene linkage betweenthe 2′ oxygen and 4′ carbon. This bridge locks the structure inplace and prevents molecule flexibility and any changes inconformation [104]. In both cases, these modifications providean increased binding affinity for target nucleic acid sequences[105]. PNA and LNAoligomers typically exhibit greater speci-ficity in binding to complementary DNA and RNA strands,with mismatches being more destabilising than in typical oli-gonucleotide duplexes [105] (Fig. 8).

PNAwas introduced into the detection of HABs in 2006 asan alternative to DNA probes in an SHA application. Sincethen the analogue has also been integrated into FISH and real-time PCR formats [106].

A modified SHA using the alternative PNA probe for thedetection of A. tamarense was performed initially. A compar-ison of the DNA oligonucleotides and the equivalent PNAsequence demonstrated that the fluorescein-labelled PNA sig-nal probe provided a higher-intensity fluorescent response.PNA analogues are, however, significantly more expensivethan the standard DNA oligonucleotides. Because of this,Connell et al. [106] suggested that the increased signal ofthe PNA versus the DNA may not be advantageous whentaking into account the cost required to achieve it.

PNA probes were then optimised for the detection ofTakayama pulchella using FISH techniques. Almost 100 %hybridisation efficiency was observed when comparing thesignal obtained using the DNA or PNA probe with thatobtained using a positive control probe. As with the study in

�Fig. 7 Process of loop-mediated isothermal amplification showing thethree main steps: non-cyclic reaction and formation of dual looped DNAstrand, cyclic amplification and elongation leading to strand displacementDNA synthesis

Detection of harmful algal blooms using oligonucleotides

2006, the fluorescent signal intensity was recorded to be 2.27times higher when the PNA was used as a probe than whenDNA was used, making it a more sensitive approach for thedetection of T. pulchella [107].

PNA detection of P. donghaiense has also been done usingthe FISH method. Several probes were trialled initially, with acomparison between DNA oligonucleotide capture probesand PNA capture probes. A higher fluorescent signal wasobtained when using the PNA probe as well as no cross-reactivity with other microalgae species. A comparison ofenumerated cell numbers using LM and the PNA probeshowed no significant difference in concentrations. TheDNA probe, however, showed a concentration statisticallydifferent from that shown by both LM and the PNA probe(p<0.05). This suggested that the PNA probe was more ef-fective for the detection of P. donghaiense than the DNAprobe [108].

Alexandrium species have also been introduced to thealternative PNA detection platform. The method used is acolorimetric bioassay that utilises a wavelength shift, andsubsequent colour change, in the cyanine dye 3,3′-diethylthiadicarbocyanine iodide once the dye aggregateswithin PNA–DNA duplexes. In a model system the applica-bility of this method for the detection of Alexandrium specieswas trialled. The method was successful and provided a rapid,specific detection method. A portable colorimeter was alsodeveloped to detect the change of wavelength, and this wasfound to correlate with the data obtained from a benchtopspectrophotometer, providing the potential for in-field analy-sis of samples [109].

An alternative DNA analogue, LNA, has been used in onlya couple of applications for the detection of HABs. The use ofLNA was first introduced to the detection of a number ofunialgal samples in an SHA format as well as in a DNAmicroarray. Here, an oligonucleotide capture probe and asignal probe as well as alternative LNA capture probes weretested and compared. The LNA, just as PNA had done previ-ously, provided an enhanced fluorescent signal. A disadvan-tage with the enhanced signal, however, was a subsequentincrease in false-positive signals when using the microarrayformat. In the SHA format there was no distinguishable dif-ference between the DNA and LNA. Because of the results

from the microarray format and the SHA format, the advan-tage from use of LNA as an alternative to DNA in this casewas negligible [110].

LNA probes have also been used for the detection ofK. brevis and K. mikimotoi in a PCR bead suspension assay.DNA oligonucleotide probes were once again compared withLNA for both the species. DNA oligonucleotide probes weretested against LNA probes specific for both species to see ifeither provided higher sensitivity. Higher affinity and fluores-cent signals were obtained from the LNA, with the resultsbeing more significant when the assay was performed attemperatures above 53 °C. This research allowed an integra-tion between LNA probes, Luminex technology and directchemical labelling (Mirys Label IT) for simple identificationand quantification of K. brevis and K. mikimotoi [111].

Conclusion

Recent developments in gene sequencing have identified re-gions in the genome of HAB-causative species that can betargeted for selective and sensitive detection. A number ofoligonucleotide sequences specific for rRNA have beenreviewed. These can be used diagnostically via a number ofstrategies, the commonest being FISH, SHA, NASBA andreal-time PCR. The simplicity of analysing rRNA in crudelysates without purification or amplification results in a po-tential for automation and in-field analysis. In most cases themethods, however, are limited to the detection of a singlespecies at a time. Very little research has focused on thedevelopment of strategies for the detection of multiple speciesin a single assay. The selectivity of the molecular probes isgenerally reported to be very high, and as such this approach ispromising for a truly multispecies detection platform.Much ofthe research presented used single-stranded oligonucleotidesequences in the solution phase. Oligonucleotides are expen-sive and can be used only once. There is significant potentialfor the development of reusable solid devices based on similaroligonucleotides that are covalently attached to surfaces.These surfaces have the potential to be developed further intosensor microarrays with different regions of the surface beingselective for different species. Dehybridisation to regenerate

Fig. 8 Structure of DNA and itssynthetic analogues lockednucleic acid (LNA) and peptidenucleic acid (PNA)

K.L. Bruce et al.

the original surface could be readily achieved by heatingabove the melting temperature and washing, returning thesurface to its original state for reuse. Detection can beachieved via traditional fluorescence and colorimetric strate-gies or via alternative techniques such as surface plasmonresonance

It is documented that the distance between the surface andthe oligonucleotide can influence the behaviour of the system,with long spacers between the surface and the oligonucleo-tides allowing them to act as if they are in the solution phaseand bind more readily than those that are bound directly to thesurface. The incorporation of covalent attachment with longspacers may allow improved sensitivity when compared withcurrent surface strategies. Further, work on the developmentof synthetic probes such as PNA, which are documented toexhibit greater selectivity, reduced non-selective binding andimproved sensitivity, may also serve to improve detection ofHAB-causative species.

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