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Journal of Virological Methods 183 (2012) 8– 13

Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods

j ourna l ho me p ag e: www.elsev ier .com/ locate / jv i romet

apid, simple influenza RNA extraction from nasopharyngeal samples

arrell P. Chandlera,∗ , Sara B. Griesemerb, Christopher G. Cooneya, Rebecca Holmberga, Nitu Thakorea,ecca Mokhibera, Phillip Belgradera, Christopher Knickerbockera, Jeanmarie Schiedc, Kirsten St. Georgeb

Akonni Biosystems, Inc., 400 Sagner Avenue, Suite 300, Frederick, MD 21701, United StatesLaboratory of Viral Diseases, Wadsworth Center, New York State Dept of Health, 120 New Scotland Avenue, Albany, NY 12208, United StatesLittle Company of Mary Hospital, Pediatric Hospitalist Program, University of Chicago, 2800 West 95th Street, Evergreen Park, IL 60805, United States

rticle history:eceived 26 October 2011eceived in revised form 24 February 2012ccepted 1 March 2012vailable online 7 March 2012

eywords:NA

a b s t r a c t

This report describes the development and pre-clinical testing of a new, random-access RNA samplepreparation system (TruTip) for nasopharyngeal samples. The system is based on a monolithic, porousnucleic acid binding matrix embedded within an aerosol-resistant pipette tip and can be operated withsingle or multi-channel pipettors. Equivalent extraction efficiencies were obtained between automatedQIAcube and manual TruTip methods at 106 gene copies influenza A per mL nasopharyngeal aspirate.Influenza A and B amended into nasopharyngeal swabs (in viral transport medium) were detected byreal-time RT-PCR at approximately 745 and 370 gene copies per extraction, respectively. RNA extrac-

ample preparationasopharyngeal swabasopharyngeal aspirateutomation

tion efficiency in nasopharyngeal swabs was also comparable to that obtained on an automated QIAcubeinstrument over a range of input concentrations; the correlation between threshold cycles (or nucleicacid recovery) for TruTip and QIAcube-purified RNA was R2 > 0.99. Preclinical testing of TruTip on blindednasopharyngeal swab samples resulted in 98% detection accuracy relative to a clinically validated easy-MAG extraction method. The physical properties of the TruTip binding matrix and ability to customizeits shape and dimensions likewise make it amenable to automation and/or fluidic integration.

. Introduction

Influenza viruses continue to be a significant long-term publicealth concern because of their genetic mutability, rapid trans-ission, and ability to move from species to species. Few drugs

re approved by the US Food and Drug Administration (FDA) forreating influenza infections, and successful treatment depends ondministering these drugs within the first 48 h of illness (Couch,000). To ensure appropriate use of antiviral drugs, a near-patientiagnostic test is therefore highly desirable (Barenfanger et al.,000). In addition, there is growing interest in deploying rapidetection technologies in resource-limited settings (Ghosh and

ogt, 2008; Hanvoravongchai et al., 2010; Kubo et al., 2010; Ortizt al., 2009; Oshitani et al., 2008) and expanding non-human

∗ Corresponding author. Tel.: +1 734 428 0713; fax: +1 301 698 0202.E-mail addresses: dchandler@akonni.com (D.P. Chandler),

bg03@health.state.ny.us (S.B. Griesemer), cooney@akonni.com (C.G. Cooney),holmberg@akonni.com (R. Holmberg), nthakore@akonni.com (N. Thakore),mokhiber@gmail.com (B. Mokhiber), pbelgrader@akonni.com (P. Belgrader),knickerbocker@akonni.com (C. Knickerbocker), jeanschied@gmail.com (J. Schied),xs16@health.state.ny.us (K. St. George).

166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2012.03.002

© 2012 Elsevier B.V. All rights reserved.

influenza surveillance activities (e.g., Antarasena et al., 2007;Samaan et al., 2011).

Nucleic acid tests involving target amplification are becomingmore prevalent for influenza diagnostics and surveillance (Abeet al., 2011; Bolotin et al., 2009; Habib-Bein et al., 2003; Han et al.,2008; Huber et al., 2011; Létant et al., 2007; Pachucki et al., 2004;Shisong et al., 2011; Wenzel et al., 2010; Wu et al., 2010; Xu et al.,2010), in part because of their excellent sensitivity and relativelyrapid turn-around times. In many cases, these molecular detectionmethods are paired with automated (robotic) sample preparationtechnology (Agüero et al., 2007; Bolotin et al., 2009; Tewari et al.,2007), typically to increase throughput in a reference laboratorysetting. Even for many upcoming molecular point-of-care devices,however, there is continued reliance upon external nucleic acidextraction methods that require some form of instrumentation (Wuet al., 2010), limiting the deployment potential of the test. Alter-natively, some influenza tests utilize unprocessed nasopharyngealsamples (e.g., Regan et al., 2010), but limits of detection may becompromised (Létant et al., 2007) because there is either no con-centration step, viral nucleic acids become inextricably bound to

the sample matrix itself (and unavailable for amplification), or sub-stances in the matrix inhibit the amplification reaction.

For molecular methods to be readily utilized in point-of-useor limited resource settings, there is a need for simple and

D.P. Chandler et al. / Journal of Virolo

Fig. 1. Basic TruTip construction and operation. The 2 mm thick, monolithic bindingmatrix is embedded in an aerosol-barrier pipette tip. Fluid flow is bi-directionaltt

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hrough the matrix. The shape and pore size of the binding matrix can be tailoredo fit different tip sizes.

ost-effective sample preparation systems that are consistent withhe user’s infrastructure, level of training, and sample through-ut. The objectives of this study were therefore to develop aample preparation system (TruTip) and methodology that onlyequires a pipettor (single- or multi-channel) and single tiper sample to operate, optimize a protocol for influenza RNAxtraction and purification from nasopharyngeal aspirate andasopharyngeal swabs (in viral transport media), and test the sys-em’s efficacy on a blinded set of clinical nasopharyngeal swabpecimens.

. Materials and methods

.1. TruTip construction

The TruTip itself contains a highly porous, rigid, monolithic, mm thick silica binding matrix of defined (yet adaptable) geom-try and pore size (Fig. 1). Of importance for nasopharyngealamples, the TruTip matrix offers less fluidic impedance than a silicaembrane, and combined with bi-directional flow, the system can

chieve rapid extraction of nucleic acids from larger volumes thanypically used in either spin-filter or bead-based sample prepara-ion systems. This study utilized 2 mL SPT tips for single-channelample preparation with a Rainin EDP3+ Electronic LTS pipette, and

mL SPT tips for a Rainin EDP3 LTS 8-channel electronic pipette100–1200 �L).

.2. Positive controls

Viruses used for these studies included influenza A/Hongong/8/68 (H3N2), influenza A/New Jersey/8/76 (H1N1), influenza/New York/1669/2009 (H1N1), influenza B/Hong Kong/5/72,

nfluenza B/Taiwan/2/62 and a 2008 influenza B isolate from Nework State. New York strains were isolated from human respira-ory samples that were submitted to the reference and surveillancerogram at the Laboratory of Viral Diseases, Wadsworth Center,

ew York State Department of Health (Albany, New York). Virusesere propagated in primary rhesus monkey kidney (pRhMK) cells

s per standard procedures (Diagnostic Hybrids Inc., Athens, OH).iral RNA was isolated from 60 �L of cultured virus using a QIAamp

gical Methods 183 (2012) 8– 13 9

Viral RNA kit on the automated QIAcube, as per the manufacturer’sinstructions (Qiagen, Valencia, CA). Viral RNA was eluted in 60 �Ltotal volume and 10-fold serially diluted in RNA Storage Solution(Applied Biosystems/Ambion, Austin, TX) to achieve RNA dilutionsfrom 0 to 1:1000.

Influenza RNA copy number in each dilution was determinedusing single-plex, real-time reverse transcriptase TaqMan assaysdesigned for the universal detection of influenza A matrix (M) geneand influenza B non-structural (NS) gene, as previously described(Ghedin et al., 2009). Both TaqMan assays are validated for diagnos-tic use. Duplicate reactions were performed at each RNA dilutionlevel. RNA standard curves were generated from influenza M andNS RNA transcripts that had been previously quantified by UV spec-trophotometry. The calculated gene copy (gc) numbers for eachdilution (and replicate) were averaged to arrive at a viral stock RNAconcentration (in RNA gc mL−1).

In order to measure extraction efficiency, quantified greenfluorescent protein (GFP) RNA transcripts were amended intonasopharyngeal swab samples prior to TruTip extraction. Tran-scripts were created from GFP-containing plasmid pTU65 (Chalfieet al., 1994) and quantified by real-time RT-PCR using previouslydescribed primers and probes (Tavakoli et al., 2007).

2.3. Clinical samples

Nasopharyngeal aspirate was obtained from routine clinicalsampling at Little Company of Mary Hospital. De-identified, resid-ual portions of specimens were stored frozen at −20 ◦C until use.Nasopharyngeal swab specimens (in viral transport medium) wereobtained from reference and surveillance samples submitted toWadsworth Center and tested for influenza A and B presenceusing NucliSENS® easyMAG® nucleic acid extraction (bioMérieux,Durham, NC) and real-time RT-PCR assays that are approved bythe New York State Department of Health for clinical use. Leftoverspecimens were stored frozen at −70 ◦C until use.

Influenza-negative nasopharyngeal specimens (nasopharyngealaspirate, or nasopharyngeal swabs in viral transport medium) werepooled and used as the sample matrix for TruTip protocol develop-ment, optimization experiments, and QIAcube comparison studies.Virus-amended matrix was serially diluted, dispensed into multi-ple aliquots, and frozen at −70 ◦C. Each frozen sample was thawedonly once, immediately before extraction.

For pre-clinical testing, 48 de-identified specimens (32influenza A – positive, 10 influenza B – positive, and 6 influenza– negative) were sent to Akonni Biosystems as a blinded panelfor pre-clinical evaluation of TruTip extraction efficacy. Pre-clinicalTruTip extractions and real-time PCR tests were also duplicated atthe Laboratory of Viral Diseases as an independent verification andrepeatability test (data not shown).

2.4. TruTip nucleic acid purification

A baseline TruTip extraction protocol was first developedusing amended nasopharyngeal aspirate samples before apply-ing the TruTip protocol to nasopharyngeal swabs (not shown).The optimized protocol included taking either 250 �L dilutednasopharyngeal aspirate (100 �L nasopharyngeal aspirate + 150 �LDEPC-treated H2O) or 250 �L nasopharyngeal swab (in viral trans-port medium) into 375 �L of a concentrated guanidine/sodiumacetate lysis buffer. Then, 375 �L of 95% ethanol was added tothe sample and mixed thoroughly. For analytical studies (only),4.4 × 104 gc of GFP transcript was added to each sample as an

internal extraction and RT-PCR inhibition control. Thereafter, aRainin EDP3+ Electronic LTS pipette fitted with a 2 mL SPT Tru-Tip was used to aspirate and dispense the lysed sample seventimes (7 cycles) through the TruTip to bind the RNA to the TruTip

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atrix. The tip was then washed with 1 mL of a guanidium/sodiumcetate/ethanol wash buffer for 5 cycles, followed by 1 mL ofthanol/acetone wash buffer for 5 cycles. The TruTip matrix wasir-dried by cycling the pipettor 15 times in an empty tube. Finally,urified RNA was eluted from the TruTip matrix by cycling 100 �Lf an RNase-free Tris–HCl elution buffer five times through theatrix.Extraction efficacy and reproducibility studies utilized 250 �L

liquots of sample that were prepared by diluting influenza A-r B-amended nasopharyngeal swab in sample matrix, and thenmending with 4.4 × 104 gc of GFP transcript. Amended samplesere extracted and eluted into 100 �L on three different days.uantitative, real-time RT-PCR was performed in duplicate on all

ample extracts. The extraction efficacy was determined as the low-st concentration of input viral RNA detected over all replicatescross three days of extraction.

The Qiagen QIAcube served as the reference RNA extractionethod for all studies, and all QIAcube samples were amendedith GFP transcript as described above. In order to compare Tru-

ip extraction efficiency to the QIAcube, 125 �L sample and 50 �Llution volumes were used on the QIAcube to match the volumeatios associated with TruTip extraction volumes (250 �L samplend 100 �L elution volumes). The QIAcube is not able to pro-ess a 250 �L sample (if performed as per the manufacturer’snstructions), because the recommended volumes for sample, lysisuffer, and ethanol would equal 2.45 mL, exceeding the capac-

ty of the 2 mL extraction vessel. QIAcube samples were thereforemended with half the RNA (gene copies) as the TruTip sam-les to maintain a constant target nucleic acid concentrationetween the two extraction systems. Whether or not the 2-foldolume difference between QIAcube and TruTip had an effect oneasured extraction efficiencies was not explicitly tested in this

tudy.

.5. Multi-channel TruTip

Multi-channel TruTip experiments utilized 1 mL SPT TruTips andhe same cycling procedure as described above, except the elutionolume was 75 �L rather than 100 �L.

.6. RT-PCR detection

Influenza RNA was detected by real-time RT-PCR on either LightCycler 480 (Roche, Indianapolis, IN) or Stratagene (nowgilent Technologies, Santa Clara, CA) MxP3005P thermal cycler.egardless of the thermal cycling instrument, 5 �L purified RNAemplate was amplified in duplicate (or triplicate) in a 25 �Leaction volume using the qScript One-Step qRT-PCR kit (QuantaioSciences, Gaithersburg, MD). Master mix components included00 nM each primer and 325 nM internal probe for influenza Aetection, and 750 nM each primer and 250 nM internal probe for

nfluenza B detection. Cycling conditions for both influenza A and real-time RT-PCR assays were 48 ◦C for 20 min; 95 ◦C for 5 min;nd 45 cycles of (95 ◦C for 15 s, 55 ◦C for 45 s). Recovered gene copyumbers were calculated relative to influenza M and NS standardurves as described above, and used to calculate a % recovery.

Cycling conditions for the GFP RNA assay were 48 ◦C for 30 min;5 ◦C for 10 min; and 45 cycles of (95 ◦C for 15 s, 60 ◦C for 1 min).ecovered gene copy numbers were calculated relative to a GFP

tandard curve, and used to calculate a % recovery. Standard curvesfor M, NS or GFP RNA targets) were generated for every RT-PCRun, with R2 values >0.99 over the range of 10–106 RNA copies pereaction (data not shown).

ical Methods 183 (2012) 8– 13

3. Results

3.1. RNA purification from nasopharyngeal aspirate

Nasopharyngeal aspirate was used as the specimen matrix fordeveloping basic TruTip manufacturing and operating parame-ters. Preliminary fluidic experiments with nasopharyngeal aspiratesamples demonstrated that sample liquefaction in lysis/bindingbuffer was sufficient to perfuse unprocessed nasopharyngeal aspi-rate over the TruTip matrix, where initial sample input volumeswere 500 �L nasopharyngeal aspirate and 500 �L lysis/bindingbuffer (not shown). Extraction of 106 influenza A gc mL−1 nasopha-ryngeal aspirate (n = 6 per day) on two replicate days resulted inaverage Ct values of 31.08 ± 0.06 and 31.98 ± 0.06 for TruTip, and31.08 ± 0.14 and 31.47 ± 0.15 for QIAcube. Given positive RT-PCRdetection at 500 �L nasopharyngeal aspirate sample input andequivalent performance between QIAcube and TruTip methods, wereduced sample volumes in a step-wise fashion to arrive at theprotocol described in Section 2.

3.2. RNA purification from nasopharyngeal swabs

Having developed a basic operating protocol for influenzaRNA recovery from nasopharyngeal aspirate, we focused opti-mization efforts on nasopharyngeal swabs (in viral transportmedium). Influenza A/New York/1669/2009(H1N1) and a 2008influenza B virus (both isolated and propagated in the Laboratoryof Viral Diseases) were used as material to amend into the pooledinfluenza-negative nasopharyngeal swabs. Amended nasopharyn-geal swab samples were dispensed into multiple 250 �L aliquots.On each of three consecutive days, one aliquot was extracted withTruTip and analyzed in duplicate at each dilution level. A fourthaliquot was processed on the QIAcube as a reference. The averageTruTip processing time on the single-channel pipettor was approx-imately 7 min per sample. The sample processing speed is primarilyrelated to the speed at which the crude lysate can be perfusedthrough the TruTip binding matrix by the Rainin electronic pipettor,and the total number of cycles per step.

Average Ct values and recovered RNA (in gene copies) fromreal-time RT-PCR assays and relationship to the QIAcube referencemethod for nasopharyngeal swabs are shown in Table 1. TruTipextraction efficacy over all three days was 745 gene copies perextraction for influenza A and 370 gene copies per reaction forinfluenza B, equivalent to that obtained by QIAcube. The Pearsoncorrelation between average TruTip and average QIAcube Ct val-ues was R2 = 0.99 for influenza A and influenza B over all positivedilutions and replicates.

All extracted samples were also amended with 4.4 × 104 GFPRNA transcript copies prior to purification on either TruTip orQIAcube as a means to detect PCR inhibition and evaluate RNAextraction efficiency from the lysed sample. The average Ct valuefor GFP transcripts was 30.84 ± 0.43 for TruTip and 30.43 ± 0.25for QIAcube. For 100% extraction efficiency and the volumetricsof the TruTip procedure, we would expect to recover 2.2 × 103

GFP gene copies per extraction. From the GFP internal control,then, the measured RNA extraction efficiency was 23% for Tru-Tip and 24% for QIAcube. From Table 1 data, the average influenzaA and B RNA extraction efficiency within the central, linear por-tion of the dilution series (106–103 gene copies) was 39% and69% for TruTip, and 50% and 81% for QIAcube, respectively. These

results demonstrate efficient and repeatable TruTip RNA extrac-tion from nasopharyngeal swabs in viral transport medium, withperformance characteristics comparable to the automated QIAcubesystem and an estimated extraction efficacy of 102–103 RNA gene

D.P. Chandler et al. / Journal of Virological Methods 183 (2012) 8– 13 11Ta

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Table 2Multi-channel TruTip extraction efficacy for influenza A RNA.

Input (gc) Average Ct (±St. Dev) Average recovery (gc)

7.45 × 106 25.71 ± 0.19 1.24 × 106

7.45 × 105 28.56 ± 0.17 1.74 × 105

7.45 × 104 32.13 ± 0.45 1.60 × 104

3 3

7.45 × 10 35.84 ± 0.57 1.32 × 107.45 × 102 38.50 ± 0.86 2.01 × 102

7.45 × 101 Not detected Not detected

copies per extraction (or 0.1–1 TCID50, assuming 1000 virions perTCID50; Chan et al., 2009).

3.3. Multi-channel TruTip extractions

Extraction efficacies for the multi-channel TruTip extractionsusing influenza A RNA are shown in Table 2. In this case, 8 sampleswere processed simultaneously within 7 min (<1 min per sample).The correlation coefficient between the real-time RT-PCR Ct andlog10(gc mL−1) was R2 = 0.997 with an extraction efficacy of 745input gene copies per extraction. Repeatability of multi-channelTruTip extraction is shown in Table 3, with results consistent withthe single-channel format. In this case, repeatability at 102 gc perextraction was not tested given that the single-channel efficacy wasdefined as 103 gc per extraction (i.e., positive detection in 6 of 6replicates instead of 5 of 6 replicates).

3.4. Blinded clinical samples

The optimized nasopharyngeal swab extraction protocol wasapplied to 48 blinded clinical nasopharyngeal swabs in viral trans-port medium. Typing of these blinded samples with the clinicallyvalidated real-time RT-PCR assays on a Roche 480 thermal cyclerresulted in 98% accuracy (Table 4) with only one false negativeresult (sample NYS-47; low titer of influenza B). The blinded sam-ples were also re-tested at Wadsworth using TruTip extraction anda Stratagene MxP3005P thermal cycler, with equivalent results(not shown). These data indicate that the TruTip nasopharyngealswab extraction method is repeatable across test sites and users,generates high-quality viral RNA that can be amplified by differ-ent thermal cyclers, and yields test results that are comparable tothose obtained with a clinically validated bioMérieux NucliSENSeasyMAG nucleic acid extraction system.

4. Discussion

Despite continued advances in influenza diagnostics and point-of-care systems, the extent to which molecular diagnostics areutilized beyond reference laboratory environments continues to belimited. Reasons for limited deployment or access vary dependingupon the intended use and user community, but include practicalconsiderations such as system and consumable cost; test complex-ity; sample volume and fluidic limitations; throughput; sampleacquisition, storage and/or shipment constraints; and flexibility ofthe underlying technology platform. Therefore, while integratedinfluenza diagnostic tests are either commercially available or indevelopment (e.g., Jenny et al., 2010; Xu et al., 2010), there is acontinued need to provide a robust, simple, low-cost and random-access sample preparation method that has the potential to bringmolecular detection systems out of a reference laboratory andcloser to the point of use. The results of this study show that avery simple, pipette-operated sample preparation technology (Tru-

Tip) is as effective as clinically validated QIAcube or easyMAGautomated sample preparation systems for extracting and puri-fying influenza RNA from nasopharyngeal samples. Whether ornot volumetric differences between TruTip and automated sample

12 D.P. Chandler et al. / Journal of Virological Methods 183 (2012) 8– 13

Table 3Inter-run repeatability of multi-channel TruTip extractions with influenza A RNA.

Input (gc) aAverage TruTip recovery in gene copies (and Ct) Averageb

Day 1 Day 2 Day 3 Day 4

7.45 × 105 3.61 × 105 1.74 × 105 – – 2.80 × 105

(28.08) (28.56) – – (28.29 ± 0.42)7.45 × 104 3.80 × 104 3.35 × 104 2.26 × 104 1.60 × 104 2.85 × 104

(31.39) (31.28) (31.58) (32.13) (31.60 ± 0.44)7.45 × 103 2.98 × 103 3.34 × 103 2.62 × 103 1.32 × 103 2.56 × 103

(35.18) (34.18) (34.56) (35.84) (34.94 ± 0.75)

a Average of three replicates per day.b

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reparation systems have an effect on measured extraction effi-iency was not explicitly tested in this study. However, becauseruTip only requires a pipette to operate and deliver PCR-qualityNA within 7 min, it provides a means to bring nucleic acid sam-le preparation into resource-limited settings. At the same time,he TruTip sample preparation scheme is also very adaptable toobotic liquid handling systems and a higher-throughput operatingnvironment.

The TruTip protocol described in this study should be considered reference or starting point for nasopharyngeal samples, especiallyamples like nasopharyngeal aspirate. That is, every nasopharyn-eal aspirate or nasopharyngeal swab sample is unique and williffer from the next in viscosity, particulates, viral load and back-round microflora. Sample NYS-47 (Table 4), for example, mightave been detectable under a more rigorous set of binding/elution

ycles. Successful influenza RNA extraction may therefore, at times,equire more vigorous up-front sample homogenization or lysisethods, or additional binding/washing/elution cycles to separate

nfluenza RNA from the sample matrix.

able 4eal-time, RT-PCR test results for TruTip-processed, blinded nasopharyngeal swab clinica

Sample FluA results FluB results Answera

Ct Call Ct Call

NYS-3 27.46 + − pdmH1

NYS-4 35.79 + − pdmH1

NYS-5 − − Negative

NYS-6 30.95 + − pdmH1

NYS-7 33.98 + − pdmH1

NYS-8 33.45 + − pdmH1

NYS-9 30.35 + − pdmH1

NYS-10 − − Negative

NYS-11 33.14 + − pdmH1

NYS-12 36.82 + − pdmH1

NYS-13 23.24 + − pdmH1

NYS-14 36.73 + − pdmH1

NYS-15 35.77 + − H1N1

NYS-16 34.99 + − H3N2

NYS-17 30.19 + − H3N2

NYS-18 − 30.19 + FluB

NYS-19 34.38 + − H1N1

NYS-20 28.06 + − H3N2

NYS-21 − 31.64 + FluB

NYS-22 − − Negative

NYS-23 28.59 + − H1N1

NYS-24 29.99 + − H3N2

NYS-25 − 37.13 + FluB

NYS-26 36.04 + − H1N1

dmH1 = pandemic influenza H1.a The answer key is derived from clinically validated NucliSENS easyMAG extraction mb The original RT-PCR result was negative, but positive upon re-test.c False negative result.

The significance of these results, however, extends beyondinfluenza testing and diagnostics. That is, the TruTip binding matrixitself solves some very important sample preparation technicalissues, especially for continued development of integrated or fullyautomated systems. For example, the binding matrix offers a lowerfluidic impedance than membranes (e.g., as in a spin-filter or Gen-eXpert cartridge), as illustrated by the ability to process bothnasopharyngeal aspirate and nasopharyngeal swab through thesame matrix. Bi-directional flow through the TruTip acceleratesnucleic acid binding kinetics relative to static (e.g., bead-based)techniques and, more importantly, is amenable to relatively largesample volumes. The latter feature is especially relevant for clos-ing the sample-to-detector volume gap. Finally, the binding matrixitself can be manufactured with different geometries (shapes) andpore sizes to fit within commercially available pipette tips (to 5 mL)or customized microfluidic architectures. We therefore anticipatethat the basic sample preparation principle, system and chemistry

described here can accelerate the development and deployment ofmolecular diagnostic devices beyond reference laboratories.

l specimens.

Sample FluA results FluB results Answera

Ct Call Ct Call

NYS-27 36.85 + − H1N1NYS-28 − − NegativeNYS-29 − − NegativeNYS-30 35.68 + − pdmH1NYS-31 − 36.32 + FluBNYS-32 28.73 + − H1N1NYS-33 31.87 + − H3N2NYS-34 24.97 + − H3N2NYS-35 − 28.50 + FluBNYS-36 29.08 + − H1N1NYS-37 − − NegativeNYS-38 26.19 + − H3N2NYS-39 28.75 + − H1N1NYS-40 − 35.00 + FluBNYS-41 25.70 + − H1N1NYS-42 − 35.04 + FluBNYS-43 27.76 + − H1N1NYS-44 − 35.58 + FluBNYS-45 29.30 + − H3N2NYS-46b − 36.49 + FluBNYS-47c − − FluBNYS-48 25.83 + − H3N2NYS-49 33.98 + − pdmH1NYS-50 32.41 + − H3N2

ethod and real-time RT-PCR assays.

Virolo

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A

A

A

B

B

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Jalal, H., Zambon, M., Lee, H.H., 2010. Nucleic acid dipstick test for molecular

D.P. Chandler et al. / Journal of

cknowledgements

This work was supported by the National Institutes of HealthNIH) under grant R 44 AI072784.

Samples at the Wadsworth Center were collected with the sup-ort of Cooperative Agreement number U50/CCU223671 from theDC. The study was approved by the New York State Departmentf Health Institutional Review Board (study number 07-022).

The authors thank Dr. Amy Dean and Daryl Lamson for helpfuliscussions and editorial assistance.

The contents of this manuscript are solely the responsibility ofhe authors and do not necessarily represent the views of the NIHr the CDC.

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