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JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2011, p. 3154–3162 Vol. 49, No. 90095-1137/11/$12.00 doi:10.1128/JCM.00599-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Evaluation and Verification of the Seeplex Diarrhea-V ACE Assay forSimultaneous Detection of Adenovirus, Rotavirus, and Norovirus
Genogroups I and II in Clinical Stool Specimens�
Rachel R. Higgins,1* Melissa Beniprashad,1 Mark Cardona,1 Steven Masney,1
Donald E. Low,1,2,3 and Jonathan B. Gubbay1,2,3,4
Ontario Agency for Health Protection and Promotion, Toronto, Ontario, Canada1; Mount Sinai Hospital, Toronto, Ontario,Canada2; University of Toronto, Ontario, Canada3; and The Hospital for Sick Children, Toronto, Ontario, Canada4
Received 24 March 2011/Returned for modification 6 May 2011/Accepted 6 July 2011
Acute viral gastroenteritis is an intestinal infection that can be caused by several different viruses. Here wedescribe the evaluation and verification of Seeplex Diarrhea-V ACE (Seeplex DV), a novel commercial multi-plex reverse transcription-PCR (RT-PCR) assay that detects 5 diarrheal pathogens, including adenovirus,rotavirus, norovirus genogroup I (GI) and GII, and astrovirus. We describe a retrospective study of 200 clinicalspecimens of which 177 were stool specimens previously tested for the presence of gastrointestinal viruses byelectron microscopy (EM) and/or real-time RT-PCR (rRT-PCR). The remaining 23 specimens comprised otherhuman pathogens of viral or bacterial origin. Discordant norovirus GI and GII results were resolved using acommercial kit; discordant adenovirus and rotavirus results were resolved using a home brew multiplexrRT-PCR assay. Diagnostic sensitivities and specificities were calculated before and after discordant analysis.After discordant analysis, estimated diagnostic sensitivities were 100% for adenovirus, rotavirus, and norovirusGI and 97% for norovirus GII. Diagnostic specificities after discordant analysis were 100% for adenovirus,rotavirus, and norovirus GI and 99.4% for norovirus GII. The 95% limits of detection were 31, 10, 2, and 1genome equivalent per reaction for adenovirus, rotavirus, and norovirus GI and GII, respectively. The resultsdemonstrate that the Seeplex DV assay is sensitive, specific, convenient, and reliable for the simultaneousdetection of several viral pathogens directly in specimens from patients with gastroenteritis. Importantly, thisnovel multiplex PCR assay enabled the identification of viral coinfections in 12 (6.8%) stool specimens.
Acute viral gastroenteritis is the second most common in-fectious disease worldwide (20, 5), affecting humans of all agegroups (23, 17) but mostly the young, the elderly, and people inenclosed communities, such as hospitals, nursing homes, mili-tary bases, and cruise ships. Known enteric viral pathogensinclude adenovirus group F serotypes 40 and 41, rotavirus,norovirus, and astrovirus, with rotavirus and norovirus beingthe predominant causative agents of gastroenteritis (3, 7, 30).In recent years the number of reported gastroenteritis out-breaks of suspected viral etiology has increased, hence theneed for fast, sensitive, and reliable diagnostic assays.
The established method for detection of enteric viruses atthe Ontario Agency for Health Protection and Promotion(OAHPP) Public Health Laboratories (PHL) relies on theconventional and labor-intensive electron microscopy (EM). Inrecent years, home brew real-time reverse transcription-PCR(rRT-PCR) assays were implemented for the detection ofnorovirus GI and GII (11). Similar home brew rRT-PCR assaysfor the detection of adenovirus (6) and rotavirus (35) havebeen described. These rRT-PCR assays enable the detection ofa specific virus by the amplification of a unique genomic se-quence within its RNA or DNA. However, the simultaneousdetection of several viruses can be accelerated and simplified
by the use of a multiplex RT-PCR assay. Multiplex PCR en-compasses multiple primer pairs in a single tube, making itpossible to amplify multiple target sequences.
Here we describe the verification and evaluation of the See-plex DV assay (Seegene, South Korea), a newly developedcommercial multiplex RT-PCR assay for the simultaneous de-tection of adenovirus, rotavirus, norovirus GI and GII, andastrovirus. The use of this novel multiplex RT-PCR assay offersthe advantage of reduced reagents and labor cost, as well as afaster turnaround time. The Seeplex DV assay integrates aninternal control, which facilitates the detection of PCR in-hibitors and reduces false-negative results. Importantly, aunique advantage of a multiplex assay is the detection andidentification of coinfecting pathogens within the same pa-tient specimen.
(Part of this work was presented at the 26th annual ClinicalMicrobiology Symposium, Daytona Beach, FL, April 2010.)
MATERIALS AND METHODS
Definitions. The following definitions were used for analysis: PHL tests, pre-vious tests done at OAHPP, namely, EM (for adenovirus and rotavirus) and/orrRT-PCR (for norovirus GI and GII); concordant specimen, agreement betweenPHL test result and Seeplex DV assay result; discordant specimen, disagreementbetween PHL test result and Seeplex DV assay result; resolved specimen, agree-ment in result between any two of the three assays (PHL test, Seeplex DV assay,and reference assay [RealStar for norovirus GI and GII and home brew multi-plex rRT-PCR for adenovirus and norovirus]).
Clinical specimens. Samples included in the study were from patients ages 2months to 96 years old, of which 54% were 5 years old or younger and 43% werefemale. Seventy-four (42%) of the clinical specimens were from 47 local out-breaks (as defined by the Ontario Ministry of Health) (25), while the remaining
* Corresponding author. Mailing address: Ontario Agency for HealthProtection and Promotion, 81 Resources Road, Toronto, ON M9P3T1, Canada. Phone: (416) 235-6042. Fax: (416) 235-6891. E-mail:[email protected].
� Published ahead of print on 20 July 2011.
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103 (58%) specimens were from sporadic cases of gastroenteritis (Table 1).Table 2 shows the distribution of specimens used in this study. A total of 200clinical specimens and bacterial cultures, including 177 randomly selected stoolspecimens submitted for testing from February 2009 until May 2010 for adeno-virus, rotavirus, norovirus GI, norovirus GII, astrovirus, and enteric bacteria,were used. Also, a specificity panel consisting of 10 nasopharyngeal (NP) swabscontaining respiratory viruses, 14 enteric stool/bacterial cultures, and 7 nonen-
teric bacterial cultures was included to measure specificities and cross-reactivitiesof primer sets. The 3 astrovirus-positive samples were not included in the studyanalysis due to the lack of sufficient samples containing this target.
Laboratory test methods. Routine testing of suspected adenovirus, rotavirus,and norovirus infections at OAHPP relies on the use of EM (Philips CM10 orFEI Morgagni 268). Outbreak specimens (as defined by the Ontario Ministry ofHealth) (25) are initially tested for norovirus by a home brew rRT-PCR assay,and if negative for norovirus, the specimens are then tested by EM (11). In thisstudy, we evaluated the Seeplex DV assay, a commercial multiplex viral diarrheaassay (Seegene, South Korea). This assay consists of reverse transcription andPCR, followed by resolution and detection of amplified DNA products by aga-rose gel (1.5%) or capillary electrophoresis using equipment such as the MCE-202 MultiNA microchip electrophoresis system (Schimadzu BioTech, Japan).Samples with discordant norovirus results were also tested with the RealStar 2.0norovirus kit (Astra Diagnostics, Hamburg, Germany), which detects and differ-entiate norovirus GI and GII. To resolve discrepant adenovirus and rotavirusresults, we developed a home brew multiplex rRT-PCR assay for these virusesand all specimens were retested in this rRT-PCR assay (R. R. Higgins, presentedat the 2011 Clinical Virology Symposium; unpublished data). We did not eval-uate the Seeplex DV assay for the detection of astrovirus due to the lack ofsufficient astrovirus-positive specimens.
RNA and DNA extraction. Total nucleic acid was extracted from all specimensusing an automated NucliSens easyMAG system (Biomeriuex, France). Stoolspecimens were homogenized (20% [wt/vol]) in sterile water and centrifuged for20 min at 3,000 rpm, and 250 �l of the clarified supernatants were subjected toautomated nucleic acid extraction. Total nucleic acid was eluted in a final volumeof 60 �l, of which 5 �l was used for PCR amplification. Nucleic acid wasextracted directly from respiratory specimens collected with nasopharyngealswabs in universal transport medium (Copan Diagnostics Inc.). Bacterial culturesgrown in suggested culture medium (Table 2) (14) were diluted to 0.5 McFarlandstandard (10) in phosphate buffered saline prior to automated extraction on theeasyMAG system. The efficiency of nucleic acid extraction was measured byreal-time PCR amplification of Bacteroides fragilis DNA (19) and/or the humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (1).
PCR amplification. Details of primers and probes used are shown in Table 3.Amplification of nucleic acid using the Seeplex DV assay was performed with theIcycler system (Bio-Rad Laboratories Inc.) according to the manufacturer’sinsert. PCR-inhibitory effects were assessed by coamplification of an internalcontrol included in the Seeplex primer mix. PCR products were resolved bycapillary electrophoresis on the MultiNA. Amplification of norovirus GI and GIItargets was performed on the ABI7900HT Fast real-time PCR system (AppliedBiosystems, California) using home brew rRT-PCR assays (11). Norovirus spec-imens, including discordant ones, were retested on the RealStar 2.0 kit fromAstra Diagnostics (Hamburg, Germany) with amplification on the ABI7500 HTFast real-time PCR instrument according to the manufacturer’s insert. Adeno-virus and rotavirus specimens, including discordant ones, were retested on theABI7500 HT Fast real-time PCR instrument using internally controlled triplexhome brew rRT-PCR assays developed for this study (Higgins, 2011 Clin. Virol.Symp.).
Analytical sensitivity, specificity, and limit of detection. The sensitivity of theSeeplex DV assay was determined in comparison to the PHL test (see definitions
TABLE 1. Seasonal distribution of 74 stool specimens from 47 outbreak investigations and 103 sporadic stool specimens submitted for testingbetween July 2007 and May 2010a
Pathogen
No. of spec
O/B Sporadic
TotalSeason
TotalSeason
W Sp Su F W Sp Su F
Adenovirus 1 0 1 0 0 28 15 6 6 1Rotavirus 2 0 2 0 0 50 2 47 1 0Norovirus GI 19 3 9 3 4 0 0 0 0 0Norovirus GII 28 3 0 4 21 9 5 1 3 0Astrovirus 0 0 0 0 0 3 1 2 0 0Negative 24 2 0 6 16 13 8 2 2 1
Total 74 8 12 13 41 103 31 58 12 2
a spec, specimens; O/B, outbreak; W, winter; Sp, spring; Su, summer; F, fall.
TABLE 2. Distribution of viral and bacterial pathogens tested
PathogenNo. of
specimenstested
Source
Adenovirus 29 StoolRotavirus 52 StoolNorovirus GIl 19 StoolNorovirus GII 37 StoolAstrovirusa 3 StoolNegative enteric specimens 29 StoolSalmonella enterica serovar Paratyphi 2 StoolSalmonella enterica serovar Typhi 1 StoolShigella dysenteriae type 2 5 StoolEscherichia coli 0:157:H71a 1 CultureSalmonella enterica serovar
EnteritidisbCulture
Shigella flexnerib 1 CultureShigella sonneib 1 CultureVibrio choleraec 1 CultureCampylobacter jejunid 1 CultureInfluenza virus B 2 Nasopharyngeal
swabsInfluenza virus A/H1N1 5 Nasopharyngeal
swabsInfluenza virus A/H3N2 1 Nasopharyngeal
swabsPicornavirus 2 Nasopharyngeal
swabsAeromonas hydrophilab 1 CultureNeisseria meningitidise 1 CultureStreptococcus pyogenesb 2 CultureStreptococcus pneumoniaeb 2 CultureStaphylococcus epidermidisb 1 Culture
Total 200
a Three astrovirus-positive specimens were tested in the Seeplex DV assay but notincluded in the evaluation study due to the lack of sufficient positive specimens.
b Cultured in blood agar at 37°C with CO2.c Cultured in plain Columbia agar at 37°C with CO2.d Cultured in charcoal-based selective medium agar at 42°C.e Cultured in New York City agar at 37°C with CO2.
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above). To determine the sensitivity of the Seeplex DV assay, results were scoredagainst the PHL test method (EM for adenovirus and rotavirus and/or rRT-PCRfor norovirus GI and GII). To resolve discordant results, these specimens wereretested using an rRT-PCR assay. For norovirus GI and GII, we employed theRealStar 2.0 rRT-PCR assay from Astra Diagnostics (Hamburg, Germany), andfor adenovirus and rotavirus, we used home brew rRT-PCR assays (Higgins,2011 Clin. Virol. Symp.) (Table 4). P values after discordant analysis weredetermined by chi-square analysis using the software program Stata 11 (Stata-Corp LP, Texas).
Analytical specificity of the Seeplex DV assay was determined by testing a set
of commonly encountered viral and bacterial pathogens (Tables 1 and 2). Viraland bacterial samples were initially characterized by culture, electron micros-copy, or home brew norovirus rRT-PCR during routine clinical testing. Toconfirm PHL culture results for enteric bacteria, these specimens were alsotested for enteric bacteria in the Seeplex Diarrhea ACE test kit.
The limit of detection (LOD) for each virus in the Seeplex DV assay wasdetermined using tissue culture fluid (TCF) for all viral targets except norovirusGI and plasmid DNA for all targets. Quantified plasmid DNA (Seegene, SouthKorea) and/or TCF (Zeptometrix Corporation) for each virus was serially diluted10-fold to 10�12 of the starting material in PCR-grade water and tested in theSeeplex DV assay. The respective LODs were assessed by testing a minimum of4 replicates of each of the 12 dilution steps. The 95% limit of detection (and 95%confidence interval) for each target was determined using probit regression(SPSS software regression package, version 18.0; SPSS, Chicago, IL).
Assay reproducibility and TAT. Inter- and intra-assay reproducibilities wereassessed using data generated from the LOD calculation and from testing 110samples on 3 different days. Concordance for inter- and intra-assay reproduc-ibility was calculated. Turnaround time (TAT) was calculated for the Seeplex DVassay and home brew rRT-PCR based on 5 runs, each of 96 samples, completedby one laboratory staff member. For EM, TAT was extrapolated from timing 3days’ work. The final TAT reflected the average time to process 96 samples,including reception, hands-on, assay, and resulting/reporting times. To directlycompare assays, TAT for each assay was divided by the number of targets testedto give a TAT per target for 96 samples.
RESULTS
Table 4 displays the results obtained for each of the 197clinical specimens and bacterial cultures included in this study(excluding astrovirus). A pictorial example of results obtainedfor a subset of clinical specimens, representing each of the four
TABLE 3. Primers and probes used for target amplification
Target Primer and probe sequencesb Amp size(bp)c Position PCR method Detection
system Reference
B. fragilis f: GAG AGG AAG GTC CC 129 296–425 rRT-PCR ABI7900 20r: CGC TAC TTG GCT GGp: FAM-CCA TTG ACC AAT ATT CCT CAC TGC TGC
CT-BHQ
Norovirus GI f: GCC ATG TTC CGI TGG ATG 76 5282–5358 rRT-PCR ABI7900 11r: TCC TTA GAC GCC ATC ATC ATp: FAM- AGA TCG CGG TCT CCT GTC CA-BHQ
Norovirus GII f: CAA GAG TCA ATG TTT AGG TGG ATG AG 77 5003–5080 rRT-PCR ABI7900 11r: TCG ACG CCA TCT TCA TTC ACAp: CY5- TGG GAG GGC GAT CGC AAT CT-BHQ
Adenovirusa f: CAG GAC GCC TCG GRG TAY CTS AG 103 —d rRT-PCR ABI7500 6r: GGA GCC ACV GTG GGR TTp: FAM-CCG GGT CTG GTG CAG TTT GCC CGC-
BHQ
Rotavirusa f:CCA TCT WCA CRT RAC CCT CTA TGA G 86 963–1049 rRT-PCR ABI7500 35r:GGT CAC ATA ACG CCC CTA TAG Cp: CY5-AGT TAA AAG CTA ACA CTG TCA AA-BHQ
Norovirus GI Astra diagnostics (RealStar assay) NA NA rRT-PCR ABI7500 NANorovirus GII Astra diagnostics (RealStar assay) NA NA rRT-PCR ABI7500 NAAdenovirus Seegene (Seeplex DV assay) 411 NA 2-Step RT-PCR multiNA NAAstrovirus Seegene (Seeplex DV assay) 650 NA 2-Step RT-PCR multiNA NARotavirus Seegene (Seeplex DV assay) 541 NA 2-Step RT PCR multiNA NANorovirus GI Seegene (Seeplex DV assay) 304 NA 2-Step RT PCR multiNA NANorovirus GII Seegene (Seeplex DV assay) 214 NA 2-Step RT PCR multiNA NA
a The primers used for both rotavirus and adenovirus are degenerate and allow for detection of multiple serotypes: R, A/G; Y, C/T; S, C/G; V, A or C or G; W, A/T.b f, forward; r, reverse; p, probe; FAM, 6-carboxyfluorescein.c Amp, amplicon.d —, base pair position for the adenovirus amplicon varies in different serotypes.
TABLE 4. Results obtained for verification of Seeplex DV assay
Pathogen testedor result
No. of specimens and cultureswith result by: No. of coinfecting
pathogensPHL testa Seeplex DV rRT-PCR
Adenovirus 29 28 29 1 (norovirus GII)b
Rotavirus 52 51 51 5 (norovirus GII),b
1 (adenovirus)c
Norovirus GI 19 18 18 0Norovirus GII 37 38 38 2 (rotavirus),c
3 (adenovirus)c
Negative 60 62 61 0
Total 197 197 197 12
a PHL test: EM for adenovirus and rotavirus; EM and/or rRT-PCR for noro-virus GI and GII.
b Coinfections with norovirus GII were confirmed by RealStar assay.c Coinfections with adenovirus or rotavirus were confirmed with home brew
rRT-PCR.
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targets evaluated, before and after analyses with the Seegeneviewer is depicted in Fig. 1A and B.
As shown in Table 4, when comparing PHL to Seeplex DVresults, there is one discordant result for each of the 4 viralpathogens tested and two discordant results in the negative
enteric pool. This indicates that the performances of all threetest methods are comparable. The Seeplex DV assay detectedcoinfecting pathogens in 12 specimens of 177 primary stoolspecimens tested (Table 4). Coinfections with norovirus GIwere not detected in this study, likely due to the small sample
FIG. 1. Detection of Seeplex DV PCR products on the MultiNA. Representative raw (A) or analyzed (B) data for Seeplex DV test results aredepicted. Fifteen specimens representing each of the 4 viral targets evaluated in this study are shown. Results with dual infections are shown inlanes 1 (adenovirus and norovirus GII), 2 (rotavirus and norovirus GII), and 3 (rotavirus and adenovirus). The identity of the pathogen is shownat the top. AD, adenovirus; RV, rotavirus; NVI, norovirus GI; NVII, norovirus GII; NPC and PPC refer to negative and positive PCR controls,respectively. Note that the Seeplex DV assay also amplifies an internal control for a total of 6. Numbers in the table reflect band intensities.
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size of norovirus GI-positive specimens tested and the lowprevalence of norovirus GI infection (12, 18, 21).
Among the coinfecting pathogens detected, there were 4cases of adenovirus/norovirus GII, 7 cases of rotavirus/norovi-rus GII, and 1 case of rotavirus/adenovirus mixed infections(Table 4). Examples of mixed infections are depicted in Fig.1A and B, showing adenovirus/norovirus GII, rotavirus/adeno-virus, and rotavirus/adenovirus in lanes 1 to 3, respectively.
Diagnostic sensitivity and specificity of the Seeplex DV as-say. To determine the diagnostic sensitivity and specificity ofthe Seeplex DV assay, results were scored against the PHL testmethod (EM for adenovirus and rotavirus and home brewrRT-PCR for norovirus GI and GII (see Tables 1, 2, and 4). Toresolve discordant results, all specimens were retested usingrRT-PCR assays: home brew multiplex for adenovirus androtavirus and RealStar 2.0 for norovirus GI and GII.
Diagnostic specificity was determined by testing a set ofprecharacterized viruses and bacterial specimens or cultures(Tables 1 and 2). No cross-reaction was observed in the spec-ificity panel including other viruses and bacteria (Tables 4, 5,and 6). In particular, no cross-reaction was observed with en-teric bacterial pathogens. PHL culture results for enteric bac-teria were concordant with those of the Seeplex BacterialDiarrhea (B1and B2) ACE multiplex assay (data not shown).
Overall there were 5 discordant results between the SeeplexDV and PHL tests (Table 7); 4 were resolved in favor of theSeeplex DV assay and 1 in favor of the PHL test. One noro-virus GII specimen tested negative in the Seeplex DV assay butpositive in both the PHL and RealStar assays. One adenovirusspecimen tested positive by EM but negative by both the See-plex DV assay and home brew adenovirus-rotavirus rRT-PCR.Two specimens, norovirus GI and rotavirus, tested positive in
the PHL test but negative in the Seeplex DV assay, possiblydue to repeated freeze-thaw cycles and/or a long storage pe-riod at �80°C. One norovirus GII specimen tested negative inthe PHL test but positive in both the Seeplex DV and RealStarassays. In total, the Seeplex DV assay detected 135 positivespecimens, compared to 137 for the PHL tests. Prior to dis-cordant analysis, the sensitivity and specificity of the SeeplexDV multiplex assay for each target were as follows: 97% and99.4% for adenovirus, 98% and 99.3% for rotavirus, 95% and99.4% for norovirus GI, and 97% and 99.4% for norovirus GII,respectively (Table 5). Following discordant analysis and usingclassification of specimens as per definitions described above,the sensitivity and specificity of the Seeplex DV multiplex assayare 100% and 100%, respectively, for adenovirus, rotavirus,and norovirus GI. The sensitivity and specificity for norovirus
TABLE 5. Calculated specificity and sensitivity of the Seeplex DVassay before discordant analysis
Result bySeeplex DV
No. of specimens withPHL test result
% sensitivitya % specificityb
EM rRT-PCR
True False True False
AdenovirusPositive 28 1Negative 1 168
Total 29 169 97 (28/29) 99.4 (168/169)
RotavirusPositive 51 1Negative 1 145
Total 52 146 98 (51/52) 99.3 (145/146)
Norovirus GIPositive 18 1Negative 1 178
Total 19 179 95 (18/19) 99.4 (178/179)
Norovirus GIIPositive 37 1Negative 1 159
Total 38 160 97 (37/38) 99.4 (159/160)
a Numbers in parentheses are no. positive by Seeplex DV/total no. “true” byPHL test.
b Numbers in parentheses are no. negative by Seeplex DV/total no. “false” byPHL test.
TABLE 6. Calculated specificity and sensitivity of the Seeplex DVassay after discordant analysis
Assay, virus, and result
No. ofspecimens
with SeeplexDV result
% sensitivitya % specificityb
True False
Home brew multiplexrRT-PCR
AdenovirusPositive 28 1Negative 1 168
Total 29 169 97 (28/29) 99.4 (168/169)
RotavirusPositive 51 0Negative 0 146
Total 51 146 100 (51/51) 100 (146/146)
RealStarNorovirus GI
Positive 18 0Negative 0 179
Total 18 179 100 (18/18) 100 (179/179)
Norovirus GIIPositive 38 0Negative 0 159
Total 38 159 100 (38/38) 100 (159/159)
a Numbers in parentheses are no. positive by reference assay/total no. “true”by Seeplex DV.
b Numbers in parentheses are no. negative by reference assay/total no. “false”by Seeplex DV.
TABLE 7. Summary of discordant results
Specimenno.
Result
PHL test Seeplex DV rRT-PCR (CTa)
S45 Negative Adenovirus Adenovirus (9)S25 Rotavirus Negative NegativeS81 Norovirus GI Negative NegativeS52 Norovirus GII Negative Norovirus GII
(33.59)S54 Negative Norovirus GII Norovirus GII
(29.32)
a Threshold cycle.
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GII however, remained at 97% and 99.4%, respectively (Table6). P values were not calculated for rotavirus, norovirus GI,and norovirus GII where there was exact agreement betweenthe Seeplex DV and PHL tests. P values for both adenovirussensitivity and specificity following discordant analysis were0.31, indicating no significant difference between the two tests.Consistent with its multiplex format, the Seeplex DV assaydetected 12 additional positive coinfecting pathogens, in-cluding 6 of norovirus GII, 4 adenoviruses, and 2 rotaviruses(Table 4).
Ninety-five percent LOD. Ninety-five percent limit of detec-tion (LOD) indicates the lowest concentration of genome tar-get at which a pathogen will be detected with a probability of95%. Ninety-five percent LODs for each of the 4 targets werederived by testing a minimum of 4 replicates of each of the 12dilution steps. Figure 2 depicts partial results (replicates notshown) of a typical LOD experiment using 10-fold serial dilu-tions of quantified plasmid DNA. As seen in Fig. 2, adenovirusand norovirus GI and GII are detected at 0.45 copies perreaction, and rotavirus is detected at 4.5 copies per reaction.Similarly, Fig. 3 depicts partial results of a typical LOD exper-iment using a 10-fold serial dilution of tissue culture fluid. Asseen in Fig. 3, detection varies among the 3 targets tested, most
likely due to variability in the titer of virus present in thedifferent culture fluids. Using tissue culture fluid, noroviruswas detected at 10�4.2, rotavirus at 10�5.6 and adenovirus at10�6.5 50% tissue culture infectious doses (TCID50)/ml. Usingplasmid DNAs, the estimated 95% LODs are as follows: 30.8genome equivalents (GEq)/reaction for adenovirus, 10.2 GEq/reaction for rotavirus, 1.7 GEq/reaction for norovirus GII, and2.2 GEq/reaction for norovirus GI (Fig. 2 and 3).
Assay reproducibility and TAT. Inter- and intra-assay repro-ducibilities were both 100%. Average TATs for processing 96samples in one run were as follows: 9 to 10 h for the SeeplexDV assay, 6 h for rRT-PCR, and 3 days for electron micros-copy. TATs per target for 96 samples were 1.8 h for the SeeplexDV assay, 3 h for rRT-PCR, and 9 h for EM.
DISCUSSION
We describe the evaluation of the Seeplex DV assay (See-gene, South Korea), a commercial kit for the detection ofpathogens associated with viral gastroenteritis in clinical stoolspecimens, which is approved by CE and Health Canada butnot the FDA. The Seeplex DV assay is the only commercialmultiplex RT-PCR product currently available for the simul-
FIG. 2. Determination of the limit of detection for the Seeplex DV assay using quantified plasmid DNA. Raw (A) or analyzed (B) data for serial10-fold dilutions of plasmid DNA for rotavirus, adenovirus, norovirus GI, and norovirus GII are depicted. Detection of amplified PCR productis evident at 45, 4.5, and 0.45 copies per reaction. Note that only a subset of the experiment representing each target is shown. In this experiment,triplicates of each dilution were tested in the same run. Note also that plasmid dilutions were retested on different days. Numbers in the table reflectband intensities.
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taneous detection of up to 5 viral targets, including adenovirus(group F serotypes 40 and 41), rotavirus, norovirus GI, noro-virus GII, and astrovirus. We show that the Seeplex DV assayhas sensitivity equivalent to those of other methods currentlyin use at PHL and reduces the TAT, especially when highvolumes of samples are being tested.
Viral gastroenteritis outbreaks occur frequently in nursinghomes, health care institutions, and cruise ships, where theycause considerable morbidity and mortality; they have alsobeen reported in schools and prisons (9, 33, 34). Althoughnorovirus is recognized as a common cause of gastroenteritis,other enteric viruses also contribute to the syndrome. Hence,there may be a role for multiplex assay formats that will detectnorovirus and in the same assay also test for other viral patho-gens. Here we reconfirmed that norovirus GII causes the ma-jority of enteric outbreaks in Ontario (11), and we have verified
this assay only for outbreaks caused by norovirus GI or GII(Table 1). Future prospective studies will allow further char-acterization of the assay and verification for outbreaks causedby adenovirus and rotavirus.
Current molecular diagnostic technologies rely on homebrew tests. These tests are considerably advanced and requireadditional funds and staff for up-front research and develop-ment, quality assurance, and laboratory accreditation (8, 13,15, 28). In recent years, commercial products such as thoseidentified in this communication have allowed for the imple-mentation of molecular testing without the added costs ofresearch and development and additional quality assurancecosts.
The Seeplex DV assay demonstrates several strengths as acommercial assay for use in a wider range of clinical laboratorysettings outside a highly resourced center. First, this assay
FIG. 3. Determination of the limit of detection for the Seeplex DV assay using quantified tissue culture fluid. Raw (A) or analyzed (B) datafor serial 10-fold dilutions of tissue culture fluid for adenovirus, rotavirus, and norovirus GII are depicted. Detection of adenovirus amplicons isevident at a 10�6 dilution of the original concentration, whereas amplicons corresponding to rotavirus are detected at a 10�5 dilution and norovirusGII at a 10�4 dilution. Note that only a subset of the experiment representing each target is shown. In this experiment, triplicates of each dilutionwere tested in the same run. Note also that dilutions were retested on different days. Numbers in the table reflect band intensities.
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offers a rapid, sensitive, and specific method for the diagnosisof adenovirus, rotavirus, norovirus GI, norovirus GII, and as-trovirus. Second, being a commercial product, the Seeplex DVassay can be readily implemented in the clinical diagnosticlaboratory (if approved by local licensing authorities). It alsooffers established quality controls and more-streamlined ongo-ing validation compared to a home brew PCR assay. Third, theSeeplex DV assay is a multiplex test, which allows the detectionof multiple viral targets in one reaction, instead of multiplereactions as when using monoplex assays. This makes the assayeasier to perform and utilizes less patient specimen. Fourth,compared to existing testing methodologies (rRT-PCR andEM), the reagent cost per target of the Seeplex DV multiplexassay is cheaper and the assay less laborious. The reagent costis approximately $25.00 CAD per test or $5.00 CAD per virustarget.
There are several limitations to this study, including its ret-rospective nature. We expected to detect more positive spec-imens with the Seeplex DV assay than with EM. Hence, thesensitivity of the Seeplex DV assay was not higher than that ofEM for detection of rotavirus and adenovirus. Nevertheless,the results of the Seeplex DV assay were confirmed with newlydeveloped home brew adenovirus/rotavirus rRT-PCR with noincrease in positivity. Another limitation of the Seeplex DV isthe TAT, which requires 9 to 10 h and is longer than that ofrRT-PCR (6 h). Yet the Seeplex DV assay offers detection of5 viral targets plus an internal control for each patient.
In the past 5 years, several rRT-PCR assays were developedfor the detection of enteric viruses. These assays were demon-strated to have better sensitivities and specificities than elec-tron microscopy or antigen-detection assays (4, 21, 22, 26, 27,29). Of these studies, two have described the use of multiplexassays for the detection of viral gastroenteritis (2, 31). How-ever, both assays lack internal controls in the PCR and there-fore are of limited use in a clinical routine diagnostic setting.The limit of detection reported (21, 22, 26, 27) varied between1 and 100 copies per reaction and is similar to the resultsshown in this study. A recent study described the application oftwo internally controlled multiplex real-time PCR assays forthe simultaneous detection of adenovirus, rotavirus, norovirusGI and GII, astrovirus, and sapovirus (32). In that study, thesensitivity and specificity of the assay were clinically comparedto those of the Vikia rotavirus/adenovirus chromatographicimmunoassay, with a significant increase in pathogen detectionfrom 14% to 45%. This multiplex PCR assay consists of twoseparate reactions; one targets astrovirus, rotavirus, and noro-virus GI, and the other targets adenovirus and norovirus GII.
While the manuscript was being reviewed, two studies ap-peared which describe home brew multiplex assays for thedetection of viral gastroenteritis (16, 24). The study by Liu etal. (24) combined a one-step rRT-PCR with microsphere-based fluorescence technology to detect 6 viral targets, includ-ing adenovirus, rotavirus, norovirus GI and GII, astrovirus,and sapovirus. The study by Khamrin et al. (16) combinestwo-step rRT-PCR and agarose gel electrophoresis to detect10 virus targets, including adenovirus, rotavirus, norovirus GI,norovirus GII, astrovirus, sapovirus, enterovirus, parechovirus,and aichi virus.
In conclusion, the Seeplex DV assay is sensitive, specific,convenient, and a reliable tool for the direct detection of en-
teric viruses in stool specimens from patients with gastroen-teritis. This assay can be effectively implemented in a clinicalmicrobiology setting within and outside reference laboratoriesand has the attractive feature of improving testing methodswhile also being more economical than previously used meth-ods, in particular those using EM and home brew rRT-PCRs.
ACKNOWLEDGMENTS
We thank Inverness Medical (Canada) for providing Seeplex kitsand loaning the MultiNA instrument. Astra Diagnostics provided Re-alStar kits for norovirus GI and GII testing.
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