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. 2013 Feb;51(2):472-80.
doi: 10.1128/JCM.02658-12. Epub 2012 Nov 21.

A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens

Jie Liu  1 , Jean Gratz, Caroline Amour, Gibson Kibiki, Stephen Becker, Lalitha Janaki, Jaco J Verweij, Mami Taniuchi, Shihab U Sobuz, Rashidul Haque, Doris M Haverstick, Eric R Houpt
Affiliations

A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens

Jie Liu et al. J Clin Microbiol. 2013 Feb.

Abstract

The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (Campylobacter jejuni/C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli strains including enteroaggregative E. coli [EAEC], enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], and Shiga-toxigenic E. coli [STEC]), Shigella/enteroinvasive E. coli (EIEC), protozoa (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica), and helminths (Ascaris lumbricoides and Trichuris trichiura), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, and DNA and RNA were extracted using the QiaAmp Stool DNA minikit and the QuickGene RNA Tissue kit, respectively, and then mixed with Ag-Path-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below 5% within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes.

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Figures

Fig 1
Fig 1
Enteric TaqMan Array Card layout. Format 48 was used.
Fig 2
Fig 2
Quantitative comparison of pathogen burdens in clinical samples that were positive or negative with conventional methods. Box plots with medians were generated with IBM SPSS software. Asterisks (*) indicate that CT was lower for conventional positive than for conventional negative samples (P < 0.05), including adenovirus (P = 0.02), rotavirus (P = 0.01), Cryptosporidium (P < 0.0001), Giardia (P < 0.001), E. histolytica (P < 0.001), LT of ETEC (P = 0.04), eae of EPEC (P = 0.02), and both aaiC and aatA of EAEC (P < 0.001 for both). A trend was observed for astrovirus (P = 0.06), ST of ETEC (P = 0.08), and bfpA of EPEC (P = 0.14), whereas correlation was poor between TAC and the Campylobacter enzyme immunoassay (EIA) (P = 0.59). Statistics were not applicable to stx1 and stx2 of STEC, Shigella/EIEC, Salmonella, V. cholerae, Ascaris, and Trichuris.
Fig 3
Fig 3
Analysis of TAC and PCR-Luminex assays. CT values from TAC are shown for both PCR-Luminex-positive and -negative samples. Likewise, relative fluorescence intensities from PCR-Luminex are shown for both TAC-positive and -negative samples. To adjust for different Luminex fluorescence intensity scales, the relative fluorescence intensity of a PCR-Luminex-positive sample is shown as the percentile versus the highest fluorescence intensity observed. Asterisks (*) indicate that CT values were lower for PCR-Luminex-positive than for PCR-Luminex-negative samples and Luminex fluorescence intensity (FI) values were higher for TAC-positive than for TAC-negative samples (P < 0.05).
Fig 4
Fig 4
Numbers of mixed infections detected with TAC versus conventional methods for 16 pathogens on samples from Tanzania (medians; P < 0.001).
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