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Engineered combined-positive-control template for real-time reverse transcription-PCR in multiple-pathogen-detection assays.

ABSTRACT: Recently we evaluated a custom TaqMan array card (TAC) detection system, formerly known as a TaqMan low-density array (TLDA) card, for simultaneous real-time PCR identification of 21 pathogens and three control targets in duplicate from respiratory specimens (M. Kodani et al., J. Clin. Microbiol. 49:2175-2182, 2011). We engineered an adaptable and expandable system of in vitro RNA transcripts to serve as a combined positive control for both DNA and RNA targets in multiple-pathogen-detection technologies based on real-time reverse transcription-PCR.

SUBMITTER: Kodani M

PROVIDER: S-EPMC3295119 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Engineered combined-positive-control template for real-time reverse transcription-PCR in multiple-pathogen-detection assays.

Kodani Maja M Winchell Jonas M JM

Journal of clinical microbiology 20111214 3

Recently we evaluated a custom TaqMan array card (TAC) detection system, formerly known as a TaqMan low-density array (TLDA) card, for simultaneous real-time PCR identification of 21 pathogens and three control targets in duplicate from respiratory specimens (M. Kodani et al., J. Clin. Microbiol. 49:2175-2182, 2011). We engineered an adaptable and expandable system of in vitro RNA transcripts to serve as a combined positive control for both DNA and RNA targets in multiple-pathogen-detection tech ...[more]

PMID: 22170926

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Project description:BackgroundEquine rhinitis viruses A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. Sero-surveillance studies have shown that these two viral infections are prevalent in many countries. Currently, the diagnosis of ERAV and ERBV infections in horses is mainly based on virus isolation (VI). However, the sensitivity of VI testing varies between laboratories due to inefficient viral growth in cell culture and lack of cytopathic effect. Therefore, the objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) to detect and distinguish ERAV from ERBV without the inherent problems traditionally associated with laboratory diagnosis of these infections.ResultsThree rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, with the two remaining assays specific for ERBV. Additionally, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were developed. Both rRT-PCR and cRT-PCR assays were evaluated using RNA extracted from 21 archived tissue culture fluid (TCF) samples previously confirmed to be positive for ERAV (n = 11) or ERBV (n = 10) with mono-specific rabbit antisera. The ERAV rRT-PCR and cRT-PCR assays could only detect ERAV isolates and not ERBV isolates. Similarly, the ERBV rRT-PCR and cRT-PCR assays could only detect ERBV isolates and not ERAV isolates. None of the rRT-PCR or cRT-PCR assays cross-reacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit of all of these assays was 1 plaque forming unit per ml (pfu/ml).ConclusionThe newly developed rRT-PCR and cRT-PCR assays provide improved diagnostic capability for the detection and differentiation of ERAV and ERBV. However, a larger number of clinical specimens will need to be tested before each assay is adequately validated for the detection of ERAV and/or ERBV in suspect cases of either viral infection.

Dataset Information

Engineered combined-positive-control template for real-time reverse transcription-PCR in multiple-pathogen-detection assays.

Publications

Engineered combined-positive-control template for real-time reverse transcription-PCR in multiple-pathogen-detection assays.

Similar Datasets

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