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Open Access Methodology article

Development and validation of TaqMan probe based real time PCR assays for the specific detection of genotype A and B small ruminant lentivirus strains

Urška Kuhar1*, Darja Barlič-Maganja2 and Jože Grom1

Author Affiliations

1 Veterinary Faculty, Institute for Microbiology and Parasitology, Virology Unit, University of Ljubljana, Gerbičeva 60, SI-1115 Ljubljana, Slovenia

2 College of Health Care, University of Primorska, Polje 42, SI-6310 Izola, Slovenia

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BMC Veterinary Research 2013, 9:172  doi:10.1186/1746-6148-9-172

Published: 3 September 2013

Abstract

Background

Small ruminant lentiviruses (SRLV) are members of the Retroviridae family and infect goats and sheep worldwide. Detection of specific antibodies using AGID and ELISA is the most commonly used means of diagnosing SRLV infection. The most frequent molecular method for detecting the provirus genome is PCR, using peripheral blood leucocytes as target cells. Real time PCR has also recently been used. The aim of this study was to develop a real time PCR for detection of SRLV in order to improve molecular diagnostics of SRLV infections in sheep and goats.

Results

Two new real time PCR assays using TaqMan probes for the specific detection of genotype A (MVV assay) and genoptype B (CAEV assay) SRLV strains and differentiation between them were developed and validated at both analytical and diagnostic levels following MIQE guidelines. The validation results showed that the new real time PCR is 100% specific, with a reliable limit of detection of 26 (CAEV assay) and 72 (MVV assay) plasmid DNA copies, while compared to ELISA the diagnostic sensitivity of both assays was 79% when tested with Slovenian SRLV field samples. Intra-assay and inter-assay coefficients of variation showed overall good repeatability and reproducibility of the new real time PCR assays, except for the highest dilutions.

Conclusions

Two new TaqMan probe based real time PCR assays for the specific detection of genotype A and B SRLV strains and differentiation between them were developed and validated. They can serve as an additional tool for confirming infection with SRLV and may also be useful for early detection of infected animals prior to seroconversion.

Keywords:
CAEV; Gag matrix; MVV; Real time PCR; Small ruminant lentiviruses