Development of TaqMan-based qPCR method for detection of caprine arthritis–encephalitis virus (CAEV) infection

Abstract

A specific and sensitive two-step TaqMan real-time PCR has been developed for rapid diagnosis of caprine arthritis-encephalitis virus (CAEV) infection by using a set of specific primers and a TaqMan probe targeting a highly conserved region within the gene encoding the viral capsid protein (CA). The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and isolated PBMCs, with a lower detection limit of 102 copies and a linear dynamic range of 105 to 1010 copies/ml. There was no cross-reaction with other animal viruses (e.g., goat pox virus, bovine leukemia virus, bovine mucosal disease virus, swine influenza virus and Nipah virus). When applied in parallel with serological AGID and conventional PCR for detection of CAEV in field samples, this assay exhibited a higher sensitivity than these traditional methods, and 7.8 % of the 308 specimens collected in the Shanxi and Tianjin regions of China from 1993 to 2011 were found to be positive. Thus, the TaqMan qPCR assay provides a fast, specific and sensitive means for detecting CAEV proviral DNA in goat specimens and should be useful for large-scale detection in eradication programs and epidemiological studies.

Advances in Diagnosis of Respiratory Diseases of Small Ruminants

“…Moreover, due to advancement in the polymerase chain reaction (PCR) technology, there has been enormous improvement in the diagnosis of respiratory diseases of small ruminants [25]. Recent advances in biotechnology and molecular biology have led to the development of a variety of diagnostic assays, namely, PCR, RT-PCR, PCR-ELISA, RAPD, AFLP, RFLP, real-time PCR, quantitative PCR, multiplex PCR, LAMP, microsatellites, gene sequencing, and phylogenetic analysis, which not only help in identification but also assist in molecular characterization of various pathogens [20,[22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. Various conventional diagnostic tests, namely, isolation, postmortem finding, and gross clinical examinations along with modernized serological and molecular tests, are enlisted in Figure 1.…”

“…This type of PCR assay especially provides a useful method to detect CAEV infection in goats [ 66 – 68 ]. A two-step TaqMan quantitative (q) PCR, which is specific as well as sensitive for the detection of infection due to CAEV by the use of a set of primers (specific), and a TaqMan probe that targets a region which is highly conserved within the gene that encodes the capsid protein of the virus have been developed [ 33 ]. In the total deoxyribonucleotide (DNA) extracts, the proviral DNA can be detected successfully by this assay.…”

“…In order to track the transmission of the disease in near future, phylogenetic analyses may be used [ 66 , 69 , 70 ]. Molecular techniques such as cloning and sequencing are also used to provide knowledge on the prevalence of specific strain of CAEV in a country or a region which may have influence on serological assay as well as corresponding CAEV antigen [ 33 , 71 ].…”

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

“…Several real time PCR assays for SRLV detection have also been published [8,21-23], but to our knowledge only four real time PCR assays for diagnostic purposes have been described to date [24-27]. …”

Development and application of real-time TaqMan RT-PCR assay for improved detection of classical swine fever virus in slaughtered pigs

“…Ciglenecki et al (2008) reported that the one-step real-time TaqMan RT-PCR assay was more appropriate for pestivirus quantification that reduces the risk of contamination and is less time consuming as compared to the conventional PCR. A TaqMan-based real-time PCR assay was also devised for the detection of porcine circovirus type 2 (Zhao et al, 2010), porcine boca-like virus (Li et al, 2011), caprine arthritisencephalitis virus (Li et al, 2013) and porcine parvovirus (Song et al, 2010;Gava et al, 2015).…”