Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with fluorescent probes

Abstract

-Routine detection of equine arteritis virus (EAV) can be achieved by virus isolation (VI) in cell culture, or by the amplification of viral genome by molecular methods. To simplify molecular diagnosis, a number of different Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and RT-nested PCR (RT-nPCR) assays were compared, and a one-tube method was developed and optimised utilizing a fluorogenic probe (TaqMan ® ). An artificial RNA template (Mimic) and associated probe were also constructed to provide in-tube validation of the RT-PCR system. To assess the utility of the RT-PCR TaqMan ® assay, 28 different isolates of EAV representing different genetic groups of American and European strains were tested. Furthermore, the ability of VI and RT-PCR TaqMan ® assay to detect EAV in different biological matrices such as semen, nasal and faecal swabs and blood was compared. All 28 EAV strains were detected by the RT-PCR TaqMan ® assay. The results of TaqMan ® and VI testing were in agreement for 30 of the 33 semen samples and all of the 50 other clinical specimens examined: the RT-PCR TaqMan ® assay detected 18 positive semen samples, three more than VI. In conclusion, the one-tube RT-PCR TaqMan ® assay is a rapid, reliable method for the detection of EAV.equine arteritis virus / RT-PCR / mimic / fluorescent probe / TaqMan ®

Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay

“…The dual-probe EAV RT-qPCR assay delimited a 399 bp-long region32 and included a pair of simple probes3233 in opposite orientation. The thermo-profile started with reverse transcription of 50 °C for 30 min and 95 °C for 15 min, followed by 45 cycles of 95 °C for 20 s, 60 °C for 1 min and 72 °C for 30 s, with a signal acquisition in the FAM channel at the end of the annealing/extension step.…”

Challenges in designing a Taqman-based multiplex assay for the simultaneous detection of herpes simplex virus types 1 and 2 and Varicella-zoster virus

“…Most IPCs have been developed for duplex assays as plasmid-based mimic controls containing an inserted foreign sequence Schwaiger and Cassinotti, 2003;Westcott et al, 2003;Widada et al, 2001). The mimic control of the assay presented here is driven by the two upstream primers of the virus specific HSV2 and the VZV amplicons already present in the mixture to reduce primer dimer formation.…”

Biotechnological tools for diagnosis of equine infectious diseases

“…The probe is tagged with a molecular marker of either radioactive (P32, I125 etc.) molecules or non-radioactive fluorescent molecules to detect the hybridization (Digoxigenin).The probe hybridization based assays have been used for diagnosis of equine infections such as equine arteritis virus (Balasuriya et al, 2002;Westcott et al, 2003). The probe hybridization assay is relatively easy to perform.…”