Development of a primer-probe energy transfer based real-time PCR for detection of Marek's disease virus

• Published in: Journal of virological methods 2010-04, Vol.165 (1), p.21-26
• Main Authors: Barfoed, Annette Malene, Østergaard, Erik, Frandsen, Peer Lyng, Nielsen, Eva Bækdahl, Sandberg, Eva, Rasmussen, Thomas Bruun
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological and medical sciences; Capsid Proteins - genetics; Energy Transfer; Fundamental and applied biological sciences. Psychology; Mardivirus - genetics; Mardivirus - isolation & purification; Marek Disease - diagnosis; Marek's disease herpesvirus; Marek's disease virus; Microbiology; Molecular Sequence Data; Oligonucleotide Probes - chemistry; Oligonucleotide Probes - genetics; Polymerase Chain Reaction - methods; Primer-probe energy transfer; Real-time PCR; Sensitivity and Specificity; Sequence Alignment; Techniques used in virology; Transition Temperature; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2009.12.012

A real-time PCR assay, which enables simultaneous detection and differentiation of all three serotypes of Marek's disease virus, without the need for post-PCR sequencing, has been developed. The assay is based on the primer-probe energy transfer real-time PCR, which has a relatively high tolerance towards point mutations in the probe region. The PCR is followed by a probe melting point analysis, which enables confirmation of identity of amplicon and differentiation of serotypes. The assay targets the MDV031 gene, encoding UL19 major capsid protein-like protein and was shown to be quantitative, with a detection limit below 10 TCID 50/ml starting material. This sensitivity is similar to the one obtained with traditional virus cultivation. However, the PCR method can provide a laboratory result within a day, while the virus cultivation method takes more than a week to perform. The new method will be useful for testing of avian live viral vaccines and screening for extraneous agents.