TY - JOUR
T1 - Development of a primer-probe energy transfer based real-time PCR for detection of Marek's disease virus
AU - Barfoed, Annette Malene
AU - Østergaard, E.
AU - Frandsen, P.L.
AU - Nielsen, E.B.
AU - Sandberg, E.
AU - Rasmussen, Thomas Bruun
PY - 2010
Y1 - 2010
N2 - 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 TCID50/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.
AB - 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 TCID50/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.
KW - Real-time PCR
KW - Marek's disease virus
KW - Primer-probe energy transfer
U2 - 10.1016/j.jviromet.2009.12.012
DO - 10.1016/j.jviromet.2009.12.012
M3 - Journal article
C2 - 20026120
SN - 0166-0934
VL - 165
SP - 21
EP - 26
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 1
ER -