A real-time TaqMan RT-PCR assay with an internal amplification control for rapid detection of transmissible gastroenteritis virus in swine fecal samples.
A TaqMan probe-based real-time RT-PCR assay was developed for simultaneous detection of RNA of transmissible gastroenteritis virus (TGEV) in pig fecal samples and RNA of enhanced green fluorescent protein (EGFP) added exogenously as an internal amplification control. The TGEV primers and probe were designed to be specific to a portion of the S gene sequence conserved in all TGEV isolates, but absent in the closely related porcine respiratory coronaviruses. The optimized TaqMan assay detected a minimum of 2.8 copies of in vitro transcribed RNA of the target S gene and RNA extracted from 1 TCID50/ml of TGEV. Using 113 clinical samples received at our diagnostic laboratory over a 4-year period, the performance of the assay was tested and compared with that of a previously described nested RT-PCR assay. All the fecal samples which tested positive for TGEV by the nested RT-PCR assay also tested positive by the TaqMan assay. However, approximately 9% of the samples that tested negative by the nested RT-PCR assay tested positive by the TaqMan assay. These results indicate that the developed TaqMan assay is a highly sensitive diagnostic test for rapid detection of TGEV in pig fecal samples.
Vemulapalli
Ramesh
R
Molecular Diagnostics Section, Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN 47907, USA. rvemulap@purdue.edu
Gulani
Jatinder
J
Santrich
Cecilia
C
D000818:Animals; D005243:Feces / Q000821:virology*; D005761:Gastroenteritis, Transmissible, of Swine / Q000175:diagnosis / Q000821:virology; D011786:Quality Control; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology; D019914:Taq Polymerase; D013997:Time Factors; D005760:Transmissible gastroenteritis virus / Q000235:genetics / Q000302:isolation & purification*
D012367:RNA, Viral; D019914:Taq Polymerase