A multiplex nested RT-PCR for the detection and differentiation of wild-type viruses from C-strain vaccine of classical swine fever virus.
A multiplex nested RT-PCR (RT-nPCR) was developed for the detection and differentiation of classical swine fever virus (CSFV). A fragment of 447 or 343 bp was amplified from the genomic RNA of C-strain or virulent Shimen strain, respectively, and two fragments of 447 and 343 bp were simultaneously amplified from the mixed samples of C-strain and Shimen. When detecting several wild-type isolates representative of different subgroups (1.1, 2.1, 2.2, and 2.3) circulating in Mainland China and samples from pigs experimentally infected with Shimen strain, the RT-nPCR resulted in an amplification pattern similar to Shimen. No amplification was achieved for uninfected cells, or cells infected with bovine viral diarrhea virus (BVDV), and other viruses of porcine origin. The RT-nPCR was able to detect as little as 0.04 pg of CSFV RNA. The restrictive fragment length polymorphism (RFLP) demonstrated unique patterns of wild-type viruses and C-strain. Among the 133 field samples, 42 were tested to contain wild-type viruses and 18 showing presence of C-strain. The RT-nPCR can be used to detect and differentiate pigs infected with wild-type CSFV from those vaccinated with C-strain vaccine, thus minimizing the risk of culling vaccinates during outbreaks.
Li
Yan
Y
Division of Swine Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, Heilongjiang, China
Zhao
Jian-Jun
JJ
Li
Na
N
Shi
Zixue
Z
Cheng
Dan
D
Zhu
Qing-Hu
QH
Tu
Changchun
C
Tong
Guang-Zhi
GZ
Qiu
Hua-Ji
HJ
D000818:Animals; D002681:China / Q000453:epidemiology; D006691:Classical Swine Fever / Q000097:blood / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology*; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D003937:Diagnosis, Differential; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D014765:Viral Vaccines
D014765:Viral Vaccines