鉴别猪瘟强毒和弱毒的反转录-复合套式聚合酶链式反应RT-nPCR.DOCVIP

鉴别猪瘟强毒和弱毒的反转录-复合套式聚合酶链式反应（RT-nPCR）检测方法的建立 李 艳1，2，仇华吉12，朱庆虎1，李 娜13，李国新1，3，童光志1（1中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室，哈尔滨 150001；2延边大学农学院，龙井 133400；3东北农业大学动物医学院，0030） A Reverse Transcription-Multiplex Nested Polymerase Chain Reaction for Detection and Differentiation of Wild-type and Vaccine Viruses of Classical Swine Fever Virus LI Yan1, 2, QIU Hua-ji1, WANG Xiu-rong1, ZHANG Shou-fa2, ZHU Qing-hu1, LI Na1, 3, LI Guo-xin1,3, TONG Guang-zhi1 (1 National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001; 2 Agricultural College of Yanbian University, Longjing 133400; 3College of Veterinary Sciences, Northeast Agricultural University, Harbin 150030 ) Abstract : 【Objective】A reverse transcription-multiplex nested polymerase chain reaction (RT-nPCR) was developed for the differentiation of wild-type classical swine fever virus (CSFV) from the C-strain vaccine. 【Method】A pair of primers corresponding to the highly conserved regions of CSFV genome was used as a common primer pair in the first round of the RT-nPCR. Another two primers specific for CSFV C-strain vaccine or highly virulent Shimen strain, respectively, were used as the forward primer together with the common reverse primer in the second round of the RT-nPCR.【Result】A fragment of 447 or 343 bp was amplified from genomic RNA of C-strain or Shimen, respectively, in the RT-nPCR, and two fragments of 447 and 343 bp were simultaneously amplified from the mixed RNA sample of C-strain and Shimen. No amplification was achieved for mock infected cells, or cells infected with other common viruses of porcine origin. The RT-nPCR could detect 0.04 pg of CSFV RNA. Fourteen of fifteen field samples suspected of CSF from Heilongjiang Province were found to be Shimen-like, and 1 to be C-strain-like by this method. Restrictive fragment length polymorphism and phylogenetic analyses confirmed the RT-nPCR results