Establishment and application of real-time TaqMan-quantitative PCR assay for detection of porcine cytomegalovirus
BAI Ting-yang1,3,ZHAO De-ming1,WU Zhi-ming2,YAN Ruo-qian2,LIU Shu-min4,ZHAO Ming-jun2,LIU Mei-fen2
1.College of Veterinary Medicine, China Agricultural University, Beijing 100094, China; 2.Henan Centre for Animal Disease Control & Prevention, Zhengzhou 450008, China; 3. Zhoukou Animal Improving Station, Zhoukou 466000, China; 4. Sterilization and Supply Center of Zhengzhou Central Hospital, Zhengzhou 450007, China
Abstract：A highly sensitive and specific real-time TaqMan-fluorescent-quantitative PCR (FQ-PCR) assay was developed to detect and quantitate porcine cytomegalovirus (PCMV) infection using PCMV DNA sequence-derived specific primer pairs and TaqMan fluorescent probe. The sensitivity, specificity and repetition assay of FQ-PCR were tested, and 15 clinic suspicious PCMV infected samples were detected by the FQ-PCR assay in contrast to the routine PCR method. Virus contents in different internal organs of six PCMV-positive swine were compared and detected. The results indicated the FQ-PCR assay as well as a quantitative standard carve with good linearity (R2=0.998) were successfully established. The developed FQ-PCR assay was able to detect as little as 1.0 copy/ L of recombinant pGEM-T/PCMV plasmid DNA, and the sensitivity of which was 100 times more than that of the routine PCR. The specificity assay exhibited that positive signals could be obtained from recombinant pGEM-T/PCMV plasmid, but not from the genomic DNA or total cDNA of the other 6 kinds of pathogenic microorganism acting as the controls. The repetition test indicated that the FQ-PCR was reproducible. Nine positive results from 15 clinic suspicious PCMV infected samples were obtained, which were consistent with the results detected by routine PCR. The organs with the highest and lowest PCMV content levels were tonsils and small intestine, respectively. PCMV FQ-PCR method was successfully established and used for the clinical diagnosis of PCMV and early detection of latent infection, which was of great significance for rapid diagnosis of PCMV, comprehensive prevention, control, and purification.
拜廷阳,赵德明,吴志明,闫若潜,刘淑敏,赵明军,刘梅芬. 猪巨细胞病毒TaqMan荧光定量PCR检测的建立[J]. 中国人兽共患病学报, 2014, 30(2): 169-174.
BAI Ting-yang,ZHAO De-ming,WU Zhi-ming,YAN Ruo-qian,LIU Shu-min,ZHAO Ming-jun,LIU Mei-fen. Establishment and application of real-time TaqMan-quantitative PCR assay for detection of porcine cytomegalovirus. Chinese Journal of Zoonoses, 2014, 30(2): 169-174.
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2014, Vol. 30 
