牛细小病毒DPO-PCR检测方法的建立
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摘要
为建立牛细小病毒的快速检测方法,以牛细小病毒VP2基因为靶基因,设计了1对双启动寡核苷酸(dual-priming oligonucleotide,DPO)引物,建立了牛细小病毒DPO-PCR快速检测方法。退火温度不敏感试验、特异性试验和灵敏度试验的结果显示,与常规PCR方法相比,DPO-PCR方法在41～65℃退火温度范围内均能够高效地扩增出目的基因,表明DPO引物对退火温度不敏感;该方法的最低检出量为3.94×10~3copies/uL;通过对牛细小病毒、牛轮状病毒、牛病毒性腹泻病毒、牛呼吸道合胞体病毒进行检测,只有牛细小病毒的检测结果为阳性,且无非特异性扩增。结果表明,建立的DPO-PCR方法同传统PCR方法相比,特异性强、灵敏度高,为牛细小病毒的快速检测提供了一种新方法。
In order to establish a rapid detection method for bovine parvovirus(BPV),a pair of dualpriming oligonucleotide(DPO) for BPV VP2 gene was designed.The DPO-PCR method to detect BPV was established.To analyze by the annealing temperature insensitivity test,specificity test and sensitivity test,the results showed that the DPO-PCR method can efficiently amplify the target gene in the annealing temperature range from 41 ℃ to 65 ℃ comparing with the conventional PCR method.This indicated that the DPO-PCR method was not sensitive to the annealing temperature.The minimum detectable amount of this method was 3.94×10~3 copies/uL.It showed that the method was specific by detecting BPV, bovine rotavirus,bovine viral diarrhea virus and bovine respiratory syncytial virus.Only the result of detecting BPV was positive and there was no non-specific amplification.Comparing with the conventional PCR method,the established DPO-PCR method was specific and sensitive,which provides a new method for rapid detection of BPV.
In order to establish a rapid detection method for bovine parvovirus(BPV),a pair of dualpriming oligonucleotide(DPO) for BPV VP2 gene was designed.The DPO-PCR method to detect BPV was established.To analyze by the annealing temperature insensitivity test,specificity test and sensitivity test,the results showed that the DPO-PCR method can efficiently amplify the target gene in the annealing temperature range from 41 ℃ to 65 ℃ comparing with the conventional PCR method.This indicated that the DPO-PCR method was not sensitive to the annealing temperature.The minimum detectable amount of this method was 3.94×10~3 copies/uL.It showed that the method was specific by detecting BPV, bovine rotavirus,bovine viral diarrhea virus and bovine respiratory syncytial virus.Only the result of detecting BPV was positive and there was no non-specific amplification.Comparing with the conventional PCR method,the established DPO-PCR method was specific and sensitive,which provides a new method for rapid detection of BPV.
引文
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[2]JOHNSON F B,FENN L B,OWENS T J,et al.Attachment of bovine parvovirus to sialic acids on bovine cell membranes[J].Journal of General Virology,2004,85(Pt 8):2199-2207.
[3]ABINANTI F R,WARFIELD M S.Recovery of a hemadsorbing virus(HADEN)from the gastrointestinal tract of calves[J].Virology,1961,14(2):288-289.
[4]INABA Y,KUROGI H,OMORI T,et al.A new serotype of bovine parvovirus[J].Microbiology&Immunology,2013,17(1):85-86.
[5]MANTEUFEL J,TRUYEN U.Animal bocaviruses:a brief review[J].Intervirology,2008,51(5):328-334.
[6]RYAN M M.Guillain-Barre syndrome in childhood[J].JournalofPaediatrics&ChildHealth,2005,41(5-6):237-241.
[7]COLWELL R R.Global climate and infectious disease:the cholera paradigm[J].Science,1996,274(5295):2025-2031.
[8]王娜,陶妍.水产品三种致病菌多重PCR检测方法的建立[J].食品与生物技术学报,2009,28(3):397-402.WANG Na,TAO Yan.Establishment of a multiplex PCRfor detection of three types of pathogen in aquatic foods[J].Journal of Food Science and Biotechnology,2009,28(3):397-402.(in Chinese)
[9]刘梅,黄新,马占鸿,等.应用DPO引物检测马铃薯病毒的多重RT-PCR技术研究[J].植物病理学报,2009,39(4):431-434.LIU Mei,HUANG Xin,MA Zhan-hong,et al.Multiplex RT-PCR for detection of potato viruses with a dual priming oligonucleotide(DPO)system[J].Acta Phytopathologica Sinica,2009,39(4):431-434.(in Chinese)
[10]徐焕洲,平芮巾,季汝武,等.应用DPO引物技术同时检测5种蚊媒病毒的多重RT-PCR方法[J].中国国境卫生检疫杂志,2012,35(2):73-77.XU Huan-zhou,PING Rui-jin,JI Ru-wu,et al.Multiplex RT-PCR method for detecting five kinds of mosquito-borne viruses with DPO system[J].Chinese Frontier Health Quarantine,2012,35(2):73-77.(in Chinese)
[11]WOO H Y,PARK H,KIM B I,et al.Evaluation of dual priming oligonucleotide-based multiplex PCR for detection of HBV YMDD mutants[J].Archives of Virology,2008,153(11):2019-2025.
[12]修金生,周伦江,陈如敬,等.猪流行性腹泻病毒SYBRⅠ实时荧光定量RT-PCR检测方法的建立[J].中国兽医科学,2012,42(2):160-165.XIU Jin-sheng,ZHOU Lun-jiang,CHEN Ru-jing,et al.Development of a real time RT-PCR assay based on SYBR GreenⅠfor rapid diagnosis of porcine epidemic diarrhea virus[J].Chinese Veterinary Science,2012,42(2):160-165.(in Chinese)
[13]张二芹,马强,王会岩,等.猪轮状病毒VP6基因的原核表达载体构建和表达[J].河南农业科学,2008,37(5):104-106.ZHANG Er-qin,MA Qiang,WANG Hui-yan,et al.Construction and expression of porcine rotavirus VP6 gene prokaryotic expression plasmid[J].Journal of Henan Agricultural Sciences,2008,37(5):104-106.(in Chinese)
[14]罗济冠.牛细小病毒检测试剂的制备及快速检测方法的建立[D].哈尔滨:东北农业大学,2012.LUO Ji-guan.Preparation of Reagent and Development of A Rapid Detection Method for Bovine Parvovirus[D].Harbin:Northeast Agricultural University,2012.(in Chinese)
[15]徐义刚,李丹丹,刘忠梅,等.基于DPO引物特异性检测小肠结肠炎耶尔森氏菌的PCR方法[J].中国人兽共患病学报,2014,30(5):507-510.XU Yi-gang,LI Dan-dan,LIU Zhong-mei,et al.DPO-based PCR method for specific detection of Yersinia enterocolitica[J].Chinese Journal of Zoonoses,2014,30(5):507-510.(in Chinese)
[16]CHUN J Y,KIM K J,HWANG I T,et al.Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene[J].Nucleic Acids Research,2007,35(6):e40.
[17]MA X,XU H,SHI L,et al.A multiplex PCR assay for the detection of five influenza viruses using a dual priming oligonucleotide system[J].BMC Infectious Diseases,2015,15(1):93.
[18]XU Y G,LIU Z M,GUAN X T,et al.Dual priming oligonucleotide(DPO)-based multiplex PCR assay for specific detection of four diarrhoeagenic Escherichia coli in food[J].Letters in Applied Microbiology,2015,61(2):146-152.
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