猪捷申病毒SYBR Green Ⅰ实时荧光定量RT-PCR方法的建立
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摘要
为建立一种快速且灵敏的猪捷申病毒(PTV)的检测方法,本研究根据登录于GenBank数据库的所有PTV毒株基因组序列信息,在其5'端非编码区的保守区域设计引物,经过一系列的试验测试和验证建立了能够广泛用于检测PTV的SYBR Green Ⅰ荧光定量RT-PCR方法。该方法特异性强,对除PTV外的其他常见猪病毒性病原的DNA或cDNA均无特异性的扩增。此外,该方法的敏感性高、重复性好,灵敏度能达到38.3 copiesμ/L,组内和组间变异系数均不超过2%。对临床样品的检测结果显示,本研究建立的荧光定量RT-PCR方法的PTV检出率为68.18%,远高于普通RT-PCR的PTV检出率(22.73%)。
To establish a quick and sensitive method for the detection of porcine teschovirus(PTV),a pair of primers,located in the conserved 5'untranslated region,was designed based on the multiple genome sequence alignment of all PTV strains deposited in GenBank.Finally,a SYBR GreenⅠ-based realtime RT-PCR method for PTV detection was established after a series of experimental test and validation.The method had the advantage of high specificity,and there were no specially amplifications for the DNA or c DNA of common porcine viral pathogens except PTV.Moreover,the method had the advantages of high sensitivity and reproducibility.Detection limits with PTV-plasmid was 38.3 copies/μL,and the variation coefficients of intra-assay and inter-assay were both less than 2%.An overall PTV-positivity rate of 68.18% was detected in the samples collected on a farm with the SYBR Green Ⅰ-based real-time RT-PCR method,which was much higher than the conventional PTV RT-PCR with positivity rate of only 22.73%.
To establish a quick and sensitive method for the detection of porcine teschovirus(PTV),a pair of primers,located in the conserved 5'untranslated region,was designed based on the multiple genome sequence alignment of all PTV strains deposited in GenBank.Finally,a SYBR GreenⅠ-based realtime RT-PCR method for PTV detection was established after a series of experimental test and validation.The method had the advantage of high specificity,and there were no specially amplifications for the DNA or c DNA of common porcine viral pathogens except PTV.Moreover,the method had the advantages of high sensitivity and reproducibility.Detection limits with PTV-plasmid was 38.3 copies/μL,and the variation coefficients of intra-assay and inter-assay were both less than 2%.An overall PTV-positivity rate of 68.18% was detected in the samples collected on a farm with the SYBR Green Ⅰ-based real-time RT-PCR method,which was much higher than the conventional PTV RT-PCR with positivity rate of only 22.73%.
引文
[1]ZELL R,DELWART E,GORBALENYA A E,et al.ICTV virus taxonomy profile:Picornaviridae[J].Journal General of Virology,2017,98(10):2421-2422.
[2]YANG T,LI R,YAO Q,et al.Prevalence of porcine teschovirus genotypes in Hunan,China:identification of novel viral species and genotypes[J].Journal General of Virology,2018,99(9):1261-1267.
[3]TREFNY L.Massive illness of swine in Teschen area[J].Zverolek Obz,1930,23:235-236.
[4]BUITRAGO D,CANO-GOMEZ C,AGUERO M,et al.A survey of porcine picornaviruses and adenoviruses in fecal samples in Spain[J].Journal of Veterinary Diagnostic Investigation,2010,22(5):763-766.
[5]SUN H,GAO H,CHEN M,et al.New serotypes of porcine teschovirus identified in Shanghai,China[J].Archives of Virology,2015,160(3):831-835.
[6]CANO-GMEZ C,PALERO F,BUITRAGO M D,et al.Analyzing the genetic diversity of teschoviruses in Spanish pig populations using complete VP1 sequences[J].Infection,Genetics and Evolution,2011,11(8):2144-2150.
[7]POWLEDGE T M.The polymerase chain reaction[J].Advances in Physiology Education,2004,28(1-4):44-50.
[8]胡峰,赵亚荣,吕超超,等.猪捷申病毒TaqMan实时定量RT-PCR方法的建立[J].中国预防兽医学报,2012,34(3):206-210.HU Feng,ZHAO Ya-rong,L譈Chao-chao,et al.Development of TaqMan real-time RT-PCR assay for detection of porcine teschovirus[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(3):206-210.(in Chinese)
[9]王建峰,倪健波,李如松,等.猪捷申病毒荧光定量RT-PCR检测方法的建立与应用[J].畜牧与兽医,2016,48(6):126-129.WANG Jian-feng,NI Jian-bo,LI Ru-song,et al.Development and application of real-time RT-PCR for detection of porcine teschovirus[J].Animal Husbandry&Veterinary Medicine,2016,48(6):126-129.(in Chinese)
[10]CANO-GOMEZ C,BUITRAGO D,FERNANDEZ-PINERO J,et al.Evaluation of a fluorogenic real-time reverse transcription-polymerase chain reaction method for the specific detection of all known serotypes of porcine teschoviruses[J].Journal of Virological Methods,2011,176(1-2):131-134.
[11]BURLAND T G.DNASTAR’s lasergene sequence analysis software[J].Methods in Molecular Biology,2000,132:71-91.
[12]YANG T,YU X,LUO B,et al.Epidemiology and molecular characterization of porcine teschovirus in Hunan,China[J].Transboundary and Emerging Disease,2018,65(2):480-490.
[13]DENG M Y,MILLIEN M,JACQUES-SIMON R,et al.Diagnosis of porcine teschovirus encephalomyelitis in the Republic of Haiti[J].Journal of Veterinary Diagnostic Investigation,2012,24(4):671-678.
[14]SALLES M W,SCHOLES S F,DAUBER M,et al.Porcine teschovirus polioencephalomyelitis in Western Canada[J].Journal of Veterinary Diagnostic Investigation,2011,23(2):367-373.
[15]CARNERO J,PRIETO C,POLLEDO L,et al.Detection of teschovirus type 13 from two swine herds exhibiting nervous clinical signs in growing pigs[J].Transboundary and Emerging Disease,2018,65(2):e489-e493.
[2]YANG T,LI R,YAO Q,et al.Prevalence of porcine teschovirus genotypes in Hunan,China:identification of novel viral species and genotypes[J].Journal General of Virology,2018,99(9):1261-1267.
[3]TREFNY L.Massive illness of swine in Teschen area[J].Zverolek Obz,1930,23:235-236.
[4]BUITRAGO D,CANO-GOMEZ C,AGUERO M,et al.A survey of porcine picornaviruses and adenoviruses in fecal samples in Spain[J].Journal of Veterinary Diagnostic Investigation,2010,22(5):763-766.
[5]SUN H,GAO H,CHEN M,et al.New serotypes of porcine teschovirus identified in Shanghai,China[J].Archives of Virology,2015,160(3):831-835.
[6]CANO-GMEZ C,PALERO F,BUITRAGO M D,et al.Analyzing the genetic diversity of teschoviruses in Spanish pig populations using complete VP1 sequences[J].Infection,Genetics and Evolution,2011,11(8):2144-2150.
[7]POWLEDGE T M.The polymerase chain reaction[J].Advances in Physiology Education,2004,28(1-4):44-50.
[8]胡峰,赵亚荣,吕超超,等.猪捷申病毒TaqMan实时定量RT-PCR方法的建立[J].中国预防兽医学报,2012,34(3):206-210.HU Feng,ZHAO Ya-rong,L譈Chao-chao,et al.Development of TaqMan real-time RT-PCR assay for detection of porcine teschovirus[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(3):206-210.(in Chinese)
[9]王建峰,倪健波,李如松,等.猪捷申病毒荧光定量RT-PCR检测方法的建立与应用[J].畜牧与兽医,2016,48(6):126-129.WANG Jian-feng,NI Jian-bo,LI Ru-song,et al.Development and application of real-time RT-PCR for detection of porcine teschovirus[J].Animal Husbandry&Veterinary Medicine,2016,48(6):126-129.(in Chinese)
[10]CANO-GOMEZ C,BUITRAGO D,FERNANDEZ-PINERO J,et al.Evaluation of a fluorogenic real-time reverse transcription-polymerase chain reaction method for the specific detection of all known serotypes of porcine teschoviruses[J].Journal of Virological Methods,2011,176(1-2):131-134.
[11]BURLAND T G.DNASTAR’s lasergene sequence analysis software[J].Methods in Molecular Biology,2000,132:71-91.
[12]YANG T,YU X,LUO B,et al.Epidemiology and molecular characterization of porcine teschovirus in Hunan,China[J].Transboundary and Emerging Disease,2018,65(2):480-490.
[13]DENG M Y,MILLIEN M,JACQUES-SIMON R,et al.Diagnosis of porcine teschovirus encephalomyelitis in the Republic of Haiti[J].Journal of Veterinary Diagnostic Investigation,2012,24(4):671-678.
[14]SALLES M W,SCHOLES S F,DAUBER M,et al.Porcine teschovirus polioencephalomyelitis in Western Canada[J].Journal of Veterinary Diagnostic Investigation,2011,23(2):367-373.
[15]CARNERO J,PRIETO C,POLLEDO L,et al.Detection of teschovirus type 13 from two swine herds exhibiting nervous clinical signs in growing pigs[J].Transboundary and Emerging Disease,2018,65(2):e489-e493.
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