牛支原体和牛病毒性腹泻病毒二重二温式PCR检测方法的建立
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摘要
为建立一种牛支原体(Mycoplasma bovis,MB)和牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)的快速鉴别诊断方法,针对MB的uvr C基因和BVDV的5'端非编码区(5'-UTR)保守基因序列,分别设计两对特异引物,并将三温式PCR扩增程序简化为二个温度梯度,建立了鉴别MB和BVDV的二重二温式PCR方法。该方法能同时扩增MB和BVDV,扩增产物大小分别为412和170 bp。特异性试验结果显示,该方法对参试的所有毒株只扩增MB和BVDV基因组,对其它牛病原体无扩增;敏感性试验结果显示,该方法最低能同时检测到104拷贝的两种目的核酸;干扰性试验结果显示,该方法能同时检测两个模板不同浓度的组合,试验结果不受模板影响。综上,本研究所建立的二重二温式PCR方法特异、敏感、快速、简便,可应用于MB和BVDV临床鉴别诊断和流行病学调查。
In order to establish a rapid identification and detection method for Mycoplasma bovis(MB)and bovine viral diarrhea virus(BVDV),two pairs of specific primers were designed based on the conserved sequences of MB uvr C gene and BVDV 5'-UTR,and a new modified two-temperature duplex PCR was developed from threetemperature conventional PCR. According to the results,the developed assay could amplify the genes of both MB and BVDV simultaneously,and the PCR products were 412 bp for MB and 170 bp for BVDV,respectively. The specificity test results showed no cross-reaction with other bovine pathogens was observed. The sensitivity test results showed that the detection limit was 10~4 copies of nucleic acids of two target genes. The interference test results showed the combination of different concentrations of the two templates could be detected by the method,and the experimental results were not affected by the template concentrations. In conclusion,the developed two-temperature duplex PCR assay was specific,sensitive,rapid and simple,and it could be applied in differential diagnosis for clinical samples and epidemiological investigation.
In order to establish a rapid identification and detection method for Mycoplasma bovis(MB)and bovine viral diarrhea virus(BVDV),two pairs of specific primers were designed based on the conserved sequences of MB uvr C gene and BVDV 5'-UTR,and a new modified two-temperature duplex PCR was developed from threetemperature conventional PCR. According to the results,the developed assay could amplify the genes of both MB and BVDV simultaneously,and the PCR products were 412 bp for MB and 170 bp for BVDV,respectively. The specificity test results showed no cross-reaction with other bovine pathogens was observed. The sensitivity test results showed that the detection limit was 10~4 copies of nucleic acids of two target genes. The interference test results showed the combination of different concentrations of the two templates could be detected by the method,and the experimental results were not affected by the template concentrations. In conclusion,the developed two-temperature duplex PCR assay was specific,sensitive,rapid and simple,and it could be applied in differential diagnosis for clinical samples and epidemiological investigation.
引文
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[2]辛九庆,李媛,郭丹,等.国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J].中国预防兽医学报,2008,30(9):661-664.
[3]高铎,孔令聪,贾博岩,等.牛支原体致病机制研究进展[J].动物医学进展,2005,36(7):90-94.
[4]胡长敏,石磊,龚瑞,等.牛支原体病研究进展[J].动物医学进展,2009,30(8):73-77.
[5]WENG X G,SONG Q J,WU Q,et al.Genetic characterization of bovine viral diarrhea virus strains in Beijing,China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle[J].Journal veterinary science,2015,16(4):491-500.
[6]SANTMAN-BERENDS I M,MARS M H,VAN D L,et al.Evaluation of the epidemiological and economic consequences of control scenarios for bovine viral diarrhea virus in dairy herds[J].Journal dairy science,2015,98(11):7699-7716.
[7]YUMIKO H,RYOKO U,WATARU Y,et al.An improved loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis[J].Journal veterinary medicine science,2016,78(8):1343-1346.
[8]NAIKARE H,BRUNO D,MAHAPATRA D.Development and evaluation of a novel Taqman real-time PCR assay for rapid detection of Mycoplasma bovis:Comparison of assay performance with a conventional PCR assay and another Taqman real-time PCR assay[J].Veterinary science,2015,2(1):32-42.
[9]FU P,SUN Z H,ZHANG Y W,et al.Development of a direct competitive ELISA for the detection of Mycoplasma bovis infection based on a monoclonal antibody of P48 protein[J].BMC veterinary research,2014,41(10):42-50.
[10]LI T S,HUANG M L,XIAO H R,et al.Selection and characterization of specific nanobody against bovine virus diarrhea virus(BVDV)E2 protein[J].PLos one,2017,12(6):e0178469.
[11]范晴,谢芝勋,谢志勤,等.牛支原体、牛病毒性腹泻病毒和牛传染性鼻气管炎病毒三重二温式PCR检测方法的建立及应用[J].动物医学进展,2018,39(2):43-48.
[12]范晴,谢芝勋,刘加波,等.牛轮状病毒二温式PCR检测方法的建立[J].广西农业科学,2010,41(10):1125-1127.
[13]范晴,谢芝勋,刘加波,等.BVDV与BRV二温式多重PCR检测方法的建立[J].西南农业学报,2011,24(5):1952-1954.
[14]ANNE T,ISABELLED D,ANNICK L,et al.Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis[J].Veterinary journal,2004,168(3):100-102.
[15]LETELLIER C,KERKHOFS P,WELLENMANS G,et al.Detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the 5'untranslated region[J].Veterinary microbiology,1999,64(9):155-167.
[16]ZENG T T,XIE Z X,XIE L J,et al.Simultaneous detection of six immunosuppressive chicken viruses by Ge XP analyser-based multiplex PCR assays[J].Virology journal,2015,12:226-231.
[2]辛九庆,李媛,郭丹,等.国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J].中国预防兽医学报,2008,30(9):661-664.
[3]高铎,孔令聪,贾博岩,等.牛支原体致病机制研究进展[J].动物医学进展,2005,36(7):90-94.
[4]胡长敏,石磊,龚瑞,等.牛支原体病研究进展[J].动物医学进展,2009,30(8):73-77.
[5]WENG X G,SONG Q J,WU Q,et al.Genetic characterization of bovine viral diarrhea virus strains in Beijing,China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle[J].Journal veterinary science,2015,16(4):491-500.
[6]SANTMAN-BERENDS I M,MARS M H,VAN D L,et al.Evaluation of the epidemiological and economic consequences of control scenarios for bovine viral diarrhea virus in dairy herds[J].Journal dairy science,2015,98(11):7699-7716.
[7]YUMIKO H,RYOKO U,WATARU Y,et al.An improved loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis[J].Journal veterinary medicine science,2016,78(8):1343-1346.
[8]NAIKARE H,BRUNO D,MAHAPATRA D.Development and evaluation of a novel Taqman real-time PCR assay for rapid detection of Mycoplasma bovis:Comparison of assay performance with a conventional PCR assay and another Taqman real-time PCR assay[J].Veterinary science,2015,2(1):32-42.
[9]FU P,SUN Z H,ZHANG Y W,et al.Development of a direct competitive ELISA for the detection of Mycoplasma bovis infection based on a monoclonal antibody of P48 protein[J].BMC veterinary research,2014,41(10):42-50.
[10]LI T S,HUANG M L,XIAO H R,et al.Selection and characterization of specific nanobody against bovine virus diarrhea virus(BVDV)E2 protein[J].PLos one,2017,12(6):e0178469.
[11]范晴,谢芝勋,谢志勤,等.牛支原体、牛病毒性腹泻病毒和牛传染性鼻气管炎病毒三重二温式PCR检测方法的建立及应用[J].动物医学进展,2018,39(2):43-48.
[12]范晴,谢芝勋,刘加波,等.牛轮状病毒二温式PCR检测方法的建立[J].广西农业科学,2010,41(10):1125-1127.
[13]范晴,谢芝勋,刘加波,等.BVDV与BRV二温式多重PCR检测方法的建立[J].西南农业学报,2011,24(5):1952-1954.
[14]ANNE T,ISABELLED D,ANNICK L,et al.Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis[J].Veterinary journal,2004,168(3):100-102.
[15]LETELLIER C,KERKHOFS P,WELLENMANS G,et al.Detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the 5'untranslated region[J].Veterinary microbiology,1999,64(9):155-167.
[16]ZENG T T,XIE Z X,XIE L J,et al.Simultaneous detection of six immunosuppressive chicken viruses by Ge XP analyser-based multiplex PCR assays[J].Virology journal,2015,12:226-231.
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