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

Development of Two-temperature Duplex PCR for Detection of Mycoplasma bovis and Bovine Viral Diarrhea Virus

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 uvrC 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 104 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.