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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

点击数:4225  时间:2019-03-01 08:58:36  来源: 中国动物检疫      作者: 谢志勤等

为建立一种牛支原体(Mycoplasma bovisMB)和牛病毒性腹泻病毒(Bovine viral diarrhea virusBVDV)的快速鉴别诊断方法,针对MBuvrC基因和BVDV5'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MBBVDV的二重二温式PCR方法。该方法能同时扩增MBBVDV,扩增产物大小分别为412170 bp。特异性试验结果显示,该方法对参试的所有毒株只扩增MBBVDV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MBBVDV临床鉴别诊断和流行病学调查。


Development of Two-temperature Duplex PCR

for Detection of Mycoplasma bovis and Bovine ViralDiarrhea Virus

In order to establish a rapididentification and detection method for Mycoplasma bovisMBand bovine viral diarrhea virusBVDV),two pairs of specific primers were designed based onthe conserved sequences of MB uvrC gene and BVDV 5'-UTRand a new modified two-temperature duplexPCR was developed from three-temperature conventional PCR. According to theresultsthe developed assay could amplify the genesof both MB and BVDV simultaneouslyand the PCR products were 412 bp for MB and 170 bp forBVDVrespectively. The specificity test resultsshowed no cross-reaction with other bovine pathogens was observed. Thesensitivity test results showed that the detection limit was 104 copies ofnucleic acids of two target genes. The interference test results showed the combinationof different concentrations of the two templates could be detected by themethodand the experimental results were notaffected by the template concentrations. In conclusionthe developed two-temperature duplex PCRassay was specificsensitiverapid and simpleand it could be applied in differential diagnosis for clinical samplesand epidemiological investigation.

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