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牛支原體和牛病毒性腹瀉病毒二重二溫式PCR檢測方法的建立

點擊數:17872  時間:2019-03-01 08:58:36  來源: 中國動物檢疫      作者: 謝志勤等

為建立一種牛支原體(Mycoplasma bovisMB)和牛病毒性腹瀉病毒(Bovine viral diarrhea virusBVDV)的快速鑒別診斷方法,針對MBuvrC基因和BVDV5'端非編碼區(5'-UTR)保守基因序列,分別設計兩對特異引物,并將三溫式PCR擴增程序簡化為二個溫度梯度,建立了鑒別MBBVDV的二重二溫式PCR方法。該方法能同時擴增MBBVDV,擴增產物大小分別為412170 bp。特異性試驗結果顯示,該方法對參試的所有毒株只擴增MBBVDV基因組,對其它牛病原體無擴增;敏感性試驗結果顯示,該方法最低能同時檢測到104拷貝的兩種目的核酸;干擾性試驗結果顯示,該方法能同時檢測兩個模板不同濃度的組合,試驗結果不受模板影響。綜上,本研究所建立的二重二溫式PCR方法特異、敏感、快速、簡便,可應用于MBBVDV臨床鑒別診斷和流行病學調查。


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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