為建立一種牛支原體（Mycoplasma bovis，MB）和牛病毒性腹瀉病毒（Bovine viral diarrhea virus，BVDV）的快速鑒別診斷方法，針對MB的uvrC基因和BVDV的5'端非編碼區(qū)（5'-UTR）保守基因序列，分別設(shè)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MB和BVDV的二重二溫式PCR方法。該方法能同時擴增MB和BVDV，擴增產(chǎn)物大小分別為412和170 bp。特異性試驗結(jié)果顯示，該方法對參試的所有毒株只擴增MB和BVDV基因組，對其它牛病原體無擴增；敏感性試驗結(jié)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(jié)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(jié)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(yīng)用于MB和BVDV臨床鑒別診斷和流行病學(xué)調(diào)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 bovis（MB）and bovine viral diarrhea virus（BVDV），two pairs of specific primers were designed based onthe conserved sequences of MB uvrC gene and BVDV 5'-UTR，and a new modified two-temperature duplexPCR was developed from three-temperature conventional PCR. According to theresults，the developed assay could amplify the genesof both MB and BVDV simultaneously，and the PCR products were 412 bp for MB and 170 bp forBVDV，respectively. 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 themethod，and the experimental results were notaffected by the template concentrations. In conclusion，the developed two-temperature duplex PCRassay was specific，sensitive，rapid and simple，and it could be applied in differential diagnosis for clinical samplesand epidemiological investigation.
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