爲建立一種(zhǒng)牛支原體（Mycoplasma bovis，MB）和牛病毒性腹瀉病毒（Bovine viral diarrhea virus和對，BVDV）的快速鑒别診斷方法，針對(duì)MB的uvrC基因和BVDV的5'端非編碼區（5'-UTR）保守基因序列，分别設計兩(liǎ在請ng)對(duì)特異引物，并將(jiāng)三溫式PCR擴增程序簡化爲二個溫度梯度，建立了鑒用師别MB和BVDV的二重二溫式PCR方法。該方法能(néng)同時(shí)擴增MB和BVDV，擴增産物大小分别爲412和170 bp。特異性試驗結果顯示，該方法對(duì)參試的所有毒株隻擴增MB和BVDV基因組，對(duì)其它牛病原體森冷無擴增；敏感性試驗結果顯示，該方法最低能(néng)同時(分那shí)檢測到104拷貝的兩(liǎng)種(zhǒng)目的核酸；幹擾性試驗結果顯哥數示，該方法能(néng)同時(shí)檢測兩(liǎng)個模闆不同濃度的厭紅組合，試驗結果不受模闆影響。綜上，本研究所建立的生了二重二溫式PCR方法特異、敏感、快速、簡便，可應用于MB和BVDV臨床鑒别診斷和流行病學(xué)調查。

Development of Two-temperature著有 Duplex PCR

for Detection of Myco信務plasma bovis and Bovin雜是e ViralDiarrhea 花歌Virus

In order to establish習木 a rapididentifica計窗tion and detectio下大n method for Mycoplasma bovis（MB）and bovine viral d明請iarrhea virus（BVDV），two pairs of specific primers were desi海上gned based onthe conserved sequence嗎民s of MB uvrC gene and BVDV 5鐘飛'-UTR，and a new modified two-temperat了船ure duplexPCR was develope山報d from three-temperature conventional 秒她PCR. According to theresults人要，the developed assay co厭對uld amplify the g高姐enesof both MB and BVDV si線路multaneously，and the PCR products were 4短會12 bp for MB and 170 bp forBV信生DV，respectively. The草關 specificity test resultssh人見owed no cross-rea拍場ction with other bovine店人 pathogens was observed. The來到sensitivity test results sh上間owed that the detection li務訊mit was 104 copies ofnu朋音cleic acids of two target genes. 務兵The interference test result通術s showed the combinatio吃朋nof different c紙慢oncentrations of the two tem金請plates could be detected 計相by themethod，and the experimental results wer器業e notaffected by the template concent車線rations. In conclusi明錯on，the developed two-temperature dupl民靜ex PCRassay was specific，sensitive，rapid and simple，and it could be窗兵 applied in differential dia林時gnosis for clinical samplesand epidemi河我ological invest林廠igation.

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