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目的建立用于牛源样本中牛病毒性腹泻病毒(BVDV)双重RT-PCR检测方法。方法选择已发表的BVDV 1型和BVDV 2型包含5’UTR区高保守区域基因作为靶基因,分别设计合成引物,建立双重BVDV RT-PCR方法,并对方法的特异性、敏感性、稳定性等进行方法学评价。同时用建立的RT-PCR方法检测了小牛血清、小牛血清去蛋白提取液、脾多肽注射液等41批次牛源性样本和64份牛血浆样本。牛源样本和64份牛临床样本。结果建立的BVDV RT-PCR检测方法与牛副流感病毒III型(BPIV3)、猪瘟病毒(CSFV)、乙型脑炎病毒(JEV)均无交叉反应;检测BVDV 1型和BVDV 2型DNA模板最低浓度分别为每微升8.87×10~2拷贝和6.31×10~2拷贝,BVDV 1型和2型c DNA在-30℃冰箱放置12个月仍可检测出目的条带。应用建立的BVDV RT-PCR方法检测41批次牛源样本和64份牛血浆样本,核酸阳性率分别为14.6%和29.7%。结论建立的BVDV RT-PCR检测方法具有快速、特异、敏感及稳定的特点,可用于牛源样本携带BVDV核酸的检测。
Objective To establish a dual RT-PCR assay for bovine viral diarrhea virus (BVDV) in bovine samples. Methods The published BVDV type 1 and type 2 BVDV genes containing the highly conserved region of 5 ’UTR region as target genes were designed and synthesized. The double BVDV RT-PCR method was established and the specificity, sensitivity and stability Methodological evaluation. At the same time, 41 batches of bovine samples and 64 samples of bovine serum were obtained by reverse transcription-polymerase chain reaction (RT-PCR), including calf serum, bovine serum albumin extract and spleen polypeptide injection. Bovine source samples and 64 bovine clinical samples. Results The established BVDV RT-PCR assay showed no cross-reactivity with BPIV3, CSFV and JEV. The BVDV type 1 and BVDV 2 DNA templates The lowest concentrations were 8.87 × 10 ~ 2 copies and 6.31 × 10 ~ 2 copies per microliter, respectively. BVDV type 1 and 2 cDNA were still detected in the refrigerator at -30 ℃ for 12 months. The BVDV RT-PCR method was used to detect 41 batches of bovine source samples and 64 bovine plasma samples. The positive rates of nucleic acids were 14.6% and 29.7% respectively. Conclusion The established BVDV RT-PCR method is rapid, specific, sensitive and stable and can be used to detect BVDV nucleic acid in bovine samples.