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首页> 外文期刊>Journal of Clinical Microbiology >Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.
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Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.

机译：巢式PCR检测技术的开发，用于检测临床样品中的牛呼吸道合胞病毒。

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Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.
机译：开发了两种嵌套式PCR检测试剂盒，用于检测牛呼吸道合胞病毒（BRSV）。引物选自编码F融合蛋白的基因（PCR-F）和编码G附着蛋白的基因（PCR-G）。选择生物素化的寡核苷酸探针，称为F和G，以杂交各自的PCR产物。 PCR-F和PCR-G分析的敏感性相似，均检测到0.1组织培养感染剂量的病毒。 PCR-F分析扩增了所有牛品系和一种人类品系（RS32）。没有观察到与九种异源呼吸道病毒的交叉反应。牛和人RSV菌株的PCR-F产物通过使用核酸内切酶限制性内切酶ScaI（BRSV的特异性裂解产物）来区分。寡核苷酸探针F也对BRSV的产物具有特异性。 PCR-G分析检测到所有牛株，没有检测到人类株。仙台病毒的产物也观察到一条微弱的电泳带。然而，探针G不与该产物杂交，仅与BRSV的产物杂交。通过免疫荧光（IF）方法以及PCR-F和PCR-G分析对从无症状牛和呼吸道疾病急性期收集的鼻拭子进行BRSV分析。通过PCR检测，在35个样本（89％）中的31个样本中检测到了病毒。通过IF方法，只有23个样品（66％）呈阳性，而通过PCR-F和PCR-G分析也均为阳性。 PCR检测出的31份阳性样本来自表现出急性呼吸道疾病临床体征的牛;这四个PCR阴性样品均来自临床无症状的邻近牛。随后对所有采样的动物进行血清转化，并对BRSV起反应。因此，通过PCR检测BRSV与临床观察相关，并且比IF灵敏得多（66比89％）。这些结果表明，两种嵌套式PCR检测都为检测牛BRSV感染提供了快速而灵敏的方法。考虑到其更高的特异性，可推荐将PCR-F分析作为BRSV临床标本分析的选择方法。

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