BVDV与BRV二温式多重PCR检测方法的建立

Study on Development of Two-temperature Multiplex PCR for BVDV and BRV

根据牛病毒性腹泻病毒(BVDV)和牛轮状病毒(BRV)的保守基因设计了两对特异引物,并将三温式PCR扩增程序简化为2个温度梯度,建立了用于同时检测BVDV和BRV的二温式多重PCR方法,扩增长度分别为244和385 bp。特异性试验和敏感性试验结果表明,该方法只对BVDV和BRV模板进行扩增,而对其他对照病毒的检测均为阴性;检测灵敏度高,最低能检测到BVDV和RBV各1 pg病毒RNA。建立的BVDV和BRV二温式多重PCR方法,是一种快速、特异、敏感的检测方法。

A two-temperature polymerase chain reaction was optimized to simultaneously detect two pathogens of BVDV and BRV.Two sets of primers were designed according to the conserved sequences of bovine viral diarrhea（BVDV） and bovine rotavirus（BRV）,yielding two specific bands of BVDV 244-bp and BRV 385-bp.A new modified two-temperature multiple PCR was developed from three-temperature conventional PCR in the study.The results showed that the specificity of the assay was high without amplification of other viruses,and the detection limit of the two-temperature multiplex PCR assay was 1 pg RNA of BVDV and BRV,respectively.This two-temperature multiplex PCR assay reported here was a valuable method with high specificity and sensitivity detection of BVDV and BRV.