摘要:
建立一种针对副溶血弧菌blaCARB-17和toxR基因的双重PCR检测方法。按照副溶血弧菌的blaCARB-17和toxR基因序列设计并合成引物,于同一反应体系中进行PCR扩增,优化退火温度、退火时间和引物比例,确定双重PCR的特异性和灵敏性,最后用实验室分离鉴定的副溶血弧菌分离株验证。结果表明:建立的双重PCR最佳退火条件是52℃ 30 s,引物比例1:1,在同一体系中特异地扩增出副溶血弧菌的blaCARB-17和toxR基因的303、350 bp片段,其它对照菌株均无扩增,表明该方法特异性良好。双重PCR的最低检测限达1×102 CFU/mL,对实验室鉴定的56个副溶血弧菌分离株验证均为阳性。建立的双重PCR方法操作简单、高效快速、特异性强,适用于副溶血弧菌的检测。
Abstract:
A duplex polymerase chain reaction(PCR)method was established to simultaneously detect the blaCARB-17 and toxR genes of Vibrio parahaemolyticus. Two sets of primers were designed and synthesized according to the blaCARB-17 and toxR gene sequences of Vibrio parahaemolyticus. PCR amplification was performed in the same reaction system. The reaction parameters(annealing temperature,annealing time and primer ratio)were optimized to determine specificity and sensitivity. The isolates of Vibrio parahaemolyticus in our laboratory were detected by this duplex PCR. The results showed that the best annealing condition of the duplex PCR was 52℃ 30 s,and the ratio of primers was 1:1.Using two pairs of primers,303 and 350 bp fragments of blaCARB-17 and toxR genes were amplified,respectively. No amplification was observed in other control bacteria,indicating that this method had good specificity. The minimum detection limit of duplex PCR was 1×102 CFU/mL,and all the 56 isolated of Vibrio parahaemolyticus were positive for detection. The established duplex PCR method is simple,efficient,rapid,and specific. It is suitable for the detection of large-scale samples of Vibrio parahaemolyticus.
百度学术
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