双重PCR快速检测百日咳杆菌

Duplex PCR assay for detecting specific gene sequence from pertussis bacillus

目的:以百日咳毒素S1亚基启动子ptxA-Pr基因和插入序列IS481为目的基因,建立敏感、特异的双重PCR快速检测百日咳杆菌方法。方法:运用百日咳杆菌ptxA-Pr和IS481基因序列特异性引物,采用双重PCR技术同时扩增百日咳杆菌的特异性基因ptxA-Pr和IS481。通过构建目的质粒获得阳性对照,测序并与GenBank比对序列验证扩增产物。百日咳标准菌株DNA10倍系列稀释为模板,采用此方法扩增双基因,检测此方法敏感性。扩增肺炎克雷伯菌、肺炎链球菌、铜绿假单胞菌、金黄色葡萄球菌、大肠杆菌及阳性样本DNA,检测此方法特异性。结果:百日咳杆菌标准株和阳性样本均能同时扩增ptxA-Pr和IS481目标序列,分别为191、145bp。构建质粒后测序结果与GenBank对比一致。每个反应体系能检测到的标准菌株核酸最小量为1.65×10-2ng。其他菌株检测未出现非特异性扩增。结论:双重PCR扩增百日咳杆菌ptxA-Pr和IS481基因的方法可用来特异快速的检测百日咳杆菌。

Objective To establish a rapid assay with high sensitivity and specificity based on the sequence for pertussis toxin S1 promoter ptxA-Pr and insertion element sequence IS481.Methods Oligonucleotide primers targeting the ptxA-Pr and IS481 were used to amplify in the same reaction.The amplicons were inserted into the plasmid,sequenced and compared in the GenBank.The sensitivity of pertussis bacillus assay was determined by amplifying pertussis bacillus standard bacterial strain DNA at 10 dilutions.The five other bacterium included Klebsiella pneumoniae,Streptococcus pneumoniae,Pseudomonas aeruginosa,Staphylococcus aureus,Escherichia coli and one positive sample were detected in the same time for determining the specificity of this duplex PCR assay.Results The bands of expected size were obtained in standard bacterial strain and positive sample PCR assay（191 bp amplicon in ptxA-Pr and 145 bp amplicon in IS481）.The sequence analysis revealed that the amplicons were the same as the sequence in the GenBank.The detection level was approximately 1.65×10-2ng per reaction.There was no cross reaction with other bacterium.Conclusion The duplex PCR assay can be used to detect pertussis bacillus rapidly with high sensitivity and specificity.