产肠毒素大肠杆菌黏附素基因多重PCR检测方法的建立与应用

Establishment and application of multiplex PCR method for detection of enterotoxigenic Escherichia coli adhesin genes

为建立一种能同时快速、灵敏、准确检测产肠毒素大肠杆菌(ETEC)的多重PCR检测方法,根据GenBank中ETEC K88(F4)、K99(F5)、987P(F6)、F41和etp A黏附素基因的保守序列设计并合成了5对特异性引物,通过多重PCR反应条件优化,建立了一种能快速检测5种黏附素基因的多重PCR方法。结果表明,该方法可同时特异性扩增出大肠杆菌K88、K99、987P、F41和etp A基因片段,对鼠伤寒沙门菌、猪链球菌、金黄色葡萄球菌、猪巴氏杆菌、猪霍乱沙门菌、单增李斯特菌均无特异性扩增。敏感性试验结果显示,用该多重PCR能检测到K88、K99、etp A、F41和987P基因的最小活菌数分别为1.1×10^(6)、1.2×10^(6)、6.3×10^(5)、1.2×10^(5)和6.2×10~4CFU/m L。K88、K99、987P、etp A和F41的基因组DNA的检测下限分别为1.17×10^(-3)、0.1、1.88×10^(-3)、2.55×10^(-3)和1.0×10^(-5)ng/μL,表明其敏感性较高。人工模拟污染粪便样品试验结果表明,人工污染粪便样品中K88、K99、F41、987P和etpA的检测限分别为2.5×10^(8)、2.5×10^(8)、2.5×10^(3)、1.9×10^(7)和1.75×10^(3)CFU/g,进一步验证了非预增菌下该多重PCR方法的临床可行性。并利用所建立的多重PCR方法对80株大肠杆菌临床分离株进行检测,K88的阳性率为12.5%(10/80),K99的阳性率为1.25%(1/80)。研究表明:建立的产肠毒素大肠杆菌黏附素基因多重PCR分型方法具有很好的特异性、敏感性,可用于临床上大肠杆菌分离菌株黏附素基因的快速鉴定。

The aim of this study was to establish a multiplex PCR method that could simultaneously detect the five adhesin genes of enterotoxigenic Escherichia coli(ETEC).Five pairs of specific primers were designed and synthesized according to K88(F4),K99 (F5),987P(F6),F41,and etp A gene sequences in Gen Bank.A multiplex PCR method that could rapidly detect five adhesin genes was established by optimizing the reaction conditions.This method could specifically amplify E.coli K88,K99,987P,F41,and etp A gene fragments,however,Salmonella typhimurium,Streptococcus suis,Staphylococcus aureus,Pasteurella suis,Salmonella choleraesuis,and Listeria monocytogenes had no specific amplification.The sensitivity test results showed that the minimum viable bacterial counts of K88,K99,etp A,F41 and 987P genes detected by the multiplex PCR were 1.1×10^(6)CFU/m L,1.2×10^(6)CFU/m L,6.3×10^(5)CFU/m L,1.2×10^(5)CFU/m L and 6.2×10~4CFU/m L,respectively.The minimum detection mass concentration of K88,K99,987P,etp A,and F41 in genomic DNA were 1.17×10^(-3)ng/μL,0.1ng/μL,1.88×10^(-3)ng/μL,2.55×10^(-3)ng/μL and 1.0×10^(-5)ng/μL,indicating high sensitivity.Furthermore,the detection limits of K88,K99,F41,987P and etp A in artificially contaminated fecal samples were 2.5×10^(8)CFU/g,2.5×10^(8)CFU/g,2.5×10^(3)CFU/g,1.9×10~7CFU/g and 1.75×10^(3)CFU/g,which further verified the clinical feasibility of the multiplex PCR method established without enrichment in fecal sample in this study.The multiplex PCR method was used to detect 80 clinical isolates of E.coli,the positive rate of K88 was 12.5%(10/80),and the positive rate of K99 was 1.25%(1/80).The data show that the multiplex PCR method of detecting adhesin genes of ETEC has good specificity and sensitivity,which could be used for the rapid identification of the adhesin gene of E.coli isolates in clinic.