荧光环介导等温扩增检测化脓性链球菌sdaB方法的建立

Development of fluorescent loop-mediated isothermal amplification for detection of sdaB of Streptococcus pyogenes

目的建立荧光环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测化脓性链球菌毒力基因sdaB的方法。方法根据GenBank公布的化脓性链球菌sdaB基因保守序列(GenBank:69901515),使用引物设计软件Primer Explorer V5.0获得LAMP引物。在LAMP体系中加入荧光染料,对引物、MgSO_(4)、甜菜碱、脱氧核糖核苷三磷酸(deoxy-ribonucleosidetriphosphate,dNTP)、Bst DNA聚合酶的使用浓度进行了筛选,MgSO_(4)浓度范围为0~12 mmol/L、甜菜碱浓度范围为0~2.4 mol/L、dNTP浓度范围为0.2~2μmol/L、上游内部引物(forward inner primer,FIP)和下游内部引物(backward inner primer,BIP)浓度范围为0.2~2μmol/L、上游外部引物(forward outer primer,F3)和下游外部引物(back⁃ward outer primer,B3)浓度范围为0.2~0.4μmol/L、Bst DNA聚合酶浓度范围为0.16~0.96 U/μL、荧光染料浓度范围为0.2~2μmol/L。以优化后体系,在ABI7500实时荧光定量PCR分析仪上评估方法学特异性和最低检出限,检测了13种标准菌株包括A群链球菌、B群链球菌、C群链球菌、G群链球菌、肺炎链球菌、草绿色链球菌、粪肠球菌、屎肠球菌、淋病奈瑟菌、嗜酸乳杆菌、大肠埃希菌、肺炎克雷伯菌和铜绿假单胞菌,最后检测了103例临床样本。结果反应体系以25μL为宜,包含25μmol/L荧光染料0.8μL、100 mmol/L MgSO_(4)1μL、5 mol/L甜菜碱6μL、25 mmol/L dNTP 1.4μL、20μmol/L FIP和BIP各2μL、20μmol/L F3和B3各0.5μL、8 U/μL Bst DNA聚合酶1μL和2μL模板,去离子水补足,63℃反应45 min即可完成;最低检出限为500 pg/μL;检测12株非化脓性链球菌株,均为阴性结果。103例临床样本结果与培养法比较,临床灵敏度为100.0%(16/16),特异性为96.6%(84/87)。结论本研究检测化脓性链球菌的特异性和灵敏度较好,且操作简单,能满足临床需求,适宜现场检测及基层推广。

Objective To establish a method for detecting sdaB virulence gene of Streptococcus pyogenes with loop mediated isothermal amplification(LAMP).Methods According to the conserved sequence of Streptococcus pyogenes sdaB gene published in GenBank(GenBank:69901515),LAMP primers were designed with Primer Explorer V5.0 software.Main components of LAMP reaction system were optimized including of fluorescent dye,MgSO_(4),betaine,deoxy-ribonucleosidetriphosphate(dNTP),and Bst DNA polymerase,with the concentration of MgSO_(4)from 0 mmol/L to 12 mmol/L,betaine from 0 mol/L to 2.4 mol/L,dNTP from 0.2μmol/L to 2μmol/L,forward inner primer(FIP)and backward inner primer(BIP)from 0.2μmol/L to 2μmol/L respetively,forward outer primer(F3)and backward outer primer(B3)from 0.2μmol/L to 0.4μmol/L,Bst DNA polymerase from 0.16 U/μL to 0.96 U/μL,fluorescent dye from 0.2μmol/L to 2μmol/L.With the optimized system,the methodological specificity and the minimum detection limit were evaluated on the ABI7500 real-time fluorescent quantitative PCR analyzer,and 13 standard strains including Group A Streptococcus,Group B Streptococcus,Group C Streptococcus,Group G Streptococcus,Streptococcus pneumoniae,Streptococcus viridis,Enterococcus faecalis,Enterococcus faecium,Neisseria gonorrhoeae,Lactobacillus acidophilus,Escherichia coli,Klebsiella pneumoniae and Pseudomonas aeruginosa were detected.Finally,103 clinical samples were tested.Results The optimized reaction system contained 25μL reaction mixture,including 0.8μL of 25μmol/L fluorescent dye,1μL of 100 mmol/L MgSO_(4),6μL of 5 mol/L betaine,1.4μL of 25 mmol/L dNTP,2μL of 20μmol/L FIP and BIP,0.5μL of 20μmol/L F3 and B3,1μL of 8 U/μL Bst DNA polymerase,and 2μL of template.After adding deionized water,the mixture was incubated at 63°C for 45 min to complete the reaction.The limit of detection(LOD)was 500 pg/μL.All 12 non-S.pyogenes strains tested were negative.Compared with the culture method,the clinical sensitivity and specificity were 100.0%(16/16)and 96.6%(84/87),respectively,for 103 clinical samples.Conclusions This LAMP assay is reliable for the detection of Streptococcus pyogenes in clinic and is suitable for field detection with good specificity and sensitivity,as well as simply operation.