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

作　　者：钟运华 李春华 刘振杰[2] ZHONG Yunhua;LI Chunhua;LIU Zhenjie(Guangzhou Weimi Bio-tech Co.Ltd,Guangdong Engineering Technology Research Center for Multiple Fluorescence Accurate Diagnosis of Neuro-immune Diseases,Guangzhou,Guangdong 510663,China;Guangdong Provincial Hospital of Chinese Medicine,Guangzhou,Guangdong 510000,China)

机构地区：[1]广州市微米生物科技有限公司,广东省神经免疫性疾病多重荧光精准诊断工程技术研究中心,广东广州510663 [2]广东省中医院,广东广州510000

出　　处：《中国热带医学》2023年第8期870-874,共5页China Tropical Medicine

摘　　要：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),res

关 键 词：化脓性链球菌 环介导等温扩增 毒力基因 

分 类 号：R446.5[医药卫生—诊断学]