荧光PCR熔解曲线法检测幽门螺杆菌克拉霉素、喹诺酮耐药基因的性能验证  

Performance validation of the fluorescence quantitative PCR melting curve method for detecting clarithromycin and quinolone resistant genes in Helicobacter pylori

作　　者：杨昕澄 范柏月 何帮顺[1] 聂珍琳[1] 韦静 万芳[1] 蔺昕[1] YANG Xincheng;FAN Boyue;HE Bangshun;NIE Zhenlin;WEI Jing;WAN Fang;LIN Xin(Department of Laboratory Medicine,Nanjing First Hospital,Nanjing Hospital Affiliated to Nanjing Medical University,Nanjing 210006,Jiangsu,China)

机构地区：[1]南京医科大学附属南京医院医学检验科,南京210006

出　　处：《临床检验杂志》2024年第11期845-850,共6页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省医学重点学科建设单位(JSDW202239)。

摘　　要：目的探讨采用荧光PCR熔解曲线法检测粪便样本中的幽门螺杆菌(Helicobacter pylori,Hp)克拉霉素(23S rRNA)及喹诺酮(gyrA)耐药相关基因突变在临床诊断中的应用价值。方法纳入胃镜检查Hp快速尿素酶阳性且未经治疗的1176例患者,收集患者胃黏膜组织及粪便样本用于对比研究,针对同一阳性病例,采用E-test法分析胃黏膜组织中Hp克拉霉素、喹诺酮耐药表型,荧光PCR熔解曲线法检测粪便样本中Hp克拉霉素、喹诺酮耐药基因型,Kappa检验分析两种方法检测结果的一致性;对于阳性病例的粪便样本提取2份核酸,分别采用荧光PCR熔解曲线法及一代测序法检测Hp 23S rRNA、gyrA耐药突变基因,并比较两种方法检测结果的一致性。结果在克拉霉素耐药研究中,最终获得有效样本934例,其中耐药阳性表型453例,耐药阳性基因型481例,阳性符合率为93.38%(95%CI:90.70%~95.32%);在喹诺酮耐药研究中,最终获得有效样本909例,其中表型耐药阳性426例,基因型耐药阳性413例,阳性符合率为86.85%(95%CI:83.31%~89.74%)。在对比研究中,23S rRNA基因检测有效样本986例,其中荧光PCR熔解曲线法检测耐药阳性514例,测序检测耐药阳性509例,阳性符合率为96.27%(95%CI:94.24%~97.60%),gyrA基因检测有效样本895例,其中荧光PCR熔解曲线法检测耐药阳性422例,测序检测耐药阳性405例,阳性符合率为95.80%(95%CI:93.38%~97.36%)。结论基于粪便样本的荧光PCR熔解曲线法检测Hp 23S rRNA、gyrA对预测Hp的耐药性具有一定的临床应用价值。Objective To investigate the application value of the fluorescence quantitative PCR(qPCR)melting curve method in the detection of clarithromycin(23S rRNA)and quinolone(gyrA)resistant genes of Helicobacter pylori(Hp)in fecal samples.Methods A total of 1176 untreated patients who underwent gastroscopy and were Hp positive proved by rapid urease test(RUT)were enrolled in the study.Their gastric mucosal and fecal samples were collected.The E-test method was used to analyze the clarithromycin and quinolone resistant phenotypes of Hp in gastric mucosal samples.The qPCR melting curve method was used to detect the clarithromycin and quinolone resistant genotypes of Hp in fecal samples.The consistency of the results obtained by the two methods was evaluated by the Kappa test.In addition,the nucleic acids were extracted from the fecal samples with Hp positive,and the Hp 23S rRNA and gyrA resistance mutation genes were detected by the qPCR melting curve method and Sanger sequencing,respectively.The consistency of the results obtained by the two methods was compared.Results In the study of clarithromycin resistance,a total of 934 valid samples were obtained.Among them,453 samples had positive resistance phenotype and 481 had positive resistance genotype,with a positive consistency rate of 93.38%(95%CI:90.70%~95.32%).In the study of quinolone resistance,a total of 909 valid samples were obtained.Among them,426 samples had positive resistance phenotype and 413 had positive resistance genotype,with a positive consistency rate of 86.85%(95%CI:83.31%-89.74%).In the comparative study,986 valid samples were detected for Hp 23S rRNA gene.Among them,514 samples were resistance positive detected by the qPCR melting curve method and 509 by Sanger sequencing,with a positive consistency rate of 96.27%(95%CI:94.24%-97.60%).Similarly,895 valid samples were detected for Hp gyrA gene.Among them,422 samples were resistance positive detected by the qPCR melting curve method and 405 by Sanger sequencing,with a positive consistency rate of 95.80

关 键 词：幽门螺杆菌 荧光PCR熔解曲线 克拉霉素 喹诺酮 耐药 基因型 

分 类 号：R446.5[医药卫生—诊断学]