Performance validation of the fluorescence quantitative PCR melting curve method for detecting clarithromycin and quinolone resistant genes in Helicobacter pylori
10.13602/j.cnki.jcls.2024.11.11
- VernacularTitle: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
- Author:
Xincheng YANG
1
;
Boyue FAN
;
Bangshun HE
;
Zhenlin NIE
;
Jing WEI
;
Fang WAN
;
Xin LIN
Author Information
1. 南京医科大学附属南京医院医学检验科,南京 210006
- Keywords:
Helicobacter pylori;
polymerase chain reaction melting curve;
clarithro-mycin;
quinolone;
drug resistance;
genotype
- From:
Chinese Journal of Clinical Laboratory Science
2024;42(11):845-850
- CountryChina
- Language:Chinese
-
Abstract:
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 1 176 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 consis-tency 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 consis-tency rate of 95.80%(95%CI:93.38%-97.36%).Conclusion The qPCR melting curve method can detect Hp 23S rRNA and gyrA in fecal samples,which has certain clinical application value for predicting the resistance of Hp.
