Qingdao Nucleic Acid Rapid Testing International Science and Technology Cooperation Base, College of Life Sciences, Department of Pathogenic Biology, School of Basic Medicine, and Department of Clinical Laboratory, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao 266071, PR China.
Shandong Provincial Key Laboratory of Biochemical Engineering, Qingdao Nucleic Acid Rapid Detection Engineering Research Center, College of Marine Science and Biological Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
J Microbiol Methods. 2022 Jan;192:106393. doi: 10.1016/j.mimet.2021.106393. Epub 2021 Dec 14.
Due to the abuse of antibiotics, the prevalence of antibiotic resistant Helicobacter pylori strains continues to increase. Therefore, antibiotic resistance assessment is now essential in addition to general H. pylori diagnosis in medical institutions to fulfill clinicians administering effective antibiotic regimens. However, the conventional antibiotic resistance assessment methods, such as in vitro antibiotic susceptibility test and E-test, are skilled-staff dependent and time-consuming. The aim of this study was to establish an easy-operating TaqMan-MGB probe multiplex real-time PCR system for one-step detection of levofloxacin and clarithromycin resistance mutations with concurrent H. pylori infection diagnosis. Through the optimization of primers, probes and reaction buffers, this proposed system could accurately distinguish the recombinant plasmids with different mutation markers. More importantly, the diagnosis results of this detection system exhibited excellent consistence with the gold standard of gastric biopsy and Sanger sequencing on the detection of H. pylori infection and relevant antibiotic resistant strains, the Kappa values of which all exceeded 0.90. In addition, the results of this detection system could also be applied for the prevalence statistics of antibiotic resistance patterns for patients by age, gender and geographical location. This simple and rapid system should be beneficial for clinicians issuing personalized treatments according to the patient's H. pylori strains and avoid the abuse of antibiotics.
由于抗生素的滥用，对抗生素耐药的幽门螺杆菌菌株的流行率持续上升。因此，除了医疗机构中常规的幽门螺杆菌诊断外，现在还必须进行抗生素耐药性评估，以满足临床医生开出有效抗生素方案的需求。然而，传统的抗生素耐药性评估方法，如体外抗生素药敏试验和 E 试验，依赖于熟练的技术人员，且耗时较长。本研究旨在建立一种易于操作的 TaqMan-MGB 探针多重实时 PCR 系统，用于一步检测左氧氟沙星和克拉霉素耐药突变，并同时进行幽门螺杆菌感染诊断。通过优化引物、探针和反应缓冲液，该系统能够准确区分具有不同突变标记的重组质粒。更重要的是，该检测系统的诊断结果与胃活检和 Sanger 测序的金标准在幽门螺杆菌感染和相关抗生素耐药株的检测上具有极好的一致性，kappa 值均超过 0.90。此外，该检测系统的结果还可用于按年龄、性别和地理位置统计患者的抗生素耐药模式流行率。这种简单快速的系统应该有助于临床医生根据患者的幽门螺杆菌菌株制定个性化的治疗方案，避免抗生素的滥用。
