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Published on: July 9, 2012
Multiplex PCR-Mass Spectrometry Mini-Sequencing Technology Detected Antibiotic Resistance of Helicobacter pylori to
Fei Zhao1, Xin Zhao1, Huifang Zhang1
1National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Abstract:
The abuse of antibiotics has led to widespread resistance to Helicobacter pylori (H. pylori) in the population. There is an urgent need to establish a method to detect multiple antibiotic resistance rapidly. This study aimed to construct a novel strategy for the high-throughput detection of H. pylori's resistance to varying antibiotics using multiplex PCR-mass spectrometry mini-sequencing (mPCR-MS mini-sequencing) technology. This study detected the resistance of H. pylori to six antibiotics using eight mutated sites (23S rRNA-2143; pbp1A-1667, 1684, 1240; gyrA-261, 271, 573; and 16S rRNA-928) of four resistance genes (pbp1A, gyrA, 23S rRNA, and 16S rRNA), and 525 were detected in all 528 results (99.43%). Then, the culture-based phenotypic drug susceptibility testing (DST) method was used as a reference for drug resistance detection. We found that the consistency rate between mPCR-MS mini-sequencing with the DST results of amoxicillin (AMX), moxifloxacin (MOX), levofloxacin (LEV), clarithromycin (CLA), azithromycin (AZI), and tetracycline (TET) were 95.5% (63/66), 77.3% (51/66), 68.2% (45/66), 93.9% (62/66), 92.4% (61/66), and 97.0% (64/66), respectively. This method was high-throughput and extensible, easily improving the entire detection system by adding new mutation sites. mPCR-MS mini-sequencing technology provides a new approach to mutation sites related to H. pylori's antibiotic resistance.
Insights
Multiplex PCR-mass spectrometry mini-sequencing rapidly detects Helicobacter pylori antibiotic resistance. This high-throughput method accurately identifies resistance genes, offering a new approach for clinical diagnostics and treatment strategies.
Area of Science:
- Microbiology and Infectious Diseases
- Molecular Diagnostics
- Genetics and Genomics
Background:
- Antibiotic resistance in Helicobacter pylori (H. pylori) is a growing global health concern.
- Rapid and accurate detection of H. pylori antibiotic resistance is crucial for effective treatment.
- Existing methods for detecting resistance can be time-consuming and may not cover multiple resistance profiles simultaneously.
Purpose of the Study:
- To develop and validate a novel high-throughput method for detecting H. pylori resistance to multiple antibiotics.
- To utilize multiplex PCR-mass spectrometry mini-sequencing (mPCR-MS mini-sequencing) technology for rapid genetic resistance profiling.
- To assess the performance of the developed method against conventional culture-based drug susceptibility testing (DST).
Main Methods:
- Constructed a multiplex PCR-mass spectrometry mini-sequencing assay targeting eight mutation sites in four key H. pylori resistance genes (pbp1A, gyrA, 23S rRNA, and 16S rRNA).
- Analyzed 528 H. pylori samples to detect resistance profiles for six antibiotics: amoxicillin (AMX), moxifloxacin (MOX), levofloxacin (LEV), clarithromycin (CLA), azithromycin (AZI), and tetracycline (TET).
- Compared the results of mPCR-MS mini-sequencing with traditional culture-based drug susceptibility testing (DST) as the reference standard.
Main Results:
- The mPCR-MS mini-sequencing method achieved a high detection rate of 99.43% across all tested samples and mutation sites.
- Consistency rates with DST were high for AMX (95.5%), CLA (93.9%), AZI (92.4%), and TET (97.0%).
- Moderate to good consistency rates were observed for MOX (77.3%) and LEV (68.2%), indicating areas for potential refinement.
Conclusions:
- Multiplex PCR-mass spectrometry mini-sequencing is a high-throughput, extensible, and accurate method for detecting H. pylori antibiotic resistance.
- This technology offers a valuable new approach for rapid genetic profiling of H. pylori resistance, aiding in personalized treatment strategies.
- The method's ability to be expanded with new mutation sites makes it adaptable for future surveillance of emerging resistance patterns.
