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Published on: September 5, 2013
A high throughput multiplex PCR assay for simultaneous detection of seven aminoglycoside-resistance genes in
Xiumei Hu1, Banglao Xu, Yinmei Yang
1Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, People's Republic of China.
Background:
The aminoglycoside-resistance genes encoding aminoglycoside modifying enzymes and 16S rRNA methyltransferases are main factors contributing to increasing resistance to aminoglycosides. Characterization and distribution of antimicrobial resistance gene profiles provide important information on the potential difficulty of treatment of bacteria. Several molecular methods have been developed to investigate the prevalence of aminoglycoside-resistance genes. These existing methods are time-consuming, labor-intensive, expensive or limited sensitivity in the epidemiological investigation. Therefore, it is necessary to develop a rapid, less-costly and high throughput and sensitive method to investigate the distribution of antimicrobial resistance gene in clinical isolates.
Results:
In this study, we developed a GeXP analyzer-based multiplex PCR assay to simultaneously detect seven aminoglycoside-resistance genes, including aac(3)-II, aac(6')-Ib, aac(6')-II, ant(3″)-I,aph(3')-VI,armA and rmtB, and to analyze the distribution of these genes in clinical Enterobacteriaceae isolates. Under optimized conditions, this assay achieved a limit-of-detection as low as 10 copies of each of the seven genes. The presented method was applied to analyze the distribution of aminoglycoside-resistance genes in 56 clinical Enterobacteriaceae isolates, and the results were compared with that of the conventional single PCR assay. Kappa values of the two methods for detecting each of the seven resistance genes were 0.831, 0.846, 0.810, 0.909, 0.887, 0.810 and 0.825, respectively.
Conclusion:
This GeXP assay is demonstrated to be a rapid, cost-effective and high throughput method with high sensitivity and specificity for simultaneously detecting seven common aminoglycoside-resistance genes.
Insights
A new multiplex PCR assay rapidly detects seven common aminoglycoside-resistance genes in clinical isolates. This cost-effective method offers high sensitivity and specificity for epidemiological surveillance of antimicrobial resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Aminoglycoside resistance is a growing concern, driven by resistance genes encoding modifying enzymes and methyltransferases.
- Understanding the distribution of these resistance genes is crucial for effective bacterial infection treatment.
- Current molecular methods for detecting aminoglycoside resistance genes are often slow, costly, and labor-intensive.
Purpose of the Study:
- To develop a rapid, cost-effective, and high-throughput method for detecting multiple aminoglycoside-resistance genes.
- To analyze the distribution of seven specific aminoglycoside-resistance genes in clinical Enterobacteriaceae isolates.
Main Methods:
- A multiplex PCR assay was developed using a GeXP analyzer.
- The assay was optimized to simultaneously detect seven aminoglycoside-resistance genes: aac(3)-II, aac(6')-Ib, aac(6')-II, ant(3″)-I, aph(3')-VI, armA, and rmtB.
- The method's limit of detection was determined to be as low as 10 copies per gene.
Main Results:
- The GeXP multiplex PCR assay demonstrated a high limit-of-detection for all seven target genes.
- The assay was successfully applied to 56 clinical Enterobacteriaceae isolates.
- Results showed high agreement (Kappa values ranging from 0.810 to 0.909) when compared to conventional single PCR methods.
Conclusions:
- The developed GeXP assay is a rapid, cost-effective, and high-throughput tool for detecting common aminoglycoside-resistance genes.
- The assay exhibits high sensitivity and specificity.
- This method facilitates efficient epidemiological investigation of antimicrobial resistance patterns.
