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Development of Two Loop-Mediated Isothermal Amplification Assays for Rapid Detection of ermB and mefA Genes in
LuLu Li1,2, JinRui Ren1,2,3, Qing Zhang1,2
1Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan, China.
Abstract:
Streptococcus suis is an important zoonotic pathogen that poses a serious threat to the pig industry and human health. The massive use of macrolides has led to the emergence of resistance in S. suis, and S. suis is suspected to be a reservoir of antimicrobial resistance genes. The mechanism to macrolide resistance in S. suis is mainly due to ermB and mefA. In this study, loop-mediated isothermal amplification (LAMP) methods were developed to detect ermB and mefA genes in S. suis through turbidimetry detection. The sensitivity and specificity of the LAMP reactions were determined. All results of LAMP and polymerase chain reaction (PCR) assay were compared to determine whether LAMP method was accurate and reliable. The results showed that all 100 nonstreptococcus clinical isolates tested negative, indicating the high specificity of LAMP assays. The detection limit of LAMP assay was 1 fg per reaction, and 102-104-fold lower than those of conventional PCR methods. Evaluation of the performance of the LAMP assay in S. suis clinical strains revealed a good consistency between LAMP and PCR assays. In conclusion, LAMP assays are specific, sensitive, and rapid methods to detect ermB and mefA in S. suis.
Insights
Loop-mediated isothermal amplification (LAMP) offers a rapid, sensitive, and specific method for detecting macrolide resistance genes (ermB and mefA) in Streptococcus suis. This technique aids in controlling antimicrobial resistance in swine and protecting public health.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Antimicrobial Resistance
Background:
- Streptococcus suis is a significant zoonotic pathogen impacting swine health and human well-being.
- Widespread macrolide use has driven resistance in S. suis, with ermB and mefA being key resistance genes.
- S. suis is implicated as a reservoir for antimicrobial resistance genes, necessitating effective detection methods.
Purpose of the Study:
- To develop and validate loop-mediated isothermal amplification (LAMP) assays for detecting ermB and mefA genes in S. suis.
- To assess the sensitivity, specificity, and reliability of the developed LAMP methods.
- To compare LAMP assay performance against conventional polymerase chain reaction (PCR) methods.
Main Methods:
- Development of turbidimetry-based loop-mediated isothermal amplification (LAMP) assays targeting ermB and mefA genes.
- Determination of the analytical sensitivity (detection limit) and specificity of the LAMP assays.
- Comparative analysis of LAMP assay results with conventional PCR assays using S. suis clinical isolates and non-streptococcal strains.
Main Results:
- The developed LAMP assays demonstrated high specificity, with all 100 non-streptococcal isolates testing negative.
- The detection limit of the LAMP assay was determined to be as low as 1 fg per reaction, significantly more sensitive than conventional PCR.
- A strong consistency was observed between the results obtained from LAMP and PCR assays when evaluating S. suis clinical strains.
Conclusions:
- Loop-mediated isothermal amplification (LAMP) provides a specific, sensitive, and rapid diagnostic tool for detecting ermB and mefA genes in Streptococcus suis.
- The developed LAMP assays are reliable alternatives to conventional PCR for identifying macrolide resistance in S. suis.
- This method can contribute to better surveillance and management of antimicrobial resistance in the pig industry and public health.
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