Single-Cell Genotyping of Single-Nucleotide Mutations Using in Situ Allele-Specific Loop-Mediated Isothermal Amplification.

The authors include Zilan Yuan, Xinmiao Liu, Sha Deng, Guiping He, Jiaqi Zhang, Qiang He, Yuanlong Chi, Xiue Jiang, Xuhan Xia, and Ruijie Deng. They are affiliated with institutions such as Sichuan University and Chongqing University, with research focuses on fields including Aptamer, food safety, Crispr-Cas12, microbiome, and food quality and safety control.

Abstract

• A novel in situ isothermal amplification method named AlleLAMP is proposed for single-nucleotide resolution imaging of bacterial genes.
• Artificial mismatched bases are used to design primers, enabling LAMP to specifically amplify genes containing SNMs.
• AlleLAMP can be used to detect SNMs in the gyrA gene of Salmonella associated with quinolone resistance and accurately quantify the resistant strains in mixtures.
• The selection of antibiotic resistance under preservative pressure is studied, revealing that resistant mutants have a survival advantage over wild-type strains under preservative pressure.
• AlleLAMP can serve as a single-cell tool to analyze the relationship between bacterial genotype and phenotype.

Introduction

• Single-nucleotide mutation (SNM) analysis is crucial for revealing complex genetic traits such as drug resistance.
• Existing SNM analysis techniques (e.g., PCR, sequencing) can only perform in vitro population testing and cannot provide information at the single-cell level.
• In situ nucleic acid imaging techniques (e.g., FISH, RCA) can provide gene expression information at the single-cell level but are difficult to resolve SNMs.
• This study proposes an in situ isothermal amplification method based on LAMP for the imaging of SNMs in bacterial genes.

Experimental Section

• Materials: Primer design, bacterial strains, reagents, etc.
• In situ Imaging: Bacterial fixation, proteinase K treatment, LAMP reaction, fluorescence microscopy imaging.
• Gel Electrophoresis Analysis: Detection of LAMP amplification products.
• Growth of Salmonella under Preservative Pressure: Determination of growth curves.
• Competition of Salmonella under Preservative Pressure: Determination of the growth of different strains at different preservative concentrations.
• RNA Extraction: Extraction of total bacterial RNA.
• RT-qPCR: Detection of gene expression levels.

Results and Discussion

• AlleLAMP Principle: Using LAMP and allele-specific primers for SNM imaging.
• SNM Detection: AlleLAMP successfully detected SNMs in the gyrA gene of Salmonella.
• Primer Design: Optimizing primer design to enhance the specificity of SNM detection.
• Quantification of MT Salmonella: AlleLAMP can accurately quantify the resistant strains in mixtures.
• Competition between MT and WT Salmonella under Preservative Pressure: Resistant mutants have a survival advantage over wild-type strains under preservative pressure.
• Adaptive Mechanism of Salmonella under Preservative Pressure: Resistant strains resist preservative pressure by expressing efflux pump proteins.

Conclusion

• AlleLAMP is an effective tool for the imaging of SNMs in bacterial genes.
• AlleLAMP can be used to study bacterial drug resistance and its relationship with phenotype.
• AlleLAMP contributes to the understanding of the selection mechanism of bacterial resistance under preservative pressure.

Q: What specific research methods were used in the paper?

• AlleLAMP technology: An isothermal amplification technique based on LAMP, using specific primers to distinguish single nucleotide mutations (SNM) and perform in situ imaging.
• Fluorescence microscopy: Used to observe the fluorescent signals of SNM within bacterial cells.
• Gel electrophoresis: Used to analyze LAMP amplification products, verifying the specificity of the AlleLAMP technology.
• Plate counting: Used to quantitatively analyze bacterial counts, verifying the accuracy of the AlleLAMP technology.
• RT-qPCR: Used to detect the expression levels of antibiotic resistance-related genes in bacteria, investigating resistance mechanisms.

Q: What are the main research findings and outcomes?

• AlleLAMP technology can effectively detect SNM in bacterial genes and perform in situ imaging.
• AlleLAMP technology can accurately quantify the proportion of drug-resistant strains within mixed bacterial populations.
• Under preservative pressure, drug-resistant strains have a survival advantage over sensitive strains.
• Drug-resistant strains resist preservative pressure by expressing efflux pump proteins.

Q: What are the current limitations of this study?

• Currently, AlleLAMP technology can only detect specific SNM in the gyrA gene.
• Further optimization of primer design is needed to improve the versatility of AlleLAMP technology.
• Further research is required to apply AlleLAMP technology to other bacteria and genes.