Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the gene in detection.

BACKGROUND

The gene codes for an important toxin produced by (), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the gene.

METHODS

Three sets of primers were designed and optimized to amplify the gene in using a LAMP assay. To evaluate the specificity of the LAMP assay, VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The gene of was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction.

RESULTS

At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the primers for 26 pathogenic bacterial strains that do not carry the gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X = 47, < 0.01).

CONCLUSION

LAMP method is an effective technique for the rapid and visual detection of the gene of , and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The -LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of infection in populations at high risk.