[Viable Bacteria Assay of by RT-qPCR Measurement of Gene Expression Levels: Establishment and Application of a New Method].

OBJECTIVE

To establish a viable bacteria assay for () by assessing the gene expression, and to develop accordingly a rapid and novel testing method for clinical precision treatment.

METHODS

Viable bacteria count was determined in bacterial cultures. The transcriptional expression level of (), the conserved gene that encodes cholesterol-α-glucosyltransferase (CGT) in , was measured by RT-PCR. The correlation between the number of colonies and gene transcription expression was analyzed and the regression model was constructed. The linear range, sensitivity, and specificity of the new method were examined accordingly. The bactericidal action of clarithromycin was assessed using this method to verify the performance of the method in determining clinical bacterial drug resistance.

RESULTS

The Ct values of for colony counts of 10, 10, 10, and 10 CFU/mL were 29.67±0.14, 23.37±0.36, 17.65±0.37, and 11.38±0.39, respectively. In the range of 10-10 CFU/mL, the regression equation for gene expression and viable bacterial counts determined by RT-qPCR was =-0.3501+12.49, with the correlation coefficient being =0.9992 and the sensitivity being 10 CFU/mL, showing no cross-reaction with 13 other bacteria. The lg values of live bacteria treated with clarithromycin at 0, 5, 10, 20, and 40 μg/mL for 12 h were 2.57±0.02, 2.45±0.01, 2.19±0.02, 1.91±0.07, and 1.33±0.05, respectively. The corresponding gene Ct values were 27.76±0.09, 28.37±0.24, 29.51±0.14, 30.11±0.12, and 31.66±0.11. By applying the gene expression in the equation, the estimated counts of viable bacteria were found to be 2.73±0.03, 2.52±0.08, 2.11±0.05, 1.89±0.02, and 1.33±0.04, showing no significant difference in statistical analysis (>0.05).

CONCLUSION

The method for assessing viable bacteria account by evaluating gene expression in was successfully established, significantly reducing the time required to determine viable bacteria count and providing a new method for clinical viable bacteria testing.