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

Abstract

Objective: To establish a viable bacteria assay for Helicobacter pylori (H. pylori) by assessing the cgt 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 cgt (hp0421), the conserved gene that encodes cholesterol-α-glucosyltransferase (CGT) in H. pylori, was measured by RT-PCR. The correlation between the number of colonies and cgt 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 cgt for H. pylori colony counts of 10², 10⁴, 10⁶, and 10⁸ 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 10¹-10⁸ CFU/mL, the regression equation for cgt gene expression and viable bacterial counts determined by RT-qPCR was y=-0.3501x+12.49, with the correlation coefficient being R ²=0.9992 and the sensitivity being 10¹ CFU/mL, showing no cross-reaction with 13 other bacteria. The lg values of live H. pylori 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 cgt 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 cgt 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 (P>0.05).

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