A qPCR method for rapid detection of gut microorganisms

Abstract:

[Background] In recent years, 16S rRNA gene sequencing and metagenomic analysis have been commonly used for detecting microbial pathogens in the gut. [Objective] To overcome the limitations of high cost and long detection time, we established a method for analyzing the composition of human gut microbiota based on real-time fluorescence quantitative PCR (qPCR), which can measure the abundance of gut microorganisms. [Methods] Ten microbial taxa commonly detected in the intestine were selected from public databases. We designed specific primers and probes for the ten targets and validated them using 20 fecal samples. Furthermore, we compared the results of qPCR and 16S rRNA gene sequencing to evaluate the performance of the established method. [Results] The ten pairs of primers and probes were specific to the targeted taxa, and the coverage of targeted bacteria in the HITdb database exceeded 70%. The coefficient of variation (CV) of sample detection results was less than 10%, indicating that the method was highly stable. The results of qPCR and 16S rRNA gene sequencing for measuring the microbial abundance were correlated (P＜0.05). [Conclusion] The results of the relative abundance of microorganisms detected in fecal samples using the primers and probes designed based on the HITdb database were consistent with those obtained by 16S rRNA gene sequencing. This study provides an accurate and cost-effective method for analyzing gut microbiota and the reference information for clinical diagnosis and treatment.