Guidelines for absolute quantitative real-time PCR for microbial determination in in vitro gastrointestinal digestion

Currently, numerous pieces of evidence have demonstrated that the health gut microbiota contributed to physiological homeostasis for the host. Accumulating methods appear to detect microbial population, including denaturing gradient gel electrophoresis (DGGE), high-throughput sequencing, and quantitative real-time polymerase chain reaction (qPCR). With characteristics of flexibility and high-sensitivity, qPCR technique plays a critical role in determining the dynamic alteration of bacterial abundances, which entirely exhibits the microbial activity in various external situations. In these guidelines, taking Akkermansia muciniphila for example, we provide experimental details in specific primer design and validation, standard plasmid construction, qPCR assay, and data analysis for microbial detection, which also can be used for bacterial determination in other environmental systems. In addition, we offer the solution preparation and operation procedures of in vitro gastrointestinal digestion system. Furthermore, these guidelines can contribute to verify the high-throughput sequencing results and reveal the underlying mechanism of the microbial shift in response to the dietary intervention or the development of diseases.