Quantification of the active microbiota will contribute to a better understanding of functional groups in environmental microbiology and can help in producing better microbiota interactions models. Real-time PCR based 16S rRNA gene-specific oligonucleotide primers has proved to be an effective method to detect target bacteria in complex ecosystems. Creative Biolabs is a biotechnical company specializing in live biotherapeutic products (LBP) development. Our professional scientists and excellent technical support team have provided full services for many customers in the gut microbiota research projects.
Recent studies have shown that intestinal flora plays a role in the pathogenesis and pathophysiology of many parenteral diseases. In infants with the atopic disease, differences in the composition of gut microbiota were found long before any clinical manifestations appeared. The health effects of flora are likely to be modulated not by specific, single species, but by global composition. A key requirement for understanding the gut microbiota is to correctly and comprehensively quantify its composition. Culture is a classical method for the identification and quantification of bacteria. Most of the available data on gut bacteria are generated by culture and counting. Although selective media and special growth conditions are available to culture intestinal flora, only a small fraction in the complex flora can be detected. Real-time fluorescence PCR using species-specific probes can provide an accurate and sensitive method for the quantification of species, flora, and total bacterial count. The detection range of real-time PCR is 10 to 108 cells. In quantitative terms, real-time PCR is more reliable than single strand conformation polymorphism analysis, temperature gradient gel electrophoresis and fluorescence in situ hybridization. Real-time PCR has been successfully used to quantify specific bacterial species in intestinal mucosa and faeces.
Fig.1 Human gut microbiota and diseases. (El-Dalatony, 2020)
It is well known that PCR is a non-quantitative technique, but its variant, real-time PCR, is also known as quantitative PCR (qPCR) and is used for microbiome analysis, especially phylogenetic analysis. Depending on the application, it can be used quantitatively and semi-quantitatively, qPCR can be used for the quantitative determination of DNA content in feces or intestinal mucosa samples. In this technique, fluorescent probes or dye molecules are used that intercalate between the double strand of DNA molecules or 16S rRNA amplicons. Primer design is the key step of RT-PCR technology. Therefore, primers must be specific to all phyla or groups or species of bacteria present in the sample. Quantitative PCR can also be used alone or in combination with other gel and non-gel techniques. The combination of these regimens is used to understand the functional microbial diversity of the gut microbiota in patients of different ages and the effects of antibiotics on gut microbiota. Due to the high fluorescence sensitivity of real-time PCR, which can accurately detect specific microbial species in samples, qRT-PCR analysis is the "gold standard method", especially in the field of molecular microbial diagnostics and probiotics.
Fig.2 The schematic workflow of a genus-specific primer design method for qRT-PCR assay. (Jeong, 2022)
The real-time PCR system has universal primers and specific probes, providing an accurate and stable method for the determination of bacterial concentration in clinical samples. It is possible to determine the relative and absolute numbers of bacteria. The scientists expect that the use of this global quantification tool could contribute to a complete understanding of gut flora. Devoted to providing professional and well-tailored custom services in LBP development for more than 10 years, Creative Biolabs has gained a global reputation. If you are interested in our real-time PCR services for gut microbiota research, please contact us for more information.
For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.