Current and future uses of real-time polymerase chain reaction and microarrays in the study of intestinal microbiota, and probiotic use and effectiveness

Abstract

Probiotics are defined as live microorganisms that confer a health benefit to the host when administered in adequate amounts. In addition to human health benefits, probiotics can improve various aspects of growth and performance in livestock and poultry, as well as control undesirable microorganisms in food animals. Studies indicate that probiotics can prevent or treat certain conditions, including atopic disease in infants, food allergy, infection after surgery, acute diarrhea, and symptoms associated with irritable bowel syndrome. Understanding the complete mechanism, effectiveness, and potential use of probiotics is limited by the availability and sensitivity of current methods (i.e., culturing techniques). In recent years, real-time polymerase chain reaction (PCR) and microarrays have become prominent and promising methods to examine quantitative changes of specific members of the microbial community and the influence of probiotics on the structure and function of human and animal intestinal ecosystems. Culture-independent studies have established that only a fraction of organisms present in feces are cultivable, therefore, results obtained by cultivation are limited. Conversely, in-depth knowledge of microbial genomes has enabled real-time PCR and microarrays to be more sensitive and has resulted in precise methods for comprehensive analysis of the complex gut microbiota. Additionally, these technologies can assess the influence of intestinal microorganisms on host metabolism, nutrient status, and disease. This paper reviews method technologies and applications of real-time PCR and microarray assays as they relate to the effect and use of probiotics on the intestinal microbiota and gastrointestinal disease.

Potential Fate of Ingested Lactobacillus plantarum and Its Occurrence in Human Feces

“…The major restricting factor of these studies may be attributed to the use of traditional plate culture methods involving only a very limited proportion of the microbial population (7)(8)(9).…”

Principles of DNA-Based Gut Microbiota Assessment and Therapeutic Efficacy of Fecal Microbiota Transplantation in Gastrointestinal Diseases

“…Thus, the pattern of fragments with unique sizes and intensity of fluorescence reflects the relative composition of the populations in the community. Real-time quantitative PCR is another powerful technique that can be used to characterize the microbiota [17]. However, it is based on the design of primers specific for individual microbial groups, from phyla to species.…”

Detection and Identification of Probiotic Microorganisms and Other Beneficial Organisms from the Human GI Tract

“…Relative quantification is calculated by comparing the cycle threshold value of a test sample with the cycle threshold value of control DNA. Cycle threshold refers to the PCR cycle at which the fluorescence raises over threshold (Carey et al 2007;Louis and Flint 2009). One commonly used florescent dye is SYBR green, which intercalates with double-stranded DNA.…”

“…If using broad specificity primers targeting phylum or class levels, a mixed standard is recommended. For comparison of different runs, the use of control sample in each run is recommended (Carey et al 2007).…”