The Study Of Detection And Quantification Bifidobacteria In Human Feces By PCR

Objective:There are a lot of active and complex bacteria in human intestine. Bifidobacteria is one of the most important bacteria that maintain the microecological balance of intestine. They are 3% in normal adult intestine and 75%-91% in baby intestine. Bifidobacteria can prevent diarrhea and intestine infections, release constipation,regulate immunological function. Quick and accurate quantification bifidobacteria of human feces can do favor to analyze the structures of complex intestine bacteria, instruct clinical dosage, maintain intestine bacteria balance, keep health and prevent people from disease. Nowadays quantification of intestine bifidobacteria method is to dilute feces samples, separate and count bacteria. But it is time consuming and labor consuming. Normal PCR that founded in the condition of DNA extraction can only qualify and semiquantify bacteria. The extraction of DNA is complex; Phenol and proteinase K can inhibit the PCR amplification. Our purpose is to use real-time PCR found a new method that can quantify bifidobacteria in feces accurately and quickly without separating bacteria after feces centrifuge and washing. Method: There are two parts in the experiment: One part is using normal PCR quantifying bifidobacteria in human feces. We design bifidobacteria specific primers basing on 16SrDNA. We use normal PCR semiquantifying bifidobacteria in feces samples without separating bacteria and DNA purifying after feces centrifuge and washing. We also compare the correlation between normal PCR bifidobacteria quantification and traditional diluting bifidobacteria quantification. The other part is using realtime PCR quantifying bifidobacteria in human feces. After series diluting, we use the same bifidobacteria specific primers and realtime PCR quantifying bifidobacteria without separating bacteria and DNA purifying after feces centrifuge and washing. We also compare the correlation between realtime PCR bifidobacteria quantification and traditional diluting bifidobacteria quantification. Samples include 20 healthy adult feces and 10 diarrhea, constipation feces.Results: (1) The preparations of feces samples include simple centrifuge, washing and diluting. The purpose is to get rid of the inhibitors in feces samples. 2% Triton-X100 can release DNA template. (2) There is no obvious difference between normal PCR quantification in feces and cultured bacteria (p>0.05); there is obvious correlation between normal PCR quantification in feces and cultured bacteria quantification (P>0.05). (3) There is no obvious difference between real-time PCR quantification and cultured bacteria (P>0.05); there is obvious correlation between real-time PCR quantification and normal PCR (P<0.05). Conclusions: Normal PCR can semiquantify bifidobacteria in human feces quickly, and real-time PCR can quantify bifidobacteria in human feces accurately and quickly.