Establishment and evaluation of detection method of bifidobacteria in human fecal using real-time fluorescence quantitative PCR / 吉林大学学报(医学版)

ABSTRACT

Objective To establish the real-time fluorescence quantitative PCR method for the detection of bifidobacteria in human fecal samples, and to provide an effective means for measuring intestinal bacteria. Methods Total DNA of bacteria was extracted from 60 cases of children's fecal samples. Three primers of bifidobacteria based on the 16S ribosomal RNA (16SrRNA)which possessed specialities of bacteria as amplified region were designed.The part of amplified 16SrRNA gene sequences was used as standard production.The serial dilution of standard was analyzed to build an absolute quantitative standard curve with SYBR GreenⅠ dye method, and the bifidobacterium contents in sixty human fecal samples were calculated. The sensitivity of the reaction was calculated by detecting the lowest detectable standard which determined the sensitivity of the reaction. The PCR products’melting curve was used to evaluate the specificity.The coefficient of variation (CV)of different batches of standard with the same concentration was used to evaluate the stability of reaction.Results The length of PCR product fragment which was used to build the standard curve was about 6 1 3 bp, the sequencing result was consist with the goals, and the standard sample of bifidobacteria was successfully established in real-time fluorescence quantitative PCR.The standard curve showed a good linear relationship with R2=0.999.The minimum detection value was 1.48×102 copies per reaction.The melting curve of real-time fluorescence quantitative PCR was a single peak.The test samples were batched and then examined by fluorescence quantitative PCR.The CV of standards’ Ct values which calculated from the standard (1.48 × 103 -1.48 × 107 copies · μL-1 )were 2.94%, 3.39%, 3.54%,3.08%,and 3.34%,respectively.The contents of bifidobacteria in fecal from 60 children was 7.77± 0.86(copies · g-1 wet fecal)transformed by logarithmic.Conclusion The established real-time fluorescence quantitative PCR method has high sensitivity, strong specificity and good repeatability, which is suitable for detection of human fecal bifidobacteria content.