关键词: 实时荧光定量聚合酶链式反应, 引物, 标准曲线, 双歧杆菌

Abstract:

Objective To establish the real-time fluorescence quantitative PCR method for the detection of bifidobacteria in human fecal samples,and to provide a
n effective means for measuring intestinal bacteria.Methods Total DNA of bacteria was extracted from 60 cases of children's fecal samples.Three primers of bif
idobacteria 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 Green Ⅰ 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 613 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 (1.48×103－1.48×107copies/ μL) 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.

Key words: real-time fluorescence quantitative polymerase chain reaction, primer, standard curve, bifidobacteria