Establishment Of A Method For Gut Microbiota Using Real-time Fluorescence Quantitative PCR And The Content Change In Children During Acute Exacerbation Of Wheezing

Objective To establish the real-time fluorescence quantitative PCR methods for thedetection of bifidobacteria and lactobacillus in human fecal samples, providing an effectivemeans for measuring intestinal bacteria. To detect the content of bifidobacteria andlactobacillus in the fecal sample from children with bronchiolitis and asthma. And tofurther explore the relationship between intestinal flora and respiratory diseases.Methods The detected fecal sample were collected from inpatients in the departmentof the first pediatrics respiratory medicine institute of pediatrics, First hospital of Jilinuniversity during October in2012to October in2013. The selected inpatients werediagnosed as bronchiolitis or asthma and both during acute exacerbation of wheezing.Among which including11cases of asthma and14caces of bronchiolitis. The controlgroup are selected from hemangiomas, hernia and other elective surgery patients in thePediatric Surgery，25cases were collected.The serial dilution of standard was analyzed tobuild an absolute quantitative standard curve with SYBR Green Ⅰ dye method, and thebifidobacteria and lactobacillus contents in fifty human fecal samples were calculated.Total DNA of bacteria was extracted from children’s fecal samples. And then We designedprimers based on the16S ribosomal RNA (16S rRNA) which possessed specialities ofbacteria as amplified region. The amplified gene fragment of16S rRNA acquired fromconventional PCR were used as standard.the sensitivity of the reaction was calculated bydetecting the lowest detectable standard which determined the sensitivity of the reaction.The PCR products’ melting curve was used to evaluate the specificity. The coefficient ofvariation (CV) of different batches of standard which with the same concentration was usedto evaluate the stability of reaction. Quantify the bifidobacteria and lactobacillus in fecalsample with the establised real time PCR method and observe the contents changes ofbifidobacteria and lactobacillus in the intestinal of children during acute exacerbation ofwheezing. And differences among the bronchiolitis group, bronchial asthma and normalcontrol group were compared respectively. Statistical software of SPSS16.0was used toanalyze the experimental results. Result1.The PCR products fragments of part16S rRNA sequencing which amplified byconventional PCR reaction were used to build the standard curve were consist with thegoals, and be used as standard in Real-time fluorescent quantitative quantitative PCR. Thestandard curve showed a good linear relationship with r2>0.99. The minimum detectionvalue was74.4copies per reaction in bifidobacteria and142copies in lactobacillus.Melting curve showed a single peak, the CV of Ct values from same concentrationstandards are small.2.The bifidobacteria in children with asthma during acute exacerbation of wheezing isreduced, and the difference was statistically significant (p=0.02); the detected oflactobacillus has no significant changes in the content(p=0.559).3.The bifidobacteria in children with bronchiolits is reduced, and the difference wasstatistically significant (p=0.036); the detected of lactobacillus also has no significantchanges in the content(p=0.935).4.In the detection of50cases of children, we found that the levels of bifidobacteriaand lactobacillius have no correlation, p=0.028, r2=0.097.Conclusion1.The established Real-time fluorescent quantitative PCR method was created withhigh sensitivity, strong specificity and good repeatability, which was suitable for thedetection of bifidobacteria and lactobacillus in human fecal sample.2.When children with asthma in wheezing attack, the structure of the intestinal flora isdisordered, which is reflected in a reduced the amount of fecal bifidobacteria than thecontrol group, while the change of lactobacillus have no statistical significance.3.The variation tendency of gut microbiota are consistent between bronchiolitis andasthma，prompt that the reduce levels of bifidobacteria may be the early targets changes inintestinal flora during bronchiolitis turned into bronchial asthma.4.There is no correlation analysis of bifidobacteria and lactobacillus in detected samples,indicate lactobacillus and bifidobacteria has no coordination or antagonism effects.