Design For DNA CutOff Program And Screening For Bacterial Pathogen In Diarrheal Samples Of Old Men Based On 16S RRNA Technology

Objectives:1 To establish the platform of 16S rRNA technology independent on microbial cultivation.2 To designe the DNA CutOff program.3 The feces samples of three old men with diarrhea will be tested with 16S rRNA technology.Methods: The procedures of 16S rRNA technology include sample collection, storage, pre-treatment and processing, DNA extraction, PCR reaction, cloning and sequencing, statistical analysis and so on. The platform of 16S rRNA technology was established in the study. About 140 colonies were randomly selected for sequencing after the PCR products of every sample cloned into T-vector. It is essential to cut off the sequenced DNA in order to analyze the 16S rRNA sequence correctly. But the workload is very large and mistakes can be made easily when sequence cut off by hand. So DNA Cutoff program was designed to resolve the above questions. In the program, 20 base pairs close to clone site by SP6 primer in T-vector were used for initial sequence, another 20 base pairs by T7 primer for terminal sequence.The feces samples of three old men with diarrhea were tested with 16S rRNA technology.Results:1) The program is tested correctly and the analysis speed increased after the DNA CutOff program applied.2) The feces samples of three old men with diarrhea were tested with 16S rRNA technology. Patient number 3 may be dysentery because Shigella was the second dominant bacterium in her feces and proved further by PCR amplification of ipaH gene which has been used as genetic marker for virulence gene of Shigella species. Meanwhile, Shigella can't be excluded to be the cause of patient number 2 because four clones of Shigella were detected and ipaH gene was also amplified in his feces. However, only conditional pathogen but disease-causing pathogen was found in feces of patient number 1. So disorders of micro flora may be the main cause resulting in diarrhea of patient 1.Contusions:1) The DNA CutOff program has been exactly designed for the cut-off not only of 16S rRNA sequence but also of other DNA sequence.2) The platform of 16S rRNA technology being independent on microbial cultivation has been established successfully in the study.3) The feces samples of three old men with diarrhea were tested with 16S rRNA technology.1 Disorders of micro flora may be the main of cause resulting in diarrhea of patient 1.2 Shigella can't be excluded to be the cause of patient number 2.3 Patient number 3 may be dysentery.