Isolation And Identification Of Pathogenic Bacterium From Diarrheal Captive Macaca Fascicularis And Establishment Of A Detection Method By Multiplex PCR

In clinic, captive macaca fascicularis diarrhea is commonly caused by bacterium. For learning about the species of them, this study used conventional method to isolate the diarrhea pathogens. Then, identified each bacteria by compared the gene sequences with 16S rRNA in gene bank. Finally, established a triplex PCR for 3 kinds of the important diarrhea pathogens, which is Salmonella, Shigella and Proteus, and further to optimize the reaction. The contents and results of this study are as follows:1. Isolated 112 strains of pathogenic bacteria from 85 faecal samples (anal swab) of diarrheal captive macaca fascicularis by conventional method. Using the universal primers of the bacterial 16S rRNA, amplified and sequenced 11 strains of them through PCR reaction, and subsequently of which 10 strains were identified by biochemical test. The results showed that 11 strains included:uncultured bacteria (1 strain), Salmonella (1 strain), Acinetobacter bacillus (2 strain), Proteus mirabilis (1 strain), Proteus vulgaris (1 strain), Proteus pennea (1 strain), Staphylococcus aureus (1 strain), Kurthia gibsonii (1 strain), Escherichia coli (1 strain) and Shigella bacteria (1 strain).2. This research chose 3 pairs of specific primers from the related references to establish a multiplex PCR detection method, which can once amplify the target genes of fimY gene of Salmonella, iPaH gene of Shigella and atpD gene of Proteus mirabilis. After optimized conditions of the reaction and verified the specificity and sensibility of each pair specific primer, this Multiple PCR system was used to detect pathogenic bacterium in 30 artificially contaminated fecal samples. The results indicate:1.40 μL optimal system consisted of 20μL EasyTapPCR Supermix, adequate amounts of upstream primer and downstream primer (Proteus mirabilis 0.7 μL, Salmonella 1 μL and Escherichia coli 1 μL, respectively),1 μL DNA of each kind bacteria, and add double distilled water up to 40 μL total volume.2. The appropriate annealing temperature is 58℃.3. The procedure of PCR is initial denaturation 5 min. at 94 ℃; then, denaturalizing 30 sec. at 94 ℃, annealing 45 sec. at 58 ℃, extending 45 sec. at 72℃, total 35 cycles; finally, extending 7 min. at 72℃ again.3.The specificity experiments of used primers in PCR showed that, none but add target gene, the lane can appear specific bands. After compared the sequencing results of PCR production in gene bank, the homology between the obtained sequence and Salmonella, Shigella spp., Proteus mirabilis was respectively 100%,100% and 99%.The minimum detectability against a single germ of optimized multiplex PCR system is Salmonella 1 pg/μL, Shigella bacteria 1 pg/μL, Proteus mirabilis 100 fg/μL. This minimum detectability against the mixture of three kinds pathogenic bacterium is 10 pg/μL.4.The detection rate of this multiplex PCR system was 100% by using to analysis the artificial contamination sample.