Isolation And Identification Of Major Intestinal Pathogens In Dairy Goats And Establishment Of Multiplex QPCR Detection Methods

Escherichia coli,Clostridium perfringens,Salmonella and Shigella are the main pathogens in dairy goat farms,and two or more mixed infections in clinical cases are common.In order to understand the main intestinal pathogenic bacteria of dairy goat farms in Shilin area and provide rapid diagnostic techniques,this experiment isolated and identified bacteria in fecal samples collected from farms in this area,and established a single pathogen qPCR detection method for Clostridium perfringens,Salmonella and Shigella,and a triple qPCR detection method for Clostridium perfringens,Salmonella and Shigella.1.Isolation and identification of major pathogenic bacteria.For 34 fecal samples from dairy goat farms,through bacterial culture characteristics,morphological identification,biochemical identification,and 16S r RNA sequencing,Escherichia coli,Clostridium perfringens,Shigella,Salmonella,Enterococcus faecalis,Enterococcus bile,Acinetobacter,and Staphylococcus aureus were mainly isolated.Through animal regression tests,Clostridium perfringens,Shigella and Salmonella were found to be the main pathogenic bacteria.2.The detection methods of Taq Man probe qPCR for three major pathogenic bacteria were establishedBy designing primers and probes for the 16S rDNA part of Clostridium perfringens,the qPCR detection method of Clostridium perfringens was established,and the optimal amplification conditions were annealing temperature of 56.3°C,annealing extension for30s,standard curve of y=-3.43x+41.14,correlation coefficient of 1.000,amplification efficiency of 95.72%,and the lowest sample detection concentration was 3.19 copies/μL.By designing primers and probes for Salmonella Fim Y gene,a Salmonella qPCR detection method was established,and the optimal amplification conditions were annealing temperature of 56.3°C,annealing extension for 30s,standard curve of y=-3.23x+40.29,correlation coefficient of 0.999,amplification efficiency of 103.92%,and the lowest sample detection concentration of 6.33 copies/μL.The best amplification conditions were annealing temperature of 56.3°C,annealing extension for 30s,standard curve was y=-3.45x+39.90,correlation coefficient was 0.998,amplification efficiency was 94.88%,and the lowest sample detection concentration was 2.96 copies/μL.The sensitivity of the above three detection methods is more than 100 times higher than that of ordinary PCR.Using the specificity of common bacterial DNA testing methods in farms,the control wells were not amplified,indicating that the specificity of this detection method was good.3.Establishment of triple qPCR detection method for Clostridium perfringens,Salmonella and ShigellaThree bacterial plasmid standards were doubled diluted to optimize primer and probe concentration combinations based on individual fluorescence quantitative detection methods.When the annealing temperature was 56℃and the annealing extension was 50s,the standard curve relationship of Clostridium perfringens was y=-3.59x+42.11,the correlation coefficient was 0.999,the amplification efficiency was 89.80%,and the minimum detection limit was 50 copies/μL.The standard curve relationship of Salmonella was y=-3.29x+38.01,the correlation coefficient was 0.999,the amplification efficiency was 101.16%,and the minimum detection limit was 10 copies/μL.The standard curve of Shigella is y=-3.32x+38.18,the correlation coefficient is 1.000,the amplification efficiency is 100.20%,and the minimum detection limit is 10 copies/μL.The triple qPCR detection method obtained good amplification efficiency and good specificity.The intergroup and intra-group replicate tests were carried out with standards with five concentration gradients of 10~4copies/μL～10~8copies/μL,and the results had good repeatability and stability.This study provides technical support for the rapid diagnosis of the main pathogenic bacteria in the intestines of dairy goats.