| Porcine proliferative enteritis,Clostridial enteritis of piglets and Swine dysentery are all bacterial diarrhea diseases that are caused by three different bacterial species:Lowsonia intracellularis,Clostridium perfringens Type C and Brachyspira hyodysenteriae,respectively.Porcine proliferative enteritis is a contagious disease transmitted through direct contact.Acute infection can result in hemorrhagic dysentery and even death in pigs.Clostridium perfringens Type C infection can cause bloody dysentery and persistent diarrhea in pigs,which is called Clostridial enteritis of piglets.Swine dysentery is a serious intestinal infectious disease,which can cause diarrhea,mucinous hemorrhagic dysentery,emaciation and other symptoms after infecting pigs.In severe cases,it can lead to hemorrhagic,catarrhal or necrotizing colitis and appendicitis.Three kinds of bacteria can cause bloody dysentery and acute death in infected pigs.From the clinical symptoms,it is impossible to make a direct differential diagnosis,and there may be mixed infection,which makes the differential diagnosis difficult.At present,using microbiological methods and serological methods to detect bacterial pathogens is time-consuming and laborious.From existing research,there is no single detection method capable of directly detecting all three types of bacteria.Multiple q PCR technologies have been developed and have demonstrated good sensitivity and specificity.There is an urgent need to establish a rapid and efficient detection method capable of distinguishing and diagnosing all three types of bacteria.Such a method would play a critical role in the treatment,prevention,and control of diseases.In this study,three single-plex fluorescent quantitative PCR methods for detection of Lowsonia intracellularis,Clostridium perfringens Type C and Brachyspira hyodysenteriae were established.Search the conserved region of gene on NCBI,design three pairs of specific primers and Taq Man probe,optimize the annealing temperature and concentration of primer probe,establish standard curve,and evaluate the sensitivity,specificity and repeatability of each method.The results show that the standard curve correlation coefficients R~2 of the three methods are all greater than 0.990,and the linear relationship is good.The lowest detection limit of Lowsonia intracellularis is 10~0copies/μL,which is 100 times more sensitive than common PCR.The lowest detection limit of Clostridium perfringens type C is 10~0copies/μL,which is 1000 times more sensitive than common PCR.The lowest detection limit of Brachyspira hyodysenteriae was 10~0copies/μL,which was 10000 times more sensitive than that of common PCR.In all the three methods,only positive standard samples showed amplification signals,while E.coli,Salmonella,S.aureus,PEDV,TGEV,Po RV,Coccidian showed no DNA amplification signals,proving that the method has good specificity.The repetitive test results show that the intra-batch coefficient of variation is less than 2%,and the inter-batch coefficient of variation is less than 2%,and the stability is good.Based on the establishment of the single-plex fluorescent quantitative PCR method,a triple fluorescence quantitative PCR method was established for simultaneous detection of Lowsonia intracellularis,Clostridium perfringens Type C and Brachyspira hyodysenteriae.The reaction conditions were optimized and the standard curve was established.The correlation coefficient R~2 was more than 0.990 and the linear relationship was good.The sensitivity test showed that the lowest detection limit of Lowsonia intracellularis was 10~1copies/μL,which was 10 times more sensitive than that of ordinary PCR.The lowest detection limit of Clostridium perfringens type C is 10~0copies/μL,which is 1000 times more sensitive than common PCR.The lowest detection limit of Brachyspira hyodysenteriae was 10~0copies/μL,which was 10000 times more sensitive than that of common PCR.The specificity test showed that all the three bacterial positive standards produced amplification signals,and there was no cross reaction among the three standards,indicating that the specificity was strong.The repetitive test results showed that the intra-and inter-batch coefficients of variation of the three bacteria were all less than 4%,and the stability was good.Ordinary PCR,single-plex fluorescent quantitative PCR and triple fluorescence quantitative PCR were used to detect 226 clinical stool samples.According to the results of ordinary PCR,the detection rate of Lowsonia intracellularis was 17.26%(39/226),the detection rate of Clostridium perfringens type C was 11.06%(25/226),and the detection rate of Brachyspira hyodysenteriae was 7.96%(18/226).The results of single-plex fluorescence quantitative PCR showed that 32.03%(73/226)of Lowsonia intracellularis,18.14%(41/226)of Clostridium perfringens type C and 12.83%(29/226)of Brachyspira hyodysenteriae.The results of triple fluorescence quantitative PCR showed that Lowsonia intracellularis was 30.53%(69/226),Clostridium perfringens type C was 18.14%(41/226),Brachyspira hyodysenteriae was 12.83%(29/226).From the results of clinical samples,it is obvious that the detection rate of fluorescence quantitative detection method is much higher than that of ordinary PCR detection method.The positive coincidence rate of triple fluorescence quantitative PCR and single-plex fluorescence quantitative PCR in the detection of Lowsonia intracellularis bacteria was 94.52%,the negative coincidence rate was 100%,and the total coincidence rate was 98.23%.The positive coincidence rate,negative coincidence rate and total coincidence rate of triple fluorescence quantitative PCR and single fluorescence quantitative PCR in the detection of Clostridium perfringens type C and Brachyspira hyodysenteriae were all 100%.In this study,we successfully established a single-plex fluorescence quantitative PCR method for Lowsonia intracellularis,Clostridium perfringens type C and Brachyspira hyodysenteriae,and a triple fluorescence quantitative PCR method for the detection of three kinds of bacteria with high specificity,high sensitivity and good stability.This method provides a technical means for the quantitative detection of clinical samples of intracellular Lawson,Clostridium perfringens type C and Porcine dysentery short spirochete,and provides a faster and more accurate differential diagnosis method for the mixed infection of three kinds of bacteria in pig farms.Make pig farms better prevention,control and treatment strategies. |
PDF Full Text Request