Establishment Of Nfo RPA Nfo RPA Detection Method For Main Pathogenic Bacteria Of Lamb Diarrhea

Lamb diarrhea is one of the most common sheep diseases in the world,which seriously affects the survival,development and growth of lambs and restricts the healthy development of the sheep breeding industry.Pathogenic microorganism infection is the main factor of diarrhea in lambs.The common bacterial pathogens that cause diarrhea in lambs are Escherichia coli,Salmonella,Clostridium perfringens.Rapid detection of bacteria has positive significance for early diagnosis and early treatment of lamb diarrhea.Recombinase polymerase amplification technology is a new in vitro nucleic acid isothermal amplification technology,which has the characteristics of rapidity,sensitivity and high efficiency.The nfo RPA technology further combines Lateral flow strip detection(LFD)with RPA technology to realize the visualization of detection results,which is widely used in various research fields.In this project,on the basis of bioinformatics analysis and combined with PCR to screen the specific marker genes of the main pathogenic bacteria of lamb diarrhea.To establish a nfo RPA detection method for Escherichia coli,Salmonella and Clostridium perfringens based on their specific marker genes,which can provide new technologies for the clinical detection of pathogenic bacteria in lamb diarrhea,and lay a foundation for the prevention,control and treatment of lamb diarrhea.The specific results are as follows:1.According to the literature review combined with bioinformatics analysis,using PCR method,1 pathogenic Escherichia coli-specific marker gene,3 Salmonella-specific marker genes,and 3 Clostridium perfringens-specific marker genes were initially screened.Take Escherichia coli orf-3737 gene,Salmonella hilA gene,and Clostridium perfringens colA gene as target genes for establishment of the nfo RPA rapid detection method.2.Based on the orf-3737 gene to establish a nfo RPA detection method for Escherichia coli.This method is at 39℃ and reacts for 10 minutes to observe the results through lateral flow chromatography test strips.It does not cross-react with other lamb diarrhea pathogens.The minimum detection limit is 4.3 × 102 copies/μL,which is 100 times more sensitive than PCR method,and has good inter-group and intra-group repeatability.3.A Salmonella nfo RPA detection method based on hilA gene was established.Under the condition of 39℃,the experimental results can be observed by lateral flow chromatography strips after reaction for 10 minutes.There is no cross-reaction with other lamb diarrhea pathogens.The minimum detection limit is 4.7×102 copies/μL,which is 100 times more sensitive than PCR method,and has good inter-group and intra-group repeatability.4.Based on the colA gene to establish a nfo RPA detection method for Clostridium perfringens.Under the condition of 39℃,the experimental results can be observed by lateral flow chromatography strips after reaction for 10 minutes.The minimum detection limit could reach 5.0×102 copies/μL,and does not cross-react with other pathogenic bacteria,with good specificity,good inter-group and intra-group repeatability.5.A total of 47 clinical samples of lambs with diarrhea in Ningxia were collected and detected by the established nfo RPA detection method for Escherichia coli,Salmonella,and Clostridium perfringens,and the detection results were compared with PCR detection results.The results show that the positive rate of E.coli nfo RPA detection method is 48.9%,the positive rate of PCR method is 40.4%;the positive rate of Salmonella nfo RPA detection method is 12.7%,the positive rate of PCR is 4.3%,and the positive rate of Clostridium perfringens nfo RPA method is 44.6%,and the positive rate of the PCR method is 38.2%.The detection rate of the established nfo RPA detection method for the three pathogens was higher than that of the PCR method.To sum up,the nfo RPA detection method for Escherichia coli,Salmonella and Clostridium perfringens established in this study is equipment-independent,simple to operate,short timeconsuming,good specificity,high sensitivity and repeatability.Well,it is suitable for on-site detection of Escherichia coli,Salmonella and Clostridium perfringens,and can provide technical support for the detection and prevention of diarrhea in lambs.