Establishment And Application Of Triple Fluorescence Quantitative PCR For Detection Of Major Pathogens In Sheep Respiratory Tract

With the rapid development of sheep industry from traditional decentralized stocking to large-scale captivity,due to the change of feeding environment,the feeding and management level is lagging behind,combined with frequent animal trade,resulting in increased morbidity and mortality of various infectious diseases.Among them,respiratory tract disease is one of the most common and serious infectious diseases,which has become an important factor that seriously affects the healthy development of the sheep industry.Mannheimia haemolytica,Mannheimia haemolytica and Mannheimia haemolytica are the three main pathogens causing respiratory diseases in sheep and are widely infected in sheep and goats in China.The clinical symptoms of respiratory tract in sick sheep after infection with these three pathogens are similar,which can not only cause respiratory diseases alone,but also often have mixed infection and secondary infection infection,thus causing difficulties in the diagnosis and treatment of respiratory diseases in sheep.Effective prevention and control of sheep respiratory diseases requires accurate identification of their pathogens.Compared with the traditional methods of pathogen isolation and identification,molecular detection methods have the advantages of rapidity,specificity and sensitivity,and have been widely used in the detection and infection diagnosis of sheep respiratory pathogens.Common PCR methods are relatively time-consuming and labor-intensive because they require reaction followed by gel electrophoresis,and the detection throughput is low.Compared with common PCR,fluorescence quantitative PCR has the advantages of rapid detection,sensitivity,accurate quantification and high throughput.At present,fluorescence quantitative PCR methods have been established for the above Mannheimia haemolytica,Mannheimia haemolytica and Mannheimia haemolytica,respectively.In order to further improve the efficiency of detection of major pathogens in the respiratory tract of sheep,shorten the detection time,and reduce the economic cost of detection,a triple fluorescent quantitative PCR method was established in this study that could simultaneously detect the three main pathogens of Mannheimia haemolytica,Mannheimia haemolytica,and Mannheimia haemolytica in a reaction,and the main methods and results were as follows.1.Establishment of triple fluorescent quantitative PCR method for the detection of three main pathogens of sheep respiratory tract.In order to establish a multiplex fluorescent quantitative PCR method for the simultaneous detection of three main pathogens of sheep respiratory tract:Mannheimia haemolytica,Mannheimia haemolytica and Mannheimia haemolytica,in order to achieve the rapid detection of sheep respiratory tract pathogens and accurate diagnosis of infection,on the basis of bioinformatics analysis,this study established a triple fluorescent quantitative PCR method for the simultaneous detection of three main pathogens of sheep respiratory tract using three pairs of specific primers and Taqman probes for Mannheimia haemolytica lkt D gene,Mannheimia haemolytica tuf gene and Mannheimia haemolytica kmt1 gene,respectively,on the basis of bioinformatics analysis.The results showed that the established triple fluorescent quantitative PCR method had good specificity,only amplifying the genomic DNA of three pathogens,Mannheimia haemolytica,Mannheimia haemolytica and Mannheimia haemolytica,but not amplifying the genomic DNA of Mycoplasma conjunctivae,Mycoplasma filiformis subspecies capricolum,Mycoplasma bovis,Mycoplasma agalactiae,Mycoplasma arginini,Escherichia coli and Staphylococcus aureus.This method has high sensitivity,with a lower limit of detection of 39 copies,94 copies,and 392 copies for genomic DNA of Mannheimia haemolytica,Mannheimia haemolytica,and Mannheimia haemolytica strains,respectively,and64.6 CFU,47.8 CFU,and 75.4 CCU for bacterial concentrations,respectively.2.Clinical application of triple fluorescent quantitative PCR.In order to further evaluate the clinical application effect of the established triple fluorescent quantitative PCR method and preliminarily master the infection of the three pathogens in sheep and goats,DNA was extracted from 300 sheep and goat nasal wipes as clinical samples in this study,and the triple fluorescent quantitative PCR method established in this study was used for detection.The results showed that the three pathogenic DNAs could be accurately detected from clinical samples by this method,and the infection rates of Mannheimia haemolytica,Mannheimia haemolytica,and Mannheimia haemolytica in sheep were 40%(86/215),43.7%(94/215),and10.2%(22/215),respectively;while the infection rates of the three pathogens in goats were 45.9%(39/85),24.7%(21/85),and 7.1%(6/85),respectively.Mixed infection was also found in the three pathogens.In conclusion,in this study,we established a triple fluorescent quantitative PCR method for the main pathogens of the respiratory tract of three sheep,Mannheimia haemolytica,Mannheimia haemolytica,and Mannheimia haemolytica,with good specificity and sensitivity,which can be used for the accurate detection of three pathogen infections in sheep and goats,thus providing a new rapid molecular diagnostic method for the diagnosis of respiratory diseases in sheep and useful data for the comprehensive prevention and control of respiratory diseases in sheep.