Application Of Multiplex Melting Curve Analysis In The Detection Of Pathogens Of Lower Respiratory Tract

Lower respiratory tract infections are one of the most serious infectious diseases of human health.According to the different clinical manifestations,it can be divided into a variety of types,including community acquired pneumonia,hospital acquired pneumonia,ventilator associated pneumonia and so on.The pathogenic spectrum of different disease types has its own characteristics,and there are extensive crossovers at the same time.Therefore,accurate detection of pathogens of lower respiratory tract infections is of great significance for clinical medication and treatment.In this paper,based on multiplex PCR,combined with multicolor melting curve analysis technology and mediator probe technology,we establish a rapid,accurate,simple,low-cost identification method to meet the needs of clinical testing.In chapter one,the background,epidemiology,pathogen interaction,and drug resistance of lower respiratory tract infections were reviewed.Several common detection methods for lower respiratory tract infections and their respective advantages and disadvantages were compared.Finally,the purpose,method and significance of this study were proposed.In chapter two,we combined with multiplex PCR technology,multicolor melting curve analysis technology and mediator probe technology,a multi-mediator probe melting curve analysis system for lower respiratory tract was designed to detect common pathogens in the lower respiratory tract,including 18 pathogens and one drug-resistance gene.Experiments were designed to examine the performance of the system,including investigations of specific,sensitivity,repeatability,mixed infection recognition ability,and the final evaluation of clinical specimens.In the third chapter,a multi-mediator probe melting curve analysis system was established.The performance was investigated using the plasmid as a reference product.The inspection results showed that the system was very specific and can distinguish each test object accurately.The sensitivity of the system reaches 50 copies/reaction,individual subjects can reach 5 copies/reaction.The repeatability was very good(CV<0.5%).In addition,the system can identify a certain kind of bacteria mixed concentration of 50 copies/reaction.Finally,we tested 299 clinical specimens with a positive rate of 61.87%and mixed infection rate was 21.40%.We randomly selected 160 specimens for single-by-one verification.The results showed that the consistency rate of the two methods was greater than 95%for each pathogen,confirming the reliability and accuracy of our system.