Application Of Multiplex Mediator Probe Melting Curve Analysis In The Detection Of Gastrointestinal Pathogens

Infectious diarrhea refers to diarrhea caused by bacteria,viruses,fungi,and parasites.Because the clinical symptoms of diarrhea patients are very similar,it is difficult to identify the pathogens leading to diarrhea according to the symptoms,which makes it impossible for clinicians to formulate targeted treatment plans.There are few studies on the simultaneous detection of bacteria and viruses in the literature that has been reported.In this paper,a rapid method for the detection of 12 kinds of bacteria and 6 kinds of viruses and a serotype was established by mediator probe melting curve analysis.The first section of the first chapter,we introduced the background,clinical manifestations,pathogenesis of infectious diarrhea and the treatment of antibacterial drugs;The second section introduced the commonly used diagnostic techniques for the detection of gastrointestinal pathogens.The third section introduced the principle of multiplex mediator probe melting curve analysis and the selection of gastrointestinal pathogen target genes.The second chapter introduced the system’s materials and methods including instruments and reagents used,the construction of plasmids,the design of primers and probes,the establishment of single and multiplex systems,sensitivity investigations,specificity investigations,repetitive investigations,the ability to identify mixed infections,and clinical evaluations.In the third chapter,we introduced the performance of the system and test results of the specimen.In this paper,a 5-color 29-plex system was established for the detection of bacteria and viruses.The detection sensitivity reaches 50 copies/reaction,and some subjects can reach 5 copies/reaction.The reproducibility of the system is good.The CV value of the melting point is the maximum 0.78%,and the others are all less than 0.5%.The system has detected 100 stool samples and 119 clinical isolates from the Shenzhen Center for Disease Control and Prevention.Using real-time fluorescence PCR as a control method,the virus detection rate was 94%(94/100).The results of the 119 clinical isolates were consistent with Shenzhen Center forDisease Control and Prevention.In addition,39 fecal specimens from the Jining Center for Disease Control and Prevention were tested and a commercial virus detection kit was used as a control method.The virus detection consistency rate reached 89.7%(35/39),and the bacterial detection results were consistent with the sequencing results.The inconsistent virus samples were verified by a uniplex real-time PCR system established in our laboratory.The verification results were consistent with the detection results of this system.