Development Of Two Novel QPCR Detection Methods Based On Respiratory Syncytial Virus And Nine Bacterial Resistance Genes

Infectious disease is a kind of infectious and epidemic disease caused by various pathogens.Viruses and bacteria are the main causative agents of most infectious diseases.Real-time quantitative PCR(q PCR)based on Taq Man probes is commonly used for the diagnosis or quantification of viruses and bacteria.However,the high mutation characteristic of the virus(especially RNA viruses)reduces the efficiency of conventional q PCR amplification and limits the detection ability of q PCR.In addition,with the increasing resistance of bacteria to commonly used antibiotics and the increasing number of multi-resistant bacteria,it is difficult for conventional q PCR to detect multiple drug-resistant genes in a single tube at the same time.Based on the above problems,two q PCR detection methods were established in this study.The main research contents are as follows:(1)Development of RSV mismatch-resistant q PCR detection method.Respiratory syncytial virus(RSV)is one of the major viral pathogens that cause acute respiratory infections in infants and young children.The timely diagnosis of RSV infection is very important for the treatment and prevention of RSV infection.In this part,based on the 3'-5' exonuclease properties of high-fidelity DNA polymerase,a new mismatch-tolerant q PCR method was developed and applied to the detection of RSV.Compared with conventional q PCR,the new method has higher amplification efficiency for mutants and has good sensitivity and specificity.The detection limit is 1.8 PFU/m L.The new method has a good consistency with the commercial RSV test kit,and 80% of RSV positive samples,the Ct value detected by the new method is relatively low.Through sequencing analysis of these samples,it is found that there is a mismatch between the samples and the 3' end of primer,which further verifies the tolerance of the new method to mismatch.The experimental results show that the mismatch-tolerant q PCR is a promising method for sensitive detection of highly mutated viruses.(2)Development of a two-dimensional multiplex q PCR detection method for bacterial resistance genes.The widespread use of cephalosporins and carbapenem antibiotics has significantly increased the number of multi-drug resistant Gram-negative bacteria,and rapid detection of multiple drug resistance genes of bacteria is essential for the treatment of multi-drug resistant infections.In this part,a single tube two-dimensional multiple q PCR method was established by combining the color of the probe fluorophore and the Tm value of the amplicon.,which can quickly perform molecular diagnosis of multiple bacterial resistance genes.It was verified by detecting 5 carbapenemase genes(ie KPC,NDM,VIM,OXA-48 and GES)and 4 cephalosporinase(Amp C enzyme)genes(ie CIT,EBC,ACC and DHA).This method has good specificity,no cross-reaction with other strains,and a sensitivity range of 30 to 3000 copies per reaction.For coexisting genes,this method can also distinguish well.This method has good consistency with the single q PCR method.The two-dimensional multiple q PCR method is simple,rapid,sensitive and specific,and may become a powerful tool for monitoring bacterial resistance.