Establishment And Application Of Multiple QPCR Method For Main Pathogenic Pathogens Of Female Reproductive System Infectious Diseases

Female reproductive system infectious diseases,namely the female reproductive system due to changes in external conditions or internal imbalance of regulation and other conditions resulting in vaginal environmental disorders or reproductive organ diseases,female reproductive system infectious diseases more than sexually mature women,women’s life and health caused varying degrees of harm,and the infection rate is rising year by year.Early and accurate diagnosis of pathogenic pathogens is the key to clinical prevention and timely and effective treatment of female reproductive system infectious diseases.At present,the physiological and biochemical indexes and microbial detection methods used in clinical practice have some limitations,and the problems of missing and false detection are more serious.The purpose of this study is to establish a fast,accurate and suitable for widespread application of multiple fluorescence quantitative PCR(Mq PCR)detection method for 16 major pathogenic pathogens of female reproductive system infectious diseases,and to detect and evaluate the specificity,sensitivity,repeatability and accuracy of this method,which can be applied to the detection of suspected female reproductive system infectious diseases samples.The detection rate of pathogens was counted,and the drug resistance site mutation of gyrA and parC genes in quinolone resistance determining regions of Ureaplasma urealyticum with high detection rate was analyzed.The main research results are as follows:Sixteen major pathogenic pathogens of female reproductive system infectious diseases were identified,including six bacteria:Mycobacterium tuberculosis(MTB),Group A Streptococcus(GAS),Group B Streptococcus(GBS),Escherichia coli(EC),Staphylococcus aureus(SA),and Gardnerella vaginalis(GV).1 fungus:Candida albicans(CA);Three kinds of mycoplasma:Mycoplasma genitalium(Mg),Ureaplasma parvum(UP),Ureaplasma urealyticum(UU);1 chlamydia:Chlamydia trachomatis(CT);Four DNA viruses:Herpes simplex virus type 2(HSV-2),Herpes simplex virus type 1(HSV-1),Epstein-Barr virus(EBV),Cytomegalovirus(CMV);1Parasite:Trichomonas vaginalis(Tv).Primers and probes were designed for specific gene fragments of 16 major pathogenic pathogens of female reproductive system infectious diseases,and the specificity and sensitivity of the primers and probes were verified by single fluorescence quantitative PCR.The results showed that the specificity of 16 primers and probes was good,no cross reaction occurred,and the sensitivity was high,up to 1copies/μL.Sixteen kinds of pathogen primers and probes were divided into five groups to verify the specificity,sensitivity,repeatability and accuracy of Mq PCR method.The results showed that the five groups of pathogen Mq PCR primers had good specificity,and there was no cross reaction between each group of pathogens or with non-specific pathogens,and the sensitivity was high,up to10~1copies/μL.The coefficient of variation between and within batches was less than5.51.High accuracy,no false positive and false negative occurred in the detection,Mq PCR method was highly consistent with the detection results of generation sequencing.A total of 1830 suspected reproductive system infectious disease samples were collected from Yunnan Province,and epidemiological analysis of their clinical information showed that the patients were mainly sexually mature women.Mq PCR method was used to detect the samples,and the detection rate of positive samples reached 67%,with the highest detection rate of UP,followed by UU.According to the statistics of detected pathogens,14 common pathogens infected by FRS were detected,but only EC and GAS were not detected.The highest pathogen detection rate was UP(38.1%),followed by UU(13.56%),and MTB(0.16%)was the lowest.Based on the analysis of the number and age of detected pathogens,UP ranked the first in the number of pathogen infections at all ages,followed by UU.According to the analysis of mixed infection of detected pathogens,the FRS infectious diseases were mainly single infection,accounting for 59.1%.The mixed infection was mainly double infection,accounting for 27.76%.In the correlation analysis of the detection rate of double infection pathogens,there was a strong correlation between UP and GV(P<0.01),and a correlation between UP and CA,UP and UU(P<0.05).The maximum multiple infection detected in this study was six multiple infections.In addition,the detection rates of UP and UU were the highest in both single infection and mixed infection.The statistics of 256 UU positive samples,63 UU single infection samples and20 UU mutant samples showed that UU infection mainly occurred in sexually mature females in age group(>28-38).The 256 UU positive samples were mainly mixed infection at all ages,and the detection rates of UP and EBV were high in mixed infection.The detection rates of SA and MTB were low.In addition,no HSV-1 was detected.The samples of ureaplasma urealyticum single infection were collected for the detection of drug resistance mutation sites of gyrA and parC genes in quinolone resistance determining regions,and the mutations of bases and amino acids were analyzed.A total of 49 mutation sites were detected,including G112A missense mutation site and 21 synonym mutation sites of gyrA gene,and S83L,T125A and T136A missense mutation site and 23 synonym mutation sites of parC gene.The mutation rate of parC gene S83L was 56.25%.Moreover,some missense mutations are accompanied by synonymous mutations.Finally,the information of 20 samples with drug-resistant site mutation and 18 samples without mutation were sorted out and analyzed.The results showed that the UU single infection mainly occurred in the age range(>29-39),and the mutation rate of drug-resistant sites was 52.63%,mainly missense mutations,and only 31.58%of the positive samples had no mutation of drug-resistant sites.In summary,this paper establishes a fast,accurate and suitable Mq PCR detection method for the main pathogenic pathogens of infectious diseases of female reproductive system.In order to provide laboratory information for the establishment of pathogen spectrum of female reproductive system infectious diseases in Yunnan province,suspected samples of female reproductive system infectious diseases were tested.The mutation analysis of bases and amino acid sequence of the drug resistance sites of gyrA and parC genes in quinolone resistance determining regions of UU was performed,which provided theoretical basis for clinical analysis of UU quinolone resistance and drug guidance.