Establishment And Application Of Fluorescent Quantitative PCR Detection Method For M.urealyticum In Reproductive Tract

Ureaplasma urealyticum?Ureaplasma urealyticum?is a minimal prokaryotic microorganism,which is between bacteria and virus and can live independently.It is also a pathogenic microorganism that causes genital tract infection and urinary tract infection.For men,it can cause prostatitis,urethritis,pyelonephritis,etc.;for women,it can cause inflammatory reactions such as vaginitis,cervicitis,pelvic inflammatory disease and other reproductive tracts.It is one of the pathogens causing infertility and abortion.The culture and serological methods commonly used in the detection of Ureaplasma urealyticum are low sensitivity,poor specificity,high false positivity and time-consuming.However,the fluorescence quantitative PCR?FQ-PCR?has been widely used in clinical microbiological detection because of its advantages of sensitive,rapid and accurate quantitative.Fluorescence quantitative PCR?FQ-PCR?detection technology combines the advantages of high sensitivity of common PCR,high specificity of DNA hybrids and high precision and quantification of spectral technology.It can achieve the advantages of more accurate detection,more sensitive specificity,less way of closed operation pollution.It directly detects the changes in the fluorescence signal during the PCR reaction to obtain the quantitative results of amplification products in a specific region.It does not require post-processing or electrophoresis detection to achieve a completely closed operation,which solves many problems of ordinary PCR technology.Objective:This study establishes and optimizes fluorescence quantitative PCR to detect Ureaplasma urealyticum reaction system,and provides a new technical means for clinical microorganism detection.Method:1.Design primers and probes by selecting highly conserved regions by comparing the existing M.urealyticum and Ureaplasma parvum,UreB gene sequences in NCBI BLAST.2.Using Uu?serover 10,ATCC33699?,Up?serover 3,ATCC700970?as positive control,clinical isolates Candida albicans,Escherichia coli,Staphylococcus aureus,Streptococcus,Enterococcus faecalis,Gardnerella,Lactobacillus,Mycoplasma hominis,human genomic DNA and other 9 strains were used as negative controls,clinical isolates Uu and Up were used as positive controls,deionized water was used as negative control,and the amount of Taq DNA polymerase in the reaction system was used.The concentration of Mg²⁺,primers and probes were optimized to establish a FQ-PCR reaction system for detecting Ureaplasma urealyticum Uu and Up.3.The optimized FQ-PCR was applied to the patients with urethritis or cervicitis and health examiners in the clinical outpatient of the Third Affiliated Hospital of Guangzhou Medical University and,and the usefulness of the fluorescence quantitative PCR method in clinical practice was analyzed.Result:1.In the qualitative PCR reaction systems what we established,PCR reaction systems had no cross reaction to 9 reference products and without amplification curve.The Uu and Up positive control groups of clinical isolates had obvious amplification curves,indicating that the Uu and Up fluorescence quantitative PCR reaction system has a high specificity for the detection of Ureaplasma.At the same time,with the detection of 9 clinical isolates of other bacterial groups and clinical isolates Uu and Up positive control group,the fluorescence quantitative PCR reaction system has high accuracy and sensitivity.2.In 369 patients with urethritis or cervicitis,Uu single positive was 55 cases,positive rate was 14.91%,Up single was 276 cases,positive rate was 74.80%,Uu and Up mixed infection 32 cases,positive rate was 8.67%,total The detection rate was 98.37%.Uu single positive was 10 cases in 200 healthy checkups,the positive rate was 5%,Up single was 77 cases,the positive rate was 38.5%,Uu and Up mixed infection in 2 cases,the positive rate was 1%,the total detection rate It is 44.5%.The results of fluorescence quantitative PCR and sequenc ing showed that the total coincidence rate was 98.59%,and the consistency was good.Conclusion:This study established an accurate and reliable fluorescence quantitative PCR rapid detection method based on experiments.This method is easy to operate,has a short detection period,high sensitivity,and good specificity.At the same time,it overcomes the shortcomings of high false positive rate of common PCR technology,and has certain clinical application value.