Establishment And Application Of Fluorescent Quantitative PCR Method For TV And CA

Research purpose:1.Establish dual fluorescence quantitative PCR detection method and detection of trichomonas vaginalis and candida albicans infection,optimized reaction conditions,the detection method and evaluate the specificity,sensitivity,repeatability of the method.It aims to provide a rapid and simultaneous detection of vaginal trichomoniasis and candida albicans in clinical diagnosis of female vaginitis and urethritis.2.Collected in August 2016 to April 2017,during the third hospital affiliated to medical university in guangzhou specimens of vaginal secretions in the patients of obstetrics and gynecology,establish good dual fluorescence quantitative PCR detection methods were used to detect specimens of trichomonas vaginalis and candida albicans infection status,analyze the clinical practicability of dual fluorescence quantitative PCR detection method.Research methods:1.The gene sequence of trichomonas vaginalis and candida albicans was synthesized and the reaction system of double fluorescence PCR was preliminarily determined.The reaction conditions of the detection method were optimized by screening the types of enzymes in the reaction system and the concentration of Mg²⁺.2.Choose to genital tract common microorganisms,including escherichia coli,neisseria gonorrhoeae,Gardner bacteria,lactic acid bacteria,mycoplasma clinical isolates DNA and genome DNA as a template,with Trichomonas vaginalis?Trichomonas vaginalis,TV?and Candida albicans,Candida albicans,CA)clinical isolates as positive control,with deionized water as a negative control,dual fluorescence quantitative PCR detection,evaluate its specificity.The sensitivity of the detection method was evaluated by double fluorescence quantitative PCR as a template for different concentrations of TV and CA.The accuracy of the detection method was evaluated 10 times by 20copies of plasmid and2000 copies TV and CA positive standard plasmid.3.Collected in August 2016 to April 2017 in the third hospital affiliated to guangzhou medical college of obstetrics and gynecology clinic patients specimens of vaginal discharge,this paper is adopted to establish the good twofold real-time fluorescent quantitative PCR detection method,detection of trichomonas vaginalis and candida albicans,and compared with ordinary PCR detection method,to determine the practicability of the method.Results:1.Specificity of the experimental results show that the method can specific amplification segments of trichomonas vaginalis and candida albicans purpose,for lactic acid bacteria,escherichia coli,neisseria gonorrhoeae,Gardner bacteria,people mycoplasma clinical isolates,genome DNA,DNA without specific amplification;The results of sensitivity test showed that the minimum detection limit was 10copies/ul for trichomonas vaginalis and candida albicans.Repetitive experimental results show that the dual fluorescence quantitative PCR detection method for trichomonas vaginalis and candida albicans test results of the repeatability is good,all samples were positive,CV value between batch in 20 copies and 2000 copies response range is less than 5%.Therefore,the double fluorescence quantitative PCR detection method of trichomonas vaginalis and candida albicans has a good specificity,sensitivity and repeatability.2.Collected in August 2016 to April 2017 issued 217 vaginal discharge specimens of obstetrics and gynecology clinic,to test the samples,the dual fluorescence quantitative PCR method for detection of trichomonas vaginalis positive rate was 1%,the positive rate was 11.06%,candida albicans and ordinary PCR sequencing method of detection of trichomonas vaginalis positive rate was 1%,positive rate was 10.60%of candida albicans,TV fluorescent quantitative polymerase chain reaction?PCR?and sequencing method,the test results of total coincidence rate was 100%,the CA's fluorescence quantitative polymerase chain reaction?PCR?and sequencing method,the test results of total coincidence rate was 99.54%,Consistency is good?Kappa=0.859,P<0.001?.Conclusion:1.This study successfully constructed the method of double fluorescence quantitative PCR for the detection of trichomonas vaginalis and candida albicans.This method has a good specificity,high sensitivity and good repeatability.2.Adopt double fluorescent quantitative PCR can rapid detection of trichomonas vaginalis in clinical samples and candida albicans,and compared with ordinary PCR sequencing method,consistency is good,for women's vaginitis diagnosis provides a means of reliable detection.