The Study On Nucleic Acid Diagnostic In Early Warning Of Acute Gout Attack And Early Screening Of Cervical Cancer

The increasing incidence of chronic diseases,infectious diseases,and tumors,exerts much pressure on ?D industry to satisfy the demand boom,particularly the molecular diagnostic market to bear the brunt,which develops rapidly.At present,nucleic acid molecular diagnostic products still occupy the mainstream position of molecular detection,of which more than 70%employ PCR technology,due to its earlier starting-point,relatively lower technical barriers,and higher degree of localization.This study will discuss how to develop and evaluate nucleic acid diagnostic products within specific projects,based on multiple real-time fluorescence quantitative PCR method.The first chapter focuses on the "molecular typing research project of hyperuricemia&gout",detailing the R&D process of acute gout attack early warning kit.The finally formed kit features as follows:for six target genes(IL1B,P2RX7,CXCL8,TLR2,TLR4,NLRP3)and two housekeeping genes(ACTB,GAPDH),the PCR amplification efficiency ranged from 92%to 98%;the detectable linear dynamic interval covered 102 to 108 copies,corresponding linear correlation coefficient R2 all above 0.99;the LOD varied from 10 to 100 copies;and accurate quantification of the target can be realized under the interference of multiple competitive reactions(Ct value change of the low-concentration target caused by high-concentration competitive targets does not exceed 1.3,even if the concentration difference is as high as 10,000 times).Moreover,kit's detection specificity was verified through four cell lines,and its performance remained stable among different instruments or reagent batches.Besides,two assorted kits(for RNA extraction and reverse transcription)have been successfully assembled and performed well compared to similar products,hence applicable.The second chapter lays stress on evaluating the CerviHPV high-risk HPV DNA testing kit,which was developed by our team during "cervical cancer screening project".Based on 437 cervical swab specimens,CerviHPV demonstrated highly comparable performance with cobasHPV-the concordance rates between the two tests were 99.5%,100%,91.7%,and 92.2%respectively for HPV16,HPV18,12HPV,and all 14 HR-HPV types,corresponding kappa values were 0.987,1.000,0.782 and 0.839.In the simulation experiment,CerviHPV detected the 12HPV types more uniformly than cobasHPV.As for the comparison between CerviHPV and careHPV based on other 93 cervical swab specimens,the overall concordance rate reached 91.4%,corresponding kappa value was 0.682.CerviHPV showed higher specificity and performance advantages in genotyping HPV16 and HPV18 than careHPV-one confirmed false positive and two possible false positive results in careHPV were identified in our study.