ABSTRACT
Objective To clone Tp0821,Tp0319,Tp0971 and Tp0663 gene,construct prokaryotic expression plasmid,the ex-pression of recombinant proteins,purification and used to detect syphilis patients serum antibodies.To explore Tp0821, Tp0971,Tp0319 and Tp0663 recombinant protein in the diagnosis of Tp infection,rich library of candidate antigens syphilis serology diagnosis.Methods From Tp library screening Tp0821,Tp0319,Tp0971 and Tp0663 four kinds of membrane pro-tein,through the analysis of the bioinformatics software selection advantage antigen epitope,connected with pQE32 carrier respectively built pQE32/Tp0821,pQE32/Tp0319,pQE32/Tp0971 and pQE32/Tp0663 prokaryotic expression recombi-nant;In vitro cloning,expression and purification and on single Tp0821,Tp0319,Tp0971,Tp0663 and four kinds of recombi-nant protein chimeric antigen (Tp0821-Tp0319-Tp0971-Tp0663)in order to establish the indirect Tp-envelope antigen ELISA method,based on TRUST and TPPA method comparison,detection collected from January 2013 to June 2014 in Shenzhen Baoan District People’s Hospital outpatient and hospitalization Tp negative patients,160 cases of positive speci-mens of 83 cases of clinical and Tp,and evaluated its application in the syphilis serology diagnosis.Results Successful con-structed of prokaryotic expression vector pQE32/Tp0821,pQE32/Tp0319,pQE32/Tp0971 and pQE32/Tp0663.Efficient expressed and purified their relative molecular mass of recombinant proteins.Seted up Tp-indirect ELISA method to detect 160 cases of Tp negativepositive clinical specimens and 83 cases of Tp,and compared with TPPA,the sensitivity were 85.5% (71/83),84.3% (70/83),89.2% (74/83),81.9% (68/83)and 95.2% (79/83)respectively,specificity was 100%(160/160),and the coincidence rate was 82.6%~95.7%.Single Tp0821,Tp0319,Tp0971 and Tp0663 recombinant protein positive rate of TP-ELISA was established (85.5%,84.3%,89.2% and 81.9%)compared with TPPA method of positive detection rate (100%)had significant difference statistically significant (χ2= 24.5~31.8,P<0.01),and four kinds of re-combinant protein chimeric antigen positive rate of TP-ELISA was established with the TPPA method the positive detection rate of comparative difference was statistically significant (χ2=7.95,P<0.05).Conclusion Preparation Tp0821,Tp0319, Tp0971 and Tp0663 recombinant protein had good immune activity,for the further study of its application in the diagnosis of syphilis serology lay a certain foundation,but in four kinds of recombinant protein chimeric antigen based Tp Tp-indirect ELISA method for serological diagnosis,can improve the detection sensitivity.
ABSTRACT
Three SNaPshot multiplex assays were developed to test 23 coding region single nucleotide polymorphisms (SNPs) and one control region SNP outside hypervariable regions (HVR)I and II, which was aimed at increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and confirming haplogroup assignments of mtDNA profiles in both human population studies and medical research. The selected SNPs targeted the East Asian phylogeny. These multiplex assays were validated by comparing with the sequencing analysis of samples chosen randomly. The mtDNA variations of 100 unrelated individuals from the Wuhan population in China were examined and classified into 31 haplotypes, and the haplotype diversity was estimated to be 0.952. The multiplex SNaPshot method is rapid and robust, and suitable for large-scale screening studies of mtDNA variability.
ABSTRACT
The absorption-elution test using low ionic strength solution (LISS) has been compared with the test using normal saline in MN typing of 258 bloodstain samples stored 1 to 6 years. The accuracy rate was 94.57％ using LISS method. The present study indicated that the LISS method is more sensitive than tests carried out in normal saline.