ABSTRACT
Objective To establish genotyping methods for vitamin K epoxide reductase complex subunit 1 (VKORC1)and cytochrome P450 2C9(CYP2C9)based on pyrosequencing technique to detection of warfarin metabolizing enzyme related gene polymorphisms.Methods A total of 50 peripheral blood samples from healthy adults were collected and the whole blood genomic DNA was extracted.A set of biotin-labeled amplifi-cation primers and sequencing primers were designed respectively for three SNP sites:VKORC1 -1639 G>A,CYP2C9 430C> T and CYP2C9 1075A>C.After PCR amplification of the samples,pyrophosphoric acid se-quencing was conducted.And then the signal peaks form were combined to analyze and determine each sample genotype.Genotyping results were verified by Sanger sequencing,and the consistency of the two sequencing methods was compared.Results Genotypes of the three SNPs can be clearly determined according to the ba-ses and height of the signal peaks.Among the 50 samples,there were 41 AA and nine AG for VKORC1 -1639G>A,accounting for 82% and 12% respectively,and there were 45 *1/*1,five *1/*3 for CYP2C9, accounting for 90% and 10% respectively,no CYP2C9*2 allele detected.Genotype results detected by pyrose-quencing and Sanger sequencing were consistent with each other.Conclusion In SNP genotyping,Pyrose-quencing has the advantages of convenience,time-saving,cheap with accurate and reliable results,which can quickly determine the genotypes of CYP2C9 and VKORC1.
ABSTRACT
OBJECTIVE@#To analyze deafness gene mutations by genechip.@*METHOD@#The peripheral blood samples were obtained and DNA templates were extracted by extraction kits. The deafness gene mutations were distinguished by genechip.@*RESULT@#Among 42 patients with non-syndromic hearing loss, GJB2 235delC was found in 11 cases (7 cases were homozygosis, 4 cases were heterozygosis); 4 cases were shown to carry the PDS IVS7-2A>G mutation.@*CONCLUSION@#The incidence of GJB2 gene and PDS IVS7-2A>G mutations among the deaf- mute children in Guiyang city is 38.10%. Molecular genetic screening for these mutations and genetic counseling are effective methods to prevent the occurrence of hereditary hearing loss.
Subject(s)
Adolescent , Child , Child, Preschool , Humans , Infant , Young Adult , China , Connexin 26 , Connexins , Genetics , Deafness , Genetics , Genetic Testing , Membrane Transport Proteins , Genetics , Mutation , Oligonucleotide Array Sequence Analysis , Sulfate Transporters , Surveys and QuestionnairesABSTRACT
OBJECTIVE To evaluate the clinical application of T-cell based IFN-? release assays(IGRA) for the rapid diagnosis of active tuberculosis.METHODS IFN-? and HIV antibody were detected by using ELISA.Antibody to Mycobacterium tuberculosis was detected by colloidal gold.At the same time,the M.tuberculosis DNA loads were examined by FQ-PCR.Statistical analysis were performed to analyze the correlation of IFN-? with M.tuberculosis antibody and DNA,respectively.RESULTS The sensitivity of TB-IGRA was 90.24%,specificity was 93.34%;the positive rate of TB-IGRA in 82 tuberculosis patients was higher than from sputum smear(64.63%),TB-PCR(76.83%) and tuberculosis antibody(40.24%).CONCLUSIONS As a replacement of TB-PCR,IFN-? can be used as a valued index to evaluate tuberculosis infectin.