ABSTRACT
Objective To establish a LNA-taqman-based real-time PCR assay for detecting gene polymorphisms of Vitamin K monooxygenase CYP4F2,vitamin K epoxide reductase complex subunit 1(VKORC1)and cytochrome P450 2C9(CYP2C9), associated with Warfarin optimal dosage.Methods A set of allele-specific PCR primers and probes was designed for each single nucleotide polymorphism(SNP)of CYP4F2-1347C>T,CYP2C9*3,VKORC1-1173C>T and VKORC1-1639G>A, and the specificity of LNA-taqman probe PCR was evaluated.Genomic DNA of peripheral blood samples from 150 patients with treated with warfarin was extracted,and the some PCR products were verified with sequencing.Results ①LNA-taq-man-based real-time PCR assay was highly specificity,no overla.②Among the 150 patients,the cases of CC,CT and TT genotype of CYP4F2-1347C>T were 87(58%),56(37.3%)and 7(4.7%).The cases of *1/*1 and *1/*3 genotype of CYP2C9*3 were 142(94.7%)and 8(5.3%),*3/*3 genotype was not detected.The cases of TT,TC and CC genotype of VKORC1 1173C>T were 127(84.7%),20(13.3%)and 3(2%).The cases of AA,AG and GG genotype of VKORC1 1639G>A were 124(82.7%),23(15.3%)and 3(2%),respectively.Conclusion The LNA-taqman-based real-time PCR as-say is convenient,inexpensive,accurate and will be useful for CYP4F2-C1347T,CYP2C9* 3,VKORC1-C1173T and VKORC1-G1639A genotyping in a clinic laboratory.
ABSTRACT
Objective To diagnose the process of hepatic injury,a method of quantitating the concentration of sH2a subunit from asialoglycoprotein receptor (ASGPR) in serum by enzyme-linked immunosorbent assay was established and clinical evaluated.Methods 210 serum samples were collected in Suzhou Kowloon Hospital.Among them,70 subjects with cirrhosis,viral hepatitis and fatty liver disease were as hepatic injury group and 140 subjects of healthy and high fat,hemolysis,jaundice and with autoimmune disease were as control group.The serum sH2a of two group were measured by ELISA kit.The results of sH2a ELISA were analyzed by four table chi-square test and SPSS20.0 software.Results sH2a protein level in liver injury group and control group were 105.92+ 53.41 ng/ml and 69.25+27.45 ng/ml,respectively.The difference between the control group and the liver injury group was statistically significant (F=14.375,t=5.397,P=0.000).The sensitivity and specificity of the sH2a ELISA kit were 68.57％ (95％CI:56.37～79.15％) and 82.86％ (95％CI:75.58％～88.70％),and the total compliance rate was 78.10％ (95％CI:71.88％～83.49％) with KAPPA coefficient:0.510 6 (95％ CI:0.3877～0.6336).Conclusion SH2a serum ELISA kit with positive and negative coincidence rate between SH2a serum level and meet the clinical requirement,which could be used as new marker for diagnosis of heptieal injury related diseases.