Rapid detection of CYP2C9, CYP2C19,CYP4F2,VKORC1 and ABCB1 gene polymorphisms by liquid phase chip technology / 中华检验医学杂志

Objective@#To establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P450 2C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology.@*Methods@#Method establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results.@*Results@#The results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4% and 10.9%, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results.@*Conclusion@#A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology.

Rapid detection of CYP2C9, CYP2C19, CYP4F2, VKORC1 and ABCB1 gene polymorphisms by liquid phase chip technology / 中华检验医学杂志

Objective To establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P4502C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology. Methods Method establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results. Results The results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4％and 10.9％, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results. Conclusion A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology.

Detection of First-Line Anti-Tuberculosis Drug Resistance Mutations by Allele-Specific Primer Extension on a Microsphere-Based Platform

BACKGROUND: Resistance of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs is almost exclusively due to spontaneous chromosomal mutations in target genes. Rapid detection of drug resistance to both first- and second-line anti-TB drugs has become a key component of TB control programs. Technologies that allow rapid, cost-effective, and high-throughput detection of specific nucleic acid sequences are needed. This study was to develop a high-throughput assay based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres to detect anti-TB drug resistance mutations. METHODS: DNA samples from 357 M. tuberculosis clinical isolates and H37Rv were amplified by multiplex PCR using four primer sets, followed by multiplex ASPE using 23 TAG-ASPE primers. The products were sorted on the TAG-ASPE array and detected by using the Luminex xMAP system. Genotypes were also determined by sequencing. RESULTS: Genetic drug susceptibility typing by the TAG-ASPE method was 100% concordant with those obtained by sequencing. Compared with phenotypic drug susceptibility testing (DST) as a reference method, the sensitivity and specificity of the TAG-ASPE method were 83% (95% confidence interval [CI], 79-88%) and 97% (95% CI, 90-100%) for isoniazid. For rifampin testing, the sensitivity and specificity were 90% (95% CI, 86-93%) and 100% (95% CI, 99-100%). Also, the sensitivity and specificity were 58% (95% CI, 51-65%) and 86% (95% CI, 79-93%) for ethambutol. CONCLUSIONS: This study demonstrated the TAG-ASPE method is suitable for highly reproducible, cost-effective, and high-throughput clinical genotyping applications.

Prenatal diagnostic testing for infantile and late-infantile neuronal ceroid lipofusinoses (NCL) using allele specific primer extension (ASPE) / 北京大学学报(医学版)

SUMMARY Infantile (INCL, NCL1) and late-infantile (LINCL, NCL2) neuronal ceroid lipofuscinoses have been found to result from genetic deficiency of genes CLN1 and CLN2, respectively. The application of molecular analyses can facilitate prenatal diagnosis for families affected by NCL1 or NCL2, in which the familial mutation(s) have been identified. Molecular testing with allele-specific primer extension and DNA sequencing was performed in nine pregnancies, four from two NCL1 families and five from five NCL2 families. Lysosomal enzyme activity assays were carried out as well.Four fetuses from three pregnancies in NCL1 families were found to be carriers for a mutation 451C-T in the CLN1 gene and one was normal. Prenatal testing of three NCL2 families who carried mutation R208X in the CLN2 gene showed that all fetuses were carriers. In NCL2 families who carried either mutation IVS5-1C or/and IVS5-1A two normal pregnancies were detected. Our studies indicate that DNA testing, which may provide definitive prenatal diagnosis for NCL, may be used in combination with lysosomal enzyme activity analyses.