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.

Human Platelet Antigen Genotyping Using a Multiplex Single-Base Primer Extension Reaction in Koreans / 대한수혈학회지

BACKGROUND: Alloimmunization of human platelet antigens (HPA) is associated with clinically significant disease, such as platelet refractoriness, neonatal alloimmune thrombocytopenia, or posttransfusion purpura. It is determined by single nucleotide polymorphism of genes for platelet membrane glycoprotein. To date, approximately 27 HPAs have been discovered, and their frequencies differ depending on ethnicity and country. METHODS: We conducted an investigation of prevalence of HPA in the Korean population using a multiplex single-base primer extension reaction (SNaPshot). With 84 specimens from healthy donors, HPA genotyping was performed on 11 different HPAs, including HPA-1, -2, -3, -4, -5, -6, -7, -8, -9, -13, and -15. RESULTS: A total of 90 blood samples were genotyped. The genotype frequencies of HPA were as follows: HPA-1a/1a: 100.0%, -2a/2a: 83.3%, -2a/2b: 14.3%, -2b/2b: 2.4%, -3a/3a: 39.3%, -3a/3b: 52.4%, -3b/3b: 8.3%, -4a/4a: 100.0%, -5a/5a: 95.2%, -5a/5b: 4.8%, -6a/6a: 94.0%, -6a/6b: 6.0%, -7a/7a: 100.0%, -8a/8a: 100.0%, -9a/9a: 97.6%, -9a/9b: 2.4%, -13a/13a: 100.0%, -15a/15a: 23.8%, -15a/15b: 51.2%, and -15b/15b: 25.0%. CONCLUSION: The SNaPshot assay was employed for detection of SNPs in various clinically significant HPA genes. In addition to well-known frequencies of previously reported HPA-1 to -8, this study showed frequencies of HPA-9, -13, and -15 in Koreans for the first time. The SNaPshot technique might be suitable for use in actual clinical testing in patients with platelet alloimmunization.

Novel multiplex primer extension and denaturing high-performance liquid chromatography for genotyping of the deafness gene mutations / 中南大学学报(医学版)

To find a rapid and accurate genotyping method for specific non-syndromic hearing loss (NSHL)-causing gene mutations for disease diagnosis in different ethnic populations.Methods We performed a novel multiplex primer extension (PE) reaction in combination with denaturing high-performance liquid chromatography (DHPLC) to simultaneously detect and genotype the 6 most common mutations in 180 patients with NSHL (GJB2-235delC,GJB2-299delAT,PDS-A2168G,PDS IVS7-2A ＞ G,mtDNA-A1555G,and mtDNA-C1494T) in Chinese population.This method involved the amplification of the target sequence,followed by a purification step,a multiplex PE reaction,and DHPLC analysis performed on the Transgenomic Wave DNA fragment analysis system under fully-denaturing conditions.Results In a blind analysis,this technique successfully and accurately genotyped 100％ of the samples simultaneously characterized by direct sequencing.Conclusion Combination of PE and DHPLC is simple,rapid,accurate,and cost-effective for genotyping common disease-causing mutations,including substitutions,insertions,and deletions in NSHL,and may be successfully used in other genetic diseases.

ABO Genotyping using a Multiplex Single-base Primer Extension Reaction / 대한수혈학회지

BACKGROUND: ABO genotyping is being widely used in the case of ABO discrepancies and in forensic medicine. We have designed a method using a multiplex single-base primer extension reaction that has allowed us to detect six single nucleotide polymorphism (SNP) sites in the ABO gene and to determine ABO genotypes. METHODS: Genomic DNA was isolated from the peripheral blood of 75 unrelated Korean subjects. Exon 6 containing nucleotides 261 and 297 and exon 7 containing nucleotides 703, 802, 803 and 1059 were amplified using two pairs of primers. Using the products as templates, a multiplex single-base primer extension reaction was performed with six typing primers of different lengths for the six SNP sites. These reactions were performed on a PTC-200 thermal cycler (MJ Research, Waltham, MA, USA) using the SNaPshot multiplex kit (Applied Biosystems, Foster City, CA, USA), and the products were analyzed using an ABI 3130xl Genetic Analyzer (Applied Biosystems). RESULTS: The ABO genotypes determined by this method (75/75) all matched the genotypes that were determined by the use of the polymerase chain reaction using sequence-specific priming (PCR-SSP). We analyzed the peak pattern detected at each of the six SNP sites for each sample. For the smaller-sized primers, peaks were shifted to the right-side compared with the expected site and for the larger-sized primers peaks was close to the expected site. In addition, the coefficients of variation (CVs) of the smaller-sized primers were higher than the CVs of the larger-sized primers. CONCLUSIONS: We are able to detect six SNP sites in the ABO gene and to determine ABO genotypes using a multiplex single-base primer extension reaction.

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.