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2.
Acta Pharmaceutica Sinica ; (12): 2862-2874, 2023.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-999037

ABSTRACT

An UPLC-Q-TOF/MSE technology coupled with UNIFI database was used to develop a rapid, high coverage, accurate and efficient chemical composition qualitative method for Xuezhikang Capsule. A UNIFI database was established utilizing compound name, formula, structure, following automatic matching with high-resolution mass numbers, isotope distributions, mass deviations, fragment ion matching, and chromatographic retention features in UNIFI database to achieve the qualitative results of natural products in Xuezhikang Capsules. Combined with manual confirmation, 82 chemical components were identified in Xuezhikang Capsules, and the MS2 fragmentation pathway of typical organic acids, flavonoids, monacrines, and monascus were analyzed to ensure accuracy of the LC-MS workflow. This study clarified the chemical substance basis of Xuezhikang Capsules by LC-MS technology, providing experimental data support for the identification of key quality attributes, quality control and consistency evaluation in the manufacturing process of Xuezhikang Capsules.

3.
J Mol Diagn ; 24(6): 655-665, 2022 06.
Article in English | MEDLINE | ID: mdl-35390515

ABSTRACT

In minimal residual disease (MRD), where there are exceedingly low target copy numbers, digital PCR (dPCR) can improve MRD quantitation. However, standards for dPCR MRD interpretation in acute lymphoblastic leukemia are lacking. Here, for immunoglobulin/T-cell receptor-based MRD, we propose an objective, statistics-based analytic algorithm. In 161 postinduction samples from 79 children with acute lymphoblastic leukemia, MRD was performed by dPCR and real-time quantitative PCR (qPCR) using the same markers and primer-probe sets. The dPCR raw data were analyzed by using an automated algorithm. dPCR and qPCR results were highly concordant (P < 0.0001): 98% (50 of 51) of qPCR positive were positive by dPCR, whereas 95% (61 of 64) of qPCR negative results were also negative by dPCR. For MRD quantitation, both qPCR and dPCR were tightly correlated (R2 = 0.94). Using more DNA (1 µg × 7 versus 630 ng × 3), dPCR improved sensitivity of MRD quantitation by one log10 (median MRD positive cutoff 1.6 × 10-5). With dPCR, 83% (29 of 35) of positive-not-quantifiable results by qPCR could be assigned positive/negative MRD status. Seven replicates of tested samples and negative controls were optimal. Compared with qPCR, dPCR could improve MRD sensitivity by one log10. We proposed an automatable, statistics-based algorithm that minimized interoperator variance for dPCR MRD.


Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma , Algorithms , Child , Genes, T-Cell Receptor , Humans , Immunoglobulins/genetics , Neoplasm, Residual/diagnosis , Neoplasm, Residual/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
5.
J Clin Pathol ; 69(9): 801-4, 2016 Sep.
Article in English | MEDLINE | ID: mdl-26896490

ABSTRACT

AIMS: PCR amplicon-based next-generation sequencing (NGS) panels are increasingly used for clinical diagnostic assays. Amplification bias is a well-known limitation of PCR amplicon-based approaches. We sought to characterise lower-performance amplicons in an off-the-shelf NGS panel (TruSight Myeloid Sequencing Panel) for myeloid neoplasms and attempted to patch the low read depth for one of the affected genes, CEBPA. METHODS: We performed targeted NGS of 158 acute myeloid leukaemia samples and analysed the amplicon read depths across 568 amplicons to identify lower-performance amplicons. We also correlated the amplicon read depths with the template GC content. Finally, we attempted to patch the low read depth for CEBPA using a parallel library preparation (Nextera XT) workflow. RESULTS: We identified 16 lower-performance amplicons affecting nine genes, including CEBPA. There was a slight negative correlation between the amplicon read depths and template GC content. Addition of the separate CEBPA library generated a minimum read depth per base across the CEBPA gene ranging from 268x to 758x across eight samples. CONCLUSIONS: The identification of lower-performance amplicons will be informative to laboratories intending to use this panel. We have also demonstrated proof-of-concept that different libraries (TruSight Myeloid and Nextera XT) can be combined and sequenced on the same flow cell to generate additional reads for CEBPA.


Subject(s)
High-Throughput Nucleotide Sequencing/methods , Leukemia, Myeloid, Acute/genetics , Polymerase Chain Reaction/methods , Humans
6.
Prenat Diagn ; 33(11): 1017-22, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23794144

ABSTRACT

OBJECTIVE: Current methods of prenatal diagnosis to detect beta-thalassemia are Sanger sequencing and reverse dot blot. These methods are time-consuming and can prolong assay turnaround time. We aim to develop a sensitive and rapid method to detect 27 beta-thalassemia mutations using pyrosequencing. METHOD: Pyrosequencing primer pairs and sequencing primers were designed to detect 27 most common beta-thalassemia mutations found in Singapore. Pyrosequencing was performed on 191 DNA samples with known beta-thalassemia mutations isolated from 143 peripheral blood and 48 prenatal samples (seven chorionic villus biopsies, 26 cultured amniocytes, 15 uncultured amniocytes). All mutations were validated with Sanger sequencing. RESULTS: Pyrosequencing identified 210 alleles with beta-thalassemia mutations and 82 alleles without mutations with 100% sensitivity (lower 95% confidence interval [CI], 97.8%) and 100% specificity (lower 95% CI, 94.4%). All pyrosequences were concordant with Sanger-based sequences. Pyrosequencing was able to detect DNA concentrations as low as 2 ng, obviating the need for cell culture in volume-restricted samples. Sample receipt-to-report assay turnaround times were 16 to 18 h (Sanger sequencing) and 4 to 6 h (pyrosequencing). CONCLUSION: Pyrosequencing is a rapid and sensitive method to detect common beta-thalassemia mutations without the need for cell culture, thus reducing the assay turnaround time.


Subject(s)
DNA Mutational Analysis/methods , Genetic Testing/methods , Prenatal Diagnosis/methods , beta-Thalassemia/diagnosis , beta-Thalassemia/genetics , Amniotic Fluid/cytology , Asia, Southeastern , Calibration , Cells, Cultured , Chorionic Villi Sampling , DNA Mutational Analysis/standards , Female , Genetic Testing/standards , Humans , Mutation , Pregnancy , Prenatal Diagnosis/standards
7.
Pharmacogenomics ; 13(5): 533-42, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22462746

ABSTRACT

UNLABELLED: AIM, MATERIALS & METHODS: We investigated the functional significance of CYP2C19*2, *3, *17 and PON1 Q192R SNPs in 89 consecutive Asian patients on clopidogrel treatment and the prevalence of functionally significant polymorphisms among 300 Chinese, Malays and Asian Indians. RESULTS: Both CYP2C19 loss-of-function alleles (*2 or *3) were associated with higher platelet reactivity while the CYP2C19 gain-of-function allele (*17) had lower platelet reactivity. For PON1, the median PRI was not significantly different between the QQ, QR and RR groups. The allele frequencies of CYP2C19*2, CYP2C19*3 and CYP2C19*17 were 0.280, 0.065 and 0.010 (rare) for Chinese, 0.310, 0.050 and 0.025 for Malays, and 0.375, 0.010 (rare) and 0.165 for Indians, respectively. CONCLUSION: Our data suggest that genotyping studies to investigate clopidogrel response should include CYP2C19*2 and *3 but not *17 polymorphisms in Chinese, and CYP2C19*2 and *17 polymorphisms but not *3 in Indians. All three polymorphisms should preferably be genotyped in Malays.


Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Aryldialkylphosphatase/genetics , Asian People/genetics , Platelet Aggregation Inhibitors/pharmacokinetics , Polymorphism, Single Nucleotide , Ticlopidine/analogs & derivatives , White People/genetics , Aged , China , Clopidogrel , Cytochrome P-450 CYP2C19 , Female , Gene Frequency , Genotype , Humans , India , Malaysia , Male , Middle Aged , Multivariate Analysis , Platelet Aggregation/drug effects , Platelet Aggregation/genetics , Platelet Aggregation Inhibitors/administration & dosage , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation Inhibitors/therapeutic use , Ticlopidine/administration & dosage , Ticlopidine/pharmacokinetics , Ticlopidine/pharmacology , Ticlopidine/therapeutic use
8.
Drug Metab Pharmacokinet ; 27(5): 559-61, 2012.
Article in English | MEDLINE | ID: mdl-22447118

ABSTRACT

A 62-year-old Chinese patient with recurrent pompholyx submitted his blood sample for pre-treatment thiopurine S-methyltransferase (TPMT) pharmacogenetic profiling, and it was found to harbour a novel single nucleotide polymorphism (SNP). The novel SNP, detected by mRNA sequencing, was a c.2T>C (g.11018T>C) transition in the start codon, causing a Met1Thr amino acid change. This finding was confirmed on a subsequent blood sample from the same patient by DNA sequencing. The patient was genotyped as TPMT*1/*29, sequentially named as such following the latest TPMT SNP (TPMT*1/*28) at the time of writing. The novel SNP is expected to result in complete lack of protein translation, similar to the impact exerted by TPMT*14, another start codon SNP of the TPMT gene.


Subject(s)
Asian People/genetics , Methyltransferases/genetics , Base Sequence , Codon, Initiator , Genotype , Humans , Male , Middle Aged , Molecular Sequence Data , Polymorphism, Single Nucleotide , Sequence Analysis, DNA/methods , Sequence Analysis, RNA/methods
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