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Rapid detection of the irinotecan-related UGT1A1 & 5-fluorouracil related DPYD polymorphism by asymmetric polymerase chain reaction melting curve analysis.
Lian, Jiabian; Liang, Yaoji; Wang, Yunling; Chen, Ying; Li, Xun; Xia, Lu.
Afiliação
  • Lian J; Center for Precision Medicine, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361003, China; Department of Laboratory Medicine, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361003, China; Xiamen
  • Liang Y; Biochee Biotech Co.,Ltd., Xiamen, 361102, China; Amogene Biotech Co.,Ltd., Xiamen, 361102, China.
  • Wang Y; Biochee Biotech Co.,Ltd., Xiamen, 361102, China.
  • Chen Y; Amogene Biotech Co.,Ltd., Xiamen, 361102, China.
  • Li X; Center for Precision Medicine, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361003, China; Department of Laboratory Medicine, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361003, China. Electr
  • Xia L; Center for Precision Medicine, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361003, China; Department of Laboratory Medicine, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361003, China; Xiamen
Clin Chim Acta ; 561: 119761, 2024 Jul 15.
Article em En | MEDLINE | ID: mdl-38848897
ABSTRACT

BACKGROUND:

Determination of DPYD and UGT1A1 polymorphisms prior to 5-fluorouracil and irinotecan therapy is crucial for avoiding severe adverse drug effects. Hence, there is a pressing need for accurate and reliable genotyping methods for the most common DPYD and UGT1A1 polymorphisms. In this study, we introduce a novel polymerase chain reaction (PCR) melting curve analysis method for discriminating DPYD c.1236G > A, c.1679 T > G, c.2846A > T, IVS14 + 1G > A and UGT1A1*1, *28, *6 (G71R) genotypes.

METHODS:

Following protocol optimization, this technique was employed to genotype 28 patients, recruited between March 2023 and October 2023, at the First Affiliated Hospital of Xiamen University. These patients included 20 with UGT1A1 *1/*1, 8 with UGT1A1 *1/*28, 4 with UGT1A1 *28/*28, 22 with UGT1A1*6 G/G, 6 with UGT1A1*6 G/A, 4 with UGT1A1*6 A/A, 27 with DPYD(c.1236) G/G, 3 with DPYD(c.1236) G/A, 2 with DPYD(c.1236) A/A, 27 with DPYD(c.1679) T/T, 2 with DPYD(c.1679) T/G, 3 with DPYD(c.1679) G/G, 28 with DPYD(c.2846A/T) A/A, 2 with DPYD(c.2846A/T) A/T, 2 with DPYD(c.2846A/T) T/T, 28 with DPYD(c.IVS14 + 1) G/G, 2 with DPYD(c.IVS14 + 1) G/G, and 2 with DPYD(c.IVS14 + 1) G/G, as well as 3 plasmid standards. Method accuracy was assessed by comparing results with those from Sanger sequencing or Multiplex quantitative PCR(qPCR). Intra- and inter-run precision of melting temperatures (Tms) were calculated to evaluate reliability, and sensitivity was assessed through limit of detection examination.

RESULTS:

The new method accurately identified all genotypes and exhibited higher accuracy than Multiplex qPCR. Intra- and inter-run coefficients of variation for Tms were both ≤1.97 %, with standard deviations ≤0.95 °C. The limit of detection was 0.09 ng/µL of input genomic DNA.

CONCLUSION:

Our developed PCR melting curve analysis offers accurate, reliable, rapid, simple, and cost-effective detection of DPYD and UGT1A1 polymorphisms. Its application can be easily extended to clinical laboratories equipped with a fluorescent PCR platform.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Reação em Cadeia da Polimerase / Glucuronosiltransferase / Di-Hidrouracila Desidrogenase (NADP) / Fluoruracila / Irinotecano Limite: Humans Idioma: En Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Reação em Cadeia da Polimerase / Glucuronosiltransferase / Di-Hidrouracila Desidrogenase (NADP) / Fluoruracila / Irinotecano Limite: Humans Idioma: En Ano de publicação: 2024 Tipo de documento: Article