Open AccessRapid detection of the irinotecan‐related UGT1A1 *28 polymorphism by asymmetric PCR melting curve analysis using one fluorescent probe
Author(s) -
Kong Xiaomu,
Xu Ye,
Gao Peng,
Liu Yi,
Wang Xuran,
Zhao Meimei,
Jiang Yongwei,
Yang Hui,
Cao Yongtong,
Ma Liang
Publication year - 2022
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.24578
Subject(s) - melting curve analysis , genotyping , irinotecan , genotype , high resolution melt , detection limit , microbiology and biotechnology , polymerase chain reaction , fluorescence , amplicon , repeatability , chromatography , biology , analytical chemistry (journal) , chemistry , genetics , physics , cancer , gene , colorectal cancer , quantum mechanics
Background Determination of UGT1A1 (TA) n polymorphism prior to irinotecan therapy is necessary to avoid severe adverse drug effects. Thus, accurate and reliable genotyping methods for (TA) n polymorphism are highly desired. Here, we present a new method for polymerase chain reaction (PCR) melting curve analysis using one fluorescent probe to discriminate the UGT1A1 *1 [(TA) 6 ] and *28 [(TA) 7 ] genotypes. Methods After protocol optimization, this technique was applied for genotyping of 64 patients (including 23 with UGT1A1 *1/*1, 22 with *1/*28, and 19 with *28/*28) recruited between 2016 and 2021 in China‐Japan Friendship Hospital. The accuracy of the method was evaluated by comparing the results with those of direct sequencing and fragment analysis. The intra‐ and inter‐run precision of the melting temperatures (T m s) were calculated to assess the reliability, and the limit of detection was examined to assess the sensitivity. Results All genotypes were correctly identified with the new method, and its accuracy was higher than that of fragment analysis. The intra‐ and inter‐run coefficients of variation for the T m s were both ≤0.27%, with standard deviations ≤0.14°C. The limit of detection was 0.2 ng of input genomic DNA. Conclusion The developed PCR melting curve analysis using one fluorescent probe can provide accurate, reliable, rapid, simple, and low‐cost detection of UGT1A1 (TA) n polymorphism, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.