Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing, China.
Engineering Research Centre of Molecular Diagnostics, Ministry of Education, State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China.
J Clin Lab Anal. 2022 Aug;36(8):e24578. doi: 10.1002/jcla.24578. Epub 2022 Jun 29.
Determination of UGT1A1 (TA) polymorphism prior to irinotecan therapy is necessary to avoid severe adverse drug effects. Thus, accurate and reliable genotyping methods for (TA) 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) ] and *28 [(TA) ] genotypes.
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 s) were calculated to assess the reliability, and the limit of detection was examined to assess the sensitivity.
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 s were both ≤0.27%, with standard deviations ≤0.14°C. The limit of detection was 0.2 ng of input genomic DNA.
The developed PCR melting curve analysis using one fluorescent probe can provide accurate, reliable, rapid, simple, and low-cost detection of UGT1A1 (TA) polymorphism, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.
在使用伊立替康治疗前确定 UGT1A1（TA）多态性是避免严重药物不良反应所必需的。因此，非常需要准确可靠的（TA）多态性基因分型方法。在这里，我们提出了一种新的聚合酶链反应（PCR）熔解曲线分析方法，使用一个荧光探针来区分 UGT1A11[(TA)]和28[(TA)]基因型。
在方案优化后，该技术应用于 2016 年至 2021 年间在中国-日本友好医院招募的 64 例患者（包括 23 例 UGT1A1*1/1、22 例1/28 和 19 例28/*28）的基因分型。通过与直接测序和片段分析的结果进行比较来评估方法的准确性。计算熔解温度（Ts）的内和运行精度以评估可靠性，并检查检测限以评估灵敏度。
所有基因型均通过新方法正确识别，其准确性高于片段分析。Ts 的内和运行变异系数均≤0.27%，标准偏差均≤0.14°C。检测限为 0.2ng 输入基因组 DNA。
使用一个荧光探针开发的 PCR 熔解曲线分析可以提供准确、可靠、快速、简单和低成本的 UGT1A1（TA）多态性检测，并且可以在具有荧光 PCR 平台的临床实验室中轻松推广使用。
