Rapid detection of the irinotecan-related UGT1A1*28 polymorphism by asymmetric PCR melting curve analysis using one fluorescent probe

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.