VALIDATION OF A REAL-TIME PCR ASSAY FOR THE DETECTION OF THE SINGLE NUCLEOTIDE POLYMORPHISM RS1495741 IN THE HUMAN NAT2 GENE ASSOCIATED WITH XENOBIOTIC ACETYLATION

Relevance. According to polymorphisms in the N-acetyltransferase 2 gene, two main acetylation types are distinguished: rapid and slow. The frequency of adverse drug reactions varies depending on the rate of metabolism. In particular, the incidence of neuro- and hepatotoxic effects of isoniazid is associated with genotype, with slow acetylators having a higher risk of adverse reactions.

Aim: To design and compare a reagent kit developed by the authors for detecting polymorphisms in the human N-acetyltransferase 2 gene using real-time Polymerase chain reaction, aimed at determining the type of xenobiotic acetylation in humans.

Materials and Methods. Based on Eurasian Patent No. 042541, a laboratory prototype of a medical diagnostic reagent kit was developed. The kit included a real-time Polymerase chain reaction mixture, forward and reverse primers specific to the N-acetyltransferase 2 gene, and probes for detecting the rs1495741 polymorphism labeled with FAM and R6G fluorophores at the 5'-end and BHQ2 RTQ1 quenchers with closed nucleotides. Positive controls were constructed as circular plasmids pJet1.2/blunt containing the N-acetyltransferase 2 gene with rs1495741A and rs1495741G alleles. Using the developed kit, 37 Deoxyribonucleic acid samples obtained from healthy individuals were analyzed and compared with the reference method. Analytical sensitivity, diagnostic sensitivity, diagnostic specificity, precision, and reproducibility were assessed to validate the method.

Results. Validation of the reagent kit for real-time Polymerase chain reaction determination of the xenobiotic acetylation type in humans was performed. The analytical sensitivity of the test, determined by titration of the positive control, was 0.1 ng/µL Deoxyribonucleic acid; diagnostic sensitivity was 0.94, and diagnostic specificity was 1. Other validation parameters met the established acceptability criteria, confirming adequate intra-laboratory precision and inter-laboratory reproducibility.

Conclusion. Compared with the reference method, the proposed approach is less labor-intensive, time-saving, and more cost-effective, while maintaining comparable sensitivity and specificity. Based on the validation results, the laboratory prototype of the real-time Polymerase chain reaction kit for detecting the rs1495741 polymorphic site in the human N-acetyltransferase 2 gene associated with xenobiotic acetylation can be recommended for use in human genetic studies.

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