Bioassay

Performance evaluation and application of fluorescent quantitative PCR dye method in HLA-B*5801 allele detection*

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  • Department of Pharmacy, People’s Hospital of Bozhou, Bozhou 236800, China

Received date: 2021-12-05

  Online published: 2024-06-26

Abstract

Objective: To evaluate the accuracy and sensitivity of fluorescent quantitative polymerase chain reaction (PCR) dye method for detecting the allele of human leukocyte antigen B locus 5801 (HLA-B*5801) and analyze the results under interference factors, and to compare it with PCR-SBT sequencing method. Methods: 45 patients with gout, hyperuricemia and 20 normal people were collected and tested with HLA-B*5801 allele detection kit, while 2/3/4 exons with highly mutated HLA-B allele were sequenced by sequencing. In the sensitivity validation study, one positive sample was randomly selected and diluted to the lowest concentration according to the instructions of HLA-B*5801 allele detection kit, and the test results were compared with the sequencing results; In the anti-interference verification study, three positive samples were randomly selected, bilirubin, lipid and aspirin were added as interference factors, PCR dye method was used for detection. The test results were aligned to the sequencing results. Results: HLA-B*5801 gene was positive in 8 of 45 patients with gout and hyperuricemia, the positive rate was 17.8%; One of the 20 normal people carried HLA-B*5801 gene, with a positive rate of 5%. The accuracy, sensitivity and specificity of PCR dye method are all 100%; Three positive patients could still be detected normally under the influence of bilirubin, lipid and other interfering substances, and their anti-interference ability was good. Conclusion: The PCR dye method can correctly detect the HLA-B*5801 gene, with good sensitivity and unaffected results in the presence of interfering substances, consistent with the sequencing results, and faster and convenient to operate.

Cite this article

LIANG Hai, XIA Ru-nan, ZHAO Huan-huan, ZHANG Peng-cheng, LU Ning, LIN Zi-wei, WU Wei . Performance evaluation and application of fluorescent quantitative PCR dye method in HLA-B*5801 allele detection*[J]. Chinese Journal of Pharmaceutical Analysis, 2022 , 42(12) : 2092 -2100 . DOI: 10.16155/j.0254-1793.2022.12.04

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