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.
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
[1] LIU R, HAN C, WU D, et al. Prevalence of hyperuricemia and gout in mainland China from 2000 to 2014: a systematic review and meta-analysis[J].Biomed Res Int, 2015, 2015: 762820
[2] MUNGALL AJ, PALMER SA, SIMS SK, et al. The DNA sequence and analysis of human chromosome 6[J].Nature, 2003, 425(6960): 805
[3] HUNG SI, CHUNG WH, LIOU LB, et al. HLA-B*5801 allele as a genetic marker for severe cutaneous adverse reactions caused by allopurinol[J].Proc Natl Acad Sci USA, 2005, 102(11): 4134
[4] KANG HR, JEE YK, KIM YS, et al. Positive and negative associations of HLA class I alleles with allopurinol-induced SCARs in Koreans[J].Pharmacogenet Genomics, 2011, 21(5): 303
[5] CHOON SE, LAI NM. An epidemiological and clinical analysis of cutaneous adverse drug reactions seen in tertiary hospital in Johor, Malaysia[J].Indian J Dermalot Venereol Leprol, 2012, 78(6):734
[6] LI X, ZHAO Z, SUN SS. Association of human leukocyte antigen variants and allopurinol-induced Stevens-Johnson syndrome and toxic epidermal necrolysis: a meta-analysis[J].Am J Health Syst Pharm, 2017, 74(9): e183
[7] 方玲, 董敏, 汪燕燕. HLA-B*5801基因检测在别嘌醇严重过敏反应评价中的应用分析[J].中国药物警戒, 2021, 18(8): 772
FANG L, DONG M, WANG YY. Applicability of HLA-B*5801 allele detection in allopurinol irritated severe allergic reactions[J].Chin J Pharmacovigil, 2021, 18(8): 772
[8] SHIINA T, BLANCHER A, INOKO H, et al. Comparative genomics of the human, macaque and mouse major histocompatibility complex[J].Immunology, 2017, 150(2): 127
[9] CHENG L, ZHANG L, GAO L, et al. Genotyping HLA-B*5801 for allopurinol-induced severe cutaneous adverse reactions: an accurate and prompt method[J].Clin Transl Sci, 2015, 8(6): 834
[10] 曾大勇, 王长连, 黄品芳, 等. 别嘌醇引发严重皮肤不良反应标志基因HLA-B*5801的检测方法研究[J].中国现代应用药学, 2015, 32(6): 700
ZENG DY, WANG CL, HUANG PF, et al. Research on detection methods for HLA-B*5801: a biomarker for allopurinol induced serious cutaneous adverse drug reactions[J].Chin J Mod Appl Pharm, 2015, 32(6): 700
[11] 裴斌, 郭晓彤, 冉鹏, 等. 多色探针熔解曲线法应用于别嘌呤醇不良反应相关基因HLA-B*58: 01的检测[J].中国输血杂志, 2018, 31(1): 37
PEI B, GUO XT, RAN P, et al. Rapid detection of HLA-B*58: 01 gene based on multiplex melting curve analysis for the monitoring of adverse drug reaction caused by allopurinol[J].Chin J Blood Transf, 2018, 31(1): 37
[12] 中华医学会. 痛风基层合理用药指南[J].中华全科医师杂志, 2021, 20(6): 631
Chinese Medical Association. Guideline for rational medication of gout[J].Chin J Gen Pract, 2021, 20(6): 631
[13] 吴洁, 伊洁, 甘勇, 等. 实时荧光定量PCR在HLA-B*5801等位基因检测中的性能评价[J].中国医学装备, 2020, 17(2): 30
WU J, YIN J, GAN Y, et al. Performance evaluation of real-time fluorescence quantitative PCR in the determination of allele HLA-B*5801[J].China Med Equip, 2020, 17(2): 30
[14] 李育敏, 阚丽娟, 张水兰, 等. 荧光定量PCR测定HBV DNA的抗干扰能力评价[J].临床检验杂志, 2020, 38(2): 142
LI YM, KAN LJ, ZHANG SL, et al. Evaluation of anti-interference ability of fluorescence quantitative PCR in detection of HBV DNA[J].Chin J Clin Lab Sci, 2020, 38(2): 142
[15] 高会广, 王彩红, 王景胜, 等. 标本因素对实时荧光定量PCR检测丙型肝炎病毒RNA的影响[J].中国误诊学杂志, 2008, 8(15):3568
GAO HG, WANG CH, WANG JS, et al. Effect of specimen factors on real-time PCR detection of hepatitis C virus RNA[J].Chin J Misdiagn, 2008, 8(15):3568
