目的: 通过检测评估荧光定量聚合酶链反应(PCR)染料法对人类白细胞抗原B位点5801(HLA-B*5801)等位基因的准确性、灵敏度及其在干扰因素下的结果分析研究,同时与PCR-SBT测序法进行对比。方法: 收集45例痛风、高尿酸血症患者和20例正常人群样本,利用HLA-B*5801等位基因检测试剂盒进行检测,同时利用测序法对HLA-B等位基因高度突变的2/3/4外显子正反测序。灵敏度验证研究,随机选取1例阳性样本,按照HLA-B*5801等位基因检测试剂盒说明书要求稀释至最低浓度,检测结果与测序结果对比;抗干扰能力验证研究,随机选取3例阳性样本,加入规定要求的胆红素、脂质、阿司匹林作为干扰因素,利用PCR染料法检测,检测结果与测序结果对比。结果: 45例痛风、高尿酸患者中有8例HLA-B*5801基因阳性,阳性率为17.8%;20例正常人群则有1例携带HLA-B*5801基因,阳性率5%。最低浓度样本也可正确检出结果,PCR染料法的准确性、灵敏性、特异性均为100%;3例阳性患者在胆红素、脂质等干扰物质影响下仍可正常检出,抗干扰能力良好。结论: PCR染料法可正确检测HLA-B*5801基因,灵敏度较好且在干扰物质存在的状况下结果不受影响,与测序结果一致,且操作更快捷方便。
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.
[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
