流式荧光点阵技术在乙型肝炎病毒基因分型中的应用

doi:10.3969/j.issn.1673-8640.2018.05.015

摘要/Abstract

摘要：

目的建立一种基于流式荧光点阵技术（又称液态芯片法）的针对乙型肝炎病毒（HBV）基因型B型和C型的检测方法。方法设计HBV B和C基因型的型特异性引物,其中正向引物的5' 端作生物素化标记。在多重聚合酶链反应（PCR）扩增后将扩增产物与连接在不同荧光编码微球上的型特异性探针在同一体系中进行杂交,再用链霉亲和素-藻红蛋白进行标记,用Luminex多功能流式点阵仪进行检测。对经测序法基因分型的67例（B型24例、C型43例）样本进行检测,比较并确定方法的有效性。结果本研究所建方法与测序法结果一致的比例为94%（63/67）。在24例测序结果为B型的患者中有1例患者用自建液态芯片法检测显示B型和C型探针双阳性信号;43例测序法结果为C型的患者中有3例患者用自建液态芯片法检测为B型和C型探针双阳性信号。结论自建液态芯片法能准确地进行HBV基因分型,尤其是当患者为HBV混合型感染时,自建液态芯片法能进一步确定不同基因型,在临床应用时更具有优势。

关键词: 乙型肝炎病毒, 基因型, 多重聚合酶链反应, 杂交, 流式荧光点阵技术

Abstract:

Objective To establish a flow fluorescent dot matrix technique （liquid microarray） for the determinations of hepatitis B virus （HBV） genotype B and C. Methods Specific primers against genotype B and C of HBV were designed to have 5' terminal labeled by biotin. After amplification by multiple polymerase chain reaction （PCR）,the products were hybridized with different genotype specific oligonucleotide probes conjugated on the beads coded with different fluorescence. The beads were stained with streptavidin-phycoerythrin,and were determined by Luminex. A total of 67 samples were determined for genotyping by sequencing （24 cases of genotype B and 43 cases of genotype C）. The efficiency of liquid microarray was evaluated. Results The results of liquid microarray were compared with those of sequencing,and the consistency rate was 94% （63/67）. In 24 cases of genotype B determined by sequencing,only 1 case got double-positive signals from genotype B and C probes determined by liquid microarray. However,in 43 cases of genotype C,there were 3 cases getting double-positive signals. Conclusions Liquid microarray is accurate and effective for HBV genotyping,and it has the advance for the further determination of mixed infection of HBV with different genotypes.

Key words: Hepatitis B virus, Genotype, Multiple polymerase chain reaction, Hybridization, Flow fluorescent dot matrix technique

中图分类号:

R446.1

朱俊, 朱宇清, 朱岭峰, 蒋玲丽, 徐翀. 流式荧光点阵技术在乙型肝炎病毒基因分型中的应用[J]. 检验医学, 2018, 33(5): 437-441.

ZHU Jun, ZHU Yuqing, ZHU Lingfeng, JIANG Lingli, XU Chong. Flow fluorescent dot matrix technique for hepatitis B virus genotyping[J]. Laboratory Medicine, 2018, 33(5): 437-441.

图/表 3

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样本	B型探针杂交的MFI值	C型探针杂交的MFI值
空白	10	22
18号（C型）	16	523
19号（C型）	35	2 180
20号（C型）	18	1 776
33号（B型）	1 935	0
34号（B型）	1 634	14
人DNA	9	30
Sau DNA 1	13	2
Sau DNA 2	13	10
Sau DNA 3	14	13
HPV DNA 1	0	3
HPV DNA 2	17	0
HPV DNA 3	17	9

样本	B型探针杂交的MFI值	C型探针杂交的MFI值
空白	10	22
18号（C型）	16	523
19号（C型）	35	2 180
20号（C型）	18	1 776
33号（B型）	1 935	0
34号（B型）	1 634	14
人DNA	9	30
Sau DNA 1	13	2
Sau DNA 2	13	10
Sau DNA 3	14	13
HPV DNA 1	0	3
HPV DNA 2	17	0
HPV DNA 3	17	9