Genotyping hepatitis C virus isolates from Spain, Brazil, China, and Macau by a simplified PCR method
Genotyping hepatitis C virus isolates from Spain, Brazil, China, and Macau by a simplified PCR method
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DOI：
10.1128/jcm.34.10.2372-2378.1996
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发表时间：
1996-10-01
影响因子：
9.4
通讯作者：
Okamoto, H
中科院分区：
文献类型：
--
作者：
Holland, PV;Barrera, JM;Okamoto, H
为改进和简化丙型肝炎病毒(丙型肝炎病毒)分离株的基因分型方法，建立了一种新的基因分型方法，将丙型肝炎病毒分离株分型为I/1a、II/1b、III/2a、IV/2b和V/3a五种常见的基因分型。通过比较丙型肝炎病毒基因组1~9不同基因型分离株的319个核心基因序列，设计了针对5种常见基因型的正、反义引物。在第一轮聚合酶链式反应中，用通用引物扩增出了代表核苷酸319~751的433bp的序列。第二轮聚合酶链式反应分别用正义和反义引物进行，一次是扩增基因Via和II/1b，另一次是扩增基因III/2a、IV/2b和V/3a。用191份已知核心基因序列的血清样本证实了基因分型的特异性：110份感染5种常见基因的丙型肝炎病毒血清样本和81份感染其他基因类型的丙型肝炎病毒血清样本。用II/1b型的正、反义引物(389和492)消除了IV/1b型的反义引物(133)与本方法发现的部分I/1a型丙型肝炎病毒分离株的交叉反应。用该方法对来自西班牙、巴西、中国和澳门的13 0株丙型肝炎病毒分离株进行了基因分型。对5株西班牙分离株和5株澳门分离株的NS5B区(8279~8607位核苷酸)进行了329个碱基的测序，并与已知基因型别的分离株进行了比较，其中2株西班牙分离株为1D型，1株为1D型，2株西班牙分离株为新的基因群2，5株澳门分离株均为6a型。
An improved and simplified method of genotyping was developed for classifying hepatitis C virus (HCV) isolates into the five common genotypes, i.e., I/1a, II/1b, III/2a, IV/2b, and V/3a, by PCR with genotype specific primers deduced from the core gene. Sense and antisense primers, specific for each of the five common genotypes, were designed by comparison of 319 core gene sequences from HCV isolates of various genotypes from genetic groups 1 to 9. In the first round of PCR, a sequence of 433 bp representing nucleotides 319 to 751 was amplified with universal primers. The second round of PCR was performed with respective sense and antisense primers in two separate reactions, one for the amplification of genotypes Via and II/1b and the other for the amplification of genotypes III/2a, IV/2b, and V/3a. The specificity of genotyping was confirmed with a panel of 191 serum samples containing HCV isolates whose core gene sequences were known: 110 serum samples infected with HCV of the five common genotypes and 81 serum samples infected with HCV of other genotypes. The use of sense and antisense primers for genotype II/1b (primers 389 and 492) abolished the cross-reaction of the antisense primer for genotype IV/1b (primer 133) with some HCV isolates of genotype I/1a found by our original method. The new method was used for genotyping 130 HCV isolates from Spain, 53 from Brazil, 106 from China, and 30 from Macau. A total of 329 bp of the NS5b region (nucleotides 8279 to 8607) of five isolates from Spain and five isolates from Macau which could not be classified as any of the five common HCV genotypes or genotype 2c were sequenced, and the sequences were compared with those of HCV isolates of known genotypes; two isolates from Spain were deduced to be of genotype 1d and one was deduced to be of genotype 1d, while the remaining two isolates from Spain had novel genotypes in genetic group 2; however, all five isolates from Macau were of genotype 6a.