Holland P V, Barrera J M, Ercilla M G, Yoshida C F, Wang Y, de Olim G A, Betlach B, Kuramoto K, Okamoto H
Sacramento Blood Center, California, USA.
J Clin Microbiol. 1996 Oct;34(10):2372-8. doi: 10.1128/jcm.34.10.2372-2378.1996.
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 I/1a 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 II/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 4d 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.
开发了一种改进且简化的基因分型方法，通过聚合酶链反应（PCR），利用从核心基因推导的基因型特异性引物，将丙型肝炎病毒（HCV）分离株分为五种常见基因型，即I/1a、II/1b、III/2a、IV/2b和V/3a。通过比较来自遗传组1至9的不同基因型HCV分离株的319个核心基因序列，设计了针对五种常见基因型各自的正义和反义引物。在第一轮PCR中，用通用引物扩增代表核苷酸319至751的433 bp序列。第二轮PCR在两个单独反应中分别用各自的正义和反义引物进行，一个用于扩增基因型I/1a和II/1b，另一个用于扩增基因型III/2a、IV/2b和V/3a。用一组191份血清样本（其中含有核心基因序列已知的HCV分离株）证实了基因分型的特异性：110份血清样本感染了五种常见基因型的HCV，81份血清样本感染了其他基因型的HCV。使用针对基因型II/1b的正义和反义引物（引物389和492）消除了我们原方法中发现的反义引物（引物133）对一些基因型I/1a的HCV分离株的交叉反应。该新方法用于对来自西班牙的130个HCV分离株、来自巴西的53个、来自中国的106个和来自澳门的30个进行基因分型。对来自西班牙的五个分离株和来自澳门的五个分离株的NS5b区域（核苷酸8279至8607）共329 bp进行了测序，这些分离株不能归类为五种常见HCV基因型或基因型2c中的任何一种，并将序列与已知基因型的HCV分离株序列进行比较；来自西班牙的两个分离株推断为基因型4d，一个推断为基因型ld，而来自西班牙的其余两个分离株在遗传组2中有新的基因型；然而，来自澳门的所有五个分离株均为基因型6a。
