Comparative analysis of two systems for HCV genotyping.
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The HCV genotype can be determined by PCR using nested primers to structural or non-structural HCV regions, followed by hybridization analysis of the amplified products. In this study, two different systems, both based on PCR and hybridization analysis, were used to determine HCV genotype in 32 HCV positive patients at the Clinic of Infectious Diseases, University of Chieti. The main difference between these commercially available systems lies in the different PCR target. Amplification of PCR targets was obtained from all samples. Hybridization analysis gave unequivocal results for all samples with both methods, yielding a 100% rate of genotype determination, with a complete correlation at the genotype level. A lower concordance at subtype level (65% concordance) was found, due only to two types of discrepancies. Both methods proved easy to use in our hands, adding evidence to their potential usefulness and reliability in clinical settings.Keywords:
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ABSTRACT Genotyping and subtyping of 225 samples with hepatitis C virus (HCV) genotype 1, 2, 3, or 6 infection were done with Versant LiPA 2.0 and Abbott RealTime HCV Genotype (GT) II by using direct sequencing of the NS5B and 5′ untranslated regions as the reference standards. The concordance rates were >99.2% for genotypes and 96.1% for subtypes 1a and 1b. Both the Abbott RealTime and Versant LiPA assays can accurately determine hepatitis C virus genotypes. (This study has been registered at ClinicalTrials.gov under registration no. NCT00979979.)
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Background and Objectives Human platelet alloantigen ( HPA ) genotyping is important for the diagnosis and prevention the alloimmune platelet disorders. In this study, a simultaneous genotyping method for HPA ‐1 to ‐28bw systems was established using multiplex PCR ‐ SBT and the frequencies of genotypes and alleles of HPA ‐1 to ‐28bw systems in the Zhejiang Han population were analysed. Materials and Methods The specific primers were designed according to the nucleotide sequences of HPA ‐1 to 28bw systems which are located in ITGB 3, GP 1 BA , ITGA 2B, ITGA 2, GP 1 BB and CD 109, respectively. The multiplex PCR amplification systems were used, and then, the amplicons were purified and sequenced. A total of 335 healthy volunteer blood donors were detected. Results The genotypes of ten reference samples from Platelet Immunology Workshop of ISBT were in concordance with the known genotypes. Among the 28 HPA systems, HPA a and b alleles were found in HPA ‐1 to 6w, HPA ‐15 and HPA ‐21w systems in the Chinese Han population, while only HPA aa genotype was detected in the other HPA systems. The frequencies of HPA ‐1a and HPA ‐1b were 0·993 and 0·007, with 0·943 and 0·057 for HPA ‐2a and HPA ‐2b, 0·527 and 0·473 for HPA ‐3a and HPA ‐3b, 0·997 and 0·003 for HPA ‐4a and HPA ‐4b, 0·991 and 0·009 for HPA ‐5a and HPA ‐5b, 0·980 and 0·020 for HPA ‐6wa and HPA ‐6wb, 0·508 and 0·492 for HPA ‐15a and HPA ‐15b and 0·994 and 0·006 for HPA ‐21wa and HPA ‐21wb. Conclusions One multiplex PCR ‐ SBT method for HPA s was established and the data of the study could help to prevent and treat for alloimmune thrombocytopenia.
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Duffy blood group genotyping is useful to ensure transfusion safety and determine the association of Duffy blood group polymorphism with diseases, and therefore has its clinical significance. In order to improve the existing methods for genotyping of Duffy blood group, which normally require post-PCR manipulation, a new method was developed by using 5'-nuclease assay (NA) with TaqMan minor groove binding (MGB) probes.Primers and TaqMan-MGB probes were designed and synthesized to genotype FY*A and FY*B alleles at Duffy blood group locus on a real-time PCR platform. A total of 120 samples were genotyped by using the new 5'-NA and conventional polymerase chain reaction with allele-specific primers (PCR-ASP). The results obtained by the two methods were compared.There was a complete concordance of results for all samples genotyped by 5'-NA and PCR-ASP. The retesting results of 5'-NA were consistent with those of the initial testing. The detection limit of 5'-NA was determined as 100 pg per reaction. The FY*A and FY*B allelic frequencies were 93.3% and 6.7% respectively in the Chinese Han population in Dalian.The 5'-NA for genotyping of Duffy blood group is simple, rapid, reliable, reproducible, sensitive, and high-throughput and is superior to PCR-ASP used in routine genotyping.
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The distribution and kinetics of hepatitis C virus (HCV) genotypes and the prevalence of mixed infections were studied in a group of 45 French patients with haemophilia A or B or von Willebrand's disease, 21 of them being anti-human immunodeficiency virus (HIV) positive; genotyping was carried out by three methods based on the core, 5′ untranslated region (5′ UTR), and the detection of type-specific NS4 antibodies. Genotyping of the 5′UTR revealed genotypes 1a (n = 10), 1b (n = 13), 2a (n = 3), 2b (n = 4), 2NC (n = 3), 3a (n = 10), and two mixed infections (1a + 1b and 3a + 2). Five of 33 patients showed a change from one HCV genotype to another. The core genotyping assay showed 8 of 45 mixed infections: 6/8 1a + 1b and 2/8 3a + 2. Sequencing of core polymerase chain reaction (PCR) products showed that mixed infection 1a + 1b could be explained by nonspecific annealing of the 1b primer to type 1a sequence. By designing new primers whose sequence was more specific to HCV types 1a and 1b, we could confirm 1a + 1b mixed infection in only one of six cases. Serotyping assay showed for 17 of 21 anti-HIV negative patients a concordance with the 5′ UTR genotype; however, only 6 of 19 anti-HIV positive patients showed detectable serological reactivity. In summary, we have observed a similar HCV genotype distribution between our haemophilic group and the French anti-HCV positive patients. The study demonstrates the difficulties of assessing with the presently available genotyping and serotyping assays the real prevalence of mixed infections in multiply transfused patients. J Med Virol 51:36–41, 1997. © 1997 Wiley-Liss, Inc.
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Minicircle
Kinetoplast
Chagas Disease
Xenodiagnosis
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Abstract Twenty‐four patients with hepatitis C virus (HCV) antibody from the Chinese North Western province of Jilin were further analysed by the immunoblot assay‐2 (RIBA‐2). reverse transcription‐polymerase chain reaction (RT‐PCR) for serum HCV RNA detection, and direct sequence‐geno‐typing. Good concordance was found between the original second generation HCV antibody ELISA, RIBA‐2, and serum HCV RNA. The occurrence of genotype I (la), a genotype not previously reported in China, is described in 5(20.8%) of 24 cases, in association with genotypes II(1b) and III(2a) which were found in 16(66.7%) and 3(12.5%) of 24 cases, respectively. Imported blood products were unlikely to be the source of infection with genotype I (1a) but could not be definitively ruled out. © 1995 Wiley‐Liss, Inc.
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Four most widely accepted genotyping methods for hepatitis C virus (HCV) were applied to 40 HCV RNA isolates obtained from Slovenian patients in order to determine the concordance and applicability of various genotyping systems. The four methods are: (i) amplification of the core region with genotype-specific primers; (ii) nested polymerase chain reaction (PCR) in the core region followed by hybridization to HCV type-specific probes; (iii) reverse hybridization with the line probe assay Inno LiPA (Innogenetics, Gent, Belgium) using type-specific probes for the 5' non-coding region (NCR); and (iv) restriction fragment length polymorphism analysis of DNA amplified from the 5' NCR. Additionally, in isolates with discordant results nucleotide sequence analysis of a part of the NS-5 region was performed. Both genotyping methods based on the analysis of the 5' NCR were found more sensitive than those methods based on the analysis of the HCV core region. None of the four genotyping methods correctly classified all Slovenian HCV RNA isolates. PCR with genotype-specific primers was identified as entirely unsuitable for genotyping of Slovenian HCV RNA isolates. The remaining genotyping methods could clearly differentiate between HCV genotypes, but were not entirely reliable for HCV subtyping. The specificity of genotyping methods, which are based on the 5' NCR or the core region, was occasionally hampered, due to a lack or excess of sequence variation in their respective target regions.
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