Single-Nucleotide DNA Fingerprinting of Hepatitis C Virus Using the BESS T-Scan™ and New BESS G-Tracker™ Mutation Detection and Localization Kits

Hepatitis C virus (HCV) is the primary cause of non-A and non-B hepatitis. Related to the Flaviviridae and Pestiviridae virus families, HCV consists of a positive-sense RNA genome that ranges between 9.4-9.5 kb in length containing an approximately 340-base highly conserved 5'-noncoding region, a single open reading frame of approximately 9,000 bases, and a 200-300 base 3'-untranslated region.1,2 The heterogeneity of the HCV genome is high and results in numerous genotypes and subtypes that previously could only be classified effectively by DNA sequencing.3 Because complete sequencing of every virus isolate would be laborious, methods that characterize only the conserved 5'-noncoding region have been used to classify HCV into genotypes and subtypes including sequencing,1 RFLP,4 SSCP,1 dideoxy fingerprinting,5 probe specific hybridization,6 Cleavase® Fragment Length Polymorphism (CFLP®),7 and serotyping.8 In this report, we demonstrate the use of the BESS T-Scan™ Mutation Detection and Localization Kit* and the new BESS G-Tracker™ Mutation Detection and Localization Kit* to perform direct, single-nucleotide analysis of the 5'-noncoding region of uncharacterized HCV isolates.

The BESS T-Scan Kit is a rapid and powerful method for detecting and localizing approximately 95% of all mutations.9 The BESS T-Scan procedure involves PCR amplification using one labeled primer in the presence of a limiting amount of dUTP. The uracil-containing PCR product is then enzymatically cleaved at the sites of deoxyuridine incorporation, resulting in a set of nested fragments that are then separated on a standard sequencing gel. However, when detecting point mutations, the BESS T-Scan Kit is limited to detecting those mutations that involve changes in deoxythymidine content (10 of 12 possible point mutations). We have recently developed a new mutation detection and localization kit, the BESS G-Tracker Kit, that specifically detects mutational changes in deoxyguanosine.

The BESS G-Tracker Kit functions similarly to the BESS T-Scan Kit. With the BESS G-Tracker Kit, deoxyguanosine is first partially oxidized by the BESS G-Tracker Modification Reagent in the presence of visible light. The resulting modified DNA strand is then cleaved into nested fragments using the BESS G-Tracker Excision Enzyme Mix. The resulting DNA ladder looks virtually identical to a dideoxyguanosine sequencing reaction except the fragments migrate through the sequencing gel approximately one base-pair faster than sequencing reaction products. This combination of the BESS T-Scan Kit and the new BESS G-Tracker Kit is the first non-sequencing mutation detection method that is capable of detecting 100% of all mutations, while localizing mutations at the single-nucleotide level.

Methods

Isolation and analysis of HCV RNA

HCV RNA analysis was performed on the 5'-noncoding region of the virus. PCR primers and human serum containing HCV were kindly provided by Dr. Yury Khudyakov and Dr. Howard Fields, Hepatitis Branch, Centers for Disease Control, Atlanta, GA. Total RNA was isolated from 50 µl of serum using the MasterPure™ Complete RNA Isolation Kit (Epicentre Technologies). RT-PCR was performed using the MasterAmp™ RT-PCR Kit (Epicentre Technologies). The sequences of the primers used were: HCVRT-F, 5'-CTGTGAGGAACTACTGTCTTC-3'; HCVRT-R, 5'-GGTGCACGGTCTACGAGACCT-3'. The primers amplify a 296 bp fragment of the 5'-noncoding region corresponding to bases 28 to 323 of the representative HCV sequence HCVJK1G (Figure 1). Each 25 µl RT-PCR contained ~100-400 copies of HCV RNA template, 1X MasterAmp RT-PCR Buffer, 3 mM MgCl2, 0.5 mM MnSO4, 2X MasterAmp PCR Enhancer (with betaine†), 400 µM each dNTP, 25 pmoles each of forward and reverse primer, and 1.25 units of RetroAmp™ RT DNA Polymerase. Cycling parameters were as follows: 60°C for 20 minutes (reverse transcription reaction), 92°C for 2 minutes, followed by 35 cycles of 92°C for 1 minute, 54°C for 1 minute, and 68°C for 1 minute, and finishing with a 4-minute incubation at 68°C. Samples were fractionated on a 2% agarose gel and visualized on a UV transilluminator. In some samples, the RT-PCR product was faint or not visible at all. Even in these situations, enough RT-PCR product was available for the following internal nested PCR.