HBV-DNA is hybridized with a HBV RNA probe. The DNA-RNA hybrids are immobilized onto a microtiter plate using anti-DNA-RNA antibodies. Antihybrid antibodies conjugated to an enzyme are used for detection in combination with appropriate chemiluminescent substrates [23,24].

For the branched DNA (bDNA) assay, HBV-specific probes are used to capture HBV DNA in the sample on the microtiter plates. Subsequently, extender probes, bDNA and dioxetane substrate, are used for detection and the bDNA molecules serve as signal amplification molecules [23,25].

The most accurate method for hepatitis B virus (HBV) genotyping [34,35] is based on phylogenetic analysis following DNA sequencing of the entire viral genome, often restricted to the S gene [36]. This is the gold standard of HBV genotyping, the technique is highly sensitive and allows the detection of new and recombinant genotypes, but it is considered to be technically challenging, time-consuming, and costly; and cannot be used in large-scale studies. Furthermore, in the case of multiple-genotype infection, only the most abundant genotype is identified [36].

A commercial direct-sequencing assay kit is available (TRUGENE HBV Genotyping Kit; Siemens Medical Solutions Diagnostics, NY, USA). Both genotypes and HBV sequence mutations can be detected in plasma or serum specimens, simultaneously [37]. TRUGENE1 HBV Genotyping Assay (RUO). Siemens Medical Solutions Diagnostics, Tarrytown, NY, USA. Information available at: http: //www.medical.-siemens.com (accessed June 2010).

A commercially available reverse-hybridization-based line probe assay (INNO LiPA HBV Genotyping assay, LiPA) is easy to perform and is also suitable for detecting mixed genotype infections [38,31]. HBV DNA is amplified by PCR using biotinylated primers complementary to a conserved sequence in the S/pre-S ORF.51. The overall success rate for the detection of all eight HBV genotypes by this method was 98%. INNO-LiPA overestimates mixed infections as a result of erroneous genotype H detection [39].

A PCR-RFLP-based (usually gen S amplification) method that involves successive digestion of amplicon with a battery of restriction enzymes to discriminate the individual genotypes. It is a simple and cost-effective method which can detect mixed genotypes. It can also determine subgenotypes and can be used in large population studies [40].

A multiplex PCR method to identify genotypes using genotype-specific primers in two reactions based on core/surface/polymerase region. Multiplex PCR had a higher accuracy (93.2%) compared to the RFLP method (87%). This method can detect mixed genotypes while having sufficient sensitivity for detecting minor species as low as 10% [41].

This method may be useful for HBV genotyping in large-scale clinical and epidemiological studies. The simplicity and rapidity of this PCR assay may reduce the cost and complexity of recognizing these genotypes. Detection sensitivity for this assay was approximately 100-200 copies/mL of blood.

In this method, HBV serotypes or subtypes are recognized using antibodies against HBsAg. In an enzyme immunoassay format, they are more cost-effective, easy to perform, and have higher throughput. This method is suitable for large epidemiological studies and population screening [41].

The MassARRAY system based on nucleic acid analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides an alternative approach to HBV genotyping. The MALDI-TOF MS MassARRAY system is capable of detecting wild-type and mutant alleles and can identify mixes if the minority type is present at >10% This technology is less costly and easy to use as it is amenable to automation and has already found application in other disease evaluations[42].

The assay is cost-effective because of its relatively high throughput, and it is approximately half the price of sequencing and is 15 times less expensive than InnoLipa. The assay is rapid, the technology allows the testing of 960 specimens a day without requiring any additional analysis, nor any data interpretation by the operator after the data collection is completed. It provides the opportunity to detect the presence of new sequence variants and automatically adds them to the reference database.

Other studies have reported the use of MALDI-TOF mass spectrometry for the determination of YMDD (tyrosine-methionine-aspartate-aspartate motif) mutations, which are linked to lamivudine drug resistance. In these studies, this method was found to be superior to DNA sequencing since it has a lower detection limit (100 copies/ml) and can also detect mutations in samples containing wild types [42].

With the development of real-time PCR, several methods have been developed for HBV quantification and genotyping in a single reaction using real-time PCR with additional melting-curve analyses. In these assays, the first step involves the use of the real-time PCR for quantification whilst the second step involves melting curve analyses for differentiating the HBV genotypes. The melting temperature (Tm) value differs among different genotypes depending on the complement between probe and target, and/or GC-content, of the hybridization sequence [43,44]. The main disadvantage of this method is that it is less able to distinguish between genotypes having close proximity between their Tm values, such as A and C.

A new method has been developed for genotyping which makes use of the TaqMan real-time PCR in a multiplex manner this is to identify all genotypes without the need for post-PCR steps. In this assay, each sample is processed in four multiplex real-time PCRs, each targeting two or three genotype-specific HBV segments. The assay is easy to use and therefore well-suited for routine application in the modern diagnostic laboratory [45].

The use of an absolute quantitative real-time PCR assay allows one to detect mixed infections of different genotypes. It would be useful to know what the relative concentrations are for genotypes B and C in the mixed infections [46].

Other methods, such as the Oligonucleotide microarray, can determine genotypes A-G. The amplified products are heat-denatured and added to silylated slides, to which genotype-specific probes are immobilized. Fifteen probes were designed, based on phylogenetic tree analysis and the alignment of 228 pre-S region sequences. Following washing and drying, fluorescence signals were captured using a scanner. Currently, DNA-Chip technology is not used routinely in the clinical laboratory although the potential is enormous. Besides the information relating to the HBV genotype, more sequence patterns related to antiviral resistance, or promoter sequence variation, can be located on a single chip. However, it should also be noted that an easy-to-use assay for this technology is not yet available and that the number of samples that can be analyzed is limited to between 8 and 12 per day [47,48].

The COBAS^® Amplicor HCV Test v2.0 is performed on the COBAS^® AMPLICOR Analyzer (Roche Molecular systems). HCV RNA is isolated from virions by lysis of viral particles and alcohol precipitation. An RNA script with primer binding identical regions to those of the HCV target is introduced into each sample and serves as an internal control. The test utilizes reverse transcription of target RNA to generate complementary DNA (cDNA), which is denatured by heating, and then the target cDNA is amplified by the Polymerase Chain Reaction (PCR) and nucleic acid is hybridized for the detection of HCV RNA in human serum or plasma. In the last phase, a coloured complex is formed, which is measured at 660 nm [86].

Transcription-mediated amplification (TMA) is an isothermal nucleic acid amplification process that involves a more complex set of reactions with the reverse transcriptase and T7 RNA polymerase.

The Versant HCV RNA Qualitative Assay (Siemens Molecular): In this method, sample preparation, target amplification and amplicon detection are carried out in a single tube. Following a lysis step, target HCV RNA is captured by magnetic particles coated with oligonucleotides complementary to the 5′UTR of the HCV genome. Monitoring of target capture and amplification is achieved by adding an internal control RNA to each sample. The target RNA is then amplified with an isothermal TMA process that requires the addition of primers, reverse transcriptase, and T7 RNA polymerase. Some of the newly synthesized RNA amplification products re-enter the TMA process and serve as templates for new rounds of amplification. To detect the amplified product, TMA-labelled oligonucleotide probes are used to emit a chemiluminescent signal [86]. The LLOD is 5 IU/ml and 10 IU/ml with a sensitivity from 96% to 100%, respectively. It is independent of HCV genotypes [86,87] and may be useful in predicting which patients are at risk from virological release after cessation of antiviral therapy, helping to define treatment response and determine acute infection [88].

In real-time PCR monitors, the fluorescence emitted during amplification can be detected during the enzymatic reaction in each PCR cycle. This differs from end-point PCR by using fluorogenic probes, primers, and amplicons. Quantitative measurements occur during the exponential phase of the amplification step; the quantity of the amplification products is proportional to the quantity of the HCV or internal standard initially present in the sample. These assays have a broad dynamic range, improving LLOD ≤ 10 IU/mL without the need for pre-dilution in addition to being specific, accurate and reproducible. The most common assays available for fluorescence detection include: hydrolysis probe assays, hybridization probes such as molecular beacons, dual-hybridization probes, scorpion primers and DNA binding dyes (SYBR green) [85,90]. There are two commercial quantitative real-time PCRs for HCV RNA:

The Abbott Real Time HCV assay (Abbott Laboratories, IL., USA) is performed on the m2000sp for sample extraction and m2000rt for amplification-quantitative processing.

The Cobas AmpliPrep/Cobas TaqMan HCV Test (Roche Molecular Systems) is integrated into the fully automated Docking Station^® modular platform. It is a probe-based method, which combines automated isolation on the COBAS AmpliPrep^TM followed by amplification-quantitative detection on the COBAS Taqman 48/96 Analyzer^TM [91]. Reverse transcription and amplification primers, as well as the probe, are targeted to the HCV 5´UTR of HCV. In each complete process, a quantitative standard is included to compensate for the inhibition effects and for process control. It has an LLOD and high performance but there are two critical points: sample overestimation with high viral loads and viral load underestimation for genotype 2 and 4 [92].

LightCycler (Biorad, CA, USA) uses hybridization probes and SYBR Green (Molecular Probe Inc) uses DNA-binding agents. SYBR Green I, however, has a number of limitations that include the inhibition of PCR, preferential binding to GC-rich sequences and effects on melting curve analysis [93].

The AMPLICOR HCV MONITOR Test v2.0 (Roche Molecular Systems) is available on the COBAS AMPLICOR^® Analyzer, which allows automated amplification, detection, and reporting for HCV RNA quantification. The primers allow RT-PCR of the 5′UTR region of the HCV genome. On the COBAS AMPLICOR Analyzer, thermal cycling and detection is carried out via a colorimetric format using suspensions of magnetic particles coated with probes specific for the HCV and QS amplicons. Absorbance measurements are made using the COBAS AMPLICOR. Although it is highly reproducible, it has low sensitivity and yield [94].

The risk of contamination is reduced by the elimination of target nucleic acid amplification. The target nucleic acid is immobilized to a support thus allowing different washes to take place so that false-positive results are kept to a minimum [95].

The most used is the Versant 3.0 Quantitative assay performed by the System 340 bDNA Analyzer (Siemens Medical Solutions Diagnostics). This is a semi-automated RNA test to quantify HCV, which uses a solid-phase oligonucleotide probe to capture the target RNA, followed by hybridization of a branched secondary (bDNA) probe. The capture probes and the target probes bind to the 5´UTR and core regions. The DNA amplifiers bind to enzyme-conjugated tertiary probes, and after substrate is added, the chemiluminescence produced is proportional to the amount of target RNA [96]. Currently, Siemens have released the VERSANT™ 440 Molecular Systems, a fully automated system [97].

The product amplified in the TRUGENE^® assay is neutralized, purified and subsequently sequenced in both directions by a capillary method (ClipTM). The obtained sequence is aligned with prototypical sequences of different genotypes and subtypes and then a computer program performs a phylogenetic analysis to determine genotype and subtype [107].

INNOLIPA HCV II is based on reverse hybridization and uses PCR products from the 5´UTR, after being denatured, it is subjected to hybridization with multiple probes and two control targets aligned and fixed to the nitrocellulose membrane by means of respective poly-(T) queues. The hybrids formed are evidenced by the addition of a conjugate (streptavidin-labelled alkaline phosphatase) followed by chromogenic substrate. The genotype is determined after alignment with the card strip test reference. LiPA II includes 22 probes of different genotypes and subtypes [108].

TRUGENE^® and INNO-LIPA assays can be performed with amplification products obtained from Roche AMPLICOR assays. Some reports have indicated that the two assays have similar abilities in determining genotypes (rates from 95-100%) but that the main limitation is the difficulty in accurately defining some subtypes of HCV strains [107].

The Invader HCV Genotyping Assay (Third Wave Technologies, Inc., Madison, Wisconsin) is based on the analysis of the 5'UTR, using DNA cleavase technology and FRET. The amplicons may be generated from different commercial methods. It can be completed in 1.5 h (after amplification) and is precise with genotypes yet imprecise with subtypes [109].

These use universal primers that are subjected to nested PCR followed by digestion of the amplicon with restriction enzymes at the genotype-specific site [110].

Other commercial assays are based on the analysis of subgenomic regions: such as the core region (GEN-ETI-K DEIA kit, Sorin, Italy) or the NS5B region (NS5B Genotyping Assay TRUGENE, Siemens Medical Solutions) or 5'UTR and core regions (VERSANT 2.0, Siemens). These have improved the discrimination between subtypes or the Abbott Real Time HCV Genotype^® (Abbott Molecular Diagnostics), where the RNA is purified from plasma and amplified using specific primers of the NS5B regions, 5'UTR and a recombinant thermostable polymerase with transcriptase and DNA polymerase activity. The cycles of amplification are realized on the ABI PRISM^® 7000 and the data obtained are analyzed using the Sequence Genotyping Software Program 2.0 (Celera Diagnostics, Alameda, California). The LOD is 1,200-1,500 U/ml. It does not accurately discriminate between genotypes 4 and 6 [111].

The PSMEA HCV genotyping test detects genotype-specific sequence differences in the 5´UTR and is considered to be a high throughput rapid genotyping assay that can reliably identify mixed HCV infections [112].

Liquid microarrays are used for HCV genotyping and are based on the analysis of the 5´UTR and NS5B regions. The xMap Technology (Luminex Corp, Austin, Texas) is based on short pieces of DNA attached to tiny plastic beads, or microspheres, floating in a sample. It is similar to a gene chip, where many nucleic acid sequences can be detected simultaneously with a LLOD of 50 copies/reaction [113].

Nanoparticles have been proposed as promising tools to develop the next generation of assays. The most common nanoparticles used are: quantum dots and gold nanoparticles (AuNPS).

AuNPS are spheres with a typical diameter of 2-50 nm and have the property known as “surface plasmon resonance”: when the particles are distributed evenly throughout a liquid, they reflect light in a way that makes them appear red; when they clump together, they look blue [114,115]. The RNA is extracted from the virion and then short pieces of DNA complementary to the HCV RNA and the gold nanoparticles are added to this solution. In the absence of HCV RNA, the primers stick to the gold nanoparticles and separate them, thus the solution appears red. If the virus is present, the primers pair with the viral RNA instead and the gold nanoparticles aggregate, turning the solution blue. The test is performed in a tube and takes only 30 minutes [115].