Genotypic susceptibility assays use PCR to detect genes known to be responsible for resistance, coupled with genetic sequencing to determine whether genome alterations associated with resistance have occurred. Geno typic assays use DNA sequencing by automated sequencers, PCR amplification and restriction enzyme digestion of the products, and hybridization to microarrays containing multiple oligonucleotide probes. These assays are rapid, because isolation of the virus in culture is not necessary for testing.
The response to an antiviral agent is also measured by quantitative monitoring of the viral load (by means of the nucleic acid concentration) in the patient’s blood. Such testing is common in patients infected with HBV, hepatitis C virus (HCV), and cytomegalovirus (CMV). The viral load should diminish significantly after addition of an antiviral agent to which the virus is susceptible. Using molecular testing (e.g., quantitative PCR) to measure the amount of virus in serum is a surrogate test for resistance to antiviral agents. The viral load rises quickly when resistance appears.
Pyrosequencing
DNA sequencing is among the most important testing methods for the study of biologic entities. Pyrosequencing, which is relatively new, is a sequence-based detection method that allows rapid, accurate quantification of sequence variation. It allows rapid acquisition of short reads (100 to 200 bp) of genomic sequence to identify known mutations. It is based on the technology of detection of released pyrophosphate (PPi) during DNA syn thesis. In a sequence of enzymatic reactions, a enzyme (polymerase) catalyzes the addition of nucleotides into a nucleic acid chain. As a result of this addition, a PPi molecule is released and converted to adenosine triphosphate (ATP) by the ATP enzyme sulfurylase. Visible light is produced when a luciferin molecule is oxidized during the luciferase reaction. The visible light or signal strength generated is proportional to the number of nucleotides incorporated into the final product.
The two types of pyrosequencing methods currently available are solid-phase pyrosequencing and liquid phase pyrosequencing. Solid-phase pyrosequencing involves a three-enzyme system that uses immobilized DNA, and a washing step is performed to remove excess substrate after each nucleotide addition. In liquid-phase pyrosequencing, a fourth nucleotide-degrading enzyme (made from potato) is added. The advantage of the liquid-phase system is that it eliminates the need for solid support and the intermediate washing step, allowing the reaction to be performed in a single tube.
Because of its rapid, accurate quantification of sequence variation, pyrosequencing is an adaptable tool that can be used for a wide range of applications.
Automation with pyrosequencing is made possible by the liquid-phase methodology. Pyrosequencing signals are quantitative, which allows a large number of people to be screened through examination of the allelic frequency in a population. This technique also is used taxonomically to group different organisms into strains or sub types, and it can be applied to bacteria, yeasts, and viruses. It is currently the fastest method for sequencing a PCR product and can be applied to the resequencing of PCR-amplified disease genes for mutation screening. It also is used to screen clinical isolates for the genes that confer resistance to antiviral therapy, such as for analysis of influenza specimens for the adamantine resistance mutation.
