In the fight against AIDS, viral drug-resistance tests are powerful instruments to aid physicians in choosing the most suitable treatment for each patient. Different kinds of assays are available: genotypic, phenotypic or mixed. The Viroseq system is an HIV-1 drug resistance genotype test and is one of only two kits in this class approved by FDA for clinical use.
Starting from plasma or serum samples, this system enables the extraction and purification of HIV-1 viral RNA, transcription to cDNA and amplification of the gag-pol region via RT-PCR. Amplified samples can then be purified and sequenced using seven primers provided with the kit. The result is a sequence of about 1300 base pairs that spans the final part of the gag gene and the first 1000 base pairs (approximately) of the pol gene. As a result, the sequence covers both the protease and the reverse transcriptase regions, where mutations that confer drug resistance are located. Analysis software is provided to assist in editing the consensus sequence obtained, producing a resistance report. Levels of resistance are estimated for every drug available, based on the mutations detected in the analyzed viral sample. In addition, recent data show that sequences produced by genotypic tests are long enough to be used effectively in phylogenetic analysis of viral subtype determination. Hence, in a single analysis two important pieces of information are gained; drug-resistance associated mutations and viral subtype.
The Viroseq is a very powerful system, yet the suggested procedure is quite long. We have optimized a protocol that takes one and a half days of work to get the sequence and good results are almost assured. Extraction of RNA from virions is the most time-consuming and delicate part of the procedure, since RNA can be easily degraded or contaminated. Provided you have good samples (with a reasonable high viral load), you should have good success rates in amplifying DNA. RT and PCR mix preparation is quite simple (composition listed in the manual) and is followed by sample loading and thermal cycler programming.
The PCR is very long (it takes about 4 hours) because it runs 40 cycles of amplification to guarantee detectable results even with very low viral loads. We sometimes got positive outcomes even with viral loads as low as 500-1000 copies/ml of HIV-1 RNA, while the kit is certified for only 2000 cp/ml. This confirms the sensitivity of this system and its usefulness with almost every kind of sample.
At the end of PCR, we purify the samples and occasionally check them with agarose gel electrophoresis (Viroseq has been so reliable that we frequently just assume success). Although no DNA purification system is provided in the kit, primers are included to use for subsequent cycle-sequencing. Cycle-sequencing is simple, as the primers are ready to use following addition of DNA, and can be carried out with any third party system. The sequences can then be analyzed with the Viroseq Sequencing Software (for PC) to produce a consensus sequence and a resistance report, readable with ease by physicians.
The Viroseq system is great. Although is does seem to be faster than other competitors, other advantages make it the best choice in my opinion. The protocol results in the production of a single continuous sequence covering PR and RT regions, while other systems give you two shorter fragments with missing. In addition, direct sequencing of DNA enables detection of all mutations, not only expected ones. The system enables in-house synthesis so you can organize your time more effectively and because cDNA is a single fragment, it can be used for further nested PCRs. Finally, RNA is so well purified with this system that it can be used for home-made reactions even on other regions of the HIV-1 genome.
The only drawback is the cost of the system. It is very expensive and may not be worth the expense if you do not process many samples each month. The accuracy and sensitivity of this kit fully justify the expense, as it would save considerable design and execution time due to its optimization. I would recommend the Viroseq HIV-1 system to anyone working on HIV-1 drug-resistance associated mutations, as well as anyone dealing with HIV-1 subtype monitoring. It is quite easy to use, has a high efficiency and the results are provided in a useful form for further research.
Dr. Andrea Galli
University of Milan, Italy