Table 7.
HIV-1 Protease and RT Sequencing Kits
| Applied Biosystems ViroSeq™ HIV-1 Genotyping System | Visible Genetics TRUGENE™ HIV-1 Genotyping Kit | |
|---|---|---|
| Sample requirements | ||
| Plasma volume | 500 ul | Depends on method used for extraction |
| Viral load | >2,000 RNA copies/ml is recommended | >1,000 RNA copies/ml is recommended |
| RNA isolation | ||
| Lysis and centrifugation using a modification of the MONITOR Ultrasensitive Method (Roche Diagnostics). | Separate kits can be purchased for RNA extraction. | |
| RT/PCR | ||
| RT | Single primer, MMuLV RT, RNase inhibitor | Single primer, MMuLV RT, RNase inhibitor |
| PCR | Non-nested, 40 cycles with Ampli- Taq Gold DNA polymerase. 1.8 kb product | Non-nested, 27 cycles with proprietary DNA polymerase. 1.3 kb product |
| Controls | Positive and negative controls included beginning with the RT/PCR step. dUTP/UNG Amperase | Positive and negative controls included beginning with the RT/PCR step. |
| Sample clean up | Microcon columns | None |
| Analysis | Agarose gel | None |
| Equipment needed | 9600 or 9700 thermal cycler | Laboratory’s preferred thermal cycler |
| Sequencing | ||
| Method | BigDye™ dideoxyterminators | CLIP bi-directional |
| Number of primers | 6–7 primers, one per reaction | 6 reactions, each containing a primer mix |
| Equipment | 9600 or 9700 thermal cycler, ABI 310, 377, 3100 | Customized equipment for gel preparation and electrophoresis |
| Throughput | 16 samples per 7 hour run on ABI 377 (96-well). 32 samples per run over 24 hours on ABI 3100 | 1 sample per 50 minute run. With 4 Long Read Towers, 28 samples in 8 hours |
| Data analysis system | ||
| Region analyzed | Protease 1-99, RT 1-320 | Protease 1-99, RT 40-247 |
| Computer platform | MacIntosh for ABI 377/ABI 310, PC or MacIntosh for ABI3100 | PC |
| Software features | Primer assembly, auto trimming, full view of electropherograms, translation and alignment to reference sequence, quick tab to positions of interest for editing. | Primer assembly, auto trimming, full view of electropherograms, translation and alignment to reference sequence, quicktab to positions of interest for editing. Includes “fingerprinting” comparison to prior runs. |
| Mutations reported | Resistance mutations, novel variants, insertions/deletions | Resistance mutations, known polymorphisms, novel variants, insertions/deletions |
| Sequence format | FASTA | FASTA (Ns may need to be deleted for data analysis) |
| Other reported information | Information about sequence quality and editing | Drug resistance interpretation |