Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.
Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART).
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20.
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.
HIV-1 infected individuals on combination antiretroviral treatment (cART) or who naturally control the infection have very low viral loads, typically around 1 copy per ml of plasma4, 11, 12, 17, 18. Viral loads in patients with natural control, often fluctuate around an individual set point1, 2, 17. The sensitivity of the assays described herein is highly dependent on the input volume of plasma. Generally, we have been working with 7 ml of plasma, but positive results can be obtained from smaller am…
The authors have nothing to disclose.
The authors wish to acknowledge the patients who participated in the many studies of HIV-1.
J.M.C. was a Research Professor of the American Cancer Society with support from the F.M. Kirby Foundation.
Name of Reagent | Company | Catalogue number | Comments |
Random hexamers (500 ug/ml) | Promega | C118A | |
Taqman buffer A | Applied Biosystems | 4304441 | |
RNAsin (40 U/ul) | Promega | N211B | |
RT enzyme (200 U/ul) | Strategene | 600107-51 | |
Amplitaq Gold DNA polymerase | Applied Biosystems | 4311814 | 6-pack |
Amplitaq 10XGold PCR buffer II | Applied Biosystems | 4311814 | comes with the enzyme |
Magnesiumcloride 25 mM | Applied Biosystems | 4311814 | comes with the enzyme |
1M Tris-HCl buffer, pH 8.0, 5 mM | Invitrogen | AM9855G | |
Superscript III reverse transcriptase enzyme, 200 U/ul | Invitrogen | 18080-044 | |
Superscript III 10X buffer | Invitrogen | comes with the enzyme | |
DTT 100 mM | Invitrogen | comes with the enzyme | |
Platinum Taq DNA polymerase High Fidelity 5 U/ul | Invitrogen | 11304-102 | |
10X High Fidelity PCR Buffer | Invitrogen | 11304-102 | comes with the enzyme |
Proteinase-K 20 mg/ml | Ambion | 2546 | |
Guanidinium Thiocyanate solution 6M | FlukaBiochemica | 50983 | |
Glycogen 20 mg/ml | Roche | 10901393001 | |
Isopropanol | Sigma-Aldrich | 19516 | |
Ethanol 70% | Sigma-Aldrich | E702-3 | |
Molecular grade water | Gibco/Invitrogen | 10977-015 | |
dNTPs (25 mmol each) | Bioline | BIO-39025 |
Name of Euipment | Company | Catalogue number | Comments |
Easy Seal Centrifuge Tubes (16x73mm) | Seaton Scientific | 6041 | |
White Delrin crowns | Seaton Scientific | 4020 | Caps for the Easy Seal Tubes |
Tube Rack for Optiseal Bell-Top Tubes, 8.9 ml | Beckman | 361642 | |
Hex driver ½ inc opening | Seaton Scientific | 4013 | |
cap removal tupe | Seaton Scientific | 4014 | |
5 ml serological pipettes | Costar | 4051 | |
Pipetboy | any | ||
15 ml Transfer pipettes | ISC Bioexpress | P-5005-7 | |
5.8 ml Dispossable transfer pipettes, fine tip | VWR | 14670-329 | |
15 ml centrifuge tubes | Falcon | ||
1 ml centrifuge tubes | any | ||
Tris buffer Saline tablets | Sigma-Aldrich | T5030-100TAB | |
Ultracentrifuge and rotor | Sorval | 90SE and T-1270 | |
96-well PCR plates | Biorad | HSS-9601 | |
50 ml Reagent reservoir | Costar | 4870 | |
Microamp optical 96-well reaction plate | Applied Biosystems | N801-0560 | |
Optical plate covers | Applied Biosystems | 4333183 | |
MicroAmp Optical Compression Pad | Applied Biosystems | 4312639 | |
Microseal A film | Biorad | MSA-5001 | |
2 ml centrifuge tubes | Any | ||
1% agarose gels (E-gel, invitrogen) | Invitrogen | G-7008-01 | |
E-base (Invitrogen) | Invitrogen | EB-M03 |
EDTA Collection tubes any brand