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Multiple viruses can co-infect the genital tract, modifying the immunologic and virologic milieu and possibly playing a role in viral transmission and pathogenesis. The aim of our studies has been to understand the complex relationships between HIV-1 RNA, and multiple human herpesviruses known to frequently replicate in the genital tract of HIV-infected men (i.e. cytomegalovirus [CMV], Epstein Bar virus [EBV], herpes simplex virus [HSV] types 1 and 2, and human herpesviruses [HHV] 6, 7 and 8) (Gianella et al., 2013a; Gianella et al., 2013b; Gianella et al., 2013c; Gianella et al., 2014). This protocol was designed to collect and process male genital secretion (GS), and to isolate and further quantify HIV RNA and DNA of seven HHV from seminal plasma using quantitative real time PCR technology.
Note: 1 M Tris requires to be diluted to 10 mM with molecular grade water.
Note: Quantification standards for the different herpes viruses were obtained using DNA plasmid preparations with known concentrations. We provided the nucleotide sequences for each standard amplicon (in a separate fasta file from 5’ to 3’) (please see Supplementary Material). Oligonucleotides can be custom synthetized (e.g. Ultramer® Oligonucleotides, Integrated DNA technologies [IDT]) and used directly as quantification standard, after measuring the amount of PCR-component template by spectrophotometry or droplet digital PCR.
If desired, amplicons can also be inserted into a plasmid using standard kits (e.g. Zero Blunt® TOPO® PCR Cloning Kit, catalog number: K2800J10) and stocks of construct can be generated with large batch cultures (using standard protocols). After purification, quantify plasmid concentration by spectrophotometry (or ddPCR) and extract clone vectors using standard procedures. Consider linearization to increase efficiency and avoid supercoil formation.
Please note other cell cryopreservation methods/canisters are acceptable as long as the cells are viably frozen slowly at −1 °C per min. Collection and storage of seminal cells (steps 7-13) is optional. Stored cells can be used for future studies but will not be used for this protocol.
Note: Duplicates are only necessary to run the entire panel of seven HHV.
Note: Do not allow the samples to sit around after the high-speed spin; the viral pellet is unstable!
Note: To increase RNA yield, incubate for 10 min at room temperature after adding the binding buffer to the sample. To further increase yield, incubate High Pure Filter Tube loaded with 50 μl heated elution buffer for 5 min. Heat elution buffer at 60 °C before elution step.
Note: To increase DNA yield incubate the QIAamp Mini column loaded with 100 μl heated AE Buffer for 10 min. Heat AE buffer at 60 °C before elution step.
|Standard||Dilution factor||Copies per well|
|Dilution||Dilution factor||Copies per μl|
|D0||Undiluted stock||2 × 1010|
|D1||25*||8 × 108|
|D2||10**||8 × 107|
|D3||10**||8 × 106|
|D4||10**||8 × 105|
|D5||10**||8 × 104|
|Dilution||Dilution factor||Copies per well (per 10 μl)|
|D5||10*||8 × 104|
|D6||10*||8 × 103|
|D7||10*||8 × 102|
|D8||10*||8 × 101|
After the run is completed, the CT fluorescence level line should be adjusted to fall within the steep part of the amplification curve (see Figure 1). Amount of unknown target (samples) can be extrapolated from the standard curve using the formula for linear regression: y = ax + b
a = slope obtained from the standard curve.
b = intercept obtained from the standard curve.
y = Threshold Ct value for seminal sample.
x = Extrapolated amount of target DNA or cDNA for seminal sample.
Note: Consider repeating samples with differences of 0.3 Cts between duplicates. Run triplicates whenever possible to improve precision. Samples at the lower limit of detection might have discordant Ct values due to Poisson distribution of templates across wells. Thus, for more reliable low copy detection, a large number of replicates are necessary to provide statistical significance and to overcome the Poisson distribution limitation.
Calculator template for the analysis and an example are provided in a separate Excel spreadsheet (“example_calculator”).
Note: Recipes for processing GS Media (stored at 4 °C). GemCell™ U.S. origin fetal bovine serum (FBS) is filtered twice; once with 0.45 μm Stericup-HV and then with 0.22 μm Stericup-GV prior to GS media preparation.
This work is supported by the Department of Veterans Affairs, the UCSD Center for AIDS Research (P30 AI36214), the James B. Pendleton Charitable Trust, AmfAR grant 108537 with support from FAIR, a CTRI pilot grant: UL1TR000100, and National Institutes of Health (NIH) awards: P30-AI027763, MH101012, 7-UM1 AI068636-07. A special appreciation goes to our belated Marek Fisher, Matt Strain, Antoine Chaillon and Stephen Espitia for all his help and input in developing and validating this protocol.