Abstract
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.
Keywords: Seminal Plasma, Taqman, HIV, HHV, Cytomegalovirus
Materials and Reagents
Equipment
Procedure
Analysis
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 excels spreadsheet (“example_calculator”). Figure 1. Amplification plot
Notes
Recipes
Note: Recipes for processing GS Media (stored at 4 °C). GemCellTM 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.
Supplementary Materials
Table 1. Primers and Probes for HIV and different Herpesviruses, adapted from Gianella et al. (2013b) Table 2. Nucleotide sequences for each herpes viruses to use for standard plasmids or Amplicons
Acknowledgments
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.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.