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Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma
精浆中HIV RNA 和人疱疹病毒DNA的量化   

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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(精浆), Taqman(TaqMan探针), HIV(艾滋病咨询门诊), HHV(HHV), Cytomegalovirus(巨细胞病毒)

Materials and Reagents

  1. For male genital secretion (GS) processing
    1. Gibco® Hank’s balanced salt solution (HBSS) (no calcium, no magnesium, no phenol red) (Life Technologies, catalog number: 14175-095 )
    2. Gibco® RPMI 1640 medium (Life Technologies, catalog number: 11875-093 )
    3. GemCellTM U.S. origin fetal bovine serum (FBS) (Gemini-Bio Products, catalog number: 100-500 )
    4. Gibco® penicillin-streptomycin (10,000 U/ml) (Life Technologies, catalog number: 15140-122 )
    5. Gibco® L-Glutamine (200 mM) (Life Technologies, catalog number: 25030-024 )
    6. Nystatin suspension (10,000 units/ml in DPBS) (Sigma-Aldrich, catalog number: N1638 )
    7. Dimethyl sulfoxide (Hibri-MaxTM, sterile-filtered) (Sigma-Aldrich, catalog number: D2650 )
    8. Waxie® bleach (Waxie Sanitary Supply, catalog number: 170016 )
    9. Viral transport medium (VTM) (see Recipes)
    10. 20% GS complete RPMI medium (see Recipes)
    11. 20% GS freeze medium (see Recipes)

  2. For RNA and DNA extraction and cDNA generation
    1. QIAamp DNA mini kit (250 or 50 QIAamp mini spin columns) (QIAGEN, catalog numbers: 51306 or 51304 )
    2. High pure viral RNA kit (Roche Diagnostics, catalog number: 11858882001 )
    3. InvitrogenTM SuperScript® III first-strand synthesis system (Life Technologies, catalog number: 18080-051 )
    4. Seal-Rite 1.5 ml microcentrifuge (flip-top) tube (natural) (USA Scientific, catalog number: 1615-5500 )
      Note: These tubes are autoclaved before use.
    5. Micro tubes 1.5 ml (type D, without skirted base and with assembled neutral screw cap) (SARSTEDT AG, catalog number: 72.692.005 )
    6. Applied Biosystems® MicroAmp® reaction tube with cap (0.2 ml) (Life Technologies, catalog number: N8010540 )
    7. 1x Dulbecco’s phosphate buffered saline (DPBS) (Corning, CellgroTM, catalog number: 21-031-CV )
    8. EDTA (0.5 M pH 8.0, molecular biology grade) (Promega Corporation, catalog number: V4231 )
    9. Molecular biology grade sterile purified water (RNase, DNase, proteinase free) (Corning, CellgroTM, catalog number: 46-000-CM )
    10. Tris hydrochloride (1 M solution pH 8.0, molecular biology grade) (Thermo Fisher Scientific, catalog number: BP1758-500 )
      Note: 1 M Tris requires to be diluted to 10 mM with molecular grade water.
    11. Ethyl alcohol pure (200 proof molecular biology grade) (Sigma-Aldrich, catalog number: E7023 )
    12. MgCl2 (50 mM solution) (Bio-Rad Laboratories, catalog number: 170-8872 )

  3. For quantitative Real Time PCR (RT-qPCR)
    1. DNA plasmids or amplicons to be used as standards for RT-qPCR
      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.
    2. HIV RNA standard can be obtained from the DAIDS Virology Quality Assurance (VQA) Program (https://www.aidsreagent.org/, 150,000 copies HIV-1 RNA/ml spiked into negative plasma, catalog number: 3443 )
    3. Molecular biology grade sterile purified water (Corning, CellgroTM, catalog number: 46-000-CM)
    4. Primers and probes (Table 1)

Equipment

  1. For male genital secretion (GS) processing
    1. Fisherbrand polyethylene hinged-lid containers (Thermo Fisher Scientific, catalog number: 03-405-40 )
    2. Curwood Parafilm MTM laboratory wrapping film (4-inch W X 125-ft L) (Thermo Fisher Scientific, catalog number: 13-374-10 )
    3. Fisherbrand Infecon specimen transport bags (Thermo Fisher Scientific, catalog number: 19-287-215 )
    4. Corning® 15 ml centrifuge tube (sterile) (Corning, catalog number: 430052 )
    5. FisherbrandTM sterile polystyrene disposable serological pipets with magnifier stripe for 1 ml, 2 ml, 5 ml, 10 ml (Thermo Fisher Scientific, catalog numbers: 13-676-10G , 13-675-3C , 13-676-10H , 13-676-10J )
    6. VWR® disposable aspirating pipets (polystyrene, sterile) (VWR International, catalog number: 414004-264 )
    7. Micro tubes (2.0 ml, Type I, with skirted base and assembled neutral screw cap) (SARSTEDT AG, catalog number: 72.694.006 )
    8. 2.0 ml Ext FS CryoElite sterile cryogenic vials (yellow-cap) (Wheaton Science Products, catalog number: W985866 )
    9. Nalgene® Mr. FrostyTM freezing container (Thermo Fisher Scientific, catalog number: 5100-0001 )
    10. Allied® 1,500 ml disposable collection canisters (Allied Healthcare Products, catalog number: 20-08-0003 )
    11. SorvallTM RC4 General Purpose Floor Model Centrifuge (Thermo Fisher Scientific, catalog number: 75004481 )
    12. Original Pipetman Aid® Pipet Controller (Drummond Scientific Company, catalog number: 4-000-111-TC )
    13. Stericup-HV, 0.45 µm, PVDF (500 ml) (EMDMILLIPORE, catalog number: SCHVU05RE )
    14. Stericup-GV, 0.22 µm, PVDF (500 ml) (EMDMILLIPORE, catalog number: SCGVU05RE )

  2. For RNA and DNA extraction and cDNA generation
    1. Fisher® vortex genie 2, analog vortex mixer (Thermo Fisher Scientific, catalog number: 02-215-365 )
    2. VWR® digital dry block heaters (or 2 heat blocks) (VWR Scientific Products, catalog number: 12621-088 )
    3. Allegra® 64R high performance Benchtop centrifuge (Beckman Coulter, item number: 367585 )
    4. Eppendorf® centrifuge (model: 5430 , 120 V, with Rotor, model: FA-45-30-11) (Eppendorf, 120V no longer available only 230V catalog number: 5427 000.216)
    5. Applied Biosystems® GeneAmp® PCR system 9700 (96-well gold-plated) (Life Technologies, catalog number: 4314878 )

  3. For quantitative Real Time PCR (RT-qPCR)
    1. Applied Biosystems® MicroAmp® Optical 96-Well reaction plate (Life Technologies, catalog number: N8010560 )
    2. Applied Biosystems® MicroAmp® optical adhesive film (Life Technologies, catalog number: 4311971 )
    3. Applied Biosystems® MicroAmp® 96-Well Tray/Retainer Set (Life Technologies, catalog number: 403081 )
    4. Corning® 50 ml centrifuge tube (sterile) (Corning, catalog number: 430290 )
    5. Applied Biosystems® TaqMan® environmental master mix 2.0 (200 reactions) (Life Technologies, catalog number: 4396838 )
    6. Applied Biosystems® 7900HT fast Real-Time PCR system with Fast 96-Well block module (Life Technologies, catalog number: 4351405 )

Procedure

  1. Protocol for collection and processing of male genital secretions
    1. After cleaning hands and penis with sanitary wipes, semen is collected by masturbation into a Fisherbrand Polyethylene Hinged-Lid Container with about 2 ml of VTM (after 72 h of sexual abstinence).
    2. Following masturbation, the semen specimen should sit for at least 30 min (but no more than 4 h) at room temperature prior to processing to allow liquefaction.
    3. Record volume, time of collection and start time of processing.
    4. Transfer ejaculate (inclusive VTM) to a sterile Corning® 15 ml centrifuge tube.
    5. Centrifuge at 700 x g room-temp for 12 min in the SorvallTM RC4 floor centrifuge.
      1. If the GS specimen is very sticky/ goopy it is recomended to break up the specimen by pipetting the sample with a sterile narrow tip opening. Pipet 5-10 times gently, holding the tip very close to the bottom of the 15 ml conical tube. Repeat centrifugation step in order to separate plasma from cells.
    6. All of the plasma should be collected without disrupting the cell pellet in 0.5-1.5 ml pre-labelled aliquots (SARSTEDT 2.0 ml Micro Tubes) and stored at -80 °C.
    7. Flick to break up the cell pellet (no vortex) and re-suspend in 10 ml of Hank’s Balanced Salt Solution.
    8. Centrifuge at 700 x g room-temp for 12 min to pellet cells.
    9. Aspirate off the HBSS wash using a sterile VWR® Disposable Aspirating Pipet and Allied® 1,500 ml Disposable Collection Canister (vacuum trap) with 10% Waxie® Bleach.
    10. Flick to break up the cell pellet (no vortex) and re-suspend seminal cells in 1 ml of 20% complete RPMI medium for GS.
    11. Pipet a few times to gently homogenize the seminal cells in 1 ml of 20% GS complete RPMI medium without vortexing.
    12. Add 1 ml of 20% GS freeze medium to bring the total volume to ~2 ml and pipet a few times to mix evenly again no vortexing. Keep the cells on ice at all times.
    13. Aliquot the 2 ml cell suspension into 4 x 500 µl aliquots (yellow-cap, 2.0 ml CryoElite Sterile Cryogenic Vials) and freeze viably in Nalgene® Mr. FrostyTM Freezing Container* at -80 °C overnight.  The next day, move to -150 °C liquid nitrogen storage.
      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 A7-13) is optional. Stored cells can be used for future studies but will not be used for this protocol.

  2. Isolation of nucleic acids
    1. Thaw 1 ml of seminal plasma at room temperature.
    2. Immediately transfer 0.2 ml of seminal plasma into 1.5 ml SARSTEDT screw cap tube containing 20 μl of 0.5M EDTA solution to inhibit DNAase activity. Mix well by pipetting, quickly vortex, and put on ice. Repeat this step to obtain a duplicate of the same sample. Then extract DNA as described below.
      Note: Duplicates are only necessary to run the entire panel of seven HHV.
    3. In preparation for RNA extraction quickly transfer 0.5 ml of seminal plasma into a 1.5 ml SARSTEDT screw cap tube containing 0.5 ml 1x DPBS and pipet gently a few times to reduce viscosity. Then immediately put on ice.
    4. To concentrate RNA, centrifuge the 1 ml seminal samples with DBPS at 4 °C for 1 h at 23,500 x g using the Allegra® 64R centrifuge. Refer to RNA extraction described below.
      Note: Do not allow the samples to sit around after the high-speed spin; the viral pellet is unstable!

      For RNA extraction (high pure viral RNA kit)
      1. Following centrifugation, carefully remove the supernatant from each tube leaving 200 μl, not disturbing the invisible RNA pellet.
      2. Extract RNA following the manufacture’s protocol (high pure viral RNA kit).
        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.
      3. After elution, transfer RNA extracts to pre-labelled 1.5 ml SARSTEDT screw cap tubes and put on ice. Immediately proceed to cDNA generation discussed below. Finally store remainder of RNA extract at -80 °C.

      For DNA extraction (QIAamp DNA mini kit)
      1. Extract DNA following the manufacturer’s protocol (QIAamp DNA mini kit).
        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.
      2. Centrifuge for 1 min at max speed (16,000 x g) in a clean seal-Rite 1.5 ml microcentrifuge tube (flip-top).
      3. Combine identical duplicates respectively and transfer to pre-labelled 1.5 ml SARSTEDT screw cap tube to be stored at 4 °C short-term or -20 °C long-term storage.

      For cDNA HIV-1 generation (InvitrogenTM SuperScript® III first-strand synthesis system)

      1. In a 0.2 ml MicroAmp® reaction tube with cap, mix 10 μl of extracted RNA (for seminal sample and HIV RNA for quantification standard) with 1 μl of 20 μM HIV-1 reverse primer (see Table 1) and 1 μl of 10mM dNTPs (primers and dNTPs can be pre-combined).
      2. Incubate at 65 °C for 5 min.
      3. Place immediately on ice for at least 1 min.
      4. To each reaction tube add (can be pre-mixed):
        1. 2 μl of 10x RT buffer from kit
        2. 2 μl MgCl2 (50 mM) *not included in kit
        3. 2 μl 0.1 M DTT from kit
        4. 1 μl RNase OUT enzyme (40 U/ml) from kit
        5. 1 μl SuperScript III RT enzyme (200 U/ml) from kit
      5. Incubate at 50 °C for 50 min and inactivate reaction at 85 °C for 5 min.
      6. Store at 4 °C short-term and -20 °C long-term storage.

  3. Quantification of cDNA and DNA viruses by RT-qPCR

    For cDNA quantification RT-qPCR
    1. Standard preparation: Each HIV RNA standard aliquot (DAIDS VQA) contains 150,000 copies HIV-1 RNA/ml spiked into negative plasma. It is important to treat the quantification standard exactly the same way as the seminal plasma samples (inclusive RNA extraction, cDNA generation and quantification). The use of RNA standards takes the variable efficiencies of the different steps into account (especially the reverse transcription step). cDNA generated during reverse transcription will be used as the template for amplification in the subsequent RT-qPCR. Example of serial dilution from cDNA using 10 mM Tris buffer:

      Standard   
      Dilution factor   
      Copies per well
        D0   
      Undiluted
      3,750
      D1
      4*
      938
      D2
      4*
      234
      D3
      4*
      59
      D4
      4*
      15
      D5
      4*
      4
      Neg ctrl
      H2O
      0
      (*5 μl in 15 μl 10 mM Tris buffer)
      All standards (including negative control) should be run at least in duplicate (or triplicate if possible).
      Note: Do not store quantification standards for prolonged periods of time and thaw only once before use.

    2. In a 96 well plate, mix 5 μl of cDNA, 25 μl TaqMan® Environmental Master Mix 2.0, HIV primers (1 μM) and probe (0.3 μM) as displayed in Table 1. Add water to adjust the volume to 50 μl total volume per reaction per well. Each sample should be run in duplicate (or in triplicates) and final result will be obtained as an average value of the replicate wells.
    3. PCR cycling conditions:



    For DNA Quantification RT-qPCR
    1. Standard preparation: Quantification standards for the different herpes viruses were obtained using plasmid preparations with known concentrations. Because plasmid sequences are highly abundant (for example 1 x 1010 - 1 x 1012 per μl), they can be sources of PCR contamination. Extreme caution must be exercised when working with these DNAs to prevent their exposure to stock PCR reagents, solvents used for the dilution of PCR reagents and laboratory equipment and surfaces. Prepare little aliquots (for example 2 μl) to avoid multiple thawing.
      Example of serial dilution (using 10 mM Tris) starting from a plasmid concentration (stock=D0) of 2 x 1010 per μl. Accurate pipetting is essential because the standards must be diluted over several orders of magnitude.

      Dilution
      Dilution factor
      Copies per µl
      D0
      Undiluted stock
      2 x 1010
      D1
      25*
      8 x 108
      D2
      10**
      8 x 107
      D3
      10**
      8 x 106
      D4
      10**
      8 x 105
      D5
      10**
      8 x 104
      (*2 μl in 50 μl 10 mM Tris buffer, **20 μl in 180 μl 10 mM Tris buffer)
    2. Dilution D5 can be used as the highest concentration for the actual standard curve. Starting from D5, perform a 1:10 dilution as follows (note: numbers of copies are now indicated per PCR well):

      Dilution
      Dilution factor
      Copies per well
      (per 10 µl)
        D5  
      10*
      8 x 104
      D6
      10*
      8 x 103
      D7
      10*
      8 x 102
      D8
      10*
      8 x 101
      D9
      10*
      8
      D10
      10*
      0.8
      D11
      10*
      0.08
      Neg ctrl
      H2O
      0
      (*5 μl in 45 μl 10 mM Tris buffer)
    3. In a 96 well plate, mix 10 μl of DNA, 25 μl TaqMan® Environmental Master Mix 2.0, specific primers (1 μM) and probe (0.3 μM) as displayed in Table 1. Add water to adjust the volume to 50 μl total volume per reaction per well. Each sample should be run in duplicate (or in triplicates whenever possible) and final result will be obtained as an average value of the replicate wells.
    4. PCR cycling conditions:

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

  1. Donor should try not to ejaculate for at least 72 h (3 days) before producing sample.
  2. Donor should produce semen on the same day that he is bringing it in. Do not ejaculate the night before and store the semen in the refrigerator.
  3. Donor should avoid any form of lubricant or saliva during masturbation.
  4. Longer arousal and masturbation before ejaculation is encouraged to produce larger volume of semen (at least 15-20 min).
  5. When handling potentially infectious materials it’s highly recommended to use personal protective equipment/gear and universal precautions.

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.

  1. Viral transport medium (VTM)
    RPMI 1640 medium
    100 U/ml pen/strep
    200 U/ml nystatin
    2 mM L-glutamine
    10% FBS filtered
  2. 20% GS complete RPMI medium
    RPMI 1640 medium
    100 U/ml pen/strep
    2 mM L-glutamine
    20% FBS filtered
  3. 20% GS freeze medium
    20% DMSO
    80% FBS filtered

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

  1. Gianella, S., Morris, S. R., Anderson, C., Spina, C. A., Vargas, M. V., Young, J. A., Richman, D. D., Little, S. J. and Smith, D. M. (2013a). Herpesviruses and HIV-1 drug resistance mutations influence the virologic and immunologic milieu of the male genital tract. AIDS (London, England) 27(1): 39.
  2. Gianella, S., Smith, D. M., Vargas, M. V., Little, S. J., Richman, D. D., Daar, E. S., Dube, M. P., Zhang, F., Ginocchio, C. C., Haubrich, R. H., Morris, S. R. and Team, C. (2013b). Shedding of HIV and human herpesviruses in the semen of effectively treated HIV-1-infected men who have sex with men. Clin Infect Dis 57(3): 441-447.
  3. Gianella, S., Morris, S. R., Vargas, M. V., Young, J. A., Callahan, B., Richman, D. D., Little, S. J. and Smith, D. M. (2013c). Role of seminal shedding of herpesviruses in HIV Type 1 Transmission. J Infect Dis 207(2): 257-261.
  4. Gianella, S., Massanella, M., Richman, D. D., Little, S. J., Spina, C. A., Vargas, M. V., Lada, S. M., Daar, E. S., Dube, M. P., Haubrich, R. H., Morris, S. R., Smith, D. M. and California Collaborative Treatment Group, T. (2014). Cytomegalovirus replication in semen is associated with higher levels of proviral HIV DNA and CD4+ T cell activation during antiretroviral treatment. J Virol 88(14): 7818-7827.

简介

多种病毒可以共感染生殖道,修改免疫学和病毒学环境,并可能在病毒传播和发病中起作用。 我们的研究的目的是了解HIV-1 RNA和已知在HIV感染的男性生殖道中经常复制的多种人疱疹病毒之间的复杂关系(即巨细胞病毒[CMV],Epstein 条形病毒[EBV],单纯疱疹病毒[HSV] 1型和2型,以及人疱疹病毒[HHV] 6,7和8)(Gianella等人,2013a; Gianella等人 。,2013b; Gianella 等人,2013c; Gianella ,2014)。 该方案设计用于收集和处理男性生殖器分泌(GS),并使用定量实时PCR技术从精浆中分离并进一步定量7个HHV的HIV RNA和DNA。

关键字:精浆, TaqMan探针, 艾滋病咨询门诊, HHV, 巨细胞病毒

材料和试剂

  1. 用于男性生殖器分泌(GS)处理
    1. Gibco Hank's平衡盐溶液(HBSS)(无钙,无镁,无酚红)(Life Technologies,目录号:14175-095)
    2. Gibco RPMI 1640培养基(Life Technologies,目录号:11875-093)
    3. GemCell TM原始牛胎血清(FBS)(Gemini-Bio Products,目录号:100-500)
    4. Gibco青霉素 - 链霉素(10,000U/ml)(Life Technologies,目录号:15140-122)
    5. Gibco L-Glutamine(200mM)(Life Technologies,目录号:25030-024)
    6. 制霉菌素悬浮液(10,000单位/ml,在DPBS中)(Sigma-Aldrich,目录号:N1638)
    7. 二甲基亚砜(Hibri-Max TM,无菌过滤)(Sigma-Aldrich,目录号:D2650)
    8. Waxie ®漂白剂(Waxie Sanitary Supply,目录号:170016)
    9. 病毒转运培养基(VTM)(见配方)
    10. 20%GS完全RPMI培养基(见配方)
    11. 20%GS冷冻介质(见配方)

  2. 用于RNA和DNA提取和cDNA生成
    1. QIAamp DNA微型试剂盒(250或50 QIAamp微型离心柱)(QIAGEN,目录号:51306或51304)
    2. 高纯病毒RNA试剂盒(Roche Diagnostics,目录号:11858882001)
    3. Invitrogen TM SuperScript III第一链合成系统(Life Technologies,目录号:18080-051)
    4. Seal-Rite 1.5ml微量离心机(flip-top)管(天然)(USA Scientific,目录号:1615-5500)
      注意:这些试管在使用前要进行高压灭菌。
    5. 微管1.5ml(D型,无裙边基底和组装的中性螺帽)(SARSTEDT AG,目录号:72.692.005)
    6. 具有盖(0.2ml)(Life Technologies,目录号:N8010540)的Applied Biosystems MicroAmp
    7. 1×Dulbecco's磷酸盐缓冲盐水(DPBS)(Corning,Cellgro TM,目录号:21-031-CV)
    8. EDTA(0.5M pH 8.0,分子生物学级)(Promega Corporation,目录号:V4231)
    9. 分子生物学级无菌纯化水(RNase,DNase,无蛋白酶)(Corning,Cellgro TM,目录号:46-000-CM)
    10. Tris盐酸盐(1M溶液pH 8.0,分子生物学级)(Thermo Fisher Scientific,目录号:BP1758-500)
      注意:1M Tris需要用分子级水稀释至10mM。
    11. 纯乙醇(200分子生物学级)(Sigma-Aldrich,目录号:E7023)
    12. MgCl 2(50mM溶液)(Bio-Rad Laboratories,目录号:170-8872)

  3. 对于定量实时PCR(RT-qPCR)
    1. DNA质粒或扩增子用作RT-qPCR的标准品 注意:使用具有已知浓度的DNA质粒制备物获得不同疱疹病毒的定量标准。我们提供了每个标准扩增子的核苷酸序列(在从5'到3'的单独的fasta文件中)(请参见补充材料)。寡核苷酸可以定制合成( Ultramer 寡核苷酸,集成DNA技术[IDT])并直接用作定量标准,的PCR组件模板通过分光光度法或液滴数字PCR。
      如果需要,也可以使用标准试剂盒将扩增子插入质粒中(

      > PCR克隆试剂盒,目录号:K2800J10),并且可以用大批量培养物(使用标准方案)产生构建体储备液。纯化后,通过分光光度法(或ddPCR)定量质粒浓度,并使用标准程序提取克隆载体。考虑线性化以提高效率并避免超螺旋形成。
    2. HIV RNA标准可以从DAIDS病毒学质量保证(VQA)计划获得( https://www.aidsreagent.org/,150,000拷贝HIV-1 RNA/ml掺入阴性血浆,目录号:3443)
    3. 分子生物学级无菌纯化水(Corning,Cellgro TM,目录号:46-000-CM)
    4. 引物和探针(表1)

设备

  1. 用于男性生殖器分泌(GS)处理
    1. Fisherbrand 聚乙烯铰链盖容器(Thermo Fisher Scientific,目录号:03-405-40)
    2. Curwood Parafilm MTM实验室包装膜(4英寸W×125英尺L)(Thermo Fisher Scientific,目录号:13-374-10)
    3. 样品运输袋(Thermo Fisher Scientific,目录号:19-287-215)的Fisherbrand
    4. 15ml离心管(无菌)(Corning,目录号:430052)
    5. Fisherbrand TM无菌聚苯乙烯一次性血清学移液管,放大器条带为1ml,2ml,5ml,10ml(Thermo Fisher Scientific,目录号:13-676-10G,13-675-3C,13 -676-10H, 13-676-10J)
    6. 一次性抽吸移液管(聚苯乙烯,无菌)(VWR International,目录号:414004-264)
    7. 微管(2.0ml,I型,带有裙形基座和组装的中性螺旋盖)(SARSTEDT AG,目录号:72.694.006)
    8. 2.0ml Ext FS CryoElite无菌低温小瓶(黄盖)(Wheaton Science Products,目录号:W985866)
    9. Nalgene ® Mr. Frosty TM 冷冻容器(Thermo Fisher Scientific,目录号:5100-0001)
    10. Allied 1,500ml一次性收集罐(Allied Healthcare Products,目录号:20-08-0003)
    11. Sorvall TM RC4通用型号离心机(Thermo Fisher Scientific,目录号:75004481)
    12. 原始Pipetman Aid Pipet控制器(Drummond Scientific Company,目录号:4-000-111-TC)
    13. Stericup-HV,0.45μm,PVDF(500ml)(EMDMILLIPORE,目录号:SCHVU05RE)
    14. Stericup-GV,0.22μm,PVDF(500ml)(EMDMILLIPORE,目录号:SCGVU05RE)

  2. 用于RNA和DNA提取和cDNA生成
    1. Fisher涡流genie 2,模拟涡流混合器(Thermo Fisher Scientific,目录号:02-215-365)
    2. VWR数字干式加热器(或2个加热块)(VWR Scientific Products,目录号:12621-088)
    3. Allegra 64R高性能台式离心机(Beckman Coulter,产品编号:367585)
    4. Eppendorf离心机(型号:5430,120V,带有Rotor,型号:FA-45-30-11)(Eppendorf,120V,不再只有230V,目录号:5427000.216)
    5. Applied Biosystems GeneAmp PCR系统9700(96孔镀金)(Life Technologies,目录号:4314878)

  3. 对于定量实时PCR(RT-qPCR)
    1. Applied Biosystems MicroAmp光学96孔反应板(Life Technologies,目录号:N8010560)
    2. Applied Biosystems MicroAmp光学粘合剂膜(Life Technologies,目录号:4311971)
    3. 96孔板/保持器组(Life Technologies,目录号:403081)的MicroAmp 96-孔盘/保持器组
    4. 50毫升离心管(无菌)(Corning,目录号:430290)
    5. Applied Biosystems TaqMan 环境主混合物2.0(200个反应)(Life Technologies,目录号:4396838)
    6. 具有快速96孔模块模块(Life Technologies,目录号:4351405)的Applied Biosystems 7900HT快速实时PCR系统,

程序

  1. 收集和处理男性生殖器分泌物的议定书
    1. 用卫生巾清洁手和阴茎后,通过手淫将精液收集到具有约2ml VTM的Fisherbrand TM聚乙烯铰链盖容器中(在72小时的性戒断后)。
    2. 在手淫后,精液样品在室温下放置至少30分钟(但不超过4小时),然后进行处理以允许液化。
    3. 记录体积,收集时间和处理开始时间。
    4. 将射精(包括VTM)转移到无菌的Corning 15ml离心管中。
    5. 在Sorvall TM RC4地板离心机中在700℃室温离心12分钟。
      1. 如果GS样品是非常粘/opy,则推荐通过用无菌的窄尖端开口移取样品来破碎样品。 吸管5-10次轻轻,保持尖端非常接近15毫升锥形管的底部。 重复离心步骤,以从细胞中分离血浆。
    6. 应该收集所有血浆而不用在0.5-1.5ml预标记的等分试样(SARSTED TM sup 2.0ml Micro Tubes)中破坏细胞沉淀并储存在-80℃。
    7. 轻轻打碎细胞沉淀(无涡旋)并重悬于10ml Hank's平衡盐溶液中。
    8. 在700×g /平方厘米室温下离心12分钟以沉淀细胞。
    9. 使用无菌VWR 一次性吸液管和Allied 1,500ml一次性收集罐(真空捕集器),用10%Waxie 吸出HBSS洗液>漂白。
    10. 轻轻打破细胞沉淀(无涡流),并重新悬浮精液细胞在1毫升的20%完全RPMI培养基的GS。
    11. 吸取几次,轻轻匀浆精液细胞在1毫升20%GS完全RPMI介质中,无需涡旋。
    12. 加入1毫升20%GS冷冻介质,使总体积〜2毫升,吸取几次,混匀,再次无涡旋。保持细胞在冰上的所有时间。
    13. 将2ml细胞悬浮液等分成4×500μl等分试样(黄帽,2.0ml CryoElite Sterile Cryogenic Vials),并在Nalgene的Frosty先生冷冻容器*在-80℃下过夜。第二天,移至-150℃液氮储存 请注意,只要细胞在-1℃/分钟缓慢冷冻,其他细胞冷冻保存方法/罐是可接受的。精选细胞的收集和储存(步骤A7-13)是可选的。存储的单元格可用于以后的研究,但不会用于此协议。

  2. 核酸的分离
    1. 在室温下解冻1ml的精浆。
    2. 立即将0.2ml的精浆转移到含有20μl0.5M EDTA溶液的1.5ml SARSTEDT 螺帽管中,以抑制DNA酶活性。 通过吸移充分混合,快速涡旋,并放在冰上。 重复此步骤以获得相同样品的副本。 然后如下所述提取DNA 注意:重复只需要运行七个HHV的整个面板。
    3. 在准备RNA提取时,将0.5ml的精浆转移到含有0.5ml 1x DPBS的1.5ml SARSTEDT 螺帽管中,轻轻吸取几次以降低粘度。 然后立即放在冰上。
    4. 为了浓缩RNA,使用Allegra 64R离心机,在4℃下,在23,500×g离心1ml具有DBPS的精液样品1小时。参见下面描述的RNA提取。
      注意:高速旋转后不要让样品坐在周围;病毒沉淀不稳定!

      用于RNA提取(高纯病毒RNA试剂盒)
      1. 离心后,小心地从每个管除去上清液留下200μl,不干扰不可见的RNA沉淀。
      2. 按照制造商的方案提取RNA(高纯病毒RNA试剂盒) 注意:为了增加RNA产量,在将结合缓冲液加入样品后在室温下孵育10分钟。为了进一步提高产量,孵育高纯度过滤管装载50μl加热的洗脱缓冲液5分钟。在洗脱步骤之前在60℃热洗脱缓冲液。
      3. 洗脱后,将RNA提取物转移到预先标记的1.5ml SARSTEDT 螺旋盖管中并置于冰上。立即进行下面讨论的cDNA生成。最后将剩余的RNA提取物储存于-80℃

      用于DNA提取(QIAamp DNA mini kit)
      1. 按照制造商的方案(QIAamp DNA mini试剂盒)提取DNA 注意:为了增加DNA产量,孵育装有100μl加热的AE缓冲液的QIAamp Mini柱10分钟。 在洗脱步骤之前,在60℃下加热AE缓冲液。
      2. 在干净的密封 - Rite 1.5ml微量离心管(翻盖)中以最大速度(16,000×g)离心1分钟。
      3. 组合相同的副本,分别转移到预标记的1.5毫升SARSTEDT螺旋盖管,以储存在4°C短期或-20°C长期储存。

      对于cDNA HIV-1代(Invitrogen TM SuperScript III第一链合成系统)
      1. 在具有盖的0.2ml MicroAmp反应管中,用1μl20μMHIV-1反向引物(参见表1)混合10μl提取的RNA(用于定量标准的精液样品和HIV RNA) )和1μl的10mM dNTP(引物和dNTP可以预先组合)。
      2. 在65℃孵育5分钟。
      3. 立即置于冰上至少1分钟。
      4. 向每个反应管中加入(可以预混合):
        1. 2μl的10x RT缓冲液从试剂盒
        2. 2微升MgCl 2(50mM)*不包括在试剂盒
        3. 2μl0.1 M DTT
        4. 1μl来自试剂盒的RNA酶OUT酶(40U/ml)
        5. 来自试剂盒的1μlSuperScript III RT酶(200U/ml)
      5. 在50°C孵育50分钟,在85°C灭活反应5分钟。
      6. 储存在4°C短期和-20°C长期储存

  3. 通过RT-qPCR定量cDNA和DNA病毒

    对于cDNA定量RT-qPCR
    1. 标准制备:每个HIV RNA标准等分试样(DAIDS VQA)含有15万拷贝HIV-1 RNA/ml,掺入阴性血浆。重要的是以与精浆血浆样品完全相同的方式处理定量标准(包括RNA提取,cDNA生成和定量)。 RNA标准品的使用考虑了不同步骤的可变效率(尤其是逆转录步骤)。在逆转录过程中产生的cDNA将用作在随后的RT-qPCR中扩增的模板。使用10mM Tris缓冲液:
      从cDNA连续稀释的实施例
      标准   
      稀释因子   
      每个孔的副本
        D0   
      未稀释
      3,750
      D1
      4 *
      938
      D2
      4 *
      234
      D3
      4 *
      59
      D4
      4 *
      15
      D5
      4 *
      4
      拒绝
      H sub 2 O
      0
      (*15μl,在15μl10mM Tris缓冲液中) 所有标准(包括阴性对照)应至少一式两份(如果可能,一式三份) 注意:不要长时间存储量化标准,并在使用前只解冻一次。

    2. 在96孔板中,混合5μlcDNA,25μlTaqMan Environmental Master Mix 2.0,HIV引物(1μM)和探针(0.3μM),如表1所示。加入水调节 体积为每孔每反应总体积50μl。 每个样品应当重复(或重复三次)运行,并且最终结果将作为重复孔的平均值获得。
    3. PCR循环条件:



    对于DNA定量RT-qPCR
    1. 标准制备:使用已知浓度的质粒制备物获得不同疱疹病毒的定量标准。因为质粒序列是高度丰富的(例如1×10 10个/μl至1×10 12个/μl),所以它们可能是PCR污染的来源。在使用这些DNA时必须非常小心,以防止它们暴露于库存PCR试剂,用于稀释PCR试剂的溶剂以及实验室设备和表面。准备少量等分(例如2微升),以避免多次解冻。
      从2×10 10个/μl的质粒浓度(储液= D 0)开始连续稀释(使用10mM Tris)的实施例。准确的移液是必要的,因为标准必须稀释几个数量级。

      稀释
      稀释系数
      每μl的份数
      D0
      未稀释的股票
      2 x 10 10
      D1
      25 *
      8 x 10 8
      D2
      10 **
      8 x 10 7
      D3
      10 **
      8 x 10 6
      D4
      10 **
      8 x 10 5
      D5
      10 **
      8 x 10 4
      (*在50μl10mM Tris缓冲液中2μl,**在180μl10mM Tris缓冲液中20μl)
    2. 稀释D5可以用作实际标准曲线的最高浓度。 从D5开始,按如下方式进行1:10稀释(注意:现在每个PCR孔指示拷贝数):

      稀释
      稀释系数
      每个孔的副本
      (每10μl)
        D5  
      10 *
      8 x 10 4
      D6
      10 *
      8 x 10 3
      D7
      10 *
      8 x 10 2
      D8
      10 *
      8 x 10 1
      D9
      10 *
      8
      D10
      10 *
      0.8
      D11
      10 *
      0.08
      拒绝
      H sub 2 O
      0
      (*5μl,在45μl10mM Tris缓冲液中)
    3. 在96孔板中,混合10μlDNA,25μlTaqMan Environmental Master Mix 2.0,特异性引物(1μM)和探针(0.3μM),如表1所示。加水调节 体积为50μl 每孔反应总体积。 每个样品应当重复运行(或者尽可能一式三份),并且最终结果将作为重复孔的平均值获得。
    4. PCR循环条件:

分析

运行完成后,CT荧光水平线应调整到落入扩增曲线的陡峭部分(见图1)。 可以使用线性回归公式从标准曲线推断未知目标(样本)的量:y = ax + b
a =从标准曲线获得的斜率。
b =从标准曲线获得的截距。
y =精液样品的阈值Ct值 x =用于精液样品的目标DNA或cDNA的外推量 注意:请考虑重复 g样品,重复之间的差异为0.3 Cts。尽可能运行三次以提高精度。处于较低检测限的样品可能由于模板在孔中的泊松分布而具有不一致的Ct值。因此,对于更可靠的低辐照检测,需要大量的重复来提供统计学意义并克服泊松分布限制。
用于分析的计算器模板和示例在单独的excels电子表格中提供(" example_calculator ")。


图1.放大图

笔记

  1. 捐赠者应尽量不要在生产样品前至少72小时(3天)射精
  2. 捐赠者应在他带来的同一天产生精液。不要在前一天晚上射精,并将精液储存在冰箱中。
  3. 捐赠者在手淫期间应避免使用任何形式的润滑剂或唾液
  4. 鼓励在射精前较长的清醒和手淫产生更大量的精液(至少15-20分钟)。
  5. 当处理潜在的传染性材料时,强烈建议使用个人防护装备/装备和通用预防措施

食谱

N ote:用于处理GS Media(储存在4°C)的配方。 将来自美国的胎牛血清(FBS)过滤两次; 一次用0.45μmStericup-HV,然后用0.22μmStericup-GV在GS培养基制备前。

  1. 病毒转运介质(VTM)
    RPMI 1640培养基
    100 U/ml pen/strep
    200 U/ml制霉菌素 2mM L-谷氨酰胺 10%FBS过滤
  2. 20%GS完全RPMI培养基
    RPMI 1640培养基
    100 U/ml pen/strep
    2mM L-谷氨酰胺 20%FBS过滤
  3. 20%GS冷冻培养基
    20%DMSO
    80%FBS过滤

补充材料

表1.针对HIV和不同的疱疹病毒的引物和探针,改编自Gianella 等 (2013b) br />

< strong>每个疱疹病毒用于标准质粒或Amplicons的核苷酸序列

致谢

这项工作得到退伍军人事务部,UCSD艾滋病研究中心(P30 AI36214),James B. Pendleton慈善信托基金,AmfAR拨款108537和FAIR的支持,CTRI飞行员资助:UL1TR000100, 和国立卫生研究院(NIH)奖项:P30-AI027763,MH101012,7-UM1AI068636-07。特别感谢我们迟来的Marek Fisher,Matt Strain,Antoine Chaillon和Stephen Espitia在开发和验证这个协议方面的帮助和帮助。

参考文献

  1. Gianella,S.,Morris,S.R.,Anderson,C.,Spina,C.A.,Vargas,M.V.,Young,J.A.,Richman,D.D.,Little,S.J.and Smith,D.M。(2013a)。 疱疹病毒和HIV-1耐药性突变影响男性生殖器的病毒学和免疫学环境 AIDS(London,England) 27(1):39
  2. Gianella,S.,Smith,DM,Vargas,MV,Little,SJ,Richman,DD,Daar,ES,Dube,MP,Zhang,F.,Ginocchio,CC,Haubrich,RH,Morris,SRand Team, (2013b)。 在有效治疗艾滋病毒1感染男性性行为的精液中排除艾滋病毒和人类疱疹病毒与男性。 Clin Infect Dis 57(3):441-447。
  3. Gianella,S.,Morris,S.R.,Vargas,M.V.,Young,J.A.,Callahan,B.,Richman,D.D.,Little,S.J.and Smith,D.M。(2013c)。 疱疹病毒在艾滋病毒1型传播中的作用。 J Inf ect Dis 207(2):257-261。
  4. Gianella,S.,Massanella,M.,Richman,DD,Little,SJ,Spina,CA,Vargas,MV,Lada,SM,Daar,ES,Dube,MP,Haubrich,RH,Morris,SR,Smith, 加利福尼亚协作治疗组,T.(2014)。 巨细胞病毒在精液中的复制与较高水平的原病毒HIV DNA和CD4 + < 在抗逆转录病毒治疗期间的T细胞活化。 J Virol 88(14):7818-7827。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vargas-Meneses, M. V., Massanella, M., Ignacio, C. C. and Gianella, S. (2015). Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma. Bio-protocol 5(9): e1465. DOI: 10.21769/BioProtoc.1465.
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