For best viewing of the website please use Mozilla Firefox or Google Chrome.
Citation: Jing LU, Li QIN, Guang-jie LIU, Si-ting ZHAO, Xiao-ping CHEN. Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique [J].VIROLOGICA SINICA, 2008, 23(3) : 189-195.  http://dx.doi.org/10.1007/s12250-008-2896-0

Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

  • Corresponding author: Xiao-ping CHEN, chen_xiaoping@gibh.ac.cn
  • Received Date: 17 August 2007
    Accepted Date: 12 December 2007
    Available online: 01 June 2008

    Fund Project: National 973 Program 2006CB504208Natural Science Foundation of Guangdong Province 07118293The Grant of Science and Technology Plans of Guangdong Province 2006B36005002

  • Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian Immunodeficiency Virus (SIV) infections. To accurately measure RNA viral loads, a one-step fluorescent quantitative assay was established based on SYBR green Real-Time reverse transcription-polymerase chain reaction technique (RT-PCR). The assay with a lower detection limit of 10 copies per reaction was successfully applied to quantification of SIVmac251 and SIVmac239 virus stocks produced on CEM×174 cells. Moreover, the performance of the SYBR green real-time PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that this technique can detect as less as 215 copies per milliliter of plasma and the dynamic pattern of viral load was similar with those based on other techniques from other reports. Our assay due to its convenience, sensitivity and accuracy could serve as a good alternative to branched-chain DNA (b-DNA) assay or real-time PCR assay based on TaqMan probes.

  • 加载中
    1. Amara R R, Villinger F, et al.2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science, 292: 69-74.
        doi: 10.1126/science.1058915

    2. Cline A N, Bess J W, Piatak M, et al. 2005. Highly sensitive SIV plasma viral load assay: practical conside-rations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS. J Med Primatol, 34: 303-312.
        doi: 10.1111/jmp.2005.34.issue-5-6

    3. Cong Z, Li Z Z, Wei Q, et al. 2006. Quantification of Simian Immunodeficiency Virus (SIV) Viral Load Using Real Time Quantitative RT-PCR with SYBR Green Ⅰ. Acta Lab Anim Sci Sin, 14: 5.

    4. Hirsch V M, Fuerst T R, Sutter G, et al.1996. Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara. J Virol, 70: 3741-3752.

    5. Hofmann-Lehmann R, Swenerton R K, Liska V, et al. 2000. Sensitive and robust one-tube real-time reverse transcriptase-polymerase chain reaction to quantify SIV RNA load: comparison of one-versus two-enzyme systems. AIDS Res Hum Retroviruses, 16: 1247-1257.
        doi: 10.1089/08892220050117014

    6. Klein D, Janda P, Steinborn R, et al. 1999. Proviral load determination of different feline immunodeficiency virus isolates using real-time polymerase chain reaction: in-fluence of mismatches on quantification. Electrophoresis, 20: 291-299.
        doi: 10.1002/(ISSN)1522-2683

    7. Leutenegger C M, Higgins J, Matthews T B, et al. 2001. Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quan-titation of simian immunodeficiency virus RNA. AIDS Res Hum Retroviruses, 17: 243-251.
        doi: 10.1089/088922201750063160

    8. Marthas M L, Lu D, Penedo M C, et al. 2001. Titration of an SIVmac251 stock by vaginal inoculation of Indian and Chinese origin rhesus macaques: transmission effic-iency, viral loads, and antibody responses. AIDS Res Hum Retroviruses, 17: 1455-1466.
        doi: 10.1089/088922201753197123

    9. Monceaux V, Viollet L, Petit F, et al. 2007. CD4+CCR5+ T-cell dynamics during SIV infection of Chinese rhesus macaques. J Virol, (In Press).

    10. Mulder J, McKinney N, Christopherson C, et al. 1994. Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: appli-cation to acute retroviral infection. J Clin Microbiol, 32: 292-300.

    11. O'Shea S, Chrystie I, Cranston R, et al. 2000. Problems in the interpretation of HIV-1 viral load assays using commercial reagents. J Med Virol, 61: 187-194.
        doi: 10.1002/(ISSN)1096-9071

    12. Piatak M, Jr Saag M S, Yang L C, et al. 1993. High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science, 259: 1749-1754.
        doi: 10.1126/science.8096089

    13. Reimann K A, Parker R A, Seaman M S, et al. 2005. Pathogenicity of simian-human immunodeficiency virus SHIV-89.6P and SIVmac is attenuated in cynomolgus macaques and associated with early T-lymphocyte responses. J Virol, 79: 8878-8885.
        doi: 10.1128/JVI.79.14.8878-8885.2005

    14. Ririe K M, Rasmussen R P, Wittwer C T. 1997. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem, 245: 154-160.
        doi: 10.1006/abio.1996.9916

    15. Saag M S, Holodniy M, Kuritzkes D R, et al. 1996. HIV viral load markers in clinical practice. Nat Med, 2: 625-629.
        doi: 10.1038/nm0696-625

    16. Suryanarayana K, Wiltrout T A, Vasquez G M, et al. 1998. Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction. AIDS Res Hum Retroviruses, 14: 183-189.
        doi: 10.1089/aid.1998.14.183

    17. Ten Haaft P, Verstrepen B, Uberla K, et al. 1998. A pathogenic threshold of virus load defined in simian immunodeficiency virus-or simian-human immunodefi-ciency virus-infected macaques. J Virol, 72: 10281-10285.

    18. Urdea M S, Wilber J C, Yeghiazarian T, et al. 1993. Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay. Aids, 7 Suppl 2: S11-14.

    19. Watson A, Ranchalis J, Travis B, et al. 1997. Plasma viremia in macaques infected with simian immunodefi-ciency virus: plasma viral load early in infection predicts survival. J Virol, 71: 284-290.

    20. Wittwer C T, Herrmann M G, Moss A A, et al. 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques, 22:130-131, 134-138.

  • 加载中

Figures(3) / Tables(1)

Article Metrics

Article views(2804) PDF downloads(4) Cited by()

Related
Proportional views
    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

      Corresponding author: Xiao-ping CHEN, chen_xiaoping@gibh.ac.cn
    • Guangzhou Institute of Biomedicine and Health GIBH, Chinese Academy of Sciences, Guangzhou 510663, China
    Fund Project:  National 973 Program 2006CB504208Natural Science Foundation of Guangdong Province 07118293The Grant of Science and Technology Plans of Guangdong Province 2006B36005002

    Abstract: Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian Immunodeficiency Virus (SIV) infections. To accurately measure RNA viral loads, a one-step fluorescent quantitative assay was established based on SYBR green Real-Time reverse transcription-polymerase chain reaction technique (RT-PCR). The assay with a lower detection limit of 10 copies per reaction was successfully applied to quantification of SIVmac251 and SIVmac239 virus stocks produced on CEM×174 cells. Moreover, the performance of the SYBR green real-time PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that this technique can detect as less as 215 copies per milliliter of plasma and the dynamic pattern of viral load was similar with those based on other techniques from other reports. Our assay due to its convenience, sensitivity and accuracy could serve as a good alternative to branched-chain DNA (b-DNA) assay or real-time PCR assay based on TaqMan probes.

    • The use of a macaque model to monitor of plasma viral RNA in acquired immune deficiency syndrome (AIDS) has become an essential way to access disease progression and evaluate the effect of prophylactic or therapeutic interventions (15, 17, 19). For these reasons, studies on viral load quantification have increased in recent years. Available technology for quantitative evaluation of HIV-1 viral load includes RT-PCR (10, 12, 18), branched DNA (b-DNA) (18), and nucleic acid sequencebased amplification (NA SBA) (11). These methods are implemented in several standard commercial kits employed for HIV-1 infection monitoring. In simian immunodeficiency virus (SIV) infected macaque models of HIV, the current commercial test for detection of SIV is the branched-chain DNA assay (Bayer, Emeryville, CA) however its drawbacks are it is expensive and its sensitive is limited to about 1 500 viral RNA copies/ mL plasma. Recently, quantitative Real-Time reverse transcription-polymerase chain reaction (RT-PCR) assays mainly based on TaqMan or SYBR green due to its highly sensitive and reliable detection have found wide application in SIV viral loads quantification (2, 3, 16). In SYBR green RT-PCR assay, as the double-stranded PCR product accumulates during cycling, more SYBR green dye binds and emits fluorescence. Thus, the fluorescence intensity increases proportionally with dsDNA concentration (20). The specificity of the PCR can be confirmed through gel analysis and dissociation curve analysis where different PCR products are reflected in the number of first derivative melting peaks (14).

      In the current study, we have succeeded in establishing a one-step assay using SYBR green as a fluorescent dye to quantify SIVmac251 and SIV-mac239 RNA purified from virus stocks produced in CEM×174 cells. The detection limit of this assay was 10 copies per reaction or 215 copies/mL of plasma. The accuracy of the assay was further confirmed in a SIVmac251 infected rhesus macaque. This method does not requiring any hydrolysis probes and can be performed conveniently and economically with a sensitivity equivalent to the TaqMan assay (2, 5).

    • A healthy Chinese rhesus macaque (Macaca mulatta) which was seronegative for SIV, simian retrovirus, and simian T cell leukemia virus type-1 and B virus was housed at the animal lab in Guangzhou Institute of Biomedicine and Health. The monkey was intravenously infected with approximate 30 TCID100 of uncloned pathogenic SIVmac251 (donation courtesy of Dr. Chuan Qin). Whole blood samples were collected from experimental animal by venepuncture in tubes containing EDTA at various time points (1, 2, 3, 4, 8, 12, 16, 20, 24 weeks post inoculation). The plasma was collected by centrifuging whole blood at 2500 r/min for 20 min and stored in aliquots at -80 ℃. SIVmac251 and SIVmac239 (donated by the NIH AIDS and Reference Reagent Program) was propagated and titrated in the CEM×174 cell line (9). The virus culture supernatants were collected and stored at -80 ℃.

    • The viral RNA in cultured SIVmac251 supernatant was purified with QIAamp viral RNA minikit (Qiagen, Valencia, CA) and viral cDNA synthesis was performed according to a standard protocol using random hexamer primers (Takara, Dalian, China). The templates were amplified from viral cDNA with the following specific PCR primers: 5'-CCCGGCGGAA AGAAAAAG-3' and 5'-CGCCTGAAATCCTGGCA CTAC-3'. Products (461bp) were ligated into PMD-20T vector (Takara). The recombinant plasmid PMD-gag461 was transformed into DH5α E.coli Strain and the inserted sequence and direction were verified by sequencing. The recombinant plasmid was purified and linearized with EcoR I (Takara) and then was used in vitro transcription as described (6). RNA transcript (544bp) was purified with RNeasy mini kit (Qiagen) and the optical density (OD) was measured to determine the concentration. The RNA was then 10-fold serially diluted in diethylpyrocarbonate (DEPC)-treated water containing carrier tRNA (transfer RNA from Escherichia coli, 30ug/mL; Sigma, St. Louis, MO). The standard RNA dilutions were immediately frozen in 10 μL aliquots at -80 ℃.

    • To avoid major mismatches due to SIV variability, two specific oligonucleotides that recognize specific and conserved sequence on the gag region were chosen: 5'-AAATACTTTCGGTCTTAGC-3' and 5'-GGGTAATTTCCTCCTCTG-3'. The amplification of this pair of oligonucleotides yielded a 232bp gag fragment.

      SYBR green real-time PCR assay was carried out in 50μL PCR mixture volume consisting of 25 μL of 2×Quantitect SYBR green RT-PCR Master Mix (Qiagen), containing HotStarTaq DNA polymerase, 2.5μL of 10 μmol/L of each oligonucleotide primer, 0.5μL of 100×QuantiTect RT Mix (containing Omniscript and Sensiscript reverse transcriptases) and 2μL of RNA extracted from samples or 2μL from ten-folds serial diluted RNA standard (from 5×107 to 5 copies/μL). SIVmac251 gag gene amplification was carried out as follows: reverse transcription at 50 ℃for 30 min; initial activation of HotStar Taq DNA Polymerase at 95 ℃ for 15 min; 45 cycles in four steps: 94 ℃ for 10 s, 56 ℃ for 30 s, 72 ℃ for 30 s. At the end of the amplification cycles, melting temperature analysis was carried out by a slow increase in temperature (0.1 ℃ /s) up to 95 ℃.

    • RNA standard (from 1×106 to 1×101 copies/μL)was amplified as described above and was repeated four times to confirm the repeatability in different runs.

      The specificity of PCR was confirmed through dissociation curve analysis where different PCR products are reflected in the number of first derivative melting peaks (14) and confirmed by sequencing.

      In addition, the amplification efficiency of PCR is evaluated by using the ten-fold serial diluted RNA from transcripts and infected cell supernatants as templates. The amplification efficiencies of serial dilutions between RNA standard and viral RNA should be approximately equal; in other words the difference between the slopes (Δs) of amplification curves should be smaller than 0.1 for the purpose of reliable quantification. The amplification efficiencies (E) were calculated as 101/-s -1 (6).

    • The 461bp amplified products from the gag gene of SIVmac251 was inserted into PMD-20T plasmid and linearized with EcoRI as the transcript template. Electrophoresis demonstrated the absence of plasmid DNA contamination in the purified RNA transcripts (544bp) (Fig. 1).

      Figure 1.  Recombinant Plasmid PMD-gag461 digested by EcoR Ι and standard RNA transcripts (544bp) after digested RNasefree DNase were electrophoresed in 1% agarose gel. 1, PMD-gag461 after EcoR Ι digestion; 2, RNA standard.

    • SYBR green real-time RT-PCR products from plasma RNA showed 100% homology with SIV-mac251 on sequencing (data not shown). The tenfold diluted RNA standards containing 1×108 copies to 10 copies were amplified to determine the sensitivity of this method. The result (Fig. 2A) indicated that the lower limit of detection was 10 copies per reaction in 50μL PCR system.

      Figure 2.  Data analysis of SYBR green Real-time RT-PCR. The threshold limit, R2 value and dissociation curve were determined with the SDSv 1.3.1 software package on the 7300 ABI Prism. A: RT-PCR was conducted with a serial 10-fold dilution of RNA transcript (108-10copies), and with no template controls (NTC). Delta Rn is normalized via the fluorescence of the passive internal dye, ROX. B: Standard curve: Starting quality of target template versus threshold cycle (CT). Linearity was observed over an 8-log magnitude. C: Dissociation curve: Derivative displays a plot of the first derivative of the rate of change in fluorescence as a function of temperature. The specific PCR products with a Tm 79.4 ℃ were determined in this figure, whereas for NTC no special curve was detected.

      Standard curve and dissociation graphs were generated by using the SDSv1.3.1 software package (Fig 2B and 2C). The standard curve indicated a high correlation coefficient (R2= 0.999) and amplification efficiency. The dissociation curve plot displaying the single amplification peaks with a Tm of 79.4℃.

      The variation within a run was determined by perform each RNA copy number 3 times at one PCR reaction and repeatability of this method was confirmed by four times' separate running with each RNA copy number (Table 1). Even in the lowest template concentration (20 copies per reaction), the coefficient of variation within runs and between runs was 0.15% and 0.39%.

      Table 1.  Precision of SYBR green Real-Time RT-PCR Approach

      Furthermore, the amplification efficiency between different samples and the RNA standard was compared. Samples and RNA standards were serially diluted and amplified in 50μL system. The slopes of three curves (-3.39, -3.35 and -3.34 respectively for RNA standard, SIVmac251 and SIVmac239 viral RNA) were comparable (Δs < 0.1), which demonstrated that the amplification efficiencies of SIVmac251viral RNA, SIVmac239 viral RNA and RNA standard were approximately equal.

    • To evaluate the utility of this method, we analyzed samples from a rhesus macaque which was inoculated with SIVmac251 virus. As shown in Fig. 3, the monkey showed a typical course of primary viremia following intravenous challenge with SIVmac251. The virus was first detected at as early as first week post-challenge of virus and plasma SIV RNA value reached a peak (3.6 ×106 copies/mL) a week later.Subsequently however, viral load declined to approximately 1×104 copies/mL by 8 weeks postchallenge, and keep steady around the level in the chronic phase.

      Figure 3.  Viral load profile for Rhesus macaque (03047) which was SIV-naÏve at the time of intravenous challenge with SIVmac251. Similar with typical plasma viremia profile for SIV naÏve animals inoculated with SIVmac251, 03047 had a peak of plasma viral RNA value (peak 3.6 ×106 copies/mL) at 2 weeks post-challenge. Subsequently however, viral load declined to 1×104 copies/mL by 8 weeks post-challenge, and keep steady around this level in chronic phase.

    • The levels of viral loads in plasma and CD4+ T cell count have been shown to be the most important indicators for evaluating prognosis of SIV infection, effects of vaccine and antiviral drug therapy (1, 4). Since most SIV infections are restricted in laboratory studies, the commercial kits to detect SIV viral load are lacking in their suitability and expensive. Here we have succeeded in establishing a relatively cheap but highly sensitive SYBR green-based RT-PCR method to quantify the SIV viral load in plasma.

      In a previous report, the SYBR green Real Time RT-PCR for SIVmac251 and SIVmac239 viral RNA detection had a limit as low as 1 000 copies per analysis (3) and the assay based on TaqMan probe can reach a lower limit of 4 copies per reaction in SIV detection (5). This assay based on SYBR green as detection dye showed a sensitivity comparable to the TaqMan method: the detection limit was as low as 10 copies per reaction and 215 copies/mL of plasma. Moreover, the primers targeted a conserved sequence on the SIVmac251 gag region and so can also exactly match with other SIV isolates including SIVmac239, SIVmac17E-Cl, SIVmac17E-Fr and Simian-Human immunodeficiency virus (SHIV) strain 89.6. We did not perform a test on 'one-copy' sensitivities since this is often not realistic and unreliable since there are limitations inherent in distribution of target sequences in the volume of sample aliquots taken for testing.

      Plasma viral load of one Chinese Rhesus was determined by our assay at different times after inoculation of SIVmac251. The dynamics of plasma viral load was highly consistent with previous published results quantified by commercial branched DNA amplification assay in the same animal model (8, 9, 13). Furthermore, by using PMD-gag461 plasmids as DNA standards, the pair of primers in this assay has also been employed in quantification of the proviral DNA of SIVmac251. The detection threshold is as little as 10 copies per reaction, and 50 copies per 106 peripheral blood mononuclear cells (data not shown).

      Some experimental interventions such as vaccination and drug treatment may lead to plasma viral load to drop below the threshold of this assay (215copies/mL). To further increase the sensitivity of plasma virus detection, concentration of virus from plasma is required (16) and the method for viral RNA extraction needs to be modified.

      In conclusion, the Real-Time RT-PCR based on SYBR green as detected dye is less expensive and more convenient than the assay based on labeled probes (7, 16) and b-DNA assay. The method has application in the accurate determination of SIV RNA viral loads and can aid in furthering our understanding of viral dynamics in animal models.

    Figure (3)  Table (1) Reference (20) Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return