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).
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).
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.