Rapid and Real-time Assays for Detection and Quantification of Chikungunya Virus

MM Parida; SR Santhosh; PK Dash; PV Lakshmana Rao

Disclosures

Future Virology. 2008;3(2):179-192. 

In This Article

SYBR Green I Real-time RT-PCR

Although 5'-3' nuclease oligoprobe (TaqMan assay) is the most widely used format owing to its commercial utility and higher specificity as a result of sequence-specific hybridization of the probe, the complicated chemistry - coupled with the requirement of expensive fluorescent probes - has limited the frequent application and routine use of the test system in the laboratory. In the simplest and most economical format, SYBR Green I, a dsDNA-specific intercalating dye is used as the reporter. Although the SYBR Green I-based real-time PCR assay is less specific than the TaqMan real-time RT-PCR, it does have certain advantages, such as reduced expense (especially for high throughput screening), ease of design and testing of primer pairs, and standardization of experiments. It is also useful for the detection of target region variants, which gives additional protection from false-negative results that arise owing to natural variants of RNA viruses.[28]

Considering the simplicity and cost-effectiveness, Santhosh et al. reported the development and evaluation of a sensitive and specific SYBR Green I-based one-step quantitative real-time RT-PCR assay for the rapid detection and quantification of CHIKV RNA using primers targeting the E1 region of the CHIKV genome.[29] Following amplification, a melting curve analysis was performed to verify the correct product by its specific melting temperature (Tm). Melting curve analysis with the curve analytical software of the Mx3000 (Stratagene, USA) showed that the CHIKV E1 gene specific amplicon melts at an average of 81.6°C (81.3-81.9°C). CHIKV-specific amplification and dissociation plots as revealed by SYBR Green I-based one-step quantitative RT-PCR using E1 gene specific primer pairs are shown in Figure 2A and 2B.[29] With comparative analysis, the sensitivity of SYBR Green I-based real-time assay was found to be tenfold more sensitive than the conventional RT-PCR with a detection limit of 0.1 PFU/ml (~10 copies) (Figure 3A and 3B).[29] A standard curve was also plotted from the resulting cycle threshold (Ct) values (against the known concentration of tenfold serial dilutions of CHIKV virus [Figure 4A]).[29]

Real-time kinetics of CHIKV E1 gene-specific SYBR® Green I-based real-time RT-PCR. Figure demonstrates the amplification and dissociation curve for the reference RNA from two isolates. (A) Amplification plot. (B) Melting curve analysis depicting dissociation plot. CHIKV: Chikungunya virus; NTC: No template control; RT: Reverse transcription.

Comparative sensitivity of SYBR® Green I real-time RT-PCR assay with conventional RT-PCR for detection of the CHIKV E1 gene. (A) Sensitivity of real-time assay as shown in the amplification plot from left to right (Repl.1 to Repl.10 as shown in figure) are the curves of decreasing concentration of virus from 107 to 0.01 plaque-forming unit (PFU)/ml in serial tenfold dilution. The detection limit for the assay was 0.1 PFU/ml. (B). Sensitivity of RT-PCR for the detection of the CHIKV E1 gene as observed by 205 bp amplicon on agarose gel analysis with a detection limit of 1 PFU/ml. Lane M: 100 bp DNA ladder (Fermentas, USA); Lane 1-10: Different concentrations of virus ranging from 107 to 0.01 PFU/ml tenfold serial dilution; Lane 11: Negative control. CHIKV: Chikungunya virus; NTC: No template control; Repl.: Replicates; RT: Reverse transcription.

Quantitative estimation of virus load in acute phase patient serum samples by CHIKV E1 gene-specific SYBR® Green I real-time RT-PCR assay. (A) Standard curve for CHIKV-specific real-time assay generated from the cycle threshold (Ct) values obtained against the known concentration of tenfold serially diluted CHIKV ranging from 107 to 1 PFU/ml. (B) Quantitative determination of viral load in clinical samples through Ct value obtained by the clinical samples. The Ct values reflect virus concentration present in the samples through the standard curve generated for CHIKV real-time assay. CHIKV: Chikungunya virus; PFU: Plaque-forming unit; RT: Reverse transcription.

The feasibility of the CHIKV-specific SYBR Green I-based one-step single tube real-time quantitative RT-PCR for clinical diagnosis was validated by evaluating with 51 acute-phase serum samples collected from confirmed Chikungunya patients during an epidemic in southern states of India in April-July, 2006. On comparative evaluation with conventional RT-PCR, SYBR Green I-based real-time RT-PCR demonstrated exceptionally higher sensitivity by detecting 43 positive samples out of 51 acute-phase serum samples, compared to 37 by conventional RT-PCR.[29] It was also observed that none of the serum samples from healthy individuals analyzed in this study showed amplification. The quantification of the viral load in the positive samples was determined on the basis of their Ct values by employing the standard curve developed using the Ct values obtained against known concentration of tenfold serially diluted CHIKV virus. Most of the samples had virus concentrations in the range of 106-108 copies/ml, although in a few samples the amount of virus was quite high, corresponding to 109 copies/ml. In addition, the real-time assay also demonstrated a high degree of specificity for CHIKV virus as demonstrated by cross-reaction studies with related Alphavirus (Ross river virus [RRV], ONNV, SFV and Sindbis virus [SINV]) and clinically similar disease-producing Flavivirus (dengue 1-4, Japanese encephalitis virus [JEV], Yellow fever virus [YFV] and West Nile virus [WNV]) and a panel of serum samples from healthy humans.[29] Thus, the SYBR Green I-based one-step quantitative real-time RT-PCR assay was found to be rapid, easy to handle, highly sensitive, quantitative and specific.

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