During the COVID-19 pandemic, demand for molecular diagnosis testing grew quickly worldwide. At present, different RT-qPCR kits used to detect SARS-CoV-2 in patient samples are being widely developed. Since TaqMan-based RT-qPCR was recommended by WHO as the gold standard for SARS-CoV-2 detection, most RT-qPCR assays commercially available on the market are based on TaqMan probe. This method is extremely effective, but not accessible to all laboratories, especially in developing countries. In this study, we have developed a low cost SYBR Green-based real time RT-qPCR assay as an alternative molecular protocol for the detection of SARS-CoV-2 in clinical samples. For this purpose, we designed a new primer pair that targets the nucleocapsid protein (N) gene of SARS-CoV-2.
Our N158 primer were designed based on the sequence from an early 2021 SARS-CoV-2 isolate (MZ026854.1) from Indonesian patients. Since viruses constantly evolve, especially during an outbreak, mutation can emerge, including within the target regions of the primer. When this occurs within the primer region, the mutation could compromise the sensitivity and specificity of RT-qPCR assay21. To investigate this issue, we conducted genetic analysis to establish whether and how frequently mutation had already occurred within the N158 primer binding region of SARS-CoV-2. Through in-silico analysis using a set of 998 SARS-CoV-2 genome sequences from infected individuals in Indonesia, including Delta and Omicron variants, our observations show that there is only a very low prevalence of genome variation in the N158 primer binding regions. This indicates that these regions are highly conserved. Our observation is line with the genetic analysis of coronavirus showing that the N gene of SARS-Cov-2 is conserved and stable. When compared to the SARS-CoV-2 S gene, for example, which has 76% amino acid similarity to the SARS-CoV S gene, the SARS-CoV-2 N gene has 90% amino acid similarity to the SARS-CoV N gene. This indicates that only a few mutations had occurred over time in the coronavirus N gene23,24,25. In general, high conservation of the coronavirus genome is facilitated by the proofreading mechanism of the non-structural protein 14 (nsp14) of the viruses. Nsp14 provides activity to remove nucleotides misincorporated by the RNA-dependent RNA polymerase (RdRp) during viral RNA synthesis and thus generates faithful replication of the large (~ 30 kb) coronavirus genome22,25,26.
Indeed, mutation or mismatch that occurs in primer binding regions does not necessarily affect assay sensitivity. This is particularly true when the genetic variants are present on the left end or 5 prime half of a primer. Specifically, in relation to the sensitivity of diagnostic primers, the 5’ half of a primer is not as important as its 3’ half. In our study, the variants in the forward and reverse primer regions were located near the 5’ end. Therefore, this might not pose a major concern in relation to the sensitivity of our new N158 assay. With regards to analytical sensitivity, our SYBR Green-based assay has been shown to be sensitive for the detection of SARS-CoV-2. With the dynamic range of the assay spanning 7 log units, it was able to detect N molecules up to 10 copy number. The N158 assay has a sensitivity that is similar than that reported in studies targeting the same SARS-CoV-2 N gene27,28.
Using a set of clinical samples, we validated our newly developed SYBR Green-based RT-qPCR assay. All the samples found to be positive by the commercial TaqMan method were confirmed by the SYBR-Green method. All the negative samples tested by TaqMan assay were also found to be negative in the SYBR-Green assay. These results were confirmed by agarose electrophoresis and melting curve analysis. From these two approaches, no primer-dimer or non-specific products were observed in either the positive or negative samples. This indicates that regarding clinical performance, our SYBR Green-based assay was specific and demonstrated similar performance to the TaqMan method in detecting SARS-CoV-2 directly from clinical samples. In addition, the reported intra- and inter-assay variations regarding the Ct values were very small (< 3% for intra-assay and < 4% for inter-assay), suggesting that the N158 assay could generate reproducible results. Therefore overall, besides being sensitive, our SYBR-Green-based RT-qPCR was shown to be specific and reproducible. We believe that our SYBR-Green assay could be used as replacement for the existing TaqMan RT-PCR for effective detection of SARS-CoV-2 in clinical practice.
Apart from its low cost and no need for probe design and synthesis, one of main advantages of SYBR Green-based assay compared to TaqMan-based assay is that it is a simpler approach to designing primers and optimization procedures29.In other words, working with the SYBR Green method is cheaper and easier than using TaqMan. Theoretically, probe-based methods such as TaqMan are superior to SYBR Green-based assay. The power of the TaqMan method is due to its unique design based on oligonucleotide double-labelled probes that significantly increase the sensitivity and specificity of the assay. In contrast, SYBR Green is an unspecific dye. It can intercalate into any double-stranded DNA (dsDNA) such as primer dimers and emits a false positive signal. This unspecific signal can finally lead to a reduction in the performance of SYBR Green-based assay30,31. However, false positive results due to SYBR properties can be avoided by incorporating melting curve analysis at the end of each PCR run. By performing this step, the accuracy of the SYBR Green-based methodology can be assured. In this study, as mentioned above, our SYBR Green-based assay was comparable to TaqMan in the detection of SARS-CoV-2 in the clinical samples. It has been demonstrated that with optimisation of the SYBR Green method, including primer design, its performance and quality could be as good as the TaqMan method.
Commercial COVID-19 RT-qPCR test kits use a variety of SARS-CoV-2 RNA gene targets. Most of the tests target the envelope (E), nucleocapsid (N), spike (S), RNA-dependent RNA polymerase (RdRp) and ORF1 genes of SARS-CoV-2. In addition, they commonly target multiple genes of the virus through the multiplexing strategy32. Apart from its capability to simultaneously detect more than one target in a single analysis, multiplex assay allows accurate diagnosis even if critical mutations occur in the primer binding site of one of the gene targets. This aspect is relevant, because the SARS-CoV-2 genome is constantly mutating, and mutation can occur throughout the virus genome. In this study, we developed an SYBR Green-based assay which targets the N gene of SARS-CoV-2. We utilized the N gene since it is highly conserved and the most sensitive target for SARS-CoV-228,33. The superiority of the N gene with respect to sensitivity is due to its presence in higher amounts in samples compared to other targets. It has been reported that the N gene exists in more abundance as messenger RNAs in coronavirus and subgenomic of mRNA of the N gene are generated during virus replication34. Here we used a single gene target of the N gene. US CDC protocol utilizes two primer sets to two different regions of N gene targets, which are tested in two separate tubes. Both targets, namely N1 and N2, have shown similar performance in the detection of the virus, with no differences between Ct or measurable viral genome copies35,36. Our previous study19 also demonstrated that singleplex amplification of the N gene was equally sensitive and specific in comparison to the commercial TaqMan assay. This suggests that only a single amplification of N sequences could be used for diagnosis of COVID-19, thus reducing testing costs. Furthermore, although our N158 primer were designed based on the sequences from the initial isolate of an Indonesian patient, our N primer are still suitable with respect to assay sensitivity for the detection of SARS-CoV-2. It proved high genetic stability of the N gene in the coronavirus family.
We also evaluated the pooling strategy for efficient detection of SARS-CoV-2. When the disease prevalence is lower, such as in the COVID-19 post-pandemic era, pooling tests might be an alternative approach to effectively enhance detection capability and reduce the testing burden. Previously, pooling strategy has been successfully used for the detection of other infectious pathogens, including the hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1 among blood donors37,38. For COVID-19, a pooling strategy could reduce costs by 69% and it requires tenfold fewer tests39,40.
Three different approaches to pooling tests can be taken: (i) the collection of more than one nasopharyngeal swab in a universal container or VTM; (ii) the mixing of several VTM samples from different patients prior to RNA extraction (pooling of VTM); and (iii) the mixing of several extracted RNA into one sample before RT-qPCR (pooling of extracted RNA). In this study, we chose the third option, as described in previous studies41,42.
Our results demonstrate, in the medium positive group (Ct < 30), there is a 100% concordance between pooling and individual testing. It indicates that 5-sample pooled testing is feasible for samples with a high or medium viral load. However, in the weak positive group, the concordance decreases. Approximately 23% of borderlines samples with Ct value > 33 failed to be detected as positive, indicating that pooling may increase the risk of false negative results when dealing with very low viral loads. Nonetheless. these results underscores the potential advantage of the pooling approach for mass screening testing or epidemiological surveillance purposes.
Furthermore, most pooling studies for SARS-CoV-2 detection have primarily relied on the TaqMan assay. Our study demonstrates that SYBR Green-based RT-PCR can be a viable alternative for implementing the pooling strategy in the detection of SARS-CoV-2. As mentioned earlier, the SYBR Green-based real-time PCR assay is more cost-effective than the TaqMan-based assay. The use of SYBR Green has reduced the material costs for diagnosis by half when compared to probe-based methods43,44,45. In the context of Indonesia, for in-house assays, the SYBR Green-based method typically costs between US $1.0–1.5 per reaction, while the cost of a TaqMan probe can range from US $2–3. These estimates cover the cost of all reagents, excluding consumables used in a PCR reaction. However, it’s important to note that these costs can vary significantly based on factors such as the specific supplier, brand, and the scale of the experiment. Additionally, when compared to conventional PCR, the SYBR method is also more cost-effective, with a fourfold reduction in expenses43. According to our calculations, the overall cost per sample for conventional PCR, excluding consumables, ranges from US $3–4. This relatively higher cost for conventional PCR is due to the need for post-PCR analysis which involves agarose electrophoresis. This post-PCR step requires additional reagents such as agarose, TBE Buffer, Gel-Red (DNA dye), Gel Loading Dye, and DNA Ladder.
Based on our experiences, conventional PCR, when using high-quality Taq polymerase enzyme, can yield results of comparable sensitivity to those obtained with SYBR Green-based methods. Despite the higher initial investment of real-time PCR instruments, real-time PCR offers numerous advantages when compared to conventional PCR for long-term application. This is especially evident when handling high-throughput samples, such as during the SARS-CoV-2 pandemic. Real-time PCR is also less time-consuming, as it eliminates the need for additional post-PCR steps, and it is less labor-intensive. Although SYBR Green-based RT-qPCR methods reduce the cost of reagents per test, the high cost of the equipment remains a barrier to entry, especially in low-income countries, posing limitations to its widespread adoption.
The sample size is one limitation of our study, primarily due to budget constraints. A more extensive testing of clinical specimens is required to further evaluate the diagnostic reliability of our new N158 assay both in individual samples and pooled samples. In addition, validation of our SYBR Green-based RT-qPCR assays using other human pathogenic coronaviruses could be the basis for future studies.