Raekiansyah Muhareva, Rahmasari Ratika, Baihaqy Fathan, Irhamsyah Muhamad, Fajriani Nurul Izza, Putri Mila Meilani, Maharani Botefilia, Sauriasari Rani, Urano Takeshi, Ngwe Tun Mya Myat, Morita Kouichi
Department of Tropical Viral Vaccine Development, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan.
Microbiology and Biotechnology Laboratory, Faculty of Pharmacy, Universitas Indonesia, Depok 16424, West Java, Indonesia.
Viruses. 2025 Aug 18;17(8):1130. doi: 10.3390/v17081130.
During the COVID-19 pandemic, the standard diagnostic assay for SARS-CoV-2 detection was RT-qPCR using TaqMan probes, with samples primarily taken through nasal and oropharyngeal swabs. The TaqMan-based method is costly, highlighting the need for a more affordable alternative for SARS-CoV-2 diagnosis. As an alternative strategy, we developed and evaluated a SYBR Green-based RT-qPCR method targeting the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2. Under optimized RT-qPCR conditions, the sensitivity and linearity of the SYBR assays were assessed by using in vitro-transcribed RNA and RNA extracted from cultured SARS-CoV-2 isolates of the Wuhan reference strain and various circulating variants. Our results demonstrated that the SYBR Green-based RT-qPCR method was successfully developed with sufficient performance. The assay could detect up to 25 copies of in vitro-transcript RNA per reaction. Meanwhile, using the RNA extracted from cultured virus, the SYBR green assay was able to detect virus concentrations at least as low as 1 PFU/mL per reaction for all the variants tested. When tested on clinically relevant samples (88 naso-oropharyngeal swabs and 47 saliva samples), comparable results with the TaqMan assay were demonstrated. The Ct values of both methods for the positively detected samples were similar, with a difference in Ct of 0.72 ± 0.83 ( = 0.392) and -0.7765 ± 0.6107 ( = 0.209) for naso-oropharyngeal swab and saliva samples, respectively. These findings suggest that the SYBR method is reliable and thus offers an alternative assay for the detection of SARS-CoV-2. In particular, using saliva specimens could allow this assay to serve as a simple approach for SARS-CoV-2 detection.
在新冠疫情期间，用于检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的标准诊断方法是使用TaqMan探针的逆转录定量聚合酶链反应（RT-qPCR），样本主要通过鼻拭子和口咽拭子采集。基于TaqMan的方法成本高昂，凸显了开发更经济实惠的SARS-CoV-2诊断替代方法的必要性。作为一种替代策略，我们开发并评估了一种基于SYBR Green的RT-qPCR方法，该方法靶向SARS-CoV-2的RNA依赖性RNA聚合酶（RdRp）基因。在优化的RT-qPCR条件下，通过使用体外转录RNA以及从武汉参考菌株和各种流行变体的培养SARS-CoV-2分离株中提取的RNA，评估了SYBR检测的灵敏度和线性。我们的结果表明，基于SYBR Green的RT-qPCR方法已成功开发，性能良好。该检测方法每个反应最多可检测25个体外转录RNA拷贝。同时，使用从培养病毒中提取的RNA，SYBR Green检测方法能够检测出所有测试变体每个反应中低至至少1个空斑形成单位/毫升的病毒浓度。在临床相关样本（88份鼻-口咽拭子和47份唾液样本）上进行测试时，得到了与TaqMan检测相当的结果。两种方法对阳性检测样本的Ct值相似，鼻-口咽拭子和唾液样本的Ct差异分别为0.72±0.83（P = 0.392）和-0.7765±0.6107（P = 0.209）。这些发现表明，SYBR方法可靠，因此为SARS-CoV-2检测提供了一种替代检测方法。特别是，使用唾液标本可使该检测方法成为一种简单的SARS-CoV-2检测方法。
