ArticleScientific reports2022
Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis.
Article in Scientific reports, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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9 citing papers in PubMed.
- First Molecular Insights into the Presence of Canine Kobuvirus in Ecuadorian Dogs Through the Standardization of a Sensitive SYBR Green RT-qPCR Assay.Veterinary sciences · 2025Article
- Validation and implementation of TaqMAMA RT-PCR for SARS-CoV-2 variant surveillance: experience from a high-volume setting.BMC infectious diseases · 2025Article
- Article
- Genetic Conservation and Diversity of SARS-CoV-2 Envelope Gene Across Variants of Concern.Journal of medical virology · 2025Article
- Assessing Different PCR Master Mixes for Ultrarapid DNA Amplification: Important Analytical Parameters.Diagnostics (Basel, Switzerland) · 2024Article
- A one-step low-cost molecular test for SARS-CoV-2 detection suitable for community testing using minimally processed saliva.Biology methods & protocols · 2024Article
- Evaluation of RT-PCR assays for detection of SARS-CoV-2 variants of concern.Scientific reports · 2023Article
- Identification of mutations in SARS-CoV-2 PCR primer regions.Scientific reports · 2022Article
- Melting Curve-based Assay as an Alternative Technique for the Accurate Detection of SARS-CoV-2.Advanced biomedical research · 2022Article
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Authors and funding
7 authors.
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Abstract
TaqMan probe-based commercial real-time (RT) PCR kits are expensive but most frequently used in COVID-19 diagnosis. The unprecedented scale of SARS-CoV-2 infections needs to meet the challenge of testing more persons at a reasonable cost. This study developed a simple and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay targeting two virus-specific genes along with a host-specific internal control. A total of 180 randomly selected samples portioning into two subsets based on crude and high-quality RNA extraction were used to compare this assay with a nationwide available commercial kit (Sansure Biotech Inc., (Hunan, China)), so that we could analyze the variation and validity of this in-house developed method. Our customized-designed primers can specifically detect the viral RNA likewise Sansure. We separately optimized SYBR Green RT-PCR reaction of N, E, S, and RdRp genes based on singleplex melting curve analysis at the initial stage. After several rounds of optimization on multiplex assays of different primer combinations, the optimized method finally targeted N and E genes of the SARS-CoV-2 virus, together with the β-actin gene of the host as an internal control. Comparing with the Sansure commercial kit, our proposed assay provided up to 97% specificity and 93% sensitivity. The cost of each sample processing ranged between ~2 and ~6 USD depending on the purification level of extracted RNA template. Overall, this one-step and one-tube method can revolutionize the COVID-19 diagnosis in low-income countries.
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