Design and evaluation of an artificial positive control construct for detection of the novel coronavirus 2019 using triplex PCR

Authors

  • Afshin Samiminemati Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Iran
  • Mehdi Zeinoddini Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Iran
  • Nasrin Xodadadi Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Iran
  • Fatemeh Sheikhi Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Iran

Keywords:

Novel coronavirus, artificial construct, triplex-PCR, detection

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is a novel-emerged strain with the person-to-person transmission that had never been found in humans. The main method for the identification of SARS-CoV2 is the genome-based reverse transcription polymerase chain reaction (RT-PCR) using expensive instrument (real time thermal cycler); therefore, viral RNA is required to perform PCR reaction. On the other hand, false-positive or negative results of RT-PCR detection of this virus are a challenge for all clinical laboratories. One smart and scientific strategy for solving this problem is designing an artificial positive control construct.

Objectives: To design and evaluate this positive control construct for the correct and standard detection of SARS-CoV2. To design an artificial construct, three specific viral genomic regions were chosen as the target, which contained open reading frame (ORF1ab), envelope (E), and nucleocapsid (N) of SARS-CoV2. Hence, 813bp-conserved regions were cloned into the EcoRV sequence of pBlueScriptII SK(+) plasmid. In the following, six specific primers were designed for the specific detection of these conserved regions. Finally, the performance of this synthetic construct, named pBlue-ORF-E-N, was simulated in triplex PCR assay using SnapGene software.

Results: The data showed that this synthetic construct was very applicable for detecting SARS-CoV2. Therefore, it is possible to apply a triplexPCR to identify the virus using this construct as a positive control and to compare with the native RT-PCR by extracted viral RNA.

Conclusion: TriplexPCR can be developed using this novel construct in the detection of the emerged virus in order to confirm the common RT-PCR detection used in all clinical laboratories without real time PCR machine.

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Published

2023-07-17

How to Cite

1.
Samiminemati A, Zeinoddini M, Xodadadi N, Sheikhi F. Design and evaluation of an artificial positive control construct for detection of the novel coronavirus 2019 using triplex PCR. Chula Med J [Internet]. 2023 Jul. 17 [cited 2024 Oct. 8];66(2). Available from: https://he05.tci-thaijo.org/index.php/CMJ/article/view/64