Design and evaluation of an artificial positive control construct for detection of the novel coronavirus 2019 using triplex PCR
Keywords:
Novel coronavirus, artificial construct, triplex-PCR, detectionAbstract
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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Lu H, Stratton CW, Tang YW. Outbreak of pneumonia of unknown etiology in Wuhan China: the Mystery and the Miracle. J Med Virol 2020;92:401-2. https://doi.org/10.1002/jmv.25678
Burki T. Outbreak of coronavirus disease 2019. Lancet Infect Dis 2020;20:292-3.
https://doi.org/10.1016/S1473-3099(20)30076-1
Peeri NC, Shrestha N, Rahman MS, Zaki R, Tan Z, Bibi S, et al. The SARS, MERS and novel coronavirus (COVID-19) epidemics, the newest and biggest global health threats: what lessons have we learned? Int J Epidemiol 2020;49:717-26. https://doi.org/10.1093/ije/dyaa033
Sohrabi C, Alsafi Z, O'Neill N, Khan M, Kerwan A, Al-Jabir A, et al. World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19). Int J Surg 2020;76:71-6.
https://doi.org/10.1016/j.ijsu.2020.02.034
Liu Z, Xiao X, Wei X, Li J, Yang J, Tan H, et al. Composition and divergence of coronavirus spike proteins and host ACE2 receptors predict potential intermediate hosts of SARS-CoV2. J Med Virol 2020;92:595-601.
https://doi.org/10.1002/jmv.25726
Cascella M, Rajnik M, Cuomo A, Dulebohn SC, Di Napoli R. Features, evaluation and treatment coronavirus (COVID-19). In: StatPearls [Internet]. Treasure Island, FL: StatPearls Publishing; 2021 [cited 2021 Dec10]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK554776/.
Kannan S, Shaik Syed Ali P, Sheeza A. COVID-19 (Novel coronavirus 2019) recent trends. Eur Rev Med Pharmacol Sci 2020;24:2006-11.
Phan T. Genetic diversity and evolution of SARSCoV-2. Infect Gen Evol 2020;81:104260.
https://doi.org/10.1016/j.meegid.2020.104260
Wang Y, Kang H, Liu X, Tong Z. Combination of RT-qPCR testing and clinical features for diagnosis of COVID-19 facilitates management of SARS-CoV-2 outbreak. J Med Virol 2020;92:538-9.
https://doi.org/10.1002/jmv.25721
Ai T, Yang Z, Hou H, Zhan C, Chen C, Lv W, et al. Correlation of chest CT and RT-PCR testing in coronavirus disease 2019 (COVID-19) in China: A report of 1014 cases. Radiology 2020:296:E32-E40.
https://doi.org/10.1148/radiol.2020200642
Chan JF, Yip CC, To KK, Tang TH, Wong SC, Leung KH, et al. Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19- RdRp/Hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens. J Clin Microbiol 2020; 58:e00310-20. https://doi.org/10.1128/JCM.00310-20
World Health Organization. Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases: interim guidance, 2 March 2020 [Internet]. 2020 [cited 2021 Dec10]. Available from: https://apps. who.int/iris/handle/10665/331329.
Kim JM, Chung YS, Jo HJ, Lee NJ, Kim MS, Woo SH, et al. Identification of coronavirus isolated from a patient in Korea with COVID-19. Osong Public Health Res Perspect 2020;11:3-7.
https://doi.org/10.24171/j.phrp.2020.11.1.02
Wikramaratna PS, Paton RS, Ghafari M, Lourenço J. Estimating the false-negative test probability of SARS-CoV-2 by RT-PCR. medRxiv 2020:2020.04.05.20053355.
https://doi.org/10.1101/2020.04.05.20053355
Martel F, Grundemann D, Schomig E. A simple method for elimination of false positive results in RT-PCR. J Biochem Mol Biol 2002;35:248-50. https://doi.org/10.5483/BMBRep.2002.35.2.248
El-Tholoth M, Bau HH, Song J. A single and twostage, closed-tube, molecular test for the 2019 novel coronavirus (COVID-19) at home, clinic, and points of entry. ChemRxiv 2020. doi: 10.26434/chemrxiv. 11860137. https://doi.org/10.26434/chemrxiv.11860137.v1
Zu ZY, Jiang MD, Xu PP, Chen W, Ni QQ, Lu GM, et al. Coronavirus disease 2019 (COVID-19): A perspective from China. Radiology 2020;296:E15-E25.
https://doi.org/10.1148/radiol.2020200490
World Health Organization. Surveillance case definitions for human infection with novel coronavirus (nCoV): interim guidance, 11 January 2020 [Internet]. 2020 [cited 2021 Dec 9]. Available from: https://apps.who.int/iris/handle/10665/330376.
Kanne JP. Chest CT findings in 2019-nCoV infections from Wuhan, China: Key points for the radiologist. Radiology 2020;295:16-7. https://doi.org/10.1148/radiol.2020200241
Shi H, Han X, Jiang N, Cao Y, Alwalid O, Gu J, et al. Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive study. Lancet Infect Dis 2020;20:425-34.
https://doi.org/10.1016/S1473-3099(20)30086-4
Xu L, Ensor V, Gossain S, Nye K, Hawkey P. Rapid and simple detection of blaCTX" M genes by multiplex PCR assay. J Med Microbiol 2005;54:1183-7. https://doi.org/10.1099/jmm.0.46160-0
Wittwer CT, Herrmann MG, Gundry CN, ElenitobaJohnson KS. Real-time multiplex PCR assays. Methods 2001;25:430-42. https://doi.org/10.1006/meth.2001.1265
Boivin G, Côté S, Déry P, De Serres G, Bergeron MG. Multiplex real-time PCR assay for detection of influenza and human respiratory syncytial viruses. J Clin Microbiol 2004;42:45-51.
https://doi.org/10.1128/JCM.42.1.45-51.2004
Pourmahdi N, Zeinoddini M, Dehghan Esmatabadi MJ, Sheikhi F. Simple and rapid detection of yersinia pestis and francisella tularensis using Multiplex-PCR. Res Mol Med 2018;6:28-37.
https://doi.org/10.18502/rmm.v6i4.4801
Caasi DR, Arif M, Payton M, Melcher U, Winder L, Ochoa-Corona FM. A multi-target, non-infectious and clonable artificial positive control for routine PCR-based assays. J Microbiol Methods 2013;95:229-34.
https://doi.org/10.1016/j.mimet.2013.08.017
Sohni Y, Kanjilal S, Kapur V. Cloning and development of synthetic internal amplification control for Bacillus anthracis real-time polymerase chain reaction assays. Diagn Microbiol Infect Dis 2008;61:471-5.
https://doi.org/10.1016/j.diagmicrobio.2008.04.005
Nemati AS, Zeinoddini M, Abadi MJDE. Design and construction of a positive control for molecular diagnosis of Coxiella burnetii. Chula Med J 2020;64:389-94.
Samiminemati A, Zeinoddini M, Esmatabadi MJ, Sheikhi F. Development of new multiplex nested PCR for rapid detection of Coxiella burnetii. Res J Biotechnol 2020;15:116-20.
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