Utility of Extraction-Free SARS-CoV-2 Detection by RT–qPCR for COVID-19 Testing in a Resource-Limited Setting
Abstract
:1. Introduction
2. Materials and Methods
2.1. Samples
2.2. Solutions and Swabs
2.3. Viral RNA Extraction
2.4. Heat Inactivation
2.5. One-Step RT–qPCR
2.6. Biosafety Conditions
2.7. Data Analysis
3. Results
3.1. Evaluating Extraction-Free RT–qPCR Using Samples in Different Media with and without Heat Inactivation
3.2. Evaluating Degradation of Targets in Extraction-Free RT–qPCR Using Samples in Different Media after Freezing and Thawing
3.3. Evaluating the Impact of Plastic Shaft and Wooden Shaft Swabs in Extraction-Free COVID-19 RT–qPCR Assay after ≤6 h Incubation in Selected Media
3.4. Evaluating Plastic Shaft Versus Wooden Shaft Swabs in Extraction-Free COVID-19 RT–qPCR Assay after 2-Day Incubation in Selected Media
3.5. Effect of Freeze–Thawing on Extraction-Free COVID-19 RT–qPCR after Heating Samples at 95 °C
3.6. Extraction-Free COVID-19 RT–qPCR Compared to RT–qPCR Following Standard Extraction of Archived Samples across a Range of Ct Values
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
SARS-CoV-2 | severe acute respiratory syndrome coronavirus 2 |
RT–qPCR | reverse-transcription quantitative polymerase chain reaction |
rRT-PCR | real-time reverse-transcription-PCR |
Cq | quantification cycle |
COVID-19 | coronavirus disease 2019 |
WHO | World Health Organization |
PCR | polymerase chain reaction |
qPCR | quantitative real-time PCR |
RT | reverse-transcription |
cDNA | complementary DNA |
Pos ctrl | positive control |
Neg ctrl | Negative control |
MIC | magnetic induction cycler |
GITC | Guanidinium isothiocyanate |
Ext ctrl | extracted control |
0.9% Sal ht | unextracted samples in 0.9% normal saline, heated at 95 °C for 5 min |
1× PBS ht | unextracted samples in 1× PBS heated for 5 min at 95 °C |
0.1× PBS ht | unextracted samples in 0.1× PBS, heated for 5 min at 95 °C |
H2O ht | unextracted samples in nuclease-free water, heated for 5 min at 95 °C |
0.1× PBS rt | unextracted samples in 0.1× PBS at room temperature |
H2O rt | unextracted samples in nuclease-free water at room temperature |
Frz | Freeze |
Ext | Extracted |
HeFRA | Health Facilities Regulatory Agency |
PPE | personal protective equipment |
BSL-2 | Biosafety Level 2 |
References
- Wu, F.; Zhao, S.; Yu, B.; Chen, Y.-M.; Wang, W.; Song, Z.-G.; Hu, Y.; Tao, Z.-W.; Tian, J.-H.; Pei, Y.-Y. A new coronavirus associated with human respiratory disease in China. Nature 2020, 579, 265–269. [Google Scholar] [CrossRef] [PubMed]
- Zhu, N.; Zhang, D.; Wang, W.; Li, X.; Yang, B.; Song, J.; Zhao, X.; Huang, B.; Shi, W.; Lu, R. A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. 2020, 382, 727–733. [Google Scholar] [CrossRef] [PubMed]
- WHO. World Health Organization. WHO Coronavirus (COVID-19) Dashboard 2023. Available online: https://covid19.who.int/ (accessed on 29 December 2023).
- Yalley, A.K.; Ahiatrogah, S.; Yalley, A.B.; Yankson, I.K.; Nii-Trebi, N.I.; Yalley, A.A. Did Ghana Do Enough? A Scientometric Analysis of COVID-19 Research Output from Ghana within the African Context. Diseases 2023, 11, 56. [Google Scholar] [CrossRef] [PubMed]
- Kenu, E.; Frimpong, J.; Koram, K. Responding to the COVID-19 pandemic in Ghana. Ghana. Med. J. 2020, 54, 72–73. [Google Scholar] [CrossRef]
- AfricaCDC. Africa Identifies First Case of Coronavirus Disease: Statement by the Director of Africa CDC: 15th February 2020. Available online: https://africacdc.org/news-item/africa-identifies-first-case-of-coronavirus-disease-statement-by-the-director-of-africa-cdc/ (accessed on 30 April 2024).
- Worldometer. Available online: https://www.worldometers.info/coronavirus/ (accessed on 23 December 2023).
- Ksiazek, T.G.; Erdman, D.; Goldsmith, C.S.; Zaki, S.R.; Peret, T.; Emery, S.; Tong, S.; Urbani, C.; Comer, J.A.; Lim, W. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 2003, 348, 1953–1966. [Google Scholar] [CrossRef]
- Yang, S.; Rothman, R.E. PCR-based diagnostics for infectious diseases: Uses, limitations, and future applications in acute-care settings. Lancet Infect. Dis. 2004, 4, 337–348. [Google Scholar] [CrossRef]
- Maddocks, S.; Jenkins, R. Chapter 4-Quantitative PCR: Things to consider. In Understanding PCR; Academic: Boston, MA, USA, 2017; pp. 45–52. [Google Scholar]
- Smyrlaki, I.; Ekman, M.; Lentini, A.; Rufino de Sousa, N.; Papanicolaou, N.; Vondracek, M.; Aarum, J.; Safari, H.; Muradrasoli, S.; Rothfuchs, A.G. Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR. Nat. Commun. 2020, 11, 4812. [Google Scholar] [CrossRef]
- Abdullahi, I.N.; Emeribe, A.U.; Akande, A.O.; Ghamba, P.E.; Adekola, H.A.; Ibrahim, Y.; Dangana, A. Roles and challenges of coordinated public health laboratory response against COVID-19 pandemic in Africa. J. Infect. Dev. Ctries. 2020, 14, 691–695. [Google Scholar] [CrossRef]
- WHO: World Health Organization. Laboratory Testing for Coronavirus Disease (COVID-19) in Suspected Human Cases; World Health Organization: Geneva, Switzerland, 2020; Available online: https://www.who.int/publications/i/item/10665-331501 (accessed on 2 January 2024).
- Castro, R.; Luz, P.M.; Wakimoto, M.D.; Veloso, V.G.; Grinsztejn, B.; Perazzo, H. COVID-19: A meta-analysis of diagnostic test accuracy of commercial assays registered in Brazil. Braz. J. Infect. Dis. 2020, 24, 180–187. [Google Scholar] [CrossRef]
- Okba, N.M.; Müller, M.A.; Li, W.; Wang, C.; GeurtsvanKessel, C.H.; Corman, V.M.; Lamers, M.M.; Sikkema, R.S.; de Bruin, E.; Chandler, F.D. Severe acute respiratory syndrome coronavirus 2−specific antibody responses in coronavirus disease patients. Emerg. Infect. Dis. 2020, 26, 1478. [Google Scholar] [CrossRef]
- Worldbank. World Bank 2023 World Bank Country and Lending Groups. Available online: https://www.worldbank.org/en/home/ (accessed on 25 December 2023).
- Hoffman, T.; Nissen, K.; Krambrich, J.; Rönnberg, B.; Akaberi, D.; Esmaeilzadeh, M.; Salaneck, E.; Lindahl, J.; Lundkvist, Å. Evaluation of a COVID-19 IgM and IgG rapid test; an efficient tool for assessment of past exposure to SARS-CoV-2. Infect. Ecol. Epidemiol. 2020, 10, 1754538. [Google Scholar] [CrossRef]
- Yalley, A.K.; Ahiatrogah, S.; Kafintu-Kwashie, A.A.; Amegatcher, G.; Prah, D.; Botwe, A.K.; Adusei-Poku, M.A.; Obodai, E.; Nii-Trebi, N.I. A systematic review on suitability of molecular techniques for diagnosis and research into infectious diseases of concern in resource-limited settings. Curr. Issues Mol. Biol. 2022, 44, 4367–4385. [Google Scholar] [CrossRef] [PubMed]
- Sharma, M.G.; Popli, H. Challenges for Lower-Middle-Income Countries in Achieving Universal Healthcare: An Indian Perspective. Cureus 2023, 15, e33751. [Google Scholar] [CrossRef] [PubMed]
- Chin, A.W.; Chu, J.T.; Perera, M.R.; Hui, K.P.; Yen, H.-L.; Chan, M.C.; Peiris, M.; Poon, L.L. Stability of SARS-CoV-2 in different environmental conditions. Lancet Microbe 2020, 1, e10. [Google Scholar] [CrossRef] [PubMed]
- Morecchiato, F.; Coppi, M.; Baccani, I.; Maggini, N.; Ciccone, N.; Antonelli, A.; Rossolini, G.M. Evaluation of extraction-free RT-PCR methods for faster and cheaper detection of SARS-CoV-2 using two commercial systems. Int. J. Infect. Dis. 2021, 112, 264–268. [Google Scholar] [CrossRef]
- Chu, A.W.-H.; Chan, W.-M.; Ip, J.D.; Yip, C.C.-Y.; Chan, J.F.-W.; Yuen, K.-Y.; To, K.K.-W. Evaluation of simple nucleic acid extraction methods for the detection of SARS-CoV-2 in nasopharyngeal and saliva specimens during global shortage of extraction kits. J. Clin. Virol. 2020, 129, 104519. [Google Scholar] [CrossRef]
- Kim, Y.K.; Chang, S.H. Clinical usefulness of extraction-free PCR assay to detect SARS-CoV-2. J. Virol. Methods 2021, 296, 114217. [Google Scholar] [CrossRef]
- Jayaprakasam, M.; Aggarwal, S.; Mane, A.; Saxena, V.; Rao, A.; Bandopadhyay, B.; Chakraborty, B.; Guha, S.K.; Taraphdar, M.; Acharya, A. RNA-extraction-free diagnostic method to detect SARS-CoV-2: An assessment from two states, India. Epidemiol. Infect. 2021, 149, e245. [Google Scholar] [CrossRef]
- Ngetsa, C.; Osoti, V.; Okanda, D.; Marura, F.; Shah, K.; Karanja, H.; Mugo, D.; Gitonga, J.; Mutunga, M.; Lewa, C. Validation of saline, PBS and a locally produced VTM at varying storage conditions to detect the SARS-CoV-2 virus by qRT-PCR. PLoS ONE 2023, 18, e0280685. [Google Scholar] [CrossRef]
- CDC: Centre for Disease Control and Prevention. 2022. Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons for Coronavirus Disease 2019 (COVID-19) URL. Available online: https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html (accessed on 29 December 2023).
- Hedman, J.; Knutsson, R.; Ansell, R.; Rådström, P.; Rasmusson, B. Pre-PCR processing in bioterrorism preparedness: Improved diagnostic capabilities for laboratory response networks. Biosecurity Bioterrorism Biodefense Strategy Pract. Sci. 2013, 11, S87–S101. [Google Scholar] [CrossRef]
- Lee, A.; Cooper, T. Improved direct PCR screen for bacterial colonies: Wooden toothpicks inhibit PCR amplification. Biotechniques 1995, 18, 225–226. [Google Scholar] [PubMed]
- Nairz, M.; Bellmann-Weiler, R.; Ladstaetter, M.; Schuellner, F.; Zimmermann, M.; Koller, A.-M.; Blunder, S.; Naschberger, H.; Klotz, W.; Herold, M. Overcoming limitations in the availability of swabs systems used for SARS-CoV-2 laboratory diagnostics. Sci. Rep. 2021, 11, 2261. [Google Scholar] [CrossRef]
- Valadan, R.; Golchin, S.; Alizadeh-Navaei, R.; Haghshenas, M.; Zargari, M.; Mousavi, T.; Ghamati, M. Differential gene expression analysis of common target genes for the detection of SARS-CoV-2 using real time-PCR. AMB Express 2022, 12, 112. [Google Scholar] [CrossRef] [PubMed]
- Vogels, C.B.; Brito, A.F.; Wyllie, A.L.; Fauver, J.R.; Ott, I.M.; Kalinich, C.C.; Petrone, M.E.; Casanovas-Massana, A.; Catherine Muenker, M.; Moore, A.J. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT–qPCR primer–probe sets. Nat. Microbiol. 2020, 5, 1299–1305. [Google Scholar] [CrossRef] [PubMed]
- Zhang, X.; Li, M.; Zhang, B.; Chen, T.; Lv, D.; Xia, P.; Sun, Z.; Shentu, X.; Chen, H.; Li, L. The N gene of SARS-CoV-2 was the main positive component in repositive samples from a cohort of COVID-19 patients in Wuhan, China. Clin. Chim. Acta 2020, 511, 291–297. [Google Scholar] [CrossRef]
- Abbasi, H.; Tabaraei, A.; Hosseini, S.M.; Khosravi, A.; Nikoo, H.R. Real-time PCR Ct value in SARS-CoV-2 detection: RdRp or N gene? Infection 2022, 50, 537–540. [Google Scholar] [CrossRef]
- Kellman, B.P.; Baghdassarian, H.M.; Pramparo, T.; Shamie, I.; Gazestani, V.; Begzati, A.; Li, S.; Nalabolu, S.; Murray, S.; Lopez, L. Multiple freeze-thaw cycles lead to a loss of consistency in poly (A)-enriched RNA sequencing. BMC Genom. 2021, 22, 69. [Google Scholar] [CrossRef]
- Delgado-Diaz, D.J.; Sakthivel, D.; Nguyen, H.H.; Farrokzhad, K.; Hopper, W.; Narh, C.A.; Richards, J.S. Strategies that facilitate extraction-free SARS-CoV-2 nucleic acid amplification tests. Viruses 2022, 14, 1311. [Google Scholar] [CrossRef] [PubMed]
- Barza, R.; Patel, P.; Sabatini, L.; Singh, K. Use of a simplified sample processing step without RNA extraction for direct SARS-CoV-2 RT-PCR detection. J. Clin. Virol. 2020, 132, 104587. [Google Scholar] [CrossRef]
- Rhoads, D.D.; Pinsky, B.A. The truth about SARS-CoV-2 cycle threshold values is rarely pure and never simple. Clin. Chem. 2022, 68, 16–18. [Google Scholar] [CrossRef]
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Yalley, A.K.; Ahiatrogah, S.; Moro, I.I.; Gmagna, P.; Yankson, I.K.; Kafintu-Kwashie, A.A.; Nii-Trebi, N.I. Utility of Extraction-Free SARS-CoV-2 Detection by RT–qPCR for COVID-19 Testing in a Resource-Limited Setting. Diseases 2024, 12, 198. https://doi.org/10.3390/diseases12090198
Yalley AK, Ahiatrogah S, Moro II, Gmagna P, Yankson IK, Kafintu-Kwashie AA, Nii-Trebi NI. Utility of Extraction-Free SARS-CoV-2 Detection by RT–qPCR for COVID-19 Testing in a Resource-Limited Setting. Diseases. 2024; 12(9):198. https://doi.org/10.3390/diseases12090198
Chicago/Turabian StyleYalley, Akua K., Selasie Ahiatrogah, Iddrisu I. Moro, Peter Gmagna, Isaac K. Yankson, Anna A. Kafintu-Kwashie, and Nicholas I. Nii-Trebi. 2024. "Utility of Extraction-Free SARS-CoV-2 Detection by RT–qPCR for COVID-19 Testing in a Resource-Limited Setting" Diseases 12, no. 9: 198. https://doi.org/10.3390/diseases12090198