Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Aug 26;12(9):198.
doi: 10.3390/diseases12090198.

Utility of Extraction-Free SARS-CoV-2 Detection by RT-qPCR for COVID-19 Testing in a Resource-Limited Setting

Akua K Yalley  1 , Selasie Ahiatrogah  2 , Iddrisu I Moro  3   4 , Peter Gmagna  3 , Isaac K Yankson  5 , Anna A Kafintu-Kwashie  1 , Nicholas I Nii-Trebi  1
Affiliations

Utility of Extraction-Free SARS-CoV-2 Detection by RT-qPCR for COVID-19 Testing in a Resource-Limited Setting

Akua K Yalley et al. Diseases. .

Abstract

The COVID-19 epidemic had a profound impact on global health and the economy and Ghana was no exception to its far-reaching consequences. Regarding detection of the causative agent-the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reverse-transcription-qPCR (RT-qPCR) is widely recognized as a very sensitive and reliable diagnostic technique used globally. There are, however, high operational costs in acquiring test kits, equipment, and accessories for RT-qPCR testing, which pose significant challenges in resource-limited settings. Hence, this proof-of-concept study set out to develop a more affordable COVID-19 protocol for use in low or lower-middle-income settings, such as Ghana, that would bypass the traditional extraction process using inexpensive reagents and evaluate the possibility of processing samples collected using wooden shaft swabs. Several less expensive media were used for the extraction-free process. Results demonstrated that direct RT-qPCR assay after 5 min heat inactivation of virus at 95 °C in 0.1× PBS or molecular grade water resulted in viral detection with quantification cycle (Cq) values that are comparable to results obtained following the extraction process. Also, wooden shaft swabs could be used for sampling if incubation times are kept to less than 6 h. The study demonstrates that extraction-free protocols are one way to minimize the cost of COVID-19 testing by RT-qPCR.

Keywords: COVID-19; RT–qPCR; extraction-free PCR; qPCR.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
A flow chart showing the procedure for comparing different media conditions in extraction-free SARS-CoV-2 RT–qPCR.
Figure 2
Figure 2
Extraction-free SARS-CoV-2 RT–qPCR results comparing different media conditions. (A–D) show bar plots of average Cq values of RT–qPCR targeting internal control (IC), E gene, RDRP gene, and N gene, respectively, in a SARS-CoV-2 positive sample in different media conditions. Data represent the mean +/− standard error of the mean for three replicates (n = 3).
Figure 2
Figure 2
Extraction-free SARS-CoV-2 RT–qPCR results comparing different media conditions. (A–D) show bar plots of average Cq values of RT–qPCR targeting internal control (IC), E gene, RDRP gene, and N gene, respectively, in a SARS-CoV-2 positive sample in different media conditions. Data represent the mean +/− standard error of the mean for three replicates (n = 3).
Figure 3
Figure 3
Evaluating degradation of gene targets in extraction-free RT–qPCR using samples in different media after freezing and thawing. (A–D) show bar plots of average Cq values of RT–qPCR targeting IC, E gene. RDRP gene and N gene, respectively, in a SARS-CoV-2 positive sample subjected to a freezing and thawing cycle. Data represent the mean +/− standard error of the mean for three replicates (n = 3) for all samples except positive and negative controls (n = 2).
Figure 3
Figure 3
Evaluating degradation of gene targets in extraction-free RT–qPCR using samples in different media after freezing and thawing. (A–D) show bar plots of average Cq values of RT–qPCR targeting IC, E gene. RDRP gene and N gene, respectively, in a SARS-CoV-2 positive sample subjected to a freezing and thawing cycle. Data represent the mean +/− standard error of the mean for three replicates (n = 3) for all samples except positive and negative controls (n = 2).
Figure 4
Figure 4
A flow chart showing the process for evaluating wooden shaft swabs in comparison with plastic shaft swabs usage in extraction-free COVID-19 RT–qPCR assay.
Figure 5
Figure 5
Effects of wooden shaft swabs in comparison with plastic shaft swab usage in extraction-free COVID-19 RT–qPCR assay following ≤6 h incubation in selected media. (A–D) show bar plots of average Cq values of RT–qPCR targeting IC, E gene, RDRP gene, and N gene, respectively, in a SARS-CoV-2 positive sample subjected to different media conditions after ≤6 h incubation in plastic shaft swabs or wooden shaft swabs. Data represent the mean +/− standard error of the mean for three replicates (n = 3) for all samples except positive and negative controls (n = 2).
Figure 6
Figure 6
Effects of wooden shaft swabs in comparison with plastic shaft swab usage in extraction-free COVID-19 RT–qPCR assay following 2-day incubation in selected media. (A–D) show bar plots of average Cq values of RT-qPCR targeting IC, E gene, RDRP gene, and N gene, respectively, in a SARS-CoV-2 positive sample subjected to different media conditions after 2-day incubation in plastic shaft swabs or wooden shaft swabs and also following post-incubation extraction. Data represent the mean +/− standard error of the mean for three replicates (n = 3) for all samples except positive and negative controls (n = 2).
Figure 7
Figure 7
Average difference in Cq values following RT–qPCR after storage of heated samples at −20 °C compared to non-stored heated samples. Data represent the mean +/− standard error of the mean for four replicates (n = 4).
Figure 8
Figure 8
A flow chart showing the process of comparing extraction-free COVID-19 RT–qPCR to RT–qPCR using standard extraction procedures.
Figure 9
Figure 9
Comparing detection and sensitivities in Extraction-free COVID-19 RT–qPCR versus RT–qPCR following standard extraction of archived samples in 0.1× PBS across a range of Cq values. (A–D) show bar plots of Cq values of RT-qPCR targeting E gene, RDRP gene, and N gene, respectively, in SARS-CoV-2 positive samples that have undergone standard extraction versus extraction-free samples in 0.1× PBS across a range of Cq values. D shows the average difference in Cq values between extraction-free samples in 0.1× PBS and standard extracted samples. Data represent the mean +/- standard error of the mean for 16 samples (n = 16).
See this image and copyright information in PMC

References

    1. 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. doi: 10.1038/s41586-020-2008-3. - DOI - PMC - PubMed
    1. 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. doi: 10.1056/NEJMoa2001017. - DOI - PMC - PubMed
    1. WHO World Health Organization. WHO Coronavirus (COVID-19) Dashboard 2023. [(accessed on 29 December 2023)]. Available online: https://covid19.who.int/
    1. 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. doi: 10.3390/diseases11020056. - DOI - PMC - PubMed
    1. Kenu E., Frimpong J., Koram K. Responding to the COVID-19 pandemic in Ghana. Ghana. Med. J. 2020;54:72–73. doi: 10.4314/gmj.v54i2.1. - DOI - PMC - PubMed

Grants and funding

This research received no external funding.

LinkOut - more resources