SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR

Carme Salvador-Garcia; Maria Dolores Ocete-Mochon; Rafael Medina-Gonzalez; Begona Fuster-Escriva; Sonia Cortes-Badenes; Maria Carmen Breso-Vila; Maria Jose Lahiguera-Abalos; Concepcion Gimeno-Cardona

doi:doi:10.11648/j.ijbecs.20210702.13

SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR

Carme Salvador-Garcia, Maria Dolores Ocete-Mochon, Rafael Medina-Gonzalez, Begona Fuster-Escriva, Sonia Cortes-Badenes, Maria Carmen Breso-Vila, Maria Jose Lahiguera-Abalos, Concepcion Gimeno-Cardona

Published in International Journal of Biomedical Engineering and Clinical Science (Volume 7, Issue 2)

Received: 24 April 2021 Accepted: 12 May 2021 Published: 4 June 2021

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Abstract

The pandemic COVID-19 needs a rapid microbiological diagnostic from Clinical Microbiology Units. Due to the fact that it is done by using a reverse transcription polymerase chain reaction (rRT-PCR) previous RNA extraction and automated equipment and reagents for RNA extraction represent an economic increase to the diagnosis, we describe an easy, cost-effective and fast alternative extraction-free SARS-CoV-2. Samples were treated with proteinase K for 10 minutes at 55°C. Then, there is a heat-process for 5 minutes at 98°C and finally, 3 minutes at -20°C before a commercial-commonly-used rRT-PCR procedure. The RNA automated-extraction was also performed with QIAsymphony RNA Kit (Qiagen) equipment. A total of 220 nasopharyngeal and oropharyngeal swabs were analyzed. 113 samples were tested positive whereas 106 samples were tested negative with RNA automated-extraction and extraction-free method, for an agreement of 99%. A total of one discordant sample was noted in which no amplified result (gene ORF1ab and N) were observed by RNA automated-extraction and gene ORF1ab (Ct 39) and gene N (Ct 37) by extraction-free. Thus, results were comparable with automated-extraction. This method is not only clinically acceptable but also confers an easy, fast, and cost-effective alternative to automated-extraction. Therefore, microbiological laboratories, with low economics resources and/or without automated-extraction equipment, could incorporate it.

Published in	International Journal of Biomedical Engineering and Clinical Science (Volume 7, Issue 2)
DOI	10.11648/j.ijbecs.20210702.13
Page(s)	30-34
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

SARS-CoV-2, COVID-19, Molecular Diagnosis, Cost-effective

References

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[3]	Wong JE, Leo YS, Tan CC. COVID-19 in Singapore. Current Experience: Critical Global Issues That Require Attention and Action. JAMA. 2020, 323 (13): 1243-4. doi: 10.1001/jama.2020.2467.
[4]	Akst J. RNA Extraction Kits for COVID-19 Tests Are in Short Supply in US [Internet]. www.thescientist.com: The Scientist.; 2020 [updated 2020 11 March]. Available from: https://www.the-scientist.com/news-opinion/rna-extraction-kits-for-covid-19-tests-are-in-shortsupply-in-us-67250.
[5]	Chu DKW, Pan Y, Cheng SMS, Hui KPY, Krishnan P, Liu Y, et al. Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia. Clin Chem.2020. 66 (4): 549-55. doi: 10.1093/clinchem/hvaa029.
[6]	Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DKW, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020, 25 (3). doi: 10.2807/1560-7917.ES.2020.25.3.2000045.
[7]	Kim D, Lee JY, Yang JS, Kim JW, Kim VN, Chang H. The architecture of SARS-CoV-2 transcriptome. Cell. 2020, 181 (4): 914–2110.
[8]	Bullard J, Dust K, Funk D, Strong JE, Alexander D, Garnett L, et al. Predicting infectious SARS-CoV-2 from diagnostic samples. Clin Infect Dis. 2020. https://doi.org/10.1093/cid/ciaa638.
[9]	La Scola B, Le Bideau M, Andreani J, Hoang VT, Grimaldier C, Colson P, et al. Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards. Eur J Clin Microbiol Infect Dis. 2020, 39 (6): 1059–61.
[10]	Zheng S, Fan J, Yu F, Feng B, Lou B, Zou Q, et al. Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China, January–March 2020: retrospective cohort study. BMJ. 2020, 369: m1443.
[11]	Huang CG, Lee KM, Hsiao MJ, Yang SL, Huang PN, Gong YN, et al. Culturebased virus isolation to evaluate potential infectivity of clinical specimens tested for COVID-19. J Clin Microbiol. 2020. https://doi.org/10.1128/ JCM.01068-20.
[12]	Folgueira MD, Luczkowiak J, Lasala F, Perez-Rivilla A, Delgado R. Persistent SARS-CoV-2 replication in severe COVID-19. medRxiv. 2020. https://doi. org/10.1101/2020.06.10.20127837.
[13]	Zou L, Ruan F, Huang M, Liang L, Huang H, Hong Z, et al. SARS-CoV-2 viral load in upper respiratory specimens of infected patients. N Engl J Med. 2020, 382 (12): 1177–9.
[14]	Fomsgaard AS and Rosenstierne MW. An alternative workflow for molecular detection of SARS-CoV-2 - escape from the NA extraction kit-shortage, Copenhagen, Denmark. March, 2020. https://doi.org/10.1101/2020.03.27.20044495.
[15]	Mallmann L, Schallenberger K, Demolliner M, Antunes Eisen AK, Saraiva Hermann B, Heldt FH, et al. Pre-treatment of the clinical sample with Proteinase K allows detection of SARS-CoV-2 in the absence of RNA extraction. 2020. https://doi.org/10.1101/2020.05.07.083139.

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Carme Salvador-Garcia, Maria Dolores Ocete-Mochon, Rafael Medina-Gonzalez, Begona Fuster-Escriva, Sonia Cortes-Badenes, et al. (2021). SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR. International Journal of Biomedical Engineering and Clinical Science, 7(2), 30-34. https://doi.org/10.11648/j.ijbecs.20210702.13

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ACS Style

Carme Salvador-Garcia; Maria Dolores Ocete-Mochon; Rafael Medina-Gonzalez; Begona Fuster-Escriva; Sonia Cortes-Badenes, et al. SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR. Int. J. Biomed. Eng. Clin. Sci. 2021, 7(2), 30-34. doi: 10.11648/j.ijbecs.20210702.13

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AMA Style

Carme Salvador-Garcia, Maria Dolores Ocete-Mochon, Rafael Medina-Gonzalez, Begona Fuster-Escriva, Sonia Cortes-Badenes, et al. SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR. Int J Biomed Eng Clin Sci. 2021;7(2):30-34. doi: 10.11648/j.ijbecs.20210702.13

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@article{10.11648/j.ijbecs.20210702.13,
  author = {Carme Salvador-Garcia and Maria Dolores Ocete-Mochon and Rafael Medina-Gonzalez and Begona Fuster-Escriva and Sonia Cortes-Badenes and Maria Carmen Breso-Vila and Maria Jose Lahiguera-Abalos and Concepcion Gimeno-Cardona},
  title = {SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR},
  journal = {International Journal of Biomedical Engineering and Clinical Science},
  volume = {7},
  number = {2},
  pages = {30-34},
  doi = {10.11648/j.ijbecs.20210702.13},
  url = {https://doi.org/10.11648/j.ijbecs.20210702.13},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijbecs.20210702.13},
  abstract = {The pandemic COVID-19 needs a rapid microbiological diagnostic from Clinical Microbiology Units. Due to the fact that it is done by using a reverse transcription polymerase chain reaction (rRT-PCR) previous RNA extraction and automated equipment and reagents for RNA extraction represent an economic increase to the diagnosis, we describe an easy, cost-effective and fast alternative extraction-free SARS-CoV-2. Samples were treated with proteinase K for 10 minutes at 55°C. Then, there is a heat-process for 5 minutes at 98°C and finally, 3 minutes at -20°C before a commercial-commonly-used rRT-PCR procedure. The RNA automated-extraction was also performed with QIAsymphony RNA Kit (Qiagen) equipment. A total of 220 nasopharyngeal and oropharyngeal swabs were analyzed. 113 samples were tested positive whereas 106 samples were tested negative with RNA automated-extraction and extraction-free method, for an agreement of 99%. A total of one discordant sample was noted in which no amplified result (gene ORF1ab and N) were observed by RNA automated-extraction and gene ORF1ab (Ct 39) and gene N (Ct 37) by extraction-free. Thus, results were comparable with automated-extraction. This method is not only clinically acceptable but also confers an easy, fast, and cost-effective alternative to automated-extraction. Therefore, microbiological laboratories, with low economics resources and/or without automated-extraction equipment, could incorporate it.},
 year = {2021}
}

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TY  - JOUR
T1  - SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR
AU  - Carme Salvador-Garcia
AU  - Maria Dolores Ocete-Mochon
AU  - Rafael Medina-Gonzalez
AU  - Begona Fuster-Escriva
AU  - Sonia Cortes-Badenes
AU  - Maria Carmen Breso-Vila
AU  - Maria Jose Lahiguera-Abalos
AU  - Concepcion Gimeno-Cardona
Y1  - 2021/06/04
PY  - 2021
N1  - https://doi.org/10.11648/j.ijbecs.20210702.13
DO  - 10.11648/j.ijbecs.20210702.13
T2  - International Journal of Biomedical Engineering and Clinical Science
JF  - International Journal of Biomedical Engineering and Clinical Science
JO  - International Journal of Biomedical Engineering and Clinical Science
SP  - 30
EP  - 34
PB  - Science Publishing Group
SN  - 2472-1301
UR  - https://doi.org/10.11648/j.ijbecs.20210702.13
AB  - The pandemic COVID-19 needs a rapid microbiological diagnostic from Clinical Microbiology Units. Due to the fact that it is done by using a reverse transcription polymerase chain reaction (rRT-PCR) previous RNA extraction and automated equipment and reagents for RNA extraction represent an economic increase to the diagnosis, we describe an easy, cost-effective and fast alternative extraction-free SARS-CoV-2. Samples were treated with proteinase K for 10 minutes at 55°C. Then, there is a heat-process for 5 minutes at 98°C and finally, 3 minutes at -20°C before a commercial-commonly-used rRT-PCR procedure. The RNA automated-extraction was also performed with QIAsymphony RNA Kit (Qiagen) equipment. A total of 220 nasopharyngeal and oropharyngeal swabs were analyzed. 113 samples were tested positive whereas 106 samples were tested negative with RNA automated-extraction and extraction-free method, for an agreement of 99%. A total of one discordant sample was noted in which no amplified result (gene ORF1ab and N) were observed by RNA automated-extraction and gene ORF1ab (Ct 39) and gene N (Ct 37) by extraction-free. Thus, results were comparable with automated-extraction. This method is not only clinically acceptable but also confers an easy, fast, and cost-effective alternative to automated-extraction. Therefore, microbiological laboratories, with low economics resources and/or without automated-extraction equipment, could incorporate it.
VL  - 7
IS  - 2
ER  -

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Author Information

Carme Salvador-Garcia

Department of Microbiology, Consorcio General University Hospital of Valencia, Valencia, Spain
Maria Dolores Ocete-Mochon

Department of Microbiology, Consorcio General University Hospital of Valencia, Valencia, Spain
Rafael Medina-Gonzalez

Department of Microbiology, Consorcio General University Hospital of Valencia, Valencia, Spain
Begona Fuster-Escriva

Department of Microbiology, Consorcio General University Hospital of Valencia, Valencia, Spain
Sonia Cortes-Badenes

Department of Microbiology, Consorcio General University Hospital of Valencia, Valencia, Spain
Maria Carmen Breso-Vila

Department of Microbiology, Consorcio General University Hospital of Valencia, Valencia, Spain
Maria Jose Lahiguera-Abalos

Department of Microbiology, Consorcio General University Hospital of Valencia, Valencia, Spain
Concepcion Gimeno-Cardona

Department of Microbiology, Consorcio General University Hospital of Valencia, Valencia, Spain

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APA Style

Carme Salvador-Garcia, Maria Dolores Ocete-Mochon, Rafael Medina-Gonzalez, Begona Fuster-Escriva, Sonia Cortes-Badenes, et al. (2021). SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR. International Journal of Biomedical Engineering and Clinical Science, 7(2), 30-34. https://doi.org/10.11648/j.ijbecs.20210702.13

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ACS Style

Carme Salvador-Garcia; Maria Dolores Ocete-Mochon; Rafael Medina-Gonzalez; Begona Fuster-Escriva; Sonia Cortes-Badenes, et al. SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR. Int. J. Biomed. Eng. Clin. Sci. 2021, 7(2), 30-34. doi: 10.11648/j.ijbecs.20210702.13

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AMA Style

Carme Salvador-Garcia, Maria Dolores Ocete-Mochon, Rafael Medina-Gonzalez, Begona Fuster-Escriva, Sonia Cortes-Badenes, et al. SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR. Int J Biomed Eng Clin Sci. 2021;7(2):30-34. doi: 10.11648/j.ijbecs.20210702.13

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@article{10.11648/j.ijbecs.20210702.13,
  author = {Carme Salvador-Garcia and Maria Dolores Ocete-Mochon and Rafael Medina-Gonzalez and Begona Fuster-Escriva and Sonia Cortes-Badenes and Maria Carmen Breso-Vila and Maria Jose Lahiguera-Abalos and Concepcion Gimeno-Cardona},
  title = {SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR},
  journal = {International Journal of Biomedical Engineering and Clinical Science},
  volume = {7},
  number = {2},
  pages = {30-34},
  doi = {10.11648/j.ijbecs.20210702.13},
  url = {https://doi.org/10.11648/j.ijbecs.20210702.13},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijbecs.20210702.13},
  abstract = {The pandemic COVID-19 needs a rapid microbiological diagnostic from Clinical Microbiology Units. Due to the fact that it is done by using a reverse transcription polymerase chain reaction (rRT-PCR) previous RNA extraction and automated equipment and reagents for RNA extraction represent an economic increase to the diagnosis, we describe an easy, cost-effective and fast alternative extraction-free SARS-CoV-2. Samples were treated with proteinase K for 10 minutes at 55°C. Then, there is a heat-process for 5 minutes at 98°C and finally, 3 minutes at -20°C before a commercial-commonly-used rRT-PCR procedure. The RNA automated-extraction was also performed with QIAsymphony RNA Kit (Qiagen) equipment. A total of 220 nasopharyngeal and oropharyngeal swabs were analyzed. 113 samples were tested positive whereas 106 samples were tested negative with RNA automated-extraction and extraction-free method, for an agreement of 99%. A total of one discordant sample was noted in which no amplified result (gene ORF1ab and N) were observed by RNA automated-extraction and gene ORF1ab (Ct 39) and gene N (Ct 37) by extraction-free. Thus, results were comparable with automated-extraction. This method is not only clinically acceptable but also confers an easy, fast, and cost-effective alternative to automated-extraction. Therefore, microbiological laboratories, with low economics resources and/or without automated-extraction equipment, could incorporate it.},
 year = {2021}
}

Copy | Download

TY  - JOUR
T1  - SARS-CoV-2 Detection: Fast and Cost-Effective Sample Processing Prior to RT-PCR
AU  - Carme Salvador-Garcia
AU  - Maria Dolores Ocete-Mochon
AU  - Rafael Medina-Gonzalez
AU  - Begona Fuster-Escriva
AU  - Sonia Cortes-Badenes
AU  - Maria Carmen Breso-Vila
AU  - Maria Jose Lahiguera-Abalos
AU  - Concepcion Gimeno-Cardona
Y1  - 2021/06/04
PY  - 2021
N1  - https://doi.org/10.11648/j.ijbecs.20210702.13
DO  - 10.11648/j.ijbecs.20210702.13
T2  - International Journal of Biomedical Engineering and Clinical Science
JF  - International Journal of Biomedical Engineering and Clinical Science
JO  - International Journal of Biomedical Engineering and Clinical Science
SP  - 30
EP  - 34
PB  - Science Publishing Group
SN  - 2472-1301
UR  - https://doi.org/10.11648/j.ijbecs.20210702.13
AB  - The pandemic COVID-19 needs a rapid microbiological diagnostic from Clinical Microbiology Units. Due to the fact that it is done by using a reverse transcription polymerase chain reaction (rRT-PCR) previous RNA extraction and automated equipment and reagents for RNA extraction represent an economic increase to the diagnosis, we describe an easy, cost-effective and fast alternative extraction-free SARS-CoV-2. Samples were treated with proteinase K for 10 minutes at 55°C. Then, there is a heat-process for 5 minutes at 98°C and finally, 3 minutes at -20°C before a commercial-commonly-used rRT-PCR procedure. The RNA automated-extraction was also performed with QIAsymphony RNA Kit (Qiagen) equipment. A total of 220 nasopharyngeal and oropharyngeal swabs were analyzed. 113 samples were tested positive whereas 106 samples were tested negative with RNA automated-extraction and extraction-free method, for an agreement of 99%. A total of one discordant sample was noted in which no amplified result (gene ORF1ab and N) were observed by RNA automated-extraction and gene ORF1ab (Ct 39) and gene N (Ct 37) by extraction-free. Thus, results were comparable with automated-extraction. This method is not only clinically acceptable but also confers an easy, fast, and cost-effective alternative to automated-extraction. Therefore, microbiological laboratories, with low economics resources and/or without automated-extraction equipment, could incorporate it.
VL  - 7
IS  - 2
ER  -

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