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Vitality of Proteinase K in rRTPCR Detection of SARS-CoV2 Bypassing RNA Extraction

This study aimed to detect the SARS-COV2 viral component directly from inoculated VTM without RNA extraction. Inoculated VTMs of already tested 50 positive and 50 negative samples were divided into three groups. Group I was treated with Proteinase K (PK) followed by 3-step-heat treatment at differen...

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Autores principales: Shukla, Alka, Gangwar, Mayank, Sharma, Gaurav, Prakash, Pradyot, Nath, Gopal
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8595283/
https://www.ncbi.nlm.nih.gov/pubmed/34804989
http://dx.doi.org/10.3389/fcimb.2021.717068
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author Shukla, Alka
Gangwar, Mayank
Sharma, Gaurav
Prakash, Pradyot
Nath, Gopal
author_facet Shukla, Alka
Gangwar, Mayank
Sharma, Gaurav
Prakash, Pradyot
Nath, Gopal
author_sort Shukla, Alka
collection PubMed
description This study aimed to detect the SARS-COV2 viral component directly from inoculated VTM without RNA extraction. Inoculated VTMs of already tested 50 positive and 50 negative samples were divided into three groups. Group I was treated with Proteinase K (PK) followed by 3-step-heat treatment at different temperatures (25°C, 60°C, and 98°C) and stored at 4°C. Group II was directly subjected to 3-step-heat treatment without PK exposure and stored at 4°C. And group III was set-up as standard group; it was processed using Qiagen’s column based QIAamp Nucleic Acid kit and the obtained nucleic acids were stored at 4°C. These stored samples were used as a template to execute real-time polymerase chain reaction, and results were noted. Group I demonstrated 96% and 88% sensitivity for N and ORF1ab genes respectively, whereas group II demonstrated 78% and 60% when compared to the results of standard group III. Overall group I showed better results than group II when compared to group III. Thus, in situations where gold-standard reagents are not available, PK exposure and heat treatment can be employed to carry out molecular detection of SARS-CoV2 viral component.
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spelling pubmed-85952832021-11-18 Vitality of Proteinase K in rRTPCR Detection of SARS-CoV2 Bypassing RNA Extraction Shukla, Alka Gangwar, Mayank Sharma, Gaurav Prakash, Pradyot Nath, Gopal Front Cell Infect Microbiol Cellular and Infection Microbiology This study aimed to detect the SARS-COV2 viral component directly from inoculated VTM without RNA extraction. Inoculated VTMs of already tested 50 positive and 50 negative samples were divided into three groups. Group I was treated with Proteinase K (PK) followed by 3-step-heat treatment at different temperatures (25°C, 60°C, and 98°C) and stored at 4°C. Group II was directly subjected to 3-step-heat treatment without PK exposure and stored at 4°C. And group III was set-up as standard group; it was processed using Qiagen’s column based QIAamp Nucleic Acid kit and the obtained nucleic acids were stored at 4°C. These stored samples were used as a template to execute real-time polymerase chain reaction, and results were noted. Group I demonstrated 96% and 88% sensitivity for N and ORF1ab genes respectively, whereas group II demonstrated 78% and 60% when compared to the results of standard group III. Overall group I showed better results than group II when compared to group III. Thus, in situations where gold-standard reagents are not available, PK exposure and heat treatment can be employed to carry out molecular detection of SARS-CoV2 viral component. Frontiers Media S.A. 2021-11-03 /pmc/articles/PMC8595283/ /pubmed/34804989 http://dx.doi.org/10.3389/fcimb.2021.717068 Text en Copyright © 2021 Shukla, Gangwar, Sharma, Prakash and Nath https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular and Infection Microbiology
Shukla, Alka
Gangwar, Mayank
Sharma, Gaurav
Prakash, Pradyot
Nath, Gopal
Vitality of Proteinase K in rRTPCR Detection of SARS-CoV2 Bypassing RNA Extraction
title Vitality of Proteinase K in rRTPCR Detection of SARS-CoV2 Bypassing RNA Extraction
title_full Vitality of Proteinase K in rRTPCR Detection of SARS-CoV2 Bypassing RNA Extraction
title_fullStr Vitality of Proteinase K in rRTPCR Detection of SARS-CoV2 Bypassing RNA Extraction
title_full_unstemmed Vitality of Proteinase K in rRTPCR Detection of SARS-CoV2 Bypassing RNA Extraction
title_short Vitality of Proteinase K in rRTPCR Detection of SARS-CoV2 Bypassing RNA Extraction
title_sort vitality of proteinase k in rrtpcr detection of sars-cov2 bypassing rna extraction
topic Cellular and Infection Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8595283/
https://www.ncbi.nlm.nih.gov/pubmed/34804989
http://dx.doi.org/10.3389/fcimb.2021.717068
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