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

• Published in: Frontiers in cellular and infection microbiology Vol. 11; p. 717068
• Main Authors: Shukla, Alka, Gangwar, Mayank, Sharma, Gaurav, Prakash, Pradyot, Nath, Gopal
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 03.11.2021
• Subjects: Cellular and Infection Microbiology; COVID – 19; Endopeptidase K; heat inactivation; Humans; Proteinase K; real time PCR; Real-Time Polymerase Chain Reaction; RNA, Viral - genetics; SARS-CoV-2; SARS-CoV-2 detection; real time PCR; COVID – 19; SARS-CoV-2 detection; heat inactivation; Proteinase K
• ISSN: 2235-2988; 2235-2988
• DOI: 10.3389/fcimb.2021.717068

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.