Peer Review Report For: Generation and Characterization of a variant of Thermostable Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) Enzyme [version 1; peer review: 1 not approved]

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Background Reverse Transcriptase (RT) enzymes have been extensively utilized, especially in Polymerase Chain Reaction (PCR). Many viruses that cause infections worldwide contain ribonucleic acid (RNA), and for their molecular detection, it is essential to convert RNA to DNA using RT. This study aimed to create and characterize a thermostable Moloney Murine Reverse Transcriptase (MMLV-RT) enzyme by using consensus sequences from the latest MMLV-RT sequence database. Methodology The six latest sequences encoding MMLV-RT were retrieved from the NCBI website, and the consensus sequence was determined and cloned into the pET28a (+) vector. The vector was overexpressed in an E. coli expression system. The enzyme obtained was purified using Talon accept resin. The purified enzyme was analyzed by SDS-PAGE and Western Blotting. The enzyme performance was evaluated by performing PCR on 89 SARS-CoV 2 positive samples and 40 negative samples both in a concentrated state and at a dilution factor of x10 −1 . The performance was compared with that of the commercial enzyme, a commercial RT enzyme kit, and Superscript (Invitrogen). Results The enzyme was successfully expressed in E. coli. The concentration of MMLV-RT used was 0.313 mg/mL. The concentrated enzyme detected 98.9% of SARS-CoV-2 RNA, whereas the diluted RT enzyme detected 92.1% of SARS-CoV-2 RNA. In contrast, the diagnostic specificity was 98% for concentrated RT and 95% for diluted RT. This showed that the recombinant in-house MMLV-RT enzyme prototype could be used for the PCR amplification of viral RNA. (Figure 3) Conclusion We successfully produced a recombinant MMLV-RT enzyme whose performance was comparable to that of standard commercial reverse transcriptase (P <0.0001).

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