Development and evaluation of in-house SARS-COV-2 RNA extraction kit at Genomics Molecular Biology Laboratory, College of Health Sciences, Makerere University

Abstract

Objective: The purpose of this research was to develope in-house RNA extraction kit for SARCOV-2 using readily available reagents at Genomics Molecular biology Laboratory. Methodology: The study was an applied experimental study aimed at developing an in-house SARS-COV-2 RNA extraction kit for Covid-19 samples at Genomics Molecular Biology Laboratory . Reagents and samples used for the study were obtained from Mak-CHS molecular biology lab with requested permission from the head of molecular biology laboratory. Nine nasopharyngeal swab samples for covid-19 that were stored under appropriate conditions were used for the study where some were positive and others were negative. SARS-CoV-2 RNA extraction from the 9 samples was done in two sets using two RNA extraction kits i.e developed in-house covid-19 RNA extraction kit and the Zymo Quick Viral RNA kit which acted as the reference kit for comparison during extraction efficiency assessment. A PCR master mix was prepared using the available commercial RT-PCR kit [Novel Coronavirus (2019- nCoV) Real Time Multiplex RT-PCRkit], and test run was performed using QuantStudio 7 Flex PCRplatform. Limitations: The study had a few limitations identified in different areas of data collection that could have possibly affected the accuracy of data interpretation and efficiency of the developed in-house covid-19 RNA extraction kit. These limitations were; Omission of the RNA-carrier during extraction step that could have probably affected the quantity and/or quality of the RNA extract . Omission of negative extraction control from the extracts that could have helped in identification of possible contamination during the extraction process as well as control for true negatives Omission of positive and No Template controls during the process of master mix preparation and sample loading on to the plate wells that could have helped in identification of possible cross contamination during master mix preparation and sample loading process as well as control for true positives. Omission of the process of autoclaving developed reagents that could have improved the purity of these reagents and prevent possible contaminations by for example the RNases etc