Development and assessment of extraction efficiency of an in-house SARS-CoV-2 RNA extraction kit at genomics and bioinformatics laboratory of Makerere College of Health Sciences

Abstract

Major objective of the study:Development and assessment of extraction efficiency of an in house SARS-CoV-2 viral RNA extraction kit. Specific objectives of the study:To define the steps and reagents used to extract RNA from Viral particles and develop an in-house SARS-CoV-2 RNA extraction kit, To describe the process of SARS-CoV-2 RNA extraction using a commercially available extraction kit, and To assess the RNA extraction efficiency of the developed in-house kit by comparing it with a commercially available RNA extraction kit.The study also presented the literature review on the steps involved in RNA extraction process and the different reagents used at each step, extraction of SARS-CoV-2 RNA using commercially available Zymo Quick Viral RNA extraction Kit and the assessment of RNA extraction efficiency of RNA extraction kit. Methodology: The study was an applied experimental study aimed at developing and evaluating the extraction efficiency of an in-house SARS-CoV-2 RNA extraction kit at Mak-CHS molecular biology laboratory between April 2023 and July 2023. Reagents and samples used for the study were obtained from MAKCHS 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 ZQVR 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-PCR] kit, and test run was performed using QuantStudio 7 Flex PCR-platform. 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 hence proving the extraction efficiency of the developed in-house covid-19 RNA extraction kit. These limitations included the following. 1. Omission of the RNA-carrier during extraction step that could have probably affected the quantity and/or quality of the RNA extract. 2 2. 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. 3. 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. 4. 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. Results: With the few limitations mentioned in mind, from the findings it can be concluded that, the developed in-house covid-19 RNA extraction kit was able to extract SARS-CoV-2 RNA from some samples that were positive for SARS-CoV-2 as evidenced by results from sample BGG 469 where positive results were obtained in both extractions by in-house kit and the reference ZQVR kit and also by results from samples BMQ 421, BMQ 422, BMQ 423, BMQ 424 and BGG 562 where negative results were obtained in both sets of samples. However, in samples where results differed between in-house kit extracts and ZQVR kit extracts, it could suggest either some degree of extraction inefficiency of the developed in-house covid-19 RNA extraction kit or possibly due to errors made during sample processing stages. Therefore, to some reasonable extent, the developed in-house covid-19 RNA extraction kit can be relied on to extract SARS-CoV-2 RNA from samples obtained from nasopharyngeal swabs with a few improvements in some areas of sample processing.