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