In-house reverse transcriptase polymerase chain reaction for detection of SARS-CoV-2 with increased sensitivity

Abstract

As the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.

Superior possibilities and upcoming horizons for nanoscience in COVID-19: noteworthy approach for effective diagnostics and management of SARS-CoV-2 outbreak

“…Moreover, they can also retrieve information from the small sample volume. Microfluidic devices are used at the early stage of diagnosis as recent researchers have found that they can detect viruses (Kalita et al 2021 ). The genetic code that forms virus consists of an envelope of proteins and a lipid ligand.…”

A low-cost molecular test for SARS-CoV-2 detection suitable for variant discrimination and community testing using saliva

“…One advantage of these tests, as opposed to RT-PCR with fluorogenic probes, is that they allow for assessment of the amplification specificity by simply examining the amplicon melting curves at the end of the PCR [18]. While most SYBR Green-based RT-PCR protocols have been established for use with RNA isolated from naso-or oropharyngeal specimens [11,16,[19][20][21][22][23][24][25][26][27], there is promising evidence for the use of saliva samples with this type of tests. Ganguly et al amplified synthetic SARS-CoV-2 RNA spiked into saliva specimens from healthy donors [28].…”