End-point RT-PCR based on a conservation landscape for SARS-COV-2 detection

Abstract

End-point RT-PCR is a suitable alternative diagnostic technique since it is cheaper than RT-qPCR tests and can be implemented on a massive scale in low- and middle-income countries. In this work, a bioinformatic approach to guide the design of PCR primers was developed, and an alternative diagnostic test based on end-point PCR was designed. End-point PCR primers were designed through conservation analysis based on kmer frequency in SARS-CoV-2 and human respiratory pathogen genomes. Highly conserved regions were identified for primer design, and the resulting PCR primers were used to amplify 871 nasopharyngeal human samples with a previous RT-qPCR based SARS-CoV-2 diagnosis. The diagnostic test showed high accuracy in identifying SARS-CoV-2-positive samples including B.1.1.7, P.1, B.1.427/B.1.429 and B.1.617.2/ AY samples with a detection limit of 7.2 viral copies/µL. In addition, this test could discern SARS-CoV-2 infection from other viral infections with COVID-19-like symptomatology. The designed end-point PCR diagnostic test to detect SARS-CoV-2 is a suitable alternative to RT-qPCR. Since the proposed bioinformatic approach can be easily applied in thousands of viral genomes and over highly divergent strains, it can be used as a PCR design tool as new SARS-CoV-2 variants emerge. Therefore, this end-point PCR test could be employed in epidemiological surveillance to detect new SARS-CoV-2 variants as they emerge and propagate.

Clinical Characteristics of Coronavirus Disease (COVID-19) in Mexican Children and Adolescents

“…In addition, the Ct values were used to determine the number of viruses for each viral load. It was calculated using the following equation, y = −3.2733 x + 38.59, as described in a previous study [ 13 ]. Low viral load was defined as Ct value 30–38 (4.2 × 10 2 –1.5 copies number/mL), moderate as Ct value 25–29 (1.4 × 10 4 –8.5 × 10 2 copies number/mL), and high as Ct value <24 (<2.8 × 10 4 copies number/mL).…”

Longitudinal anti-SARS-CoV-2 antibody immune response in acute and convalescent patients

“…In this study, we did not find any relationship between levels of IgM and IgG and the Ct values. The Ct value is inversely related to the viral load, and every ~3.3 increase in the Ct value reflects a 10fold reduction in starting material (Cruz-Rangel et al, 2022). However, other studies reported that levels of IgG and IgM response could be driven by the viral load (Long et al, 2020;Masiáet al, 2021;Young et al, 2021), but these contrasting results can be explained by the heterogeneity of the studies related to the different characteristics of the study population and/or the methodology used to quantify SARS-CoV-2 and the quality of the sampling (Codina et al, 2021).…”

“…The SARS-CoV-2 RT-qPCR test provides real-time quantification by first reverse transcribing SARS-CoV-2 RNA into cDNA (RT step) and then performing qPCR, during which a fluorescence signal increases proportionally to the amount of amplified nucleic acid, enabling accurate quantitation of the RNA in the sample (Tom and Mina, 2020). Many qPCR assays involve a Ct cut-off 34 to consider the test positive, allowing the detection of very few starting RNA molecules (Cruz-Rangel et al, 2022).…”

Toward Integrated Molecular Lateral Flow Diagnostic Tests Using Advanced Micro- and Nanotechnology

“…When the results of the PCR are analyzed at the end of the reaction it is called end-point PCR (or end-point RT-PCR , ). Typically, this is performed using an agarose gel electrophoresis, where the samples are run together with a ladder to check if the amplicon size (i.e., the number of base pairs, bp) obtained is the one expected .…”