An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

Davda, Jayeshkumar Narsibhai; Frank, Keith; Prakash, Sivakumar; Purohit, Gunjan; Vijayashankar, Devi Prasad; Vedagiri, Dhiviya; Tallapaka, Karthik Bharadwaj; Harshan, Krishnan Harinivas; Siva, Archana Bharadwaj; Mishra, Rakesh; Dhawan, Jyotsna; Siddiqi, Imran

doi:10.1101/2020.06.08.139477

Cited by 5 publications

(5 citation statements)

References 18 publications

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“…Nearly similar IC 50 values were also noted in the growth inhibition of the B.6 variant (GISAID ID: EPI_ISL_458075; virus ID-hCoV-19/India/TG-CCMB-O2-P1/2020) of SARS-CoV-2 (earlier in May 2020) isolated at BSL-3 facility, CSIR-CCMB, Hyderabad (Supp Fig. 2) [14]. Inhibition of SARS-CoV-2 multiplication by 2-DG was also measured by estimating the nucleocapsid protein of the virus in Vero E6 cell lysate.…”

Section: -Dg Inhibits Sars-cov-2 Replicationsupporting

confidence: 53%

Glycolytic inhibitor 2-Deoxy-D-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions

Bhatt

Kumar

Rai

et al. 2021

Preprint

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The COVID-19 pandemic is an ongoing public health emergency of international concern. Millions of people lost their lives to this pandemic. While a lot of efforts are being invested in vaccinating the population, there is also an emergent requirement to find potential therapeutics to effectively counter this fast mutating SARS-CoV-2 virus-induced pathogenicity. Virus-infected host cells switch their metabolism to a more glycolytic phenotype. This switch induced by the virus is needed for faster production of ATP and higher levels of glycolytic intermediates, which are required for anabolic processes such as fatty acid synthesis and nucleotide generation for new virion synthesis and packaging. In this study, we used 2-Deoxy-D-glucose (2-DG) to target and inhibit the metabolic reprogramming induced by SARS-CoV-2 infection. Our results showed that virus infection induces glucose influx and glycolysis resulting in selective high accumulation of the fluorescent glucose/2-DG analogue, 2-NBDG in these cells. Subsequently, 2-DG reduces the virus multiplication and alleviates the cells from infection-induced cytopathic effect (CPE) and cell death. Herein, we demonstrate that progeny virions produced from 2-DG treated cells are defective with compromised infectivity potential. Further, it was also observed that mannose inhibits 2-NBDG uptake at a very low concentration, suggesting that 2-DG uptake in virus-infected cells might be exploiting the specific mannose transporter or high-affinity glucose transporter, GLUT3, which was found to be increased on SARS-CoV-2 infection. In conclusion, our findings suggest that 2-DG effectively inhibits the SARS-CoV-2 multiplication and can be used as a treatment regimen. Based on these preliminary in-vitro findings this molecule reached clinical trial in COVID patients.

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Section: -Dg Inhibits Sars-cov-2 Replicationsupporting

confidence: 53%

Glycolytic inhibitor 2-Deoxy-D-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions

Bhatt

Kumar

Rai

et al. 2021

Preprint

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“…Our results are in line with the results from Davda et al's study. They reported 13% false-negative among samples with negative real-time PCR results (17). Also, Wang et al reported 14 positive real-time PCR results among 156 samples (8.97%) with negative real-time PCR results (9).…”

Section: Resultsmentioning

confidence: 99%

Evaluation of False Negative Among SARS-COV-2 Patients with Negative Real-time PCR Result Using Nested-RT PCR

Alinezhadi

Neisi

Rasti

et al. 2022

Jundishapur J Microbiol

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Background: Fast and precise detection of SARS-CoV-2 RNA in clinical samples and subsequent quarantine are two critical factors in preventing virus transmission and distribution through the community. The false-negative result is a major problem in the SARS-CoV-2 detection because of the kind of sample (swab sample), sampling error, and sensitivity of PCR test, which can be reduced by a much more sensitive test such as nested PCR. Objectives: This study aimed to evaluate the false-negative rate among samples that were negative by a real-time PCR test using RT-nested PCR. Methods: One hundred eighty-four negative samples were included in the study, and nucleic acid was extracted using a commercial kit based on a silica filter column and then subjected to RT-nested PCR using three sets of primers targeting Orf1ab, N, and RdRp regions. Results: Among 184 negative swab samples for SARS-CoV-2, 27 (14.6%) cases were positive for the Orf1ab gene using RT-nested PCR. The samples were tested using N and RdRp primer sets. Also, seven (3.8%) cases were positive for the N gene, and four (2.1%) cases were positive for the RdRp gene. Conclusions: The results indicated that RT-nested PCR could be more sensitive than real-time PCR and reduce the false-negative rate.

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“…The coronavirus disease 2019 (COVID-19) pandemic has caused a flurry of activity regarding the detection of SARS-CoV-2, in particular a substantial amount of new RT-PCR primers were developed for this specific purpose [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. A number of factors can influence the reliability of the PCR detection, such as sample contamination [20], cross-reactivity with other viruses [14], contamination of reagents [21], non-specific annealing [22] and poor amplification efficiency [23].…”

Section: Introductionmentioning

confidence: 99%

“…Here, we analyse how and if mismatches do influence the melting temperatures of primer/probes hybridised to SARS-CoV-2 genomes. We collected 19 PCR primer/probe sets (297 primers and 43 probes) which cover seven different gene regions of SARS-CoV-2 genome (N, E, S, M, ORF1ab, RdRp and nsp2 genes) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. These primer/probes were aligned to 21665 genomes of SARS-CoV-2 and 323 genomes of other coronaviruses.…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes

Miranda

Weber²

2020

Preprint

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Background: DNA mismatches can affect the efficiency of PCR techniques if the intended target has mismatches in primers or probes regions. The accepted rule is that mismatches are detrimental as they reduce the hybridization temperatures, yet a more quantitative assessment is rarely performed. Methods: We calculate the hybridization temperatures of primer/probe sets after aligning to SARS-COV-2, SARS-COV-1 and non-SARS genomes, considering all possible combinations of single, double and triple consecutive mismatches. We consider the mismatched hybridization temperature within a range of 5 °C to the fully matched reference temperature. Results: We obtained the alignments of 19 PCR primers sets that were recently reported for the detection of SARS-CoV-2 and to 21665 SARS-CoV-2 genomes as well as 323 genomes of other viruses of the coronavirus family of which 10 are SARS-CoV-1. We find that many incompletely aligned primers become fully aligned to most of the SARS-CoV-2 when mismatches are considered. However, we also found that many cross-align to SARS-CoV-1 and non-SARS genomes. Conclusions: Some primer/probe sets only align substantially to most SARS-CoV-2 genomes if mismatches are taken into account. Unfortunately, by the same mechanism, almost 75% of these sets also align to some SARS-CoV-1 and non-SARS viruses. It is therefore recommended to consider mismatch hybridization for the design of primers whenever possible, especially to avoid undesired cross-reactivity.

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An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

Cited by 5 publications

References 18 publications

Glycolytic inhibitor 2-Deoxy-D-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions

Glycolytic inhibitor 2-Deoxy-D-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions

Evaluation of False Negative Among SARS-COV-2 Patients with Negative Real-time PCR Result Using Nested-RT PCR

Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes

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