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      Recent advances and challenges of RT-PCR tests for the diagnosis of COVID-19

      review-article
      a , a , ** , b , c , d , e , f , g , h , i , *** , j , ** , k , *
      Pathology, Research and Practice
      Elsevier GmbH.
      cDNA, Complementary DNA, CLIA, Chemiluminescence immunoassay, COVID-19, Coronavirus Disease 2019, CRISPR, Clustered regularly interspaced short palindromic repeats, CT, Computed tomography scan, DNA, Deoxyribonucleic acid, EDTA, Ethylene-diamine-tetra-acetic acid, ELISA, Enzyme-linked immunosorbent assay, IgG, Immunoglobulin G, iLACO, Isothermal LAMP-based method for COVID-19, NP, Nasopharyngeal, NPV, Negative predictive value, OP, Oropharyngeal, RBD, Receptor binding domain of the virus, RNA, Ribonucleic acid, RT-LAMP, Reverse transcription loop-mediated isothermal amplification, RT–PCR, Reverse transcription of polymerase chain reaction, SARS-CoV-2, Severe acute respiratory syndrome coronavirus, SRT, Sampling-to-result time, TCEP, Tris (2-carboxyethyl) phosphine, SARS-CoV-2, COVID-19, Reverse transcription polymerase chain reaction, Diagnostic tests, Reverse transcription loop-mediated isothermal amplification, serologic tests

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          Abstract

          Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the control of virus spread has remained challenging given the pitfalls of the current diagnostic tests. Nevertheless, RNA amplification techniques have been the gold standard among other diagnostic methods for monitoring clinical samples for the presence of the virus. In the current paper, we review the shortcomings and strengths of RT-PCR (real-time polymerase chain reaction) techniques for diagnosis of coronavirus disease (COVID)-19. We address the repercussions of false-negative and false-positive rates encountered in the test, summarize approaches to improve the overall sensitivity of this method. We discuss the barriers to the widespread use of the RT-PCR test, and some technical advances, such as RT-LAMP (reverse-transcriptase-loop mediated isothermal amplification). We also address how other molecular techniques, such as immunodiagnostic tests can be used to avoid incorrect interpretation of RT-PCR tests.

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          Most cited references47

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          Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

          Summary Background Coronavirus disease 2019 (COVID-19) causes severe community and nosocomial outbreaks. Comprehensive data for serial respiratory viral load and serum antibody responses from patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not yet available. Nasopharyngeal and throat swabs are usually obtained for serial viral load monitoring of respiratory infections but gathering these specimens can cause discomfort for patients and put health-care workers at risk. We aimed to ascertain the serial respiratory viral load of SARS-CoV-2 in posterior oropharyngeal (deep throat) saliva samples from patients with COVID-19, and serum antibody responses. Methods We did a cohort study at two hospitals in Hong Kong. We included patients with laboratory-confirmed COVID-19. We obtained samples of blood, urine, posterior oropharyngeal saliva, and rectal swabs. Serial viral load was ascertained by reverse transcriptase quantitative PCR (RT-qPCR). Antibody levels against the SARS-CoV-2 internal nucleoprotein (NP) and surface spike protein receptor binding domain (RBD) were measured using EIA. Whole-genome sequencing was done to identify possible mutations arising during infection. Findings Between Jan 22, 2020, and Feb 12, 2020, 30 patients were screened for inclusion, of whom 23 were included (median age 62 years [range 37–75]). The median viral load in posterior oropharyngeal saliva or other respiratory specimens at presentation was 5·2 log10 copies per mL (IQR 4·1–7·0). Salivary viral load was highest during the first week after symptom onset and subsequently declined with time (slope −0·15, 95% CI −0·19 to −0·11; R 2=0·71). In one patient, viral RNA was detected 25 days after symptom onset. Older age was correlated with higher viral load (Spearman's ρ=0·48, 95% CI 0·074–0·75; p=0·020). For 16 patients with serum samples available 14 days or longer after symptom onset, rates of seropositivity were 94% for anti-NP IgG (n=15), 88% for anti-NP IgM (n=14), 100% for anti-RBD IgG (n=16), and 94% for anti-RBD IgM (n=15). Anti-SARS-CoV-2-NP or anti-SARS-CoV-2-RBD IgG levels correlated with virus neutralisation titre (R 2>0·9). No genome mutations were detected on serial samples. Interpretation Posterior oropharyngeal saliva samples are a non-invasive specimen more acceptable to patients and health-care workers. Unlike severe acute respiratory syndrome, patients with COVID-19 had the highest viral load near presentation, which could account for the fast-spreading nature of this epidemic. This finding emphasises the importance of stringent infection control and early use of potent antiviral agents, alone or in combination, for high-risk individuals. Serological assay can complement RT-qPCR for diagnosis. Funding Richard and Carol Yu, May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Michael Tong, Marina Lee, Government Consultancy Service, and Sanming Project of Medicine.
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            CRISPR-Cas12–based detection of SARS-CoV-2

            An outbreak of betacoronavirus SARS-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from US patients, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US CDC SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.. SARS-CoV-2 in patient samples is detected in under an hour using a CRISPR-based lateral flow assay.
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              Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19)

              Abstract Background The emergence of coronavirus disease 2019 (COVID-19) is a major healthcare threat. The current method of detection involves a quantitative polymerase chain reaction (qPCR)–based technique, which identifies the viral nucleic acids when present in sufficient quantity. False-negative results can be achieved and failure to quarantine the infected patient would be a major setback in containing the viral transmission. We aim to describe the time kinetics of various antibodies produced against the 2019 novel coronavirus (SARS-CoV-2) and evaluate the potential of antibody testing to diagnose COVID-19. Methods The host humoral response against SARS-CoV-2, including IgA, IgM, and IgG response, was examined by using an ELISA-based assay on the recombinant viral nucleocapsid protein. 208 plasma samples were collected from 82 confirmed and 58 probable cases (qPCR negative but with typical manifestation). The diagnostic value of IgM was evaluated in this cohort. Results The median duration of IgM and IgA antibody detection was 5 (IQR, 3–6) days, while IgG was detected 14 (IQR, 10–18) days after symptom onset, with a positive rate of 85.4%, 92.7%, and 77.9%, respectively. In confirmed and probable cases, the positive rates of IgM antibodies were 75.6% and 93.1%, respectively. The detection efficiency by IgM ELISA is higher than that of qPCR after 5.5 days of symptom onset. The positive detection rate is significantly increased (98.6%) when combining IgM ELISA assay with PCR for each patient compared with a single qPCR test (51.9%). Conclusions The humoral response to SARS-CoV-2 can aid in the diagnosis of COVID-19, including subclinical cases.
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                Author and article information

                Journal
                Pathol Res Pract
                Pathol Res Pract
                Pathology, Research and Practice
                Elsevier GmbH.
                0344-0338
                1618-0631
                14 April 2021
                14 April 2021
                : 153443
                Affiliations
                [a ]Natural Products and Medicinal Plants Research Center, North Khorasan University of Medical Sciences, Bojnurd, Iran
                [b ]Geriatric Care Research Center, Department of Geriatric Nursing, School of Nursing and Midwifery, North Khorasan University of Medical Sciences, Bojnurd, Iran
                [c ]Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
                [d ]Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran
                [e ]Department of Medical Biotechnology, School of Medicine, Arak University of Medical Sciences, Arak, Iran
                [f ]Laser Research Centre, Faculty of Health Science, University of Johannesburg, Doornfontein, South Africa
                [g ]Department of Medical Biotechnology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran
                [h ]Department of Clinical Biochemistry, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran
                [i ]Student Research Committee, Kerman University of Medical Sciences, Kerman, Iran
                [j ]Chronic Respiratory Diseases Research Center (CRDRC), National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
                [k ]Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran
                Author notes
                [* ]Corresponding author.
                [** ]Corresponding authors.
                [*** ]Corresponding authors at: Department of Clinical Biochemistry, Afzalipour School of Medicine, Kerman University of Medical Sciences, Iran.
                Article
                S0344-0338(21)00104-7 153443
                10.1016/j.prp.2021.153443
                8045416
                33930607
                421b1e63-1729-4f75-bd4a-f12afb7ffe11
                © 2021 Elsevier GmbH. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 9 March 2021
                : 10 April 2021
                : 11 April 2021
                Categories
                Review

                cdna, complementary dna,clia, chemiluminescence immunoassay,covid-19, coronavirus disease 2019,crispr, clustered regularly interspaced short palindromic repeats,ct, computed tomography scan,dna, deoxyribonucleic acid,edta, ethylene-diamine-tetra-acetic acid,elisa, enzyme-linked immunosorbent assay,igg, immunoglobulin g,ilaco, isothermal lamp-based method for covid-19,np, nasopharyngeal,npv, negative predictive value,op, oropharyngeal,rbd, receptor binding domain of the virus,rna, ribonucleic acid,rt-lamp, reverse transcription loop-mediated isothermal amplification,rt–pcr, reverse transcription of polymerase chain reaction,sars-cov-2, severe acute respiratory syndrome coronavirus,srt, sampling-to-result time,tcep, tris (2-carboxyethyl) phosphine,sars-cov-2,covid-19,reverse transcription polymerase chain reaction,diagnostic tests,reverse transcription loop-mediated isothermal amplification,serologic tests

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