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1.
Exp Mol Med ; 52(6): 963-977, 2020 06.
Article in English | MEDLINE | ID: covidwho-601243

ABSTRACT

SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , DNA Primers , Pneumonia, Viral/virology , Polymerase Chain Reaction/methods , Coronavirus Infections/diagnosis , HEK293 Cells , Humans , Multiplex Polymerase Chain Reaction/methods , Pandemics , Pharynx/virology , Pneumonia, Viral/diagnosis , RNA Replicase/genetics , Real-Time Polymerase Chain Reaction/methods , SARS Virus/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics , Viral Proteins/genetics
2.
Emerg Microbes Infect ; 9(1): 1489-1496, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-599991

ABSTRACT

In December 2019, Wuhan, China suffered a serious outbreak of a novel coronavirus infectious disease (COVID) caused by novel severe acute respiratory syndrome-related coronavirus (SARS-CoV 2). To quickly identify the pathogen, we designed and screened primer sets, and established a sensitive and specific qRT-PCR assay for SARS-CoV 2; the lower limit of detection (LOD) was 14.8 (95% CI: 9.8-21) copies per reaction. We combined this qRT-PCR assay with an automatic integration system for nucleic acid extraction and amplification, thereby establishing an automatic integrated gene detection system (AIGS) for SARS-CoV 2. Cross reactive analysis performed in 20 other respiratory viruses and 37 nasopharyngeal swabs confirmed a 100% specificity of the assay. Using two fold diluted SARS-CoV 2 culture, the LOD of AIGS was confirmed to be 365 copies/ml (95% CI: 351-375), which was Comparable to that of conventional q RT-PCR (740 copies/ml, 95% CI: 689-750). Clinical performances of AIGS assay were assessed in 266 suspected COVID-19 clinical respiratory tract samples tested in parallel with a commercial kit. The clinical sensitivity of the AIGS test was 97.62% (95% CI: 0.9320-0.9951) based on the commercial kit test result, and concordance analysis showed a high agreement in SARS-CoV-2 detection between the two assays, Pearson R was 0.9623 (95% CI: 0.9523-0.9703). The results indicated that this AIGS could be used for rapid detection of SARS-CoV 2. With the advantage of simple operation and less time consuming, AIGS could be suitable for SARS-CoV2 detection in primary medical institutions, thus would do a great help to improve detection efficiency and control the spread of COVID-19.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , Automation, Laboratory , China , DNA Primers , Humans , Limit of Detection , Pandemics , RNA, Viral/analysis , Sensitivity and Specificity , Virus Cultivation
3.
PLoS One ; 15(6): e0234682, 2020.
Article in English | MEDLINE | ID: covidwho-595220

ABSTRACT

Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Point-of-Care Testing , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Coronavirus Infections/virology , DNA Primers , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/instrumentation , Pandemics , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/economics , Real-Time Polymerase Chain Reaction/instrumentation , Sensitivity and Specificity , Time Factors
4.
J Mol Diagn ; 22(6): 729-735, 2020 06.
Article in English | MEDLINE | ID: covidwho-477970

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. RT-LAMP assays reported in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.


Subject(s)
Betacoronavirus/genetics , Nucleic Acid Amplification Techniques/methods , Colorimetry/methods , DNA Primers , Gentian Violet , RNA, Viral/genetics , Reverse Transcription
5.
Emerg Microbes Infect ; 9(1): 1175-1179, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-361278

ABSTRACT

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Multiplex Polymerase Chain Reaction , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction , DNA Primers , DNA Probes , Humans , Multiplex Polymerase Chain Reaction/methods , Pandemics , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Sensitivity and Specificity , Viral Load
6.
Emerg Infect Dis ; 26(8): 1944-1946, 2020 08.
Article in English | MEDLINE | ID: covidwho-326416
7.
Emerg Infect Dis ; 26(8)2020 Aug.
Article in English | MEDLINE | ID: covidwho-245493

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent associated with coronavirus disease, which emerged in late 2019. In response, we developed a diagnostic panel consisting of 3 real-time reverse transcription PCR assays targeting the nucleocapsid gene and evaluated use of these assays for detecting SARS-CoV-2 infection. All assays demonstrated a linear dynamic range of 8 orders of magnitude and an analytical limit of detection of 5 copies/reaction of quantified RNA transcripts and 1 x 10-1.5 50% tissue culture infectious dose/mL of cell-cultured SARS-CoV-2. All assays performed comparably with nasopharyngeal and oropharyngeal secretions, serum, and fecal specimens spiked with cultured virus. We obtained no false-positive amplifications with other human coronaviruses or common respiratory pathogens. Results from all 3 assays were highly correlated during clinical specimen testing. On February 4, 2020, the Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of this panel.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Nucleocapsid Proteins/genetics , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Biomarkers/analysis , Centers for Disease Control and Prevention, U.S. , Coronavirus Infections/virology , DNA Primers/chemical synthesis , DNA Primers/genetics , Feces/virology , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Humans , Limit of Detection , Nasopharynx/virology , Pandemics , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Sputum/virology , United States
8.
Int J Mol Sci ; 21(8)2020 Apr 24.
Article in English | MEDLINE | ID: covidwho-145091

ABSTRACT

Testing for the presence of coronavirus is an essential diagnostic tool for monitoring and managing the current COVID-19 pandemic. The only reliable test in current use for testing acute infection targets the genome of SARS-CoV-2, and the most widely used method is quantitative fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR). Despite its ubiquity, there is a significant amount of uncertainty about how this test works, potential throughput and reliability. This has resulted in widespread misrepresentation of the problems faced using this test during the current COVID-19 epidemic. This primer provides simple, straightforward and impartial information about RT-qPCR.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , DNA Primers , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification
9.
Microb Biotechnol ; 13(4): 950-961, 2020 07.
Article in English | MEDLINE | ID: covidwho-116666

ABSTRACT

The pandemic coronavirus SARS-CoV-2 in the world has caused a large infected population suffering from COVID-19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. We applied a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to achieve the detection of SARS-CoV-2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS-CoV-2 in the regions of orf1ab, S gene and N gene. A colorimetric change was used to report the results, which enables the outcome of viral RNA amplification to be read by the naked eye without the need of expensive or dedicated instrument. The sensitivity can be 80 copies of viral RNA per ml in a sample. We validated the RT-LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 negatives. The testing results are consistent with the conventional RT-qPCR. In addition, we also show that one-step process without RNA extraction is feasible to achieve RNA amplification directly from a sample. This rapid, simple and sensitive RT-LAMP method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Betacoronavirus/classification , Betacoronavirus/genetics , China , Coronavirus Infections/virology , DNA Primers/genetics , Humans , Pandemics , Pneumonia, Viral/virology , Sensitivity and Specificity , Viral Proteins/genetics
10.
Med Hypotheses ; 141: 109786, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-116581

ABSTRACT

Recently, a novel coronavirus (SARS-CoV-2; coronavirus disease 2019, COVID-19) has emerged, rapidly spreading and severely straining the capacity of the global health community. Many nations are employing combinations of containment and mitigation strategies, where early diagnosis of COVID-19 is vital in controlling illness progression and limiting viral spread within the population. Thus, rapid and accurate methods of early detection are vital to contain COVID-19 and prevent further spread and predicted subsequent infectious waves of viral recurrence in future. Immediately after its initial characterization, Chinese and American Centers for Disease Control and Prevention (CDCs) rapidly employed molecular assays for detection of COVID-19, mostly employing real-time polymerase chain reaction (RT-PCR) methods. However, such methods require specific expensive items of equipment and highly trained analysts, requiring upwards of 4-8 h to process. These requirements coupled with associated financial pressures may prevent effective deployment of such diagnostic tests. Loop mediated isothermal amplification(LAMP) is method of nucleic acid amplification which exhibits increased sensitivity and specificity are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection. Studies have shown the successful application of LAMP assays in various forms to detect coronavirus RNA in patient samples, demonstrating that 1-10 copies of viral RNA template per reaction are sufficient for successful detection, ~100-fold more sensitive than conventional RT-PCR methods. Importantly, studies have also now demonstrated the effectiveness of LAMP methodology in the detection of SARS-CoV-2 RNA at significantly low levels, particularly following numerous improvements to LAMP assay protocols. We hypothesise that recent advancements in enhanced LAMP protocols assay perhaps represent the best chance for a rapid and robust assay for field diagnosis of COVID-19, without the requirement of specialized equipment and highly trained professionals to interpret results. Herein, we present our arguments with a view to disseminate such findings, to assist the combat of this virus that is proving so devastating. We hope that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Pandemics , Pneumonia, Viral/diagnosis , Betacoronavirus/genetics , Clinical Laboratory Techniques/economics , Coronavirus Infections/economics , Coronavirus Infections/virology , DNA Primers , Early Diagnosis , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/methods , Pandemics/economics , Pneumonia, Viral/economics , Pneumonia, Viral/virology , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/economics , Sensitivity and Specificity , Time Factors
11.
Rev Med Virol ; 30(3): e2106, 2020 05.
Article in English | MEDLINE | ID: covidwho-71880

ABSTRACT

Emerging and reemerging infectious diseases are global public concerns. With the outbreak of unknown pneumonia in Wuhan, China in December 2019, a new coronavirus, SARS-CoV-2 has been attracting tremendous attention. Rapid and accurate laboratory testing of SARS-CoV-2 is essential for early discovery, early reporting, early quarantine, early treatment, and cutting off epidemic transmission. The genome structure, transmission, and pathogenesis of SARS-CoV-2 are basically similar to SARS-CoV and MERS-CoV, the other two beta-CoVs of medical importance. During the SARS-CoV and MERS-CoV epidemics, a variety of molecular and serological diagnostic assays were established and should be referred to for SARS-CoV-2. In this review, by summarizing the articles and guidelines about specimen collection, nucleic acid tests (NAT) and serological tests for SARS-CoV, MERS-CoV, and SARS-CoV-2, several suggestions are put forward to improve the laboratory testing of SARS-CoV-2. In summary, for NAT: collecting stool and blood samples at later periods of illness to improve the positive rate if lower respiratory tract specimens are unavailable; increasing template volume to raise the sensitivity of detection; putting samples in reagents containing guanidine salt to inactivate virus as well as protect RNA; setting proper positive, negative and inhibition controls to ensure high-quality results; simultaneously amplifying human RNase P gene to avoid false-negative results. For antibody test, diverse assays targeting different antigens, and collecting paired samples are needed.


Subject(s)
Clinical Laboratory Techniques/methods , Communicable Diseases, Emerging/virology , Antibodies, Viral/isolation & purification , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Communicable Diseases, Emerging/diagnosis , Coronavirus Infections/diagnosis , DNA Primers , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Ribonuclease P/genetics , Ribonuclease P/isolation & purification , SARS Virus/genetics , SARS Virus/isolation & purification , Serologic Tests/methods
12.
J Mol Diagn ; 22(6): 729-735, 2020 06.
Article in English | MEDLINE | ID: covidwho-35118

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. RT-LAMP assays reported in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.


Subject(s)
Betacoronavirus/genetics , Nucleic Acid Amplification Techniques/methods , Colorimetry/methods , DNA Primers , Gentian Violet , RNA, Viral/genetics , Reverse Transcription
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