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1.
J Clin Lab Anal ; 36(2): e24211, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1589067

ABSTRACT

BACKGROUND: Presently, the global spread of COVID-19 is still going on, with more than 0.6 million new cases confirmed per day (as of November 20, 2021). However, since China entered a post-epidemic phase in mid-March 2020, the daily number of new domestic infections in the Chinese mainland has been maintained at almost zero or single digits, which was attributed to a series of effective measures for COVID-19 prevention and control adopted by the Chinese government. Among these measures, SARS-CoV-2 nucleic acid testing holds key role for the timely confirmation and isolation of the infections to prevent further transmission. METHODS: Referring to the national policy requirements, since April 30, 2020, The Affiliated Hospital of Qingdao University has conducted SARS-CoV-2 nucleic acid testing in its PCR laboratory for patients and social workers, as well as for environmental monitoring and employee screening. As of mid-November 2020, the daily amount of single-tube samples for nucleic acid testing rose above 4,000. RESULTS: In this article, a rapid and highly effective approach for SARS-CoV-2 nucleic acid daily testing is presented, allowing five technicians to complete nucleic acid testing in 6,500 single-tube samples in one day with a high level of quality. Using this approach, since the samples entered the PCR laboratory, all testing results were reported in 2.5-3 h with satisfactory quality control and precise reporting criterion as prerequisites. CONCLUSION: This testing approach provides a referable workflow for other testing institutions and is expected to play an important role in COVID-19 prevention and control.


Subject(s)
COVID-19 , Nucleic Acid Amplification Techniques , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/prevention & control , China , High-Throughput Nucleotide Sequencing , Humans , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Nucleic Acid Amplification Techniques/statistics & numerical data , Quality Control , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Time Factors
2.
Viruses ; 13(9)2021 09 19.
Article in English | MEDLINE | ID: covidwho-1430979

ABSTRACT

The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its emergence of variants needs rapid and point-of-care testing methods for a broad diagnosis. The regular RT-qPCR is time-consuming and limited in central laboratories, so a broad and large-scale screening requirement calls for rapid and in situ methods. In this regard, a reverse transcription recombinase-aided amplification (RT-RAA) is proposed here for the rapid and point-of-care detection of SARS-CoV-2. A set of highly conserved primers and probes targeting more than 98% of SARS-CoV-2 strains, including currently circulating variants (four variants of concerns (VOCs) and three variants of interest (VOIs)), was used in this study. With the preferred primers, the RT-RAA assay showed a 100% specificity to SARS-CoV-2 from eight other respiratory RNA viruses. Moreover, the assay here is of a high sensitivity and 0.48 copies/µL can be detected within 25 min at a constant temperature (42 °C), which can be realized on portable equipment. Furthermore, the RT-RAA assay demonstrated its high agreement for the detection of SARS-CoV-2 in clinical specimens compared with RT-qPCR. The rapid, simple and point-of-care RT-RAA method is expected to be an appealing detection tool to detect SARS-CoV-2, including variants, in clinical diagnostic applications.


Subject(s)
COVID-19/diagnosis , COVID-19/virology , Nucleic Acid Amplification Techniques/methods , Point-of-Care Testing , SARS-CoV-2/genetics , High-Throughput Nucleotide Sequencing , Humans , Nucleic Acid Amplification Techniques/standards , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
3.
Sci Rep ; 11(1): 16193, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1351975

ABSTRACT

We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-µL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8-94.7%) and specificity of 92.4% (95% CI 83.2-97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , COVID-19 Nucleic Acid Testing/standards , Humans , Molecular Diagnostic Techniques/standards , Nucleic Acid Amplification Techniques/standards , Sensitivity and Specificity
4.
Sci Rep ; 11(1): 13378, 2021 06 28.
Article in English | MEDLINE | ID: covidwho-1286471

ABSTRACT

The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved a positive percent agreement of 100% [95% CI 88.43% to 100.0%] and a negative percent agreement of 96.7% [95% CI 82.78-99.92%] relative to a reference standard test. Saliva-Dry LAMP can be completed in 105 min. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , Saliva/virology , COVID-19/virology , Fluorometry , Humans , Limit of Detection , Molecular Diagnostic Techniques/instrumentation , Molecular Diagnostic Techniques/standards , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acid Amplification Techniques/standards , RNA, Viral/standards , Reference Standards , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Temperature
5.
Am J Trop Med Hyg ; 105(2): 375-377, 2021 Jun 15.
Article in English | MEDLINE | ID: covidwho-1270184

ABSTRACT

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading rapidly all over the world. In the absence of effective treatments or a vaccine, there is an urgent need to develop a more rapid and simple detection technology of COVID-19. We describe a WarmStart colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2. The detection limit for this assay was 1 copy/µL SARS-CoV-2. To test the clinical sensitivity and specificity of the assay, 37 positive and 20 negative samples were used. The WarmStart colorimetric RT-LAMP had 100% sensitivity and specificity. End products were detected by direct observation, thereby eliminating the need for post-amplification processing steps. WarmStart colorimetric RT-LAMP provides an opportunity to facilitate virus detection in resource-limited settings without a sophisticated diagnostic infrastructure.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Colorimetry/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , Colorimetry/standards , Humans , Molecular Diagnostic Techniques/standards , Nasopharynx/virology , Nucleic Acid Amplification Techniques/standards , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
6.
Biochem Biophys Res Commun ; 567: 195-200, 2021 08 27.
Article in English | MEDLINE | ID: covidwho-1263226

ABSTRACT

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.


Subject(s)
DNA, Viral/analysis , DNA-Directed DNA Polymerase/metabolism , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , RNA, Viral/analysis , Recombinases/metabolism , SARS-CoV-2/genetics , Statistics as Topic , DNA Primers/metabolism , DNA-Binding Proteins/metabolism , Membrane Proteins/metabolism , SARS-CoV-2/isolation & purification , Viral Proteins/metabolism
7.
J Virol Methods ; 294: 114182, 2021 08.
Article in English | MEDLINE | ID: covidwho-1225321

ABSTRACT

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires fast and accurate high-throughput diagnostic tools. To evaluate the analytical performance of the Hologic Aptima transcription-mediated amplification (TMA) assay for detection of SARS-CoV-2 RNA from respiratory samples we analysed 103 clinical and proficiency panel samples pre-tested by real-time RT-PCR (Altona, RealStar) and found a positive percent agreement (sensitivity) of 95.7 % and a negative percent agreement (specificity) of 100 %. The limit of detection of the Aptima test was 150 copies/mL determined as 95 % detection probability. To further assess the Aptima assay's specificity we prospectively analysed 7545 clinical specimens from the upper and lower respiratory tract sent for the purpose of routine SARS-CoV-2 screening. SARS-CoV-2 RNA was detected in 16/7545 (0.2 %) samples by the TMA assay and confirmed independently by the Xpert SARS-CoV-2 RT-PCR (Cepheid); in one case a previous discrepant result was confirmed as true SARS-CoV-2 infection in a subsequent sample from the same patient. Results from the Aptima SARS-CoV-2 TMA assay agreed well with RT-PCR and showed an excellent specificity in a large number of routine specimens despite the low prevalence at that time of the pandemic, indicating that this assay can be used even for screening purposes.


Subject(s)
COVID-19 Testing/standards , Nucleic Acid Amplification Techniques/standards , RNA, Viral/genetics , Reagent Kits, Diagnostic/standards , SARS-CoV-2/genetics , Asymptomatic Infections , COVID-19 Testing/methods , Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , Humans , Limit of Detection , Nasopharynx/virology , Nucleic Acid Amplification Techniques/methods , Prospective Studies , Retrospective Studies , Sensitivity and Specificity
8.
Front Public Health ; 9: 568603, 2021.
Article in English | MEDLINE | ID: covidwho-1145593

ABSTRACT

The declaration of COVID-19 as a global pandemic has warranted the urgent need for technologies and tools to be deployed for confirming diagnosis of suspected cases. Diagnostic testing for COVID-19 is critical for understanding epidemiology, contract-tracing, case management, and to repress the transmission of the SARS-CoV-2. Currently, the Nucleic Acid Amplification Test (NAAT)-based RT-PCR technique is a gold standard test used for routine diagnosis of COVID-19 infection. While there are many commercially available RT-PCR assay kits available in the market, selection of highly sensitive, specific, and validated assays is most crucial for the accurate diagnosis of COVID-19 infection. Laboratory diagnosis of SARS-CoV-2 is extremely important in the disease and outbreak management. Development of rapid point of care tests with better sensitivity and specificity is the critical need of the hour as this will help accurate diagnosis and aid in containing the spread of SARS-CoV-2 infection. Early detection of viral infection greatly enhances implementation of specific public health intervention, such as infection control, environmental decontamination, and the closure of specific high-risk zones. Large-scale sequencing of SARS-CoV-2 genome isolated from affected populations across the world needs to be carried to monitor mutations that might affect performance of molecular tests. Creation of genome repositories and open-source genetic databases for use by global researchers is clearly the way forward to manage COVID-19 outbreak and accelerate vaccine development. This review summarizes various molecular diagnostics methods, technical guidelines, and advanced testing strategies adopted in India for laboratory diagnosis of COVID-19.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19 Testing , COVID-19/diagnosis , Clinical Laboratory Techniques , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques/standards , COVID-19/prevention & control , COVID-19/transmission , Humans , India , Infection Control , Mutation/genetics , Point-of-Care Testing
9.
Vopr Virusol ; 66(1): 17-28, 2021 03 07.
Article in Russian | MEDLINE | ID: covidwho-1121949

ABSTRACT

This review presents the basic principles of application of the loop-mediated isothermal amplification (LAMP) reaction for the rapid diagnosis of coronavirus infection caused by SARS-CoV-2. The basic technical details of the method, and the most popular approaches of specific and non-specific detection of amplification products are briefly described. We also discuss the first published works on the use of the method for the detection of the nucleic acid of the SARS-CoV-2 virus, including those being developed in the Russian Federation. For commercially available and published LAMP-based assays, the main analytical characteristics of the tests are listed, which are often comparable to those based on the method of reverse transcription polymerase chain reaction (RT-PCR), and in some cases are even superior. The advantages and limitations of this promising methodology in comparison to other methods of molecular diagnostics, primarily RT-PCR, are discussed, as well as the prospects for the development of technology for the detection of other infectious agents.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/standards , Nucleic Acid Amplification Techniques/standards , RNA, Viral/genetics , SARS-CoV-2/genetics , Artifacts , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , DNA Primers/genetics , DNA Primers/metabolism , DNA Probes/genetics , DNA Probes/metabolism , Humans , Reagent Kits, Diagnostic , Sensitivity and Specificity
10.
Biomacromolecules ; 22(3): 1231-1243, 2021 03 08.
Article in English | MEDLINE | ID: covidwho-1062725

ABSTRACT

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and inexpensive isothermal alternative to the current gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, unlike RT-qPCR, there are no consensus detection regions or optimal RT-LAMP methods, and most protocols do not include internal controls to ensure reliability. Naked RNAs, plasmids, or even RNA from infectious COVID-19 patients have been used as external positive controls for RT-LAMP assays, but such reagents lack the stability required for full-process control. To overcome the lack of proper internal and external positive controls and the instability of the detection RNA, we developed virus-like particles (VLPs) using bacteriophage Qß and plant virus cowpea chlorotic mottle virus (CCMV) for the encapsidation of target RNA, namely a so-called SARS-CoV-2 LAMP detection module (SLDM). The target RNA is a truncated segment of the SARS-CoV-2 nucleocapsid (N) gene and human RNase P gene (internal control) as positive controls for RT-qPCR and RT-LAMP. Target RNAs stably encapsidated in Qß and CCMV VLPs were previously shown to function as full-process controls in RT-qPCR assays, and here we show that SLDMs can fulfill the same function for RT-LAMP and swab-to-test (direct RT-LAMP with heat lysis) assays. The SLDM was validated in a clinical setting, highlighting the promise of VLPs as positive controls for molecular assays.


Subject(s)
Bromovirus , COVID-19 Nucleic Acid Testing/standards , COVID-19 , Molecular Diagnostic Techniques/standards , Nucleic Acid Amplification Techniques/standards , SARS-CoV-2/genetics , Bromovirus/chemistry , Bromovirus/genetics , COVID-19/diagnosis , COVID-19/genetics , Humans
11.
J Virol Methods ; 290: 114083, 2021 04.
Article in English | MEDLINE | ID: covidwho-1051816

ABSTRACT

In the current pandemic of SARS-CoV-2, rapid identification of infected individuals is crucial for management and control of the outbreak. However, transport of samples, sample processing and RT-qPCR analysis in laboratories are time-consuming. Here we present a prototype of a novel nucleic acid-based test format - pulse controlled amplification - that allows detection of SARS-CoV-2 directly from up to eight swab samples simultaneously without the need for RNA extraction within 25 min with a sensitivity of 100 % for samples with a viral load of ≥ 1.6 × 10e3 copies/µl This new principle might pave the way to rapid and sensitive point of care testing.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/isolation & purification , COVID-19 Nucleic Acid Testing/standards , Humans , Nucleic Acid Amplification Techniques/standards , Point-of-Care Testing , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
12.
Emerg Infect Dis ; 27(1)2021 Jan.
Article in English | MEDLINE | ID: covidwho-922785

ABSTRACT

Pooled nucleic acid amplification tests for severe acute respiratory syndrome coronavirus 2 could increase availability of testing at decreased cost. However, the effect of dilution on analytical sensitivity through sample pooling has not been well characterized. We tested 1,648 prospectively pooled specimens by using 3 nucleic acid amplification tests for severe acute respiratory syndrome coronavirus 2: a laboratory-developed real-time reverse transcription PCR targeting the envelope gene, and 2 commercially available Panther System assays targeting open reading frame 1ab. Positive percent agreement (PPA) of pooled versus individual testing ranged from 71.7% to 82.6% for pools of 8 and from 82.9% to 100.0% for pools of 4. We developed and validated an independent stochastic simulation model to estimate effects of dilution on PPA and efficiency of a 2-stage pooled real-time reverse transcription PCR testing algorithm. PPA was dependent on the proportion of tests with positive results, cycle threshold distribution, and assay limit of detection.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/isolation & purification , COVID-19/virology , Clinical Laboratory Techniques/methods , False Negative Reactions , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/standards , Prospective Studies , SARS-CoV-2/genetics , Sensitivity and Specificity , Specimen Handling , Stochastic Processes
13.
Clin Microbiol Infect ; 27(3): 341-351, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-921865

ABSTRACT

BACKGROUND: Management and control of coronavirus disease 2019 (COVID-19) relies on reliable diagnostic testing. OBJECTIVES: To evaluate the diagnostic test accuracy (DTA) of nucleic acid amplification tests (NAATs) for the diagnosis of coronavirus infections. DATA SOURCES: PubMed, Web of Science, the Cochrane Library, Embase, Open Grey and conference proceeding until May 2019. PubMed and medRxiv were updated for COVID-19 on 31st August 2020. STUDY ELIGIBILITY: Studies were eligible if they reported on agreement rates between different NAATs using clinical samples. PARTICIPANTS: Symptomatic patients with suspected upper or lower respiratory tract coronavirus infection. METHODS: The new NAAT was defined as the index test and the existing NAAT as reference standard. Data were extracted independently in duplicate. Risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 tool. Confidence regions (CRs) surrounding summary sensitivity/specificity pooled by bivariate meta-analysis are reported. Heterogeneity was assessed using meta-regression. RESULTS: Fifty-one studies were included, 22 of which included 10 181 persons before COVID-19 and 29 including 8742 persons diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The overall summary sensitivity was 89.1% (95%CR 84.0-92.7%) and specificity 98.9% (95%CR 98.0-99.4%). Nearly all the studies evaluated different PCRs as both index and reference standards. Real-time RT PCR assays resulted in significantly higher sensitivity than other tests. Reference standards at high risk of bias possibly exaggerated specificity. The pooled sensitivity and specificity of studies evaluating SARS-COV-2 were 90.4% (95%CR 83.7-94.5%) and 98.1% (95%CR 95.9-99.2), respectively. SARS-COV-2 studies using samples from the lower respiratory tract, real-time RT-PCR, and tests targeting the N or S gene or more than one gene showed higher sensitivity, and assays based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), especially when targeting only the RNA-dependent RNA polymerase (RdRp) gene, showed significantly lower sensitivity compared to other studies. CONCLUSIONS: Pooling all studies to date shows that on average 10% of patients with coronavirus infections might be missed with PCR tests. Variables affecting sensitivity and specificity can be used for test selection and development.


Subject(s)
Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus/isolation & purification , Nucleic Acid Amplification Techniques/methods , Respiratory Tract Infections/diagnosis , COVID-19/diagnosis , Clinical Laboratory Techniques/standards , Coronavirus/classification , Coronavirus/genetics , Evaluation Studies as Topic , Humans , Nucleic Acid Amplification Techniques/standards , Respiratory Tract Infections/virology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
14.
Am J Trop Med Hyg ; 103(6): 2350-2352, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-887652

ABSTRACT

A simple and rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of SARS-CoV-2. The RT-LAMP assay was highly specific for SARS-CoV-2 and was able to detect one copy of transcribed SARS-CoV-2 RNA within 24 minutes. Assay validation performed using 50 positive and 32 negative clinical samples showed 100% sensitivity and specificity. The RT-LAMP would be valuable for clinical diagnosis and epidemiological surveillance of SARS-CoV-2 infection in resource-limited areas as it does not require the use of sophisticated and costly equipment.


Subject(s)
COVID-19 Testing/standards , COVID-19/diagnosis , COVID-19/epidemiology , Molecular Diagnostic Techniques/standards , Nucleic Acid Amplification Techniques/standards , Pandemics , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/mortality , COVID-19/transmission , COVID-19 Testing/methods , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Reverse Transcription , SARS-CoV-2/pathogenicity , Sensitivity and Specificity
15.
EBioMedicine ; 61: 103036, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-844322

ABSTRACT

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.


Subject(s)
Bacterial Proteins/metabolism , Betacoronavirus/genetics , CRISPR-Associated Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Endodeoxyribonucleases/metabolism , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/pathology , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Humans , Limit of Detection , Nasal Cavity/virology , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/diagnosis , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Polyproteins , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , Reference Standards , SARS-CoV-2 , Viral Proteins/genetics , Viral Proteins/metabolism
16.
J Clin Virol ; 132: 104632, 2020 11.
Article in English | MEDLINE | ID: covidwho-765030

ABSTRACT

BACKGROUND: Due to the emergence of the coronavirus disease 2019 (COVID-19) pandemic there is an urgent need for rapid and accurate testing on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVES: The aim of this study was to assess the diagnostic performance of the GeneFinderTMCOVID-19 Plus RealAmp Kit on the ELITe InGenius sample-to-result platform, which is a commercial nucleic acid amplification test (NAT) targeting genes of SARS-CoV-2. STUDY DESIGN: Patients were eligible between March 18 and May 27, 2020, when they had respiratory symptoms that were suspected for COVID-19. The InGenius platform was compared to routine in-house NAT that was validated according to the national reference. RESULTS: Of 128 randomly selected patients, 58 (45 %) tested positive and 55 (43 %) tested negative in both platforms. Sensitivity of the InGenius platform was 100 % (95 % confidence interval 94-100). In the remaining 15 (12 %) cases E and RdRp genes were not detected in both platforms but the nucleoprotein (N) gene was tested positive by the InGenius platform. All solitary N gene positive cases were confirmed by a N-gene specific in-house validated NAT, and most of these patients could also be considered positive based on other recently available COVID-19 positive respiratory samples or highly suspected radiological findings. CONCLUSION: The InGenius platform for SARS-CoV-2 detection has excellent sensitivity, is easy to use and provides fast results. The inclusion of the N gene as a third gene target may further increase sensitivity for the diagnosis of COVID-19 in comparison to the national reference method.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , Nucleic Acid Amplification Techniques , SARS-CoV-2/genetics , COVID-19 Testing/methods , COVID-19 Testing/standards , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Reproducibility of Results , Sensitivity and Specificity
17.
J Clin Microbiol ; 58(9)2020 08 24.
Article in English | MEDLINE | ID: covidwho-636249

ABSTRACT

The clinical performances of six molecular diagnostic tests and a rapid antigen test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were clinically evaluated for the diagnosis of coronavirus disease 2019 (COVID-19) in self-collected saliva. Saliva samples from 103 patients with laboratory-confirmed COVID-19 (15 asymptomatic and 88 symptomatic) were collected on the day of hospital admission. SARS-CoV-2 RNA in saliva was detected using a quantitative reverse transcription-PCR (RT-qPCR) laboratory-developed test (LDT), a cobas SARS-CoV-2 high-throughput system, three direct RT-qPCR kits, and reverse transcription-loop-mediated isothermal amplification (RT-LAMP). The viral antigen was detected by a rapid antigen immunochromatographic assay. Of the 103 samples, viral RNA was detected in 50.5 to 81.6% of the specimens by molecular diagnostic tests, and an antigen was detected in 11.7% of the specimens by the rapid antigen test. Viral RNA was detected at significantly higher percentages (65.6 to 93.4%) in specimens collected within 9 days of symptom onset than in specimens collected after at least 10 days of symptoms (22.2 to 66.7%) and in specimens collected from asymptomatic patients (40.0 to 66.7%). Self-collected saliva is an alternative specimen option for diagnosing COVID-19. The RT-qPCR LDT, a cobas SARS-CoV-2 high-throughput system, direct RT-qPCR kits (except for one commercial kit), and RT-LAMP showed sufficient sensitivities in clinical use to be selectively used in clinical settings and facilities. The rapid antigen test alone is not recommended for an initial COVID-19 diagnosis because of its low sensitivity.


Subject(s)
Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Immunoassay , Nucleic Acid Amplification Techniques , Pneumonia, Viral/diagnosis , Saliva/virology , Adolescent , Adult , Aged , Aged, 80 and over , Antigens, Viral/analysis , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , Clinical Laboratory Techniques/statistics & numerical data , Female , Humans , Immunoassay/methods , Immunoassay/standards , Immunoassay/statistics & numerical data , Male , Middle Aged , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Nucleic Acid Amplification Techniques/statistics & numerical data , Pandemics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity , Specimen Handling , Young Adult
18.
Biotechniques ; 69(3): 178-185, 2020 09.
Article in English | MEDLINE | ID: covidwho-636778

ABSTRACT

Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Guanidine , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , COVID-19 Testing , Clinical Laboratory Techniques/standards , Colorimetry , Coronavirus Infections/diagnosis , Humans , Molecular Diagnostic Techniques/standards , Nucleic Acid Amplification Techniques/standards , Phenolsulfonphthalein , SARS-CoV-2
19.
Emerg Microbes Infect ; 9(1): 998-1007, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-88525

ABSTRACT

The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 102 RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.


Subject(s)
Coronavirus Infections/diagnosis , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Humans , Limit of Detection , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/standards , Nucleocapsid Proteins/genetics , Pandemics , Phosphoproteins , Pneumonia, Viral/virology , SARS-CoV-2 , Time Factors
20.
Int J Infect Dis ; 93: 264-267, 2020 Apr.
Article in English | MEDLINE | ID: covidwho-2506

ABSTRACT

An ongoing outbreak of severe respiratory pneumonia associated with the 2019 novel coronavirus has recently emerged in China. Here we report the epidemiological, clinical, laboratory and radiological characteristics of 19 suspect cases. We compared the positive ratio of 2019-nCoV nucleic acid amplification test results from different samples including oropharyngeal swab, blood, urine and stool with 3 different fluorescent RT-PCR kits. Nine out of the 19 patients had 2019-nCoV infection detected using oropharyngeal swab samples, and the virus nucleic acid was also detected in eight of these nine patients using stool samples. None of positive results was identified in the blood and urine samples. These three different kits got the same result for each sample and the positive ratio of nucleic acid detection for 2019-nCoV was only 47.4% in the suspect patients. Therefore, it is possible that infected patients have been missed by using nucleic acid detection only. It might be better to make a diagnosis combining the computed tomography scans and nucleic acid detection.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Nucleic Acid Amplification Techniques , Pneumonia, Viral/diagnosis , Blood/virology , COVID-19 , China/epidemiology , Coronavirus Infections/virology , Feces/virology , Female , Humans , Nucleic Acid Amplification Techniques/standards , Pandemics , Pharynx/virology , Pneumonia, Viral/virology , SARS-CoV-2 , Urine/virology , Young Adult
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