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1.
PLoS One ; 16(11): e0258263, 2021.
Article in English | MEDLINE | ID: covidwho-1700786

ABSTRACT

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.


Subject(s)
COVID-19/virology , Ribonuclease P/genetics , SARS-CoV-2/genetics , Waste Water/virology , DNA Primers/genetics , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Specimen Handling/methods , Wastewater-Based Epidemiological Monitoring
2.
Sci Rep ; 12(1): 2853, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1700332

ABSTRACT

Corona Virus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative sensitivity, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, due to the rapid mutation rate of the viral genome and the emergence of new variants, existing protocols need to be updated and improved. Designing a fast and accurate PCR-based assay is of great importance for the early detection of this virus and more efficient control of the spread of this disease. This study describes a fast, reliable, easy-to-use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N and RdRP) and a human gene (RP) simultaneously. The performance and the sensitivity of the assay were tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.40 and 0.81 copies/µL or 35.13 and 20.31 copies/reaction for RdRP and N genes, respectively. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Coronavirus Nucleocapsid Proteins/genetics , Real-Time Polymerase Chain Reaction/methods , Ribonuclease P/genetics , SARS-CoV-2/isolation & purification , Humans
3.
Int J Mol Sci ; 23(4)2022 Feb 09.
Article in English | MEDLINE | ID: covidwho-1690219

ABSTRACT

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/physiology , Virus Replication/physiology , Coronavirus Nucleocapsid Proteins/analysis , Giant Cells/drug effects , Giant Cells/virology , HEK293 Cells , Humans , Limit of Detection , Nasopharynx/virology , Phosphoproteins/analysis , Phosphoproteins/genetics , RNA, Antisense/pharmacology , RNA, Viral , Ribonuclease P/genetics , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Sensitivity and Specificity , Social Isolation , Viral Load , Viroporin Proteins/genetics , Virus Replication/drug effects
4.
PLoS One ; 16(11): e0258263, 2021.
Article in English | MEDLINE | ID: covidwho-1511816

ABSTRACT

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.


Subject(s)
COVID-19/virology , Ribonuclease P/genetics , SARS-CoV-2/genetics , Waste Water/virology , DNA Primers/genetics , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Specimen Handling/methods , Wastewater-Based Epidemiological Monitoring
5.
Sci Rep ; 11(1): 21658, 2021 11 04.
Article in English | MEDLINE | ID: covidwho-1503936

ABSTRACT

More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/genetics , COVID-19/genetics , COVID-19 Nucleic Acid Testing/economics , COVID-19 Testing/methods , Coronavirus Nucleocapsid Proteins/genetics , DNA Primers , Electrophoresis, Agar Gel/methods , Humans , Laboratories , Nasopharynx/virology , RNA, Viral/genetics , Ribonuclease P/genetics , Ribonuclease P/metabolism , SARS-CoV-2/pathogenicity , Sensitivity and Specificity
6.
Mol Cell Probes ; 58: 101744, 2021 08.
Article in English | MEDLINE | ID: covidwho-1253367

ABSTRACT

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Clinical Laboratory Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , DNA Primers/genetics , DNA Probes/genetics , Humans , Molecular Diagnostic Techniques/methods , Reproducibility of Results , Ribonuclease P/genetics , SARS-CoV-2/physiology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics
7.
J Virol Methods ; 294: 114174, 2021 08.
Article in English | MEDLINE | ID: covidwho-1226316

ABSTRACT

There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4 % of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19 Nucleic Acid Testing/standards , RNA, Viral/genetics , SARS-CoV-2/genetics , Specimen Handling/standards , COVID-19/diagnosis , COVID-19/virology , Gene Dosage , Genes, Essential , Humans , Limit of Detection , RNA, Viral/isolation & purification , Reference Standards , Ribonuclease P/genetics , Specimen Handling/methods , Viral Load
8.
J Virol Methods ; 294: 114171, 2021 08.
Article in English | MEDLINE | ID: covidwho-1226315

ABSTRACT

Respiratory syncytial virus (RSV) is a common cause of acute respiratory disease worldwide, especially in young children. The World Health Organization (WHO) has initiated an RSV Surveillance Pilot program that aims to perform worldwide RSV surveillance, requiring the development of reliable and rapid molecular methods to detect and identify RSV. A duplex real-time RT-PCR assay developed for simultaneous detection of both A and B subtypes of RSV was included as part of this program. This duplex assay targeted a conserved region of the RSV polymerase gene and was validated for analytical sensitivity, specificity, reproducibility and clinical performance with a wide range of respiratory specimens. The assay was highly specific for RSV and did not react with non-RSV respiratory pathogens, including the SARS-CoV-2 virus.


Subject(s)
Molecular Diagnostic Techniques/methods , RNA, Viral/isolation & purification , Respiratory Syncytial Virus, Human/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , DNA Primers/genetics , Humans , Limit of Detection , Nasopharynx/virology , RNA-Dependent RNA Polymerase/genetics , Reproducibility of Results , Ribonuclease P/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
9.
Virol Sin ; 35(6): 758-767, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1217478

ABSTRACT

Reverse transcription-polymerase chain reaction (RT-PCR) is an essential method for specific diagnosis of SARS-CoV-2 infection. Unfortunately, false negative test results are often reported. In this study, we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens. Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercial fluorescent RT-PCR kit targeting the ORF1ab and N regions of SARS-CoV-2 genome. The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30 (RPP30) in these specimens was also assessed by RT-PCR. Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed. By using the ROC-AUC analysis, we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity (95.03%-95.26%) and specificity (83.72%-98.55%) for respective combination of specimen type and amplification reaction. Using these Ct cutoff values, false negative results could be reliably identified. Therefore, the presence of cellular materials, likely infected host cells, are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens. RPP30 could serve as an indicator for cellular content, or a surrogate indicator for specimen quality. In addition, our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Viral/genetics , SARS-CoV-2/genetics , Autoantigens/genetics , COVID-19/epidemiology , COVID-19/virology , China/epidemiology , Humans , Negative Results , Polyproteins/genetics , RNA, Viral/isolation & purification , Reference Standards , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction/methods , Ribonuclease P/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Viral Proteins/genetics
10.
J Med Virol ; 93(4): 2439-2445, 2021 04.
Article in English | MEDLINE | ID: covidwho-996237

ABSTRACT

Diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) cases is based on the count of real-time reverse transcription-plymerase chain reaction (RT-PCR) positive people. Viral load by real-time RT-PCR has been suggested as a biomarker of the SARS-CoV-2 infection. However, the association of viral load and severity of the disease is not yet resolved. Nasopharyngeal samples from 458 patients were tested by RT-PCR for SARS-CoV-2 diagnosis. Relative quantitation was made by the comparative threshold cycle (ΔΔCt ) formula between ORF1ab viral and RNase P housekeeping genes. Absolute viral load was calculate using a reference positive control. Most prevalent clinical signs were cough (75.8%), myalgia (66.7%), and fever (48.5%). Hypertension (18.2%), neurological diseases (15.1%), and asthma and hypothyroidism (12.1%) were most frequent comorbidities. Fever, either as an exclusive symptom or combined with others, was associated with high viral loads ( 2 - ∆ ∆ C t range, 35.65-155.16; 4.25-4.89 log10 RNA copies/test]). During the first week after onset of symptoms in mild patients up to 60 years-old was detected the peak of viral load. Children under 10 years old have a high viral load (313.84; 2.50) in the first 2 days postinfection with a sharp decline thereafter. Cases between 10 and 49 years old mostly showed low and moderate viral load during the first 2 days postinfection (range, 0.03 to 17.24; -1.50 to 1.24). Patients over 60 years old have high viral load up to the second week after the onset of symptoms (range, 25.32-155.42; 1.40-2.19), indicating the longer presence of the virus in them. These findings suggest the viral load in nasopharyngeal swabs would help to monitor the SARS-CoV-2 infection in mild coronavirus disease 2019 cases.


Subject(s)
COVID-19 Testing/methods , COVID-19/virology , Nasopharynx/virology , RNA, Viral/isolation & purification , SARS-CoV-2/genetics , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/diagnosis , Child , Child, Preschool , Diagnostic Tests, Routine , Female , Humans , Infant , Male , Middle Aged , Polyproteins/genetics , Real-Time Polymerase Chain Reaction/methods , Ribonuclease P/genetics , SARS-CoV-2/isolation & purification , Viral Load , Viral Proteins/genetics , Young Adult
11.
Eur J Clin Microbiol Infect Dis ; 40(4): 807-813, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-891909

ABSTRACT

The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , DNA/analysis , Multiplex Polymerase Chain Reaction/methods , RNA, Messenger/analysis , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Autoantigens/genetics , Coronavirus Envelope Proteins/genetics , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Genes, Essential , Glucuronidase/genetics , Humans , Phosphoproteins/genetics , Real-Time Polymerase Chain Reaction , Ribonuclease P/genetics , SARS-CoV-2 , Sensitivity and Specificity
12.
Clin Chem Lab Med ; 58(9): 1579-1586, 2020 08 27.
Article in English | MEDLINE | ID: covidwho-612047

ABSTRACT

Objectives: The direct identification of SARS-CoV-2 RNA in nasopharyngeal swabs is recommended for diagnosing the novel COVID-19 disease. Pre-analytical determinants, such as sampling procedures, time and temperature storage conditions, might impact on the end result. Our aim was to evaluate the effects of sampling procedures, time and temperature of the primary nasopharyngeal swabs storage on real-time reverse-transcription polymerase chain reaction (rRT-PCR) results. Methods: Each nasopharyngeal swab obtained from 10 hospitalized patients for COVID-19 was subdivided in 15 aliquots: five were kept at room temperature; five were refrigerated (+4 °C); five were immediately mixed with the extraction buffer and refrigerated at +4 °C. Every day and for 5 days, one aliquot per condition was analyzed (rRT-PCR) for SARS-CoV-2 gene E and RNaseP and threshold cycles (Ct) compared. To evaluate manual sampling, 70 nasopharyngeal swabs were sampled twice by two different operators and analyzed separately one from the other. Results: A total of 6/10 swabs were SARS-CoV-2 positive. No significant time or storage-dependent variations were observed in SARS-CoV-2 Ct. Re-sampling of swabs with SARS-CoV-2 Ct lower than 33 resulted in highly reproducible results (CV=2.9%), while a high variability was observed when Ct values were higher than 33 (CV=10.3%). Conclusions: This study demonstrates that time and temperature of nasopharyngeal swabs storage do not significantly impact on results reproducibility. However, swabs sampling is a critical step, and especially in case of low viral load, might be a potential source of diagnostic errors.


Subject(s)
Betacoronavirus/chemistry , Nasopharynx/virology , RNA, Viral/analysis , Specimen Handling/methods , Aged , Aged, 80 and over , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Envelope Proteins , Coronavirus Infections/diagnosis , Diagnostic Tests, Routine , Female , Humans , Male , Middle Aged , Pandemics , Pneumonia, Viral/diagnosis , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Ribonuclease P/genetics , SARS-CoV-2 , Temperature , Time Factors , Viral Envelope Proteins/genetics
13.
Int J Mol Sci ; 21(9)2020 May 01.
Article in English | MEDLINE | ID: covidwho-155285

ABSTRACT

In this work, hybridization chain reactions (HCRs) toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) nucleocapsid phosphoproteins gene loci and human RNase P are proposed to provide an isothermal amplification screening tool. The proposed chain reactions target the complementary DNA (cDNA) of SARS-CoV-2, with loci corresponding to gold-standard polymerase chain reaction (PCR) loci. Four hybridization chain reaction reactions are demonstrated herein, targeting N1/N2/N3 loci and human RNase P. The design of the hybridization chain reaction, herein, is assisted with an algorithm. The algorithm helps to search target sequences with low local secondary structure and high hybridization efficiency. The loop domain of the fuel hairpin molecule H1 and H2, which are the tunable segments in such reactions, are used as an optimization parameter to improve the hybridization efficiency of the chain reaction. The algorithm-derived HCR reactions were validated with gel electrophoresis. All proposed reactions exhibit a hybridization complex with a molecular mass >1.5k base pairs, which is clear evidence of chain reaction. The hybridization efficiency trend revealed by gel electrophoresis corresponds nicely to the simulated data from the algorithm. The HCR reactions and the corresponding algorithm serve as a basis to further SARS-CoV-2 sensing applications and facilitate better screening strategies for the prevention of on-going pandemics.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Mass Screening/methods , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , COVID-19 , Computer Simulation , Coronavirus Infections/virology , DNA, Complementary/genetics , Humans , Pandemics , Pneumonia, Viral/virology , Polymerase Chain Reaction/methods , Ribonuclease P/genetics , SARS-CoV-2
14.
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 , COVID-19 Testing , 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 , SARS-CoV-2 , Serologic Tests/methods
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