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1.
Bull Exp Biol Med ; 172(4): 495-498, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1756827

ABSTRACT

The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies.


Subject(s)
COVID-19/metabolism , Cell-Free Nucleic Acids/genetics , DNA, Mitochondrial/genetics , NADH Dehydrogenase/genetics , Real-Time Polymerase Chain Reaction/standards , Adult , Aged , COVID-19/virology , Case-Control Studies , Cell-Free Nucleic Acids/blood , DNA Primers/chemical synthesis , DNA, Mitochondrial/blood , Female , Humans , Male , Middle Aged , Mitochondria/genetics , Mitochondria/virology , NADH Dehydrogenase/blood , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/pathogenicity
2.
Microbiol Spectr ; 10(1): e0059121, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1691413

ABSTRACT

Coronavirus disease 2019 (COVID-19) is a mild to severe respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnostic accuracy of the Centers for Disease Control and Prevention (CDC)- or World Health Organization (WHO)-recommended real-time PCR (RT-qPCR) primers in clinical practice remains unproven. We conducted a prospective study on the accuracy of RT-qPCR using an in-house-designed primer set (iNP) targeting the nucleocapsid protein as well as various recommended and commercial primers. The accuracy was assessed by culturing or seroconversion. We enrolled 12 confirmed COVID-19 patients with a total of 590 clinical samples. When a cutoff value of the cycle threshold (Ct) was set to 35, RT-qPCRs with WHO RdRp primers and CDC N1, N2, and N3 primers showed sensitivity of 42.1% to 63.2% and specificity of 90.5% to 100% in sputum, and sensitivity of 65.2% to 69.6% and specificity of 65.2% to 69.6% in nasopharyngeal samples. The sensitivity and specificity of iNP RT-qPCR in sputum and nasopharyngeal samples were 94.8%/100% and 69.6%/100%, respectively. Sputum testing had the highest sensitivity, followed by nasopharyngeal testing (P = 0.0193); self-collected saliva samples yielded better characteristics than oropharyngeal samples (P = 0.0032). Our results suggest that iNP RT-qPCR has better sensitivity and specificity than RT-PCR with WHO (P < 0.0001) or CDC (N1: P = 0.0012, N2: P = 0.0013, N3: P = 0.0012) primers. Sputum RT-qPCR analysis has the highest sensitivity, followed by nasopharyngeal, saliva, and oropharyngeal assays. Our study suggests that considerable improvement is needed for the RT-qPCR WHO and CDC primer sets for detecting SARS-CoV-2. IMPORTANCE Numerous research campaigns have addressed the vast majority of clinical and diagnostic specificity and sensitivity of various primer sets of SARS-CoV2 viral detection. Despite the impressive progress made to resolve the pandemic, there is still a need for continuous and active improvement of primers used for diagnosis in clinical practice. Our study significantly exceeds the scale of previously published research on the specificity and sensitivity of different primers comparing with different specimens and is the most comprehensive to date in terms of constant monitoring of primer sets of current usage. Henceforth, our results suggest that sputum samples sensitivity is the highest, followed by nasopharyngeal, saliva, and oropharyngeal samples. The CDC recommends the use of oropharyngeal specimens, leading to certain discrepancy between the guidelines set forth by the CDC and IDSA. We proved that the oropharyngeal samples demonstrated the lowest sensitivity for the detection of SARS-CoV-2.


Subject(s)
COVID-19/diagnosis , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/isolation & purification , Adult , Aged , COVID-19/virology , Cross Reactions , Female , Humans , Male , Middle Aged , Nasopharynx/virology , Oropharynx/virology , SARS-CoV-2/genetics , Saliva/virology , Sensitivity and Specificity , Sputum/virology , Viral Load , Young Adult
3.
J Clin Lab Anal ; 36(2): e24242, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1616016

ABSTRACT

BACKGROUND: Currently, SARS-CoV-2 RNA detection using real-time reverse-transcription PCR (rRT-PCR) is the standard diagnostic test for COVID-19 infection. Various rRT-PCR assays are currently used worldwide, targeting different genes of the SARS-CoV-2. Here, we compared the analytical sensitivity and clinical performance (sensitivity and specificity) of Allplex SARS-CoV-2/FluA/FluB/RSV assay (Seegene), Standard M nCoV real-time detection kit (SD Biosensor), and U-TOP COVID-19 detection kit (Seasun Biomaterials) for SARS-CoV-2 detection. METHODS: Two hundred and forty-nine nasopharyngeal swab samples were evaluated to compare the clinical performance of the rRT-PCR assays. For the analytical performance evaluation, two RNA controls with known viral loads-SARS-CoV-2 RNA control and SARS-COV-2 B.1.351 RNA control-were used to investigate the potential impact of SARS-CoV-2 variants, particularly the B.1.351 lineage. RESULTS: Limits of detection ranged from 650 to 1300 copies/ml for rRT-PCR assays, and the mean differences in cycle threshold (Ct ) values of the two RNA controls were within 1.0 for each target in the rRT-PCR assays (0.05-0.73), without any prominent Ct value shift or dropouts in the SARS-COV-2 B.1.351 RNA control. Using the consensus criterion as the reference standard, 89 samples were positive, whereas 160 were negative. The overall clinical performance of rRT-PCR assays was comparable (sensitivity 98.88%-100%; specificity 99.38%-100%), whereas the sensitivities of each target gene were more variable. CONCLUSIONS: The three rRT-PCR assays showed comparable analytical sensitivity and clinical performance. The analytical and clinical sensitivities of each target gene were influenced more by the primer and probe design than the target gene itself.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques , Reagent Kits, Diagnostic/virology , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Female , Humans , Male , Middle Aged , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/standards , Nasopharynx/virology , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Viral Load , Young Adult
4.
J Clin Lab Anal ; 36(2): e24226, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1611241

ABSTRACT

INTRODUCTION: RT-PCR is widely used as a diagnostic test for the detection of SARS-CoV-2. In this study, we aim to describe the clinical utility of serial PCR testing in the final detection of COVID-19. METHOD: We collected multiple nasopharyngeal swab samples from patients who had negative RT-PCR test on the first day after hospitalization. RT-PCR tests were performed on the second day for all patients with initial negative result. For the patients with secondary negative results on day 2, tertiary RT-PCR tests were performed on day 3 after hospitalization. RESULT: Among 68 patients with initial negative test results, at the end of follow-up, the mortality number was 20 (29.4%). About 33.8% of patients had subsequent positive PCR test results for the second time and 17.4% of the patients who performed third PCR test had positive result. CONCLUSION: Based on this study, serial RT-PCR testing is unlikely to yield additional information.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Aged , Aged, 80 and over , False Negative Reactions , Female , Humans , Male , Middle Aged , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/standards , Molecular Diagnostic Techniques/statistics & numerical data , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Real-Time Polymerase Chain Reaction/statistics & numerical data , SARS-CoV-2/isolation & purification
5.
J Med Virol ; 93(12): 6575-6581, 2021 12.
Article in English | MEDLINE | ID: covidwho-1530180

ABSTRACT

Reliable and rapid detection of severe acute respiratory syndrome coronavirus 2 in laboratory setting is critical to control the pandemic. We aimed to an evaluated polymerase chain reaction (PCR) efficiency of nasopharyngeal swabs stored in viral transport medium (VTM) in different temperatures. Ninety swabs taken into VTM were analyzed at the first hour, then divided into two groups with similar numbers of positive and negative samples. Positive samples of each group were also subgrouped according to Fam CT values as low CT (<25), medium CT (25-32), and high CT (32-38) groups. One group was stored at 4°C, while the other was stored at room temperature, PCR analyses were repeated every 24 h for 5 days and on Day 12. There was a total of 30 positive samples (12 low CT, 11 medium CT, and 7 high CT). The CT values of both groups remained unchanged in first 3 days while the CT values of the room temperature group increased after the third day. All of the positive samples remained positive in both groups for the first 5 days. On the 12th day, the total number of positives decreased to 8 in the room temperature group and 11 in the 4°C groups. All the low CT samples remained positive in both groups. In conclusion, it is safe to store positive samples in room temperature for up to 5 days. Only samples with high viral loads remain positive for 12 days, regardless of whether stored at room temperature or 4°C. Negative samples don't turn to invalid if stored in VTM.


Subject(s)
COVID-19 Testing/methods , Real-Time Polymerase Chain Reaction/methods , Specimen Handling/methods , COVID-19/diagnosis , Humans , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2 , Specimen Handling/standards , Time Factors
6.
PLoS One ; 16(11): e0260087, 2021.
Article in English | MEDLINE | ID: covidwho-1528723

ABSTRACT

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Real-Time Polymerase Chain Reaction/methods , Adult , COVID-19 Nucleic Acid Testing/standards , Female , Humans , Male , Nucleic Acid Denaturation , Oligonucleotides/chemistry , Real-Time Polymerase Chain Reaction/standards , Respiratory Mucosa/virology , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Saliva/virology , Sensitivity and Specificity
7.
Pan Afr Med J ; 39: 244, 2021.
Article in English | MEDLINE | ID: covidwho-1468747

ABSTRACT

Numerous genetic tests for the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, including those based on the ever-popular real-time polymerase chain reaction (RT-qPCR) technique, have been reported. These diagnostic tests give false negatives particularly during the early and late stages of COVID-19 clearly indicating inadequate test sensitivity. The entire COVID-19 diagnostic workflow is often overlooked and given very little attention. Herein, we propose that volumetric modifications to COVID-19 workflows would significantly improve detection limits. We would therefore encourage researchers to adopt a holistic approach, in which all the steps of a COVID-19 diagnostic workflow, are carefully scrutinised, particularly those upstream factors at the viral sampling and pre-analytical stages.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Real-Time Polymerase Chain Reaction/methods , COVID-19 Testing/standards , False Negative Reactions , Humans , Limit of Detection , Real-Time Polymerase Chain Reaction/standards , Sensitivity and Specificity , Specimen Handling
8.
PLoS One ; 16(6): e0252507, 2021.
Article in English | MEDLINE | ID: covidwho-1388918

ABSTRACT

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Subject(s)
COVID-19 Testing/standards , COVID-19/diagnosis , COVID-19/epidemiology , Diagnostic Tests, Routine/standards , Indicators and Reagents/standards , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Testing/methods , Cameroon/epidemiology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Geobacillus stearothermophilus/genetics , Geobacillus stearothermophilus/metabolism , Ghana/epidemiology , Humans , Indicators and Reagents/chemistry , Indicators and Reagents/metabolism , Indicators and Reagents/supply & distribution , Molecular Diagnostic Techniques , Plasmids/chemistry , Plasmids/metabolism , Real-Time Polymerase Chain Reaction/methods , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Synthetic Biology/methods , Transformation, Bacterial , United Kingdom/epidemiology
9.
Viruses ; 13(9)2021 08 28.
Article in English | MEDLINE | ID: covidwho-1374538

ABSTRACT

The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/virology , Polymerase Chain Reaction , SARS-CoV-2 , COVID-19 Nucleic Acid Testing/standards , Humans , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/standards , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Load
10.
Sci Rep ; 11(1): 14817, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1319044

ABSTRACT

A real-time reverse transcription polymerase chain reaction (RT-qPCR) assay that does not require Emergency Use Authorization (EUA) reagents was tested and validated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the early stages of the outbreak of coronavirus disease 2019 (COVID-19) in the Republic of Korea. Early diagnosis of COVID-19 enables timely treatment and the implementation of public health measures. We validated the sensitivity, specificity, precision, linearity, accuracy, and robustness of the RT-qPCR assay for SARS-CoV-2 detection and compared its performance with that of several EUA-approved kits. Our RT-qPCR assay was highly specific for SARS-CoV-2 as demonstrated by not amplifying 13 other viruses that cause respiratory diseases. The assay showed high linearity using a viral isolate from a patient with known COVID-19 as well as plasmids containing target SARS-CoV-2 genes as templates. The assay showed good repeatability and reproducibility with a coefficient of variation of 3%, and a SARS-CoV-2 limit of detection of 1 PFU/mL. The RT-qPCR-based assay is highly effective and can facilitate the early diagnosis of COVID-19 without the use of EUA-approved kits or reagents in the Republic of Korea.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/epidemiology , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , Chlorocebus aethiops , Humans , Limit of Detection , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Republic of Korea , Reverse Transcriptase Polymerase Chain Reaction/standards , Vero Cells
11.
Diagn Microbiol Infect Dis ; 100(4): 115381, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1269260

ABSTRACT

To compare the practicability (usability and satisfaction) and analytical performances of VitaPCR™ Flu A&B Assay (Credo Diagnostics Biomedical Pte. Ltd., Singapore, Republic of Singapore) and Xpert® Xpress Flu/RSV kit (Cepheid, Sunnyvale, USA), two rapid point-of-care (POC) nucleic acid amplification tests (NAATs) by reference to multiplex RT-PCR for respiratory viruses. Nasopharyngeal swabs (n=117) were collected from patients with influenza-like illness in Paris, France. Thawed specimens were further analyzed with both NAATs. The usability was comparable for both NAATs. Satisfaction questionnaire was better for the VitaPCR™ platform for the short time of test result in 20 minutes. Both NAATs showed comparable sensitivities (VitaPCRTM: 95.0%; Xpert® Xpress: 97.5%) and specificities (100%) for influenza A/B RNA detection, with excellent reliability and accuracy between both NAATs. Both VitaPCR™ and Xpert® Xpress NAATs can be implemented in hospital setting as POC NAATs to rapidly detect influenza A/B RNA in symptomatic patients.


Subject(s)
Molecular Diagnostic Techniques/instrumentation , Reagent Kits, Diagnostic/standards , Real-Time Polymerase Chain Reaction/instrumentation , Viruses/genetics , Humans , Influenza A virus/genetics , Influenza, Human/diagnosis , Influenza, Human/virology , Molecular Diagnostic Techniques/methods , Nasopharynx/virology , Point-of-Care Testing/standards , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Sensitivity and Specificity , Viruses/classification , Viruses/isolation & purification
12.
West J Emerg Med ; 22(3): 543-546, 2021 May 19.
Article in English | MEDLINE | ID: covidwho-1266890

ABSTRACT

Some experts have promoted the use of rapid testing for COVID-19. However, with the current technologies available, continuing to replace laboratory-based, real-time reverse transcription polymerase chain reaction tests with rapid (point-of-care) tests may lead to an increased number of false negative tests. Moreover, the more rapid dissemination of false negative results that can occur with the use of rapid tests for COVID-19 may lead to increased spread of the novel coronavirus if patients do not understand the concept of false negative tests. One means of combatting this would be to tell patients who have a "negative" rapid COVID-19 test that their test result was "indeterminate."


Subject(s)
COVID-19 Testing/standards , COVID-19/diagnosis , Point-of-Care Testing/standards , COVID-19/epidemiology , False Negative Reactions , Humans , Male , Pandemics , Predictive Value of Tests , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2
13.
Biomed Res Int ; 2021: 6653950, 2021.
Article in English | MEDLINE | ID: covidwho-1263958

ABSTRACT

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 µL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 µL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.


Subject(s)
COVID-19/diagnosis , Coronavirus Envelope Proteins/genetics , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , 2-Propanol/chemistry , Biomarkers/analysis , COVID-19/virology , DNA Primers/chemical synthesis , DNA Primers/genetics , Humans , Nasopharynx/virology , Oropharynx/virology , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/economics , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Sensitivity and Specificity
14.
RNA Biol ; 18(12): 2218-2225, 2021 12.
Article in English | MEDLINE | ID: covidwho-1221426

ABSTRACT

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.


Subject(s)
Benzothiazoles/chemistry , COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Diamines/chemistry , Intercalating Agents/chemistry , Quinolines/chemistry , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , DNA/analysis , DNA/biosynthesis , DNA Primers/chemistry , DNA Primers/metabolism , Humans , Nasopharynx/virology , Real-Time Polymerase Chain Reaction/standards , Sensitivity and Specificity
15.
J Mol Diagn ; 23(6): 691-697, 2021 06.
Article in English | MEDLINE | ID: covidwho-1179822

ABSTRACT

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, SARS-CoV-2 detectability by RT-PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2-positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on RT-PCR detection. SARS-CoV-2 RNA was still reliably detected by RT-PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The maximum observed change in cycle threshold value per day was 0.046 (±0.019) at 35°C, and the maximum observed change in cycle threshold value per freeze-thaw cycle per day was 0.197 (±0.06). Compared with storage at 4°C, viral RNA levels deviated little but significantly when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels are relatively stable at higher temperatures and repetitive freeze-thawing.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , Specimen Handling/methods , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Freezing , Humans , Nasopharynx/virology , RNA Stability , Switzerland/epidemiology , Temperature , Time Factors
17.
J Mol Diagn ; 23(6): 665-670, 2021 06.
Article in English | MEDLINE | ID: covidwho-1171781

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading all over the world and has caused millions of deaths. Several sample-to-answer platforms, including Cepheid Xpert Xpress SARS-CoV-2 (Xpert Xpress), have received emergency use authorization for SARS-CoV-2 nucleic acid detection as a point of care test in the United States. However, their application niche is unclear when compared with real-time RT-PCR assays cleared by the National Medical Products Administration in China. In this study, the clinical performance, sensitivity, and workflow of Xpert Xpress and two real-time RT-PCR kits (BioGerm kit and Sansure kit) were evaluated by the specimens from 86 symptomatic patients. The positive percent agreement of Xpert Xpress was 100% compared with 96.15% for the BioGerm kit and 90% for the Sansure kit. The negative percent agreement was 100% for all three assays. The limit of detection is 100 copies/mL for Xpert Xpress and 500 copies/mL for the BioGerm kit and Sansure kit. By serially diluting five positive specimens, the Xpert Xpress had better detection capability. In the workflow and throughput analysis, the turnaround time was 51 minutes for Xpert Xpress, 150 minutes for the BioGerm kit, and 210 minutes for the Sansure kit. This study provides some indication for diagnosis methods selection.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , RNA, Viral/genetics , Reagent Kits, Diagnostic/standards , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , Benchmarking , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , China/epidemiology , Humans , Limit of Detection , Point-of-Care Testing , United States/epidemiology
18.
J Mol Diagn ; 23(6): 691-697, 2021 06.
Article in English | MEDLINE | ID: covidwho-1155539

ABSTRACT

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, SARS-CoV-2 detectability by RT-PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2-positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on RT-PCR detection. SARS-CoV-2 RNA was still reliably detected by RT-PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The maximum observed change in cycle threshold value per day was 0.046 (±0.019) at 35°C, and the maximum observed change in cycle threshold value per freeze-thaw cycle per day was 0.197 (±0.06). Compared with storage at 4°C, viral RNA levels deviated little but significantly when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels are relatively stable at higher temperatures and repetitive freeze-thawing.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , Specimen Handling/methods , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Freezing , Humans , Nasopharynx/virology , RNA Stability , Switzerland/epidemiology , Temperature , Time Factors
19.
Viruses ; 13(4)2021 03 26.
Article in English | MEDLINE | ID: covidwho-1154533

ABSTRACT

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-CoV-2 RNA is an essential test to monitor the occurrence of COVID-19. A methodology is proposed for the determination of maximum pool size and adjustments of cut-off values of cycle threshold (Ct in RT-qPCR pool testing, to compensate for the dilution caused by pooling. The trade-off between pool size and test sensitivity is stated explicitly. The procedure was designed to ensure that samples that would be detectable in individual testing remain detectable in pool testing. The proposed relaxation in cut-off is dependent on the pool size, allowing a relatively tight correction to avoid loss of detection of positive samples. The methodology was evaluated in a study of pool testing of adults attending a public emergency care unit, reference for COVID-19 in Belo Horizonte, Brazil, and presenting flu-like symptoms. Even samples on the edge of detectability in individual testing were detected correctly. The proposed procedure enhances the consistency of RT-qPCR pool testing by enforcing that the scales of detectability in pool processing and in individual sample processing are compatible. This may enhance the contribution of pool testing to large-scale testing for COVID-19.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/virology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Adult , Aged , Aged, 80 and over , COVID-19/diagnosis , COVID-19 Nucleic Acid Testing/standards , Female , Humans , Male , Middle Aged , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Young Adult
20.
J Mol Diagn ; 23(6): 683-690, 2021 06.
Article in English | MEDLINE | ID: covidwho-1121530

ABSTRACT

Fast, accurate, and reliable diagnostic tests are critical for controlling the spread of the coronavirus disease 2019 (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The current gold standard for testing is real-time PCR; however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 minutes), user-friendly, and accurate microchip real-time PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips preloaded with COVID-19 primers and probes for the N gene accommodate 1.2-µL reaction volumes, lowering the required reagents by 10-fold compared with tube-based real-time PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip real-time PCR provides a significantly lower resource alternative to the Centers for Disease Control and Prevention-approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , Lab-On-A-Chip Devices , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , Benchmarking , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Canada/epidemiology , Humans , Limit of Detection , Nasopharynx/virology , Point-of-Care Testing , Reagent Kits, Diagnostic/supply & distribution
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