scholarly journals Comparison of SARS-CoV-2 RT-PCR on a high-throughput molecular diagnostic platform and the cobas SARS-CoV-2 test for the diagnostic of COVID-19 on various clinical samples

2020 ◽  
Vol 78 (8) ◽  
Author(s):  
Onya Opota ◽  
René Brouillet ◽  
Gilbert Greub ◽  
Katia Jaton
Keyword(s):  
High Throughput ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Test Results ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Rapid Spread ◽  
Bronchial Aspirate ◽  
Very High

ABSTRACT Objectives:In order to cope with the rapid spread of the COVID-19 pandemic, we introduced on our in-house high-throughput molecular diagnostic platform (MDx Platform) a real-time reverse transcriptase PCR (RT-PCR) to detect the SARS-CoV-2 from any clinical specimens. The aim of this study was to compare the RT-PCR results obtain with the MDx Platform and the commercial assay cobas SARS-CoV-2 (Roche) on nasopharyngeal swab and other clinical specimens including sputum, bronchial aspirate, bronchoalveolar lavage and anal swabs. Methods: Samples received in our laboratory from patients suspected of COVID-19 (n = 262) were tested in parallel with our MDx platform SARS-CoV-2 PCR and with the cobas SARS-CoV-2 test. Results: The overall agreement between the two tests for all samples tested was 99.24% (260/262), which corresponded to agreements of 100% (178/178) on nasopharyngeal swabs, 95.45% (42/44) on lower respiratory tract specimen with discordant resultS obtained for very high cycle threshold (Ct) value and 100% (40/40) on anorectal swabs. The Ct values for nasopharyngeal swabs displayed an excellent correlation (R2 > 96%) between both tests. Conclusions: The high agreements between the cobas SARS-CoV-2 test and the MDx platform supports the use of both methods for the diagnostic of COVID-19 on various clinical samples. Very few discrepant results may occur at very low viral load.

scholarly journals Clinical Performance of the Luminex NxTAG CoV Extended Panel for SARS-CoV-2 Detection in Nasopharyngeal Specimens from COVID-19 Patients in Hong Kong

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Jonathan Hon-Kwan Chen ◽  
Cyril Chik-Yan Yip ◽  
Jasper Fuk-Woo Chan ◽  
Rosana Wing-Shan Poon ◽  
Kelvin Kai-Wang To ◽  
...  
Keyword(s):  
Hong Kong ◽  
High Throughput ◽  
Clinical Performance ◽  
Diagnostic System ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Gene Target ◽  
E Gene

ABSTRACT In December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and later spread all over the world. There was an urgent need of a high-throughput molecular test for screening the COVID-19 patients in the community. The Luminex NxTAG CoV extended panel is a high-throughput FDA emergency use-authorized molecular diagnostic assay for SARS-CoV-2 detection. This system targets three genes (ORF1ab, N, and E genes) of SARS-CoV-2, the ORF1ab region of SARS-CoV, and the ORF5 region of MERS-CoV. In this study, we evaluated the diagnostic performance of this system with nasopharyngeal swab specimens of 214 suspected COVID-19 patients in Hong Kong. The results were compared with our routine COVID-19 reverse transcription-PCR (RT-PCR) protocol with a LightMix SarbecoV E-gene kit and an in-house RdRp/Hel RT-PCR assay. The NxTAG CoV extended panel demonstrated 97.8% sensitivity and 100% specificity to SARS-CoV-2 in nasopharyngeal specimens. On low-viral load specimens, the sensitivity of the NxTAG panel could still maintain at 85.71%. Strong agreement was observed between the NxTAG panel and the routine COVID-19 RT-PCR protocol (kappa value = 0.98). Overall, the E gene target of the NxTAG panel demonstrated the highest sensitivity among the three SARS-CoV-2 targets, while the N gene targets demonstrated the least. In conclusion, the NxTAG CoV extended panel is simple to use, and it has high diagnostic sensitivity and specificity to SARS-CoV-2 in nasopharyngeal specimens. We recommend this diagnostic system for high-throughput COVID-19 screening in the community.

scholarly journals Pooling of Nasopharyngeal (NP) Swab Samples to Overcome Global Shortage of rRT-PCR COVID-19 Test Kits

2021 ◽  
Author(s):  
Sunil More ◽  
Sai Narayanan ◽  
Girish Patil ◽  
Parna Ghosh ◽  
Samuel Pushparaj ◽  
...  
Keyword(s):  
Large Scale ◽  
Rapid Screening ◽  
Clinical Samples ◽  
Test Results ◽  
Rapid Testing ◽  
Efficient Utilization ◽  
Clinical Specimens ◽  
Large Scale Testing ◽  
Rapid Spread ◽  
Testing Protocols

The global outbreak and rapid spread of SARS-CoV-2 created an urgent need for large scale testing of populations. There is a demand for high throughput testing protocols that can be used for efficient and rapid testing of clinical specimens. We evaluated a pooled-PCR protocol for testing nasopharyngeal swabs using known positive/negative and untested clinical samples that were assigned to pools of 5 or 10. Six-hundred and thirty (630) samples were used in this study. Individual positive samples with Ct values as high as 33 could be consistently detected when pooled with 4 negative samples (pool of 5) and individual positive samples with Ct values up to 31 could be consistently detected when pooled with 9 negative samples (pool of 10). Pooling of up to 5 samples can be employed in laboratories for the diagnosis of COVID-19 for efficient utilization of resources, rapid screening of a greater number of people, and faster reporting of test results.

scholarly journals Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach

2021 ◽  
Author(s):  
Robin Struijk ◽  
Anton van den Ouden ◽  
Brian McNally ◽  
Theun de Groot ◽  
Bert Mulder ◽  
...  
Keyword(s):  
Large Scale ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Percent Agreement ◽  
Qpcr Method ◽  
Agreement Score ◽  
Pcr Method

The surging COVID19 pandemic has underlined the need for quick, sensitive, and high-throughput SARS-CoV-2 detection assays. Although many different methods to detect SARS-CoV-2 particles in clinical material have been developed, none of these assays are successful in combining all three of the above characteristics into a single, easy-to-use method that is suitable for large-scale use. Here we report the development of a direct RT-PCR SARS-CoV-2 detection method that can reliably detect minute quantities of SARS-CoV-2 gRNA in nasopharyngeal swab samples as well as the presence of human genomic DNA. An extraction-less validation protocol was carried out to determine performance characteristics of the assay in both synthetic SARS-CoV-2 RNA as well as clinical specimens. Feasibility of the assay and analytical sensitivity was first determined by testing a dilution series of synthetic SARS-CoV-2 RNA in two different solvents (water and AMIES VTM), revealing a high degree of linearity and robustness in fluorescence readouts. Following analytical performance using synthetic RNA, the limit of detection was determined at equal to or less than 1 SARS-CoV-2 copy/ul of sample in a commercially available sample panel that contains surrogate clinical samples with varying SARS-CoV-2 viral load. Lastly, we benchmarked our method against a reference qPCR method by testing 87 nasopharyngeal swab samples. The direct endpoint ultra-fast RT-PCR method exhibited a positive percent agreement score of 98.5% and a negative percent agreement score of 100% as compared to the reference method, while RT-PCR cycling was completed in 27 minutes/sample as opposed to 60 minutes/sample in the reference qPCR method. In summary, we describe a rapid direct RT-PCR method to detect SARS-CoV-2 material in clinical specimens which can be completed in significantly less time as compared to conventional RT-PCR methods, making it an attractive option for large-scale SARS-CoV-2 screening applications.

scholarly journals Diagnosis of SARS-CoV-2 infection with LamPORE, a high-throughput platform combining loop-mediated isothermal amplification and nanopore sequencing

2021 ◽  
Author(s):  
Leon Peto ◽  
Gillian Rodger ◽  
Daniel P Carter ◽  
Karen L Osman ◽  
Mehmet Yavuz ◽  
...  
Keyword(s):  
High Throughput ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Nanopore Sequencing ◽  
Rt Pcr ◽  
Symptomatic Infection ◽  
Clinical Specimens ◽  
Loop Mediated Isothermal Amplification

Background LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. Methods We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. Findings The limit of detection of LamPORE was ten genome copies/μl of extracted RNA, which is above the limit achievable by RT-PCR but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among these LamPORE had a diagnostic sensitivity of 99.1% (226/228 [95% CI 96.9–99.9%]). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279 [98.0–100.0%]). Overall, 1.4% (7/514 [0.5–2.9%]) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494 [94.8–98.1%]). Interpretation LamPORE has a similar performance to RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients, and offers a promising approach to high-throughput testing.

scholarly journals Diagnosis of SARS-CoV-2 infection with LamPORE, a high-throughput platform combining loop-mediated isothermal amplification and nanopore sequencing

2020 ◽  
Author(s):  
Leon Peto ◽  
Gillian Rodger ◽  
Daniel P Carter ◽  
Karen L Osman ◽  
Mehmet Yavuz ◽  
...  
Keyword(s):  
High Throughput ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Nanopore Sequencing ◽  
Rt Pcr ◽  
Symptomatic Infection ◽  
Clinical Specimens ◽  
Loop Mediated Isothermal Amplification

LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA that combines loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and from stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 7-10 genome copies/microlitre of extracted RNA. This is above the limit achievable by RT-PCR but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among these LamPORE had a diagnostic sensitivity of 99.1% (226/228 [95% CI 96.9-99.9%]). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279 [98.0-100.0%]). Overall, 1.4% (7/514 [0.5-2.9]) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494 [94.8-98.1]). This indicates that LamPORE has a similar performance to RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients, and offers a promising approach to high-throughput testing.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

scholarly journals Comparison of commercial RT-PCR diagnostic kits for COVID-19

2020 ◽  
Author(s):  
Puck B. van Kasteren ◽  
Bas van der Veer ◽  
Sharon van den Brink ◽  
Lisa Wijsman ◽  
Jørgen de Jonge ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Cross Reactivity ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Specific Method ◽  
Preliminary Evaluation ◽  
Rt Pcr ◽  
Accurate Identification ◽  
Clinical Sensitivity ◽  
Pcr Efficiency

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

scholarly journals High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next-generation sequencing

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247115
Author(s):  
Rahul C. Bhoyar ◽  
Abhinav Jain ◽  
Paras Sehgal ◽  
Mohit Kumar Divakar ◽  
Disha Sharma ◽  
...  
Keyword(s):  
High Throughput ◽  
Genetic Epidemiology ◽  
Confirmatory Test ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Additional Advantage ◽  
Depth Analysis ◽  
High Concordance ◽  
High Throughput Detection ◽  
First Time

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2.

scholarly journals Algorithmic Approach to High-Throughput Molecular Screening for Alpha Interferon-Resistant Genotypes in Hepatitis C Patients

1998 ◽  
Vol 36 (7) ◽  
pp. 1895-1901 ◽  
Author(s):  
Srinand Sreevatsan ◽  
Jack B. Bookout ◽  
Fidel M. Ringpis ◽  
Mridula R. Pottathil ◽  
David J. Marshall ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Dna Sequencing ◽  
High Throughput ◽  
Incubation Temperature ◽  
Alpha Interferon ◽  
Clinical Samples ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Hcv Genotypes ◽  
Genotype Frequencies

This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5′ untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634–639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.

scholarly journals Development, Evaluation of the PNA RT-LAMP Assay for Rapid Molecular Detection of SARS-CoV-2

2021 ◽  
Author(s):  
Chinbayar Bat-Ochir ◽  
Yeon-Sook Kim ◽  
Han Gyeul Kim ◽  
See Sok Lee ◽  
Han Woo Lee ◽  
...  
Keyword(s):  
Real Time ◽  
Lamp Assay ◽  
Study Data ◽  
Molecular Diagnostic ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Clinical Sensitivity ◽  
Clinical Accuracy ◽  
Absolute Detection Limit ◽  
Development Evaluation

Abstract Dual-labeled PNA probe used RT-LAMP molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study. Data showed that this assay is highly specific for SARS-CoV-2, and the absolute detection limit is 1 genomic copy per microliter of viral RNA which can be considered to be comparable to gold-standard molecular diagnostic method real-time reverse transcriptase PCR. Both clinical sensitivity and specificity against a commercial real-time RT-PCR assay were determined as identical. In conclusion, the PNA RT-LAMP assay showed high analytical and clinical accuracy which are identical to real-time RT-PCR which has been routinely used for the detection of SARS-CoV-2.

Sign in / Sign up

Export Citation Format

Share Document