Rapid and Sensitive Detection of SARS-CoV-2 Infection Using Quantitative Peptide Enrichment LC-MS/MS Analysis

Reliable, robust, large-scale molecular testing for SARS-CoV-2 is essential for monitoring the ongoing Covid-19 pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immunoaffinity enrichment combined with liquid chromatography - mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in PBS swab media from combined throat/nasopharynx/saliva samples.<br />The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their corresponding RT-PCR readout (r=0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative readout of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

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Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

10.21203/rs.3.rs-28883/v1 ◽

2020 ◽

Cited By ~ 8

Author(s):

Karina Helena Morais Cardozo ◽

Adriana Lebkuchen ◽

Guilherme Goncalves Okai ◽

Rodrigo Andrade Schuch ◽

Luciana Godoy Viana ◽

...

Keyword(s):

Real Time ◽

Large Scale ◽

Low Cost ◽

Large Population ◽

Virus Detection ◽

Standard Test ◽

Clinical Samples ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Fast Processing

Abstract The current outbreak of severe acute respiratory syndrome associated with coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

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Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

10.21203/rs.3.rs-28883/v2 ◽

2020 ◽

Cited By ~ 1

Author(s):

Karina Helena Morais Cardozo ◽

Adriana Lebkuchen ◽

Guilherme Goncalves Okai ◽

Rodrigo Andrade Schuch ◽

Luciana Godoy Viana ◽

...

Keyword(s):

Real Time ◽

Large Scale ◽

Low Cost ◽

Large Population ◽

Virus Detection ◽

Standard Test ◽

Clinical Samples ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Fast Processing

Abstract The current outbreak of severe acute respiratory syndrome associated with coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

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High speed large scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled with high content screening

10.1101/2020.05.14.097295 ◽

2020 ◽

Cited By ~ 2

Author(s):

Rania Francis ◽

Marion Le Bideau ◽

Priscilla Jardot ◽

Clio Grimaldier ◽

Didier Raoult ◽

...

Keyword(s):

High Speed ◽

Large Scale ◽

Vaccine Development ◽

Work Load ◽

Drug Susceptibility Testing ◽

High Content Screening ◽

Clinical Samples ◽

Rt Pcr ◽

Positive Elements

AbstractSARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination.

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Saving Resources: SARS-CoV-2 Diagnostics by Real-Time RT-PCR Using Reduced Reaction Volumes

Diseases ◽

10.3390/diseases9040084 ◽

2021 ◽

Vol 9 (4) ◽

pp. 84

Author(s):

Sabine Bock ◽

Bernd Hoffmann ◽

Martin Beer ◽

Kerstin Wernike

Keyword(s):

Real Time ◽

Viral Genome ◽

Virus Detection ◽

World Health ◽

Clinical Samples ◽

Rt Pcr ◽

Reaction Volume ◽

Reaction Volumes ◽

Health Organization ◽

The Individual

Since the beginning of 2020, the betacoronavirus SARS-CoV-2 is causing a global pandemic of an acute respiratory disease termed COVID-19. The diagnostics of the novel disease is primarily based on direct virus detection by RT-PCR; however, the availability of test kits may become a major bottleneck, when millions of tests are performed per week. To increase the flexibility of SARS-CoV-2 diagnostics, three real-time RT-PCR assays listed on the homepage of the World Health Organization were selected and investigated regarding their compatibility with three different RT-PCR kits. Furthermore, the reaction volume of the PCR chemistry was reduced up to half of the original protocol to make the individual reactions more cost- and resource-effective. When testing dilution series of culture-grown virus, nearly identical quantification cycle values (Cq) were obtained for all RT-PCR assay/chemistry combinations. Regarding the SARS-CoV-2 detection in clinical samples, agreeing results were obtained for all combinations for virus negative specimens and swabs containing high to medium viral genome loads. In cases of very low SARS-CoV-2 genome loads (Cq > 36), inconsistent results were observed, with some test runs scoring negative and some positive. However, no preference of a specific target within the viral genome (E, RdRp, or N) or of a certain chemistry was seen. In summary, a reduction of the reaction volume and the type of PCR chemistry did not influence the PCR sensitivity.

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Validation of rapid antibody (IgG-IgM) test kit for SARS COV-2 infection in Qatar

Journal of Public Health Research ◽

10.4081/jphr.2021.2421 ◽

2021 ◽

Author(s):

Jesha Mundodan ◽

Samina Hasnain ◽

Hayat Khogali ◽

Soha Shawqi Al Bayat ◽

Dina Ali ◽

...

Keyword(s):

Predictive Value ◽

Local Population ◽

Rapid Test ◽

Antibody Test ◽

Molecular Testing ◽

Rt Pcr ◽

Test Kit ◽

Asymptomatic Individuals ◽

Sensitivity Specificity ◽

Rapid Antibody

Background: In response to the growing coronavirus disease 2019 (COVID-19) pandemic and the shortage of laboratory based molecular testing capacity and reagents, multiple diagnostic test manufacturers have developed rapid and easy to use devices to facilitate testing outside laboratory settings. These kits are either based on detection of proteins from SARS-CoV-2 virus or detection of antigen or human antibodies generated in response to the infection. However, it is important to understand their performance characteristics and they must be validated in the local population setting.Design and Methods: The objective is to assess the validity of the rapid test for IgG and IgM immunoglobulins compared to the current gold standard reverse transcription polymerase chain reaction (RT-PCR) test. A total of 16951 asymptomatic individuals were tested by the Ministry of Public Health track-and-trace team using both rapid immunodiagnostic test and RT-PCR as part of screening across various random settings with potential risk of community interaction prior to gradual lifting of restrictions in Qatar. Rapid test was considered to be posiive if both IgG and IgM are positive, while only IgG/IgM positive was considered as rapid test negative. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated.Results: The sensitivity of rapid test kit was found to be 0.9%, whereas the specificity was found to be 97.8%. the PPV was found to be 0.3% whereas the NPV was found to be 99.4%.Conclusion: Based on the outcome and results of the study, it appears that the sensitivity and PPV of the rapid antibody test are low. As such, this test is not recommended for use to assist in taking clinic-based decisions or decisions related to quarantine/isolation.

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SARS-CoV-2 N Gene G29195T Point Mutation May Affect Diagnostic Reverse Transcription-PCR Detection

Microbiology Spectrum ◽

10.1128/spectrum.02223-21 ◽

2022 ◽

Author(s):

Karrie K. K. Ko ◽

Nurdyana Binte Abdul Rahman ◽

Shireen Yan Ling Tan ◽

Kenneth X. L. Chan ◽

Sui Sin Goh ◽

...

Keyword(s):

Nucleic Acid ◽

Large Scale ◽

N Gene ◽

Clinical Samples ◽

Rt Pcr ◽

Viral Genomes ◽

Reverse Transcription Pcr ◽

Pcr Assays ◽

Diagnostic Detection ◽

Complementary Binding

Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples.

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Prospective analytical performance evaluation of the QuickNavi™-COVID19 Ag for asymptomatic individuals

10.1101/2021.04.01.21254813 ◽

2021 ◽

Author(s):

Yoshihiko Kiyasu ◽

Yuto Takeuchi ◽

Yusaku Akashi ◽

Daisuke Kato ◽

Miwa Kuwahara ◽

...

Keyword(s):

Real Time ◽

Diagnostic Performance ◽

False Positives ◽

Clinical Samples ◽

Lower Sensitivity ◽

Antigen Test ◽

Rt Pcr ◽

Asymptomatic Group ◽

Asymptomatic Volunteers ◽

Asymptomatic Individuals

AbstractIntroductionAntigen testing may help screen for and detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in asymptomatic individuals. However, limited data regarding the diagnostic performance of antigen tests for this group are available.MethodsWe used clinical samples to prospectively evaluate the analytical and clinical performance of the antigen test QuickNavi™-COVID19 Ag. This study was conducted at a PCR center between October 7, 2020 and January 9, 2021. Two nasopharyngeal samples per patient were obtained with flocked swabs; one was used for the antigen test, and the other for real-time reverse transcription PCR (RT-PCR). The diagnostic performance of the antigen test was compared between asymptomatic and symptomatic patients, and the RT-PCR results were used as a reference.ResultsAmong the 1,934 collected samples, SARS-CoV-2 was detected by real-time RT-PCR in 188 (9.7%); 76 (40.4%) of these samples were from asymptomatic individuals. Over half of the total samples (1,073; 55.5%) were obtained from asymptomatic volunteers. The sensitivity of the antigen test was significantly lower for asymptomatic group than for symptomatic patients (67.1% vs 89.3%, p < 0.001). The specificity was 100% for both groups, and no false positives were observed among all 1,934 samples. The median Ct value for the asymptomatic group was significantly higher than that of the symptomatic group (24 vs 20, p < 0.001).ConclusionsThe QuickNavi™-COVID19 Ag showed a lower sensitivity for asymptomatic group than for symptomatic patients. However, its specificity was consistently high, and no false positives were found in this study.

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Evaluation of seven different rapid methods for nucleic acid detection of SARS-COV-2 virus

10.1101/2021.04.15.21255533 ◽

2021 ◽

Author(s):

Sally Mahmoud ◽

Esra Ibrahim ◽

Subhashini Ganesan ◽

Bhagyashree Thakre ◽

Juliet Teddy ◽

...

Keyword(s):

Nucleic Acid ◽

Gold Standard ◽

Large Scale ◽

Virus Detection ◽

Detection Methods ◽

Nucleic Acid Detection ◽

Test Accuracy ◽

Rt Pcr ◽

Rapid Methods ◽

Kappa Value

Background In the current COVID-19 pandemic there is mass screening of SARS-CoV-2 happening round the world due to the extensive spread of the infections. There is a high demand for rapid diagnostic tests to expedite identification of cases and to facilitate early isolation and control spread. Hence this study evaluates seven different rapid nucleic acid detection assays that are commercially available for SARS- CoV- 2 virus detection. Methods Nasopharyngeal samples were collected from 4859 participants and were tested for SARS-CoV-2 virus by the gold standard RT-PCR method along with one of these seven rapid methods of detection. Evaluation of the rapid nucleic acid detection assays was done by comparing the results of these rapid methods with the gold standard RT-qPCR results for SARS-COV-2 detection. Results AQ-TOP had the highest sensitivity (98%) and strong kappa value of 0.943 followed by Genechecker and Abbot ID NOW. The POCKIT (ii RT-PCR) assay had the highest test accuracy of 99.29% followed by Genechecker and Cobas Liat. Atila iAMP showed the highest percentage of invalid reports (35.5%) followed by AQ-TOP with 6% and POCKIT with 3.7% of invalid reports. Conclusion Genechecker system, Abbott ID NOW and Cobas Liat, were found to have best performance and agreement when compared to the standard RT-PCR for COVID-19 detection. With further research, these rapid tests have the potential to be employed in large scale screening of COVID-19.

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Comparative Evaluation of SARS-CoV-2 IgG Assays in India

10.1101/2020.08.12.20173856 ◽

2020 ◽

Cited By ~ 1

Author(s):

◽

Shinjini Bhatnagar

Keyword(s):

Comparative Evaluation ◽

Spike Protein ◽

Clinical Samples ◽

Plasma Samples ◽

Rt Pcr ◽

High Agreement ◽

Asymptomatic Individuals ◽

Low Prevalence ◽

Sera Panel

IgG immunoassays have been developed and used widely for clinical samples and serosurveys for SARS-CoV-2. We compared the performance of three immunoassays, an in-house RBD assay, and two commercial assays, the Diasorin LIAISON SARS-CoV-2 IgG CLIA which detects antibodies against S1/S2 domains of the Spike protein and the Zydus Kavach assay based on inactivated virus using a well-characterized sera-panel. 379 sera/plasma samples from RT-PCR positive individuals >20 days of illness in symptomatic or RT-PCR positivity in asymptomatic individuals and 184 pre-pandemic samples were used. The sensitivity of the assays were 84.7, 82.6 and 75.7 respectively for RBD, LIAISON and Kavach. Kavach and the in-house RBD ELISA showed a specificity of 99.5% and 100%, respectively. The RBD and LIAISON (S1/S2) assays showed high agreement (94.7%;95%CI:92.0,96.6) and were able to correctly identify more positives than Kavach. All three assays are suitable for serosurveillance studies, but in low prevalence sites, estimation of exposure may require adjustment based on our findings.

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