scholarly journals Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test

2020 ◽  
Author(s):  
Qing Sun ◽  
Jonathan Li ◽  
Hui Ren ◽  
Larry Pastor ◽  
Yulia Loginova ◽  
...  
Keyword(s):  
Clinical Evaluation ◽  
High Throughput ◽  
Molecular Detection ◽  
Limit Of Detection ◽  
Saliva Sample ◽  
Clinical Samples ◽  
Rank Test ◽  
Percentage Agreement ◽  
Testing Specimen ◽  
Significant Difference

AbstractBackgroundSensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patient. Here we report the performance of QuantiVirus™ SARS-CoV-2 multiplex test using saliva as the testing specimens with or without pooling.MethodsDevelopment and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical evaluation studies were performed with the QuantiVirus™ SARS-CoV-2 multiplex test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus™ SARS-CoV-2 multiplex test were also obtained and analyzed. Moreover, saliva pooling with 6 and 12 samples together were also evaluated.ResultsThe LOD for the QuantiVirus™ SARS-CoV-2 multiplex test was confirmed to be 100-200 copies/mL. The clinical evaluation using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus™ SARS-CoV-2 multiplex test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus™ SARS-CoV-2 multiplex test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus ™SARS CoV-2 multiplex test had 80% concordance rate and no significant difference (p=0.13) in paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from the communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%).ConclusionThe studies demonstrated that the QuantiVirus™ SARS-CoV-2 multiplex test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirus™SARS-CoV-2 multiplex test offers a highly desirable test during the ongoing COVID-19 pandemic.

scholarly journals Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0243183 ◽  
Author(s):  
Qing Sun ◽  
Jonathan Li ◽  
Hui Ren ◽  
Larry Pastor ◽  
Yulia Loginova ◽  
...  
Keyword(s):  
High Throughput ◽  
Performance Studies ◽  
Molecular Detection ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Saliva Sample ◽  
Clinical Samples ◽  
Percentage Agreement ◽  
Testing Specimen ◽  
Significant Difference

Background Sensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patients. Here we report the performance of QuantiVirus™ SARS-CoV-2 test using saliva as the testing specimens with or without pooling. Methods Development and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical performance studies were performed with the QuantiVirus™ SARS-CoV-2 test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus™ SARS-CoV-2 test were also obtained and analyzed. Additionally, testing of pooled saliva samples was evaluated. Results The LOD for the QuantiVirus™ SARS-CoV-2 test was confirmed to be 100–200 copies/mL. The clinical performance studies using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus™ SARS-CoV-2 test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus™ SARS-CoV-2 test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus™ SARS CoV-2 test had 80% concordance rate and no significant difference (p = 0.13) between paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from local communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples (1:6 pooling) for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%). Conclusion The studies demonstrated that the QuantiVirus™ SARS-CoV-2 test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirus™ SARS-CoV-2 test offers a highly desirable testing platform during the ongoing COVID-19 pandemic.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Validation of a real-time PCR assay for high-throughput detection of Avibacterium paragallinarum in chicken respiratory sites

2019 ◽  
Vol 31 (5) ◽  
pp. 714-718 ◽  
Author(s):  
Kristin A. Clothier ◽  
Simone Stoute ◽  
Andrea Torain ◽  
Beate Crossley
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Bacterial Flora ◽  
Clinical Samples ◽  
Single Tube ◽  
Avibacterium Paragallinarum ◽  
Normal Bacterial Flora ◽  
Close Relatives

Avibacterium paragallinarum is the causative agent of infectious coryza, a highly contagious respiratory disease in chickens. Given its fastidious nature, this bacterium is difficult to recover and identify, particularly from locations colonized by normal bacterial flora. Standard PCR methods have been utilized for detection but are labor-intensive and not feasible for high-throughput testing. We evaluated a real-time PCR (rtPCR) method targeting the HPG-2 region of A. paragallinarum, and validated a high-throughput extraction for this assay. Using single-tube extraction, the rtPCR detected 4 A. paragallinarum (ATCC 29545T and 3 clinical) isolates with a limit of detection (LOD) of 10 cfu/mL and a PCR efficiency of 89–111%. Cross-reaction was not detected with 33 non– A. paragallinarum, all close relatives from the family Pasteurellaceae. Real-time PCR testing on extracts of 66 clinical samples (choana, sinus, or trachea) yielded 98.2% (35 of 36 on positives, 30 of 30 on negatives) agreement with conventional PCR. Duplicate samples tested in a 96-well format extraction in parallel with the single-tube method produced equivalent LOD on all A. paragallinarum isolates, and 96.8% agreement on 93 additional clinical samples extracted with both procedures. This A. paragallinarum rtPCR can be utilized for outbreak investigations and routine monitoring of susceptible flocks.

scholarly journals High throughput Next-Generation Sequencing Respiratory Viral Panel: A Diagnostic and Epidemiologic Tool for SARS-CoV-2 and Other Viruses.

2021 ◽  
Author(s):  
Nikhil Shri Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Zachary Petty ◽  
Jiani Chen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Performance Metrics ◽  
Limit Of Detection ◽  
Virus Transmission ◽  
Respiratory Viruses ◽  
Clinical Samples ◽  
Current Phase ◽  
Next Generation ◽  
Generation Sequencing

Background: In the current phase of the COVID-19 pandemic, we are facing two serious public health challenges that include deficits in SARS-CoV-2 variant monitoring and neglection of other co-circulation respiratory viruses. Additionally, accurate assessment of the evolution, extent, and dynamics of the outbreak are required to understand the transmission of the virus amongst seemingly unrelated cases and provide critical epidemiological information. To address these challenges, we evaluated a new high-throughput next-generation sequencing (NGS) panel that includes 40 viral pathogens to analyze viral subtypes, mutational variants of SARS-CoV-2, model to understand the spread of the virus in the state of Georgia, USA, and to assess other circulating viruses in the same population. Methods This study evaluated a total of 522 samples that included 483 patient samples and 42 synthetic positive control materials. The performance metrics were calculated for both clinical and reference control samples by comparing detection results with the RT-PCR assay. The limit of detection (LoD) studies were conducted as per the FDA guidelines. Inference and visualization of the phylogeny of the SARS-CoV-2 sequences were performed through the Nextstrain Command-Line Interface (CLI) tool, utilizing the associated augur and auspice toolkits. Result The performance metric was calculated using both the clinical samples and the reference control with a PPA, NPA, and accuracy of 95.98%, 85.96%, and 94.4%, respectively. The LoD was determined to be 10 copies/ml with all 25 replicates detected across two different runs. The clade for pangolin lineage B contains certain distant variants, including P4715L in ORF1ab, Q57H in ORF 3a and, S84L in ORF8 covarying with the D614G spike protein mutation were found to be prevalent in the early pandemic in Georgia, USA. Isolates from the same county formed paraphyletic groups in our analysis, which indicated virus transmission between counties. Conclusion The study demonstrates the clinical utility of the NGS panel to identify novel variants that can provide actionable information to prevent or mitigate emerging viral threats, models that provide insights into viral transmission patterns and predict transmission/ resurgence of regional outbreaks and provide critical information on co-circulating respiratory viruses that might be independent factors contributing to the global disease burden.

scholarly journals Foundation and Clinical Evaluation of a New Method for Detecting SARS-CoV-2 Antigen by Fluorescent Microsphere Immunochromatography

2020 ◽  
Vol 10 ◽  
Author(s):  
Chunyan Zhang ◽  
Lei Zhou ◽  
Kang Du ◽  
Ying Zhang ◽  
Jing Wang ◽  
...  
Keyword(s):  
Clinical Evaluation ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Test Strip ◽  
Healthy Controls ◽  
Percentage Agreement ◽  
N Protein ◽  
Fluorescent Microsphere ◽  
Methodological Evaluation ◽  
Detection Reagent

PurposeTo develop a rapid detection reagent for SARS-CoV-2 antigen for the auxiliary diagnosis of new coronary pneumonia (COVID-19), and perform the methodological evaluation and clinical evaluation of the reagent.MethodSARS-CoV-2 N-protein test strip was created by combining fluorescent microsphere labeling technology and immunochromatographic technology, based on the principle of double antibody sandwich. Then we evaluated the analytical capability and clinical application of the strips.ResultThe limit of detection of the strips for recombinant N protein was 100 ng/ml and for activated SARS -CoV-2 virus was 1 × 103 TCID50/ml. The strips also have high analytical specificity and anti-interference capability. According to the predetermined cut-off value, the specificity of the test strip in healthy controls and patients with other respiratory disease was 100.00 and 97.29%, the sensitivity in COVID-19 cases at progress stage and cured stage was 67.15 and 7.02%. The positive percentage agreement and negative percentage agreement of antigen strip to RNA test were 83.16 and 94.45%.ConclusionSARS-CoV-2 fluorescence immunochromatographic test strip can achieve fast, sensitive and accurate detection, which can meet the clinical requirements for rapid detection of viruses on the spot.

scholarly journals OS1.1 Plasma PD-L1 levels over time differ between glioblastoma and lower-grade glioma patients

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii5-iii5
Author(s):  
M Mair ◽  
A Ilhan-Mutlu ◽  
S Pajenda ◽  
G Widhalm ◽  
K Dieckmann ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Rank Test ◽  
Lower Grade ◽  
Who Grade ◽  
University Of Vienna ◽  
Markers Of Inflammation ◽  
Significant Difference ◽  
Medical University ◽  
Recurrent Gbm

Abstract BACKGROUND Immune modulating therapies are a promising treatment approach in patients with primary brain tumors, however their clinical efficacy is still under investigation as the systemic inflammatory responses are potentially challenged by the brain’s tight immune regulatory mechanisms. Here, we aimed to investigate soluble PD-L1 (sPD-L1) in the plasma of recurrent glioblastoma (GBM) and recurrent WHO grade II - III glioma (LGG) patients over their course of disease. MATERIAL AND METHODS 30 patients with recurrent GBM and 10 patients with recurrent LGG were treated with bevacizumab (400mg, q14 days) as a salvage therapy at the Medical University of Vienna. EDTA plasma samples were drawn prior to each treatment cycle. sPD-L1 was analyzed using a sandwich ELISA (Millipore ABF133 and clone 5H1). The lower limit of detection was 0.050 ng/ml as determined by serial dilutions of recombinant human PD-L1. RESULTS sPD-L1 values could be retrieved in a median of 6 timepoints per patient (range: 2 - 24). No significant difference in sPD-L1 concentrations between GBM and LGG patients was present at baseline (p > 0.05). After 2 weeks of bevacizumab treatment, LGG patients presented with statistically significant lower sPD-L1 concentrations as compared to baseline (p = 0.036, Wilcoxon signed rank test), while no difference in sPD-L1 concentration was observed in GBM patients. Significantly higher sPD-L1 intra-patient variability was evident in LGG patients compared to GBM patients (p = 0.012, Mann-Whitney-U). In LGG patients, sPD-L1 correlated with leukocyte count (Spearman’s r = 0.397, p = 0.001) as well as with C-reactive protein level (Spearman’s r = -0.424, p = 0.001), while in GBM patients no correlation of sPD-L1 and systemic markers of inflammation was evident (-0.3 < Spearman’s r < 0.3). CONCLUSION sPD-L1 as a systemic marker of inflammation was lower in recurrent LGG patients compared to recurrent GBM patients after bevacizumab-based treatment, arguing that the glioma-immune system interactions differ between LGG and GBM. SUPPORT/DISCLOSURE This project was funded by the research budget of the Medical University of Vienna.

scholarly journals High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

2021 ◽  
Author(s):  
Sarah Leah Downs ◽  
Shabir Ahmed Madhi ◽  
Lara van Der Merwe ◽  
Marta Coelho Nunes ◽  
Courtney Paige Olwagen
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Taqman Assay ◽  
Vaccine Serotypes ◽  
Serotype Replacement ◽  
The Impact

Abstract Current real-time Polymerase Chain Reaction (qPCR) methods are unable to distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009-11), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92–101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/ml of sample. There was high concordance (Kappa = 0.73–0.92); sensitivity (85–100%) and specificity (96–100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

scholarly journals High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. L. Downs ◽  
S. A. Madhi ◽  
L. Van der Merwe ◽  
M. C. Nunes ◽  
C. P. Olwagen
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Taqman Assay ◽  
Vaccine Serotypes ◽  
Serotype Replacement ◽  
The Impact

AbstractCurrent real-time high-throughput Polymerase Chain Reaction (qPCR) methods do not distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009–2011), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92–101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/mL of sample. There was high concordance (Kappa = 0.73–0.92); sensitivity (85–100%) and specificity (96–100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

scholarly journals COVIDFast: A high-throughput and RNA extraction-free method for SARS-CoV-2 detection in swab (SwabFAST) or saliva (SalivaFAST)

2022 ◽  
Author(s):  
Yue Qiu ◽  
Ling Lu ◽  
Amanda Halven ◽  
Rachel Terrio ◽  
Sydney Yuldelson ◽  
...  
Keyword(s):  
High Throughput ◽  
Nasal Swab ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Diagnostic Process ◽  
Conventional Pcr ◽  
Extraction Step ◽  
Working Together ◽  
Bottle Neck

There is an urgent need of having a rapid, high throughput, yet accurate SARS-COV-2 PCR testing to control the COVID19 pandemic. However, the RNA extraction step in conventional PCR creates a major bottle neck in the diagnostic process. In this paper we modified the CDC COVID-19 assay and developed an RNA-extraction free RT-qPCR assay for SARS-CoV-2, i.e. COVIDFast. Depending on sample types, the assay is further divided into SwabFAST, which uses anterior nares nasal swab, and SalivaFAST, which uses saliva. By utilizing the proprietary buffer for either swab or saliva samples, the performance of SwabFAST or SalivaFAST is equivalent to RNA-extraction SARS-CoV-2 RT-qPCR in both contrived and clinical samples. The limit of detection of either assay is 4 copies/uL. We further developed a semi-automatic system, which is easy to adapt by clinical lab for implementation of a high-throughput SARS-CoV-2 test. Working together with the COVIDCheck Colorado, we have tested over 400,000 samples using COVIDFast (83.62% SwabFAST and 16.38% SalivaFAST) in less than a year, resulting in significant clinical contribution in the battle against COVID-19 during the pandemic.

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.

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