Performance characteristics of a high throughput automated transcription mediated amplification test for SARS-CoV-2 detection

ABSTRACTThe COVID-19 pandemic caused by the new SARS-CoV-2 coronavirus has imposed severe challenges on laboratories in their effort to achieve sufficient diagnostic testing capability for identifying infected individuals. In this study we report the analytical and clinical performance characteristics of a new, high-throughput, fully automated nucleic acid amplification test system for the detection of SARS-CoV-2. The assay utilizes target capture, transcription mediated amplification, and acridinium ester-labeled probe chemistry on the automated Panther System to directly amplify and detect two separate target sequences in the ORF1ab region of the SARS-CoV-2 RNA genome. The probit 95% limit of detection of the assay was determined to be 0.004 TCID50/ml using inactivated virus, and 25 c/ml using synthetic in vitro transcript RNA targets. Analytical sensitivity (100% detection) was confirmed to be 83 – 194 c/ml using three commercially available SARS-CoV-2 nucleic acid controls. No cross reactivity or interference was observed with testing six related human coronaviruses, as well as 24 other viral, fungal, and bacterial pathogens, at high titer. Clinical nasopharyngeal swab specimen testing (N=140) showed 100%, 98.7%, and 99.3% positive, negative, and overall agreement, respectively, with a validated reverse transcription PCR NAAT for SARS-CoV-2 RNA. These results provide validation evidence for a sensitive and specific method for pandemic-scale automated molecular diagnostic testing for SARS-CoV-2.

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Performance Characteristics of a High-Throughput Automated Transcription-Mediated Amplification Test for SARS-CoV-2 Detection

Journal of Clinical Microbiology ◽

10.1128/jcm.01669-20 ◽

2020 ◽

Vol 58 (10) ◽

Cited By ~ 3

Author(s):

Jimmykim Pham ◽

Sarah Meyer ◽

Catherine Nguyen ◽

Analee Williams ◽

Melissa Hunsicker ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Diagnostic Testing ◽

Test System ◽

Nucleic Acid Amplification Test ◽

Performance Characteristics ◽

Nucleic Acid Amplification ◽

Molecular Diagnostic ◽

Specific Method ◽

Analytical Sensitivity

ABSTRACT The COVID-19 pandemic caused by the new SARS-CoV-2 coronavirus has imposed severe challenges on laboratories in their effort to achieve sufficient diagnostic testing capability for identifying infected individuals. In this study, we report the analytical and clinical performance characteristics of a new, high-throughput, fully automated nucleic acid amplification test system for the detection of SARS-CoV-2. The assay utilizes target capture, transcription-mediated amplification, and acridinium ester-labeled probe chemistry on the automated Panther system to directly amplify and detect two separate target sequences in the open reading frame 1ab (ORF1ab) region of the SARS-CoV-2 RNA genome. The probit 95% limit of detection of the assay was determined to be 0.004 50% tissue culture infective dose (TCID50)/ml using inactivated virus and 25 copies/ml (c/ml) using synthetic in vitro transcript RNA targets. Analytical sensitivity (100% detection) was confirmed to be 83 to 194 c/ml using three commercially available SARS-CoV-2 nucleic acid controls. No cross-reactivity or interference was observed with testing of six related human coronaviruses, as well as 24 other viral, fungal, and bacterial pathogens, at high titers. Clinical nasopharyngeal swab specimen testing (n = 140) showed 100%, 98.7%, and 99.3% positive, negative, and overall agreement, respectively, with a validated reverse transcription-PCR nucleic acid amplification test (NAAT) for SARS-CoV-2 RNA. These results provide validation evidence for a sensitive and specific method for pandemic-scale automated molecular diagnostic testing for SARS-CoV-2.

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Comparative performance of four nucleic acid amplification tests for SARS-CoV-2 virus

10.1101/2020.03.26.010975 ◽

2020 ◽

Cited By ~ 2

Author(s):

Yujuan Xiong ◽

Zhen-Zhen Li ◽

Qi-Zhen Zhuang ◽

Yan Chao ◽

Fei Li ◽

...

Keyword(s):

Nucleic Acid ◽

Limit Of Detection ◽

High Specificity ◽

Nucleic Acid Amplification ◽

Virus Concentration ◽

Analytical Sensitivity ◽

Comparative Performance ◽

Consistency Test ◽

Nucleic Acid Amplification Tests ◽

Selection Of

AbstractCoronavirus disease 2019 (COVID-19) can be screened and diagnosed through the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription polymerase chain reaction. SARS-CoV-2 nucleic acid amplification tests (NAATs) have been rapidly developed and quickly applied to clinical testing during the pandemic. However, studies evaluating the performance of these NAAT assays are limited. We evaluated the performance of four NAATs, which were marked by the Conformité Européenne and widely used in China during the pandemic. Results showed that the analytical sensitivity of the four assays was significantly lower than that claimed by the NAAT manufacturers. The limit of detection (LOD) of Daan, Sansure, and Hybribio NAATs was 3000 copies/mL, whereas the LOD of Bioperfectus NAATs was 4000 copies/mL. The results of the consistency test using 46 samples showed that Daan, Sansure, and Hybribio NAATs could detect the samples with a specificity of 100% (30/30) and a sensitivity of 100% (16 /16), whereas Bioperfectus NAAT detected the samples with a specificity of 100% (30/30) and a sensitivity 81.25% (13/16). The sensitivity of Bioperfectus NAAT was lower than that of the three other NAATs; this finding was consistent with the result that Bioperfectus NAAT had a higher LOD than the three other kinds of NAATs. The four above mentioned reagents presented high specificity; however, for the detection of the samples with low virus concentration, Bioperfectus reagent had the risk of missing detection. Therefore, the LOD should be considered in the selection of SARS-CoV-2 NAATs.

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Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared to endpoint assessment

10.1101/2021.01.13.21249412 ◽

2021 ◽

Author(s):

Lena M. Diaz ◽

Brandon E. Johnson ◽

Daniel M. Jenkins

Keyword(s):

Nucleic Acid ◽

Quantitative Method ◽

Limit Of Detection ◽

Detection System ◽

Diagnostic Testing ◽

Rna Extraction ◽

Lamp Assay ◽

Nucleic Acid Amplification ◽

Clinical Samples ◽

Ph Indicator

AbstractControlling the course of the COVID-19 pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel coronavirus SARS-CoV-2. Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current FDA-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than two hours to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS- CoV-2 at either 1X (50 genomes/reaction) or 2X (100 genomes/reaction) of the LOD. Importantly the quantitative method was based on dynamic optical changes during the reaction so was able to correctly classify samples that were misclassified by endpoint observation of color.

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Analytical performance characteristics of a new transcription-mediated amplification assay for Treponema pallidum

Journal of Clinical Microbiology ◽

10.1128/jcm.00511-21 ◽

2021 ◽

Author(s):

Damon Getman ◽

Mike Lin ◽

Nesreen Barakat ◽

Rhonda Skvoretz ◽

Charmie Godornes ◽

...

Keyword(s):

Diagnostic Testing ◽

Transmitted Disease ◽

Treponema Pallidum ◽

Dark Field ◽

Secondary Syphilis ◽

Performance Characteristics ◽

Specific Method ◽

Analytical Sensitivity ◽

Sexually Transmitted ◽

New Research

This study evaluated the performance characteristics of a new research use transcription-mediated amplification (TMA) assay for detection of ribosomal RNA from Treponema pallidum (Tp). Analytical sensitivity determined using dark field quantitated Tp was 1.4 organisms/ml (95% CI: 0.7-6.33). Dilution of Tp IVT RNA in STM yielded 100% positivity (n=3) at 10 copies/ml (4 copies/reaction). Analytical specificity testing of non-target microorganisms (N=59), including the closely related non-syphilis treponemes T. denticola and T. phagedenis, yielded 0% positivity. TMA testing of mucosal swab specimens collected from men who have sex with men (MSM) attending a sexually transmitted disease clinic yielded 1.8% (17/944) TMA positive results. A collection of 56 serum specimens obtained from a separate cohort of patients with known rapid plasma reagin (RPR) status and syphilis clinical diagnosis were 19.6% (11/56) TMA positive overall, 29.7% (11/37) positive among subjects with syphilis diagnosis, including 8 (36.3%) of 22 persons with primary or secondary syphilis, 2 (20%) of 10 persons with early latent syphilis and 1 (20%) of 5 persons with late latent or unstaged syphilis. None (0%) of 18 RPR positive sera from patients with a history of treated syphilis was TMA positive. These results show TMA is an analytically sensitive and specific method for detection of Tp rRNA and is compatible with serum specimens in addition to pharyngeal and rectal mucocutaneous swab specimens. Automated real time TMA testing for T. pallidum may be useful as an adjunctive method with serology for screening and diagnostic testing of selected patient populations for syphilis.

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Prospective study to find out the role of gastric aspirate examination by Ziehl-Neelsen staining (ZN staining) and cartridge based nucleic acid amplification test (CB-NAAT) as a diagnostic method in childhood tuberculosis

International Journal of Contemporary Pediatrics ◽

10.18203/2349-3291.ijcp20182574 ◽

2018 ◽

Vol 5 (4) ◽

pp. 1609

Author(s):

Akansha Arora ◽

Anil Jain ◽

B. S. Karnawat ◽

Rakesh Kumawat

Keyword(s):

Nucleic Acid ◽

Predictive Value ◽

Nucleic Acid Amplification Test ◽

Nucleic Acid Amplification ◽

Mantoux Test ◽

P Value ◽

Specific Method ◽

Gastric Aspirate ◽

Tuberculosis In Children

Background: Tuberculosis in children has been relatively neglected mainly because clinical diagnosis has low specificity, radiological interpretation is subject to inter-observer variability and the tuberculin skin test is a marker of exposure, not disease. The recent introduction of Cartridge based nucleic acid amplification test has significantly transformed the diagnostics of tuberculosis in adults but its application for Paediatric Tuberculosis is under evaluation. Therefore, authors conducted a study on role of gastric aspirate examination by ZN stain and Cartridge based nucleic acid amplification test in the diagnosis of childhood Tuberculosis.Methods: Authors did a prospective hospital-based study from Nov 2016 to Nov 2017 consisting of 100 randomly selected patients suspected of tuberculosis who had their gastric aspirate tested for CBNAAT and ZN stain for acid fast bacilli (AFB) along with Mantoux test and other routine investigations. Chi square test was used.Results: Culture positive tuberculosis was found in 21 out of 100 children. The sensitivity, specificity, positive predictive value and negative predictive value for CBNAAT were 76.1%, 98.7%, 94.1% and 93.9% and for ZN stain were 47.6%, 98.7%, 90.9% and 87.6% respectively. Positive history of contact (p value 0.0217), reactive Mantoux test (p value < 0.001) and low socioeconomic status were independently associated with a positive CBNAAT result.Conclusions: Analysis of gastric aspirate samples with CBNAAT is a sensitive and specific method for rapid diagnosis of pulmonary tuberculosis in children who cannot produce sputum. Compared with microscopy, CBNAAT offers better sensitivity and its scale up will improve access to tuberculosis diagnostics in children.

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Limited-Resource Preparable Chitosan Magnetic Particles for Extracting Amplification-Ready Zeptomole-Range Nucleic Acid from Complex Biofluid

10.26434/chemrxiv.14579127 ◽

2021 ◽

Author(s):

Sayantan Tripathy ◽

Arunansu Talukdar ◽

Goutam Pramanik ◽

P. V. Rajesh ◽

Souradyuti Ghosh

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Magnetic Particles ◽

Limited Resource ◽

Nucleic Acid Amplification ◽

Acid Extraction ◽

Analytical Sensitivity ◽

Nucleic Acid Extraction ◽

Spin Column

<b>Layman Summary: </b>Nucleic acid extraction is a key prerequisite for any nucleic acid amplification test (NAAT) or isothermal NAAT (iNAAT) based molecular diagnosis assays.<b> </b>Existing methods utilizes spin column system for nucleic acid extraction which are unsuitable for limited resource settings. Our work explores two methods for chitosan coated magnetic particle preparation that can be executed within 6 h from commonly available chemicals with nothing but a magnetic stirrer and water bath and doable by a minimally trained person. We will also investigated the compatibility of the extracted nucleic acid with downstream NAATs such as real time LAMP, colorimetric LAMP, and real time PCR. In the process, we established the analytical sensitivity of the overall method.<div><br><div><b>Characterization methods</b>: SEM, XRD, EDX, FT-IR</div><div><br></div><div><b>Bioanalytical methods:</b> Real time LAMP, Colorimetric LAMP, Real time PCR</div></div>

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Clinical Evaluation of Three Sample-to-Answer Platforms for Detection of SARS-CoV-2

Journal of Clinical Microbiology ◽

10.1128/jcm.00783-20 ◽

2020 ◽

Vol 58 (8) ◽

Cited By ~ 68

Author(s):

Wei Zhen ◽

Elizabeth Smith ◽

Ryhana Manji ◽

Deborah Schron ◽

Gregory J. Berry

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Drug Administration ◽

Clinical Evaluation ◽

Limit Of Detection ◽

Clinical Performance ◽

Molecular Diagnostic ◽

Analytical Sensitivity ◽

Reference Standard ◽

Slight Improvement ◽

Percent Agreement

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now spread across the globe. As part of the worldwide response, many molecular diagnostic platforms have been granted emergency use authorization (EUA) by the Food and Drug Administration (FDA) to identify SARS-CoV-2 positive patients. Our objective was to evaluate three sample-to-answer molecular diagnostic platforms (Cepheid Xpert Xpress SARS-CoV-2 [Xpert Xpress], Abbott ID NOW COVID-19 [ID NOW], and GenMark ePlex SARS-CoV-2 Test [ePlex]) to determine analytical sensitivity, clinical performance, and workflow for the detection of SARS-CoV-2 in nasopharyngeal swabs from 108 symptomatic patients. We found that Xpert Xpress had the lowest limit of detection (100% detection at 100 copies/ml), followed by ePlex (100% detection at 1,000 copies/ml), and ID NOW (20,000 copies/ml). Xpert Xpress also had highest positive percent agreement (PPA) compared to our reference standard (98.3%) followed by ePlex (91.4%) and ID NOW (87.7%). All three assays showed 100% negative percent agreement (NPA). In the workflow analysis, ID NOW produced the lowest time to result per specimen (∼17 min) compared to Xpert Xpress (∼46 min) and ePlex (∼1.5 h), but what ID NOW gained in rapid results, it lost in analytical and clinical performance. ePlex had the longest time to results and showed a slight improvement in PPA over ID NOW. Information about the clinical and analytical performance of these assays, as well as workflow, will be critical in making informed and timely decisions on testing platforms.

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IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

Clinical Chemistry ◽

10.1373/clinchem.2012.193664 ◽

2013 ◽

Vol 59 (2) ◽

pp. 436-439 ◽

Cited By ~ 6

Author(s):

Martin Jensen Søe ◽

Mikkel Rohde ◽

Jens Mikkelsen ◽

Peter Warthoe

Keyword(s):

Nucleic Acid ◽

Candida Glabrata ◽

Simultaneous Detection ◽

Lamp Assay ◽

Isothermal Amplification ◽

Qpcr Assay ◽

Nucleic Acid Amplification ◽

Molecular Diagnostic ◽

Nucleic Acid Detection ◽

Amplification Method

BACKGROUND Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. METHODS The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. RESULTS IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. CONCLUSIONS IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

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Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acid Amplification for Detecting Acute HIV Infection

Clinical Chemistry ◽

10.1373/clinchem.2015.243287 ◽

2015 ◽

Vol 61 (11) ◽

pp. 1372-1380 ◽

Cited By ~ 18

Author(s):

Carlos Cabrera ◽

Lei Chang ◽

Mars Stone ◽

Michael Busch ◽

David H Wilson

Keyword(s):

Nucleic Acid ◽

Early Detection ◽

Hiv Infection ◽

Single Molecule ◽

Low Cost ◽

Nucleic Acid Amplification ◽

Analytical Sensitivity ◽

Acute Hiv Infection ◽

Acute Hiv ◽

Viral Isolates

Abstract BACKGROUND Nucleic acid testing (NAT) has become the standard for high sensitivity in detecting low levels of virus. However, adoption of NAT can be cost prohibitive in low-resource settings where access to extreme sensitivity could be clinically advantageous for early detection of infection. We report development and preliminary validation of a simple, low-cost, fully automated digital p24 antigen immunoassay with the sensitivity of quantitative NAT viral load (NAT-VL) methods for detection of acute HIV infection. METHODS We developed an investigational 69-min immunoassay for p24 capsid protein for use on a novel digital analyzer on the basis of single-molecule-array technology. We evaluated the assay for sensitivity by dilution of standardized preparations of p24, cultured HIV, and preseroconversion samples. We characterized analytical performance and concordance with 2 NAT-VL methods and 2 contemporary p24 Ag/Ab combination immunoassays with dilutions of viral isolates and samples from the earliest stages of HIV infection. RESULTS Analytical sensitivity was 0.0025 ng/L p24, equivalent to 60 HIV RNA copies/mL. The limit of quantification was 0.0076 ng/L, and imprecision across 10 runs was <10% for samples as low as 0.09 ng/L. Clinical specificity was 95.1%. Sensitivity concordance vs NAT-VL on dilutions of preseroconversion samples and Group M viral isolates was 100%. CONCLUSIONS The digital immunoassay exhibited >4000-fold greater sensitivity than contemporary immunoassays for p24 and sensitivity equivalent to that of NAT methods for early detection of HIV. The data indicate that NAT-level sensitivity for acute HIV infection is possible with a simple, low-cost digital immunoassay.

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