Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods

AbstractThe COVID-19 global pandemic is an unprecedented health emergency. Insufficient access to testing has hampered effective public health interventions and patient care management in a number of countries. Furthermore, the availability of regulatory-cleared reagents has challenged widespread implementation of testing. We rapidly developed a qRT-PCR SARS-CoV-2 detection assay using a 384-well format and tested its analytic performance across multiple nucleic acid extraction kits. Our data shows robust analytic accuracy on residual clinical biospecimens. Limit of detection sensitivity and specificity was confirmed with currently available commercial reagents. Our methods and results provide valuable information for other high-complexity laboratories seeking to develop effective, local, laboratory-developed procedures with high-throughput capability to detect SARS-CoV-2.

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A Method to evaluate analytical sensitivity of qRT-PCR kits for SARS-CoV-2 detection

10.21203/rs.3.rs-57942/v1 ◽

2020 ◽

Author(s):

Di Wang ◽

Zhidong Wang ◽

Xiao Wu ◽

Yongzhuo Zhang ◽

Yunhua Gao

Keyword(s):

Reference Material ◽

Limit Of Detection ◽

False Negative ◽

Detection Sensitivity ◽

Amplification Efficiency ◽

Analytical Sensitivity ◽

Limit Of Quantification ◽

Qrt Pcr ◽

Pcr Products ◽

Pcr Assays

Abstract Background: The ongoing Coronavirus disease 2019 (COVID-19) pandemic has spread across the globe and is representing a huge challenge for all human population. Many commercial qRT-PCR assays have been developed to detect SARS-CoV-2, but related method validation especially the sensitivity evaluation has been insufficient, resulting in some false-negative cases have been reported. Methods: The analytical sensitivity of nine brands of qRT-PCR kits for detecting SARS-CoV-2 was evaluated in parallel based on a newly developed certified reference material, which was derived from genomic RNA of SARS-CoV-2 from clinical positive specimens. After validation of the the reference material by digital PCR, the detection sensitivity of these kits was preliminarily tested using the serially diluted reference material, resulting in three kits with two significantly different sensitivity levels were selected for further evaluation. We sequenced the qRT-PCR products for assay specificity evaluation, and used serial dilutions of the reference material to calculate amplification efficiency and estimate the limit of quantification as well as 95% limit of detection..Results: The results indicated that the analytical sensitivity varied markedly among these kits. For the three types of qRT-PCR kits (Kit-1, Kit-2 and Kit-7), specificity of the PCR products was confirmed by sequence alignment, in which the target amplicons completely matched the corresponding parts of the genome of SARS-CoV-2. The resulting limit of detection from replicate tests for the Kit-1 and Kit-2 was 5.6 copies (N), 3.5 copies (ORF 1ab), and 6.4 copies (N), 4.6 copies (ORF 1ab), respectively, at 95% probability. Compared with Kit-7, the limit of detection as well as limit of quantification of Kit-1 and Kit-2 were significantly lower, further supporting that the both kits worked well to detect low abundance of SARS-CoV-2.Conclusions: Considering that most of the tested kits have been approved for in vitro diagnostics (IVD) in China, the established method here provides a reliable tool to evaluate the sensitivity performance of various qRT-PCR kits for SARS-CoV-2 detection and thus enhance quality control of qRT-PCR assays, improving the laboratory diagnostic capability for fighting the COVID-19 pandemic.

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Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab and saliva patient samples

10.1101/2020.08.30.20184796 ◽

2020 ◽

Author(s):

Dawn M Dudley ◽

Christina M. Newman ◽

Andrea M Weiler ◽

Mitchell D. Ramuta ◽

Ceclia G. Shortreed ◽

...

Keyword(s):

Infectious Virus ◽

Limit Of Detection ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Lower Sensitivity ◽

Acid Extraction ◽

Short Supply ◽

Screening Programs ◽

Qrt Pcr ◽

The Us

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed samples, albeit with approximately 100-fold lower sensitivity than qRT-PCR. We demonstrate that adding lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Overall, the limit of detection (LOD) of RT-LAMP using direct nasopharyngeal swab or saliva samples without RNA extraction is 1x105-1x106 copies/ml. This LOD is sufficient to detect samples from which infectious virus can be cultured. Therefore, samples that test positive in this assay contain levels of virus that are most likely to perpetuate transmission. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR and we provide a revised method to work directly with saliva as starting material. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 screening programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

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Ultrasensitive Detection of SARS-CoV-2 Spike Proteins Using the Thio-NAD Cycling Reaction: A Preliminary Study before Clinical Trials

Microorganisms ◽

10.3390/microorganisms9112214 ◽

2021 ◽

Vol 9 (11) ◽

pp. 2214

Author(s):

Yuta Kyosei ◽

Mayuri Namba ◽

Daiki Makioka ◽

Ayumi Kokubun ◽

Satoshi Watabe ◽

...

Keyword(s):

Limit Of Detection ◽

Ultraviolet B ◽

Detection Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Antigen Test ◽

Specific Test ◽

Nucleocapsid Proteins ◽

Global Pandemic ◽

Testing Accuracy ◽

Preliminary Study

To help control the global pandemic of coronavirus disease 2019 (COVID-19), we developed a diagnostic method targeting the spike protein of the virus that causes the infection, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We applied an ultrasensitive method by combining a sandwich enzyme-linked immunosorbent assay (ELISA) and the thio-nicotinamide adenine dinucleotide (thio-NAD) cycling reaction to quantify spike S1 proteins. The limit of detection (LOD) was 2.62 × 10−19 moles/assay for recombinant S1 proteins and 2.6 × 106 RNA copies/assay for ultraviolet B-inactivated viruses. We have already shown that the ultrasensitive ELISA for nucleocapsid proteins can detect ultraviolet B-inactivated viruses at the 104 RNA copies/assay level, whereas the nucleocapsid proteins of SARS-CoV-2 are difficult to distinguish from those in conventional coronaviruses and SARS-CoV. Thus, an antigen test for only the nucleocapsid proteins is insufficient for virus specificity. Therefore, the use of a combination of tests against both spike and nucleocapsid proteins is recommended to increase both the detection sensitivity and testing accuracy of the COVID-19 antigen test. Taken together, our present study, in which we incorporate S1 detection by combining the ultrasensitive ELISA for nucleocapsid proteins, offers an ultrasensitive, antigen-specific test for COVID-19.

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Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European BatLyssavirusType 1

BioMed Research International ◽

10.1155/2015/839518 ◽

2015 ◽

Vol 2015 ◽

pp. 1-18 ◽

Cited By ~ 13

Author(s):

Evelyne Picard-Meyer ◽

Carine Peytavin de Garam ◽

Jean Luc Schereffer ◽

Clotilde Marchal ◽

Emmanuelle Robardet ◽

...

Keyword(s):

Real Time ◽

Limit Of Detection ◽

Evaluation Criteria ◽

Sybr Green ◽

Detection Sensitivity ◽

Analytical Sensitivity ◽

Reaction Efficiency ◽

Qrt Pcr ◽

One Step ◽

Pcr Assays

This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency,R2values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.

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Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples

PLoS ONE ◽

10.1371/journal.pone.0244882 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0244882

Author(s):

Dawn M. Dudley ◽

Christina M. Newman ◽

Andrea M. Weiler ◽

Mitchell D. Ramuta ◽

Cecilia G. Shortreed ◽

...

Keyword(s):

Limit Of Detection ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Lower Sensitivity ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Short Supply ◽

Qrt Pcr ◽

The Us ◽

Surveillance Programs

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies/μl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

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Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

10.26434/chemrxiv.5662315.v1 ◽

2017 ◽

Author(s):

Bo Tian ◽

Peter Svedlindh ◽

Mattias Strömberg ◽

Erik Wetterskog

Keyword(s):

Magnetic Nanoparticles ◽

Ferromagnetic Resonance ◽

Limit Of Detection ◽

Point Of Care ◽

Rolling Circle Amplification ◽

Resonance Field ◽

Isothermal Amplification ◽

Detection Sensitivity ◽

Serum Samples ◽

Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, i.e., rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg2P2O7 (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.

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A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

Genome Biology ◽

10.1186/s13059-021-02316-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Wendell Jones ◽

Binsheng Gong ◽

Natalia Novoradovskaya ◽

Dan Li ◽

Rebecca Kusko ◽

...

Keyword(s):

Quality Control ◽

Allele Frequency ◽

Liquid Biopsy ◽

Limit Of Detection ◽

Reference Sample ◽

Digital Pcr ◽

Detection Sensitivity ◽

Analytical Accuracy ◽

And Performance ◽

Reference Samples

Abstract Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

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Comparison of saliva with healthcare workers- and patient-collected swabs in the diagnosis of COVID-19 in a large cohort

BMC Infectious Diseases ◽

10.1186/s12879-021-06343-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Mitnala Sasikala ◽

Yelamanchili Sadhana ◽

Ketavarapu Vijayasarathy ◽

Anand Gupta ◽

Sarala Kumari Daram ◽

...

Keyword(s):

Healthcare Workers ◽

Early Stage ◽

Cost Effective ◽

Hospitalized Patients ◽

Detection Sensitivity ◽

Nasopharyngeal Swab ◽

Predictive Values ◽

Qrt Pcr ◽

Asymptomatic Patients ◽

Sensitivity Specificity

Abstract Background A considerable amount of evidence demonstrates the potential of saliva in the diagnosis of COVID-19. Our aim was to determine the sensitivity of saliva versus swabs collected by healthcare workers (HCWs) and patients themselves to assess whether saliva detection can be offered as a cost-effective, risk-free method of SARS-CoV-2 detection. Methods This study was conducted in a hospital involving outpatients and hospitalized patients. A total of 3018 outpatients were tested. Of these, 200 qRT-PCR-confirmed SARS-CoV-2-positive patients were recruited for further study. In addition, 101 SARS-CoV-2-positive hospitalized patients with symptoms were also enrolled in the study. From outpatients, HCWs collected nasopharyngeal swabs (NPS), saliva were obtained. From inpatients, HCWs collected swabs, patient-collected swabs, and saliva were obtained. qRT-PCR was performed to detect SARS-CoV-2 by TAQPATH assay to determine the sensitivity of saliva detection. Sensitivity, specificity and positive/negative predictive values (PPV, NPV) of detecting SARS-CoV-2 were calculated using MedCalc. Results Of 3018 outpatients (asymptomatic: 2683, symptomatic: 335) tested by qRT-PCR, 200 were positive (males: 140, females: 60; aged 37.9 ± 12.8 years; (81 asymptomatic, 119 symptomatic). Of these, saliva was positive in 128 (64%); 39 of 81 asymptomatic (47%),89 of 119 symptomatic patients (74.8%). Sensitivity of detection was 60.9% (55.4–66.3%, CI 95%), with a negative predictive value of 36%(32.9–39.2%, CI 95%).Among 101 hospitalized patients (males:65, females: 36; aged 53.48 ± 15.6 years), with HCW collected NPS as comparator, sensitivity of saliva was 56.1% (47.5–64.5, CI 95%), specificity 63.5%(50.4–75.3, CI95%) with PPV of 77.2% and NPV of 39.6% and that of self-swab was 52.3%(44–60.5%, CI95%), specificity 56.6% (42.3–70.2%, CI95%) with PPV 77.2% and NPV29.7%. Comparison of positivity with the onset of symptoms revealed highest detection in saliva on day 3 after onset of symptoms. Additionally, only saliva was positive in 13 (12.8%) hospitalized patients. Conclusion Saliva which is easier to collect than nasopharyngeal swab is a viable alternate to detect SARS-COV-2 in symptomatic patients in the early stage of onset of symptoms. Although saliva is currently not recommended for screening asymptomatic patients, optimization of collection and uniform timing of sampling might improve the sensitivity enabling its use as a screening tool at community level.

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SaQuant: a real-time PCR assay for quantitative assessment of Staphylococcus aureus

BMC Microbiology ◽

10.1186/s12866-021-02247-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Colin Wood ◽

Jason Sahl ◽

Sara Maltinsky ◽

Briana Coyne ◽

Benjamin Russakoff ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Sensitivity And Specificity ◽

Pathogen Detection ◽

Limit Of Detection ◽

Future Research ◽

Limit Of Quantification ◽

Pathogen Prevalence ◽

Single Well ◽

Effective Public Health ◽

Detection And Quantification

Abstract Background Molecular assays are important tools for pathogen detection but need to be periodically re-evaluated with the discovery of additional genetic diversity that may cause assays to exclude target taxa or include non-target taxa. A single well-developed assay can find broad application across research, clinical, and industrial settings. Pathogen prevalence within a population is estimated using such assays and accurate results are critical for formulating effective public health policies and guiding future research. A variety of assays for the detection of Staphylococcus aureus are currently available. The utility of commercial assays for research is limited, given proprietary signatures and lack of transparent validation. Results In silico testing of existing peer-reviewed assays show that most suffer from a lack of sensitivity and specificity. We found no assays that were specifically designed and validated for quantitative use. Here we present a qPCR assay, SaQuant, for the detection and quantification of S. aureus as might be collected on sampling swabs. Sensitivity and specificity of the assay was 95.6 and 99.9 %, respectively, with a limit of detection of between 3 and 5 genome equivalents and a limit of quantification of 8.27 genome equivalents. The presence of DNA from non-target species likely to be found in a swab sample, did not impact qualitative or quantitative abilities of the assay. Conclusions This assay has the potential to serve as a valuable tool for the accurate detection and quantification of S. aureus collected from human body sites in order to better understand the dynamics of prevalence and transmission in community settings.

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Identification for adulteration of beef with chicken based on single primer-triggered isothermal amplification

International Journal of Food Engineering ◽

10.1515/ijfe-2019-0239 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Jiao Chen ◽

Pansong Zhang ◽

Haixia Wang ◽

Yanjing Shi

Keyword(s):

Limit Of Detection ◽

Chicken Meat ◽

Isothermal Amplification ◽

Work Flow ◽

Acid Extraction ◽

Specificity And Sensitivity ◽

Total Dna ◽

Meat Species ◽

Species Specific ◽

Single Primer

Abstract Adulteration of beef with cheap chicken has become a growing problem worldwide. In this study, a quick, single primer-triggered isothermal amplification (SAMP) combined with a fast nucleic acid extraction method was employed to detect the chicken meat in adulterated beef. Chicken from adulterated beef was identified using the chicken species-specific primer designed according to the Gallus gallus mitochondrial conserved sequences. Our SAMP method displayed good specificity and sensitivity in detecting chicken and beef meat DNA–the limit of detection (LOD) of SAMP is 0.33 pg/μL of chicken and beef total DNA and 2% w/w chicken meat in beef. The whole work flow from DNA extraction to signal detection can be finished within 1 h, fulfilling the requirement of on-site meat species identification.

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