scholarly journals IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2

2020 ◽  
Author(s):  
Elizabeth C. Stahl ◽  
Connor A. Tsuchida ◽  
Jennifer R. Hamilton ◽  
Enrique Lin-Shiao ◽  
Shana L. McDevitt ◽  
...  
Keyword(s):  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
N Gene ◽  
Negative Results ◽  
False Negative Results ◽  
Single Well ◽  
Sample Pooling ◽  
One Step ◽  
Viral Target

AbstractCommonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.Graphical Abstract

scholarly journals LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0258263
Author(s):  
Elizabeth C. Stahl ◽  
Allan R. Gopez ◽  
Connor A. Tsuchida ◽  
Vinson B. Fan ◽  
Erica A. Moehle ◽  
...  
Keyword(s):  
Large Scale ◽  
Local Community ◽  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
N Gene ◽  
Negative Results ◽  
Clinical Surveillance ◽  
Sample Pooling ◽  
Wastewater Samples

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2–3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

scholarly journals Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes

2021 ◽  
Author(s):  
Enos C Kline ◽  
Nuttada Panpradist ◽  
Ian T Hull ◽  
Qin Wang ◽  
Amy K Oreskovic ◽  
...  
Keyword(s):  
Molecular Diagnostics ◽  
Detection System ◽  
Point Of Care ◽  
False Negative ◽  
Cost Effective ◽  
Internal Amplification Control ◽  
Robust Detection ◽  
Negative Results ◽  
False Negative Results ◽  
Target Redundancy

AbstractThe increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multi-target redundancy and an internal amplification control. A convenient and cost-effective target specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-COV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multi-target redundancy. When challenged with extracted human samples the multiplexed assay showed 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction, and 100% specificity. These results are further evidence that conventional laboratory methodologies can be leveraged at the point-of-care for robust performance and diagnostic stability over time.

scholarly journals False-negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

2020 ◽  
Author(s):  
Marcelo Fruehwirth ◽  
Açucena Veleh Rivas ◽  
Andressa Faria Rahyn Fitz ◽  
Aline Cristiane Cechinel Assing Batista ◽  
Cleypson Vinicius Silveira ◽  
...  
Keyword(s):  
Internal Control ◽  
False Negative ◽  
Rna Extraction ◽  
N Gene ◽  
Gold Standard Method ◽  
Negative Results ◽  
False Negative Results ◽  
Average Variation ◽  
Wrong Diagnosis ◽  
Inhibitor Compounds

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®). rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Cq between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.80% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

scholarly journals Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

2012 ◽  
Vol 54 (5) ◽  
pp. 245-248 ◽  
Author(s):  
Maria Cristina Carvalho do Espírito-Santo ◽  
Mónica Viviana Alvarado-Mora ◽  
Pedro Luiz Silva Pinto ◽  
Flair José Carrilho ◽  
João Renato Rebello Pinho ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
False Negative ◽  
Public Health Problem ◽  
Parasite Load ◽  
Detection Sensitivity ◽  
Major Public Health Problem ◽  
Guanidine Isothiocyanate ◽  
Negative Results ◽  
False Negative Results ◽  
Low Prevalence

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

scholarly journals False-Negative Results in Taqman One-Step RT-PCR Test: Evaluation of Endogenous Internal Control Function Used in SARS-CoV-2 Detection Tests

2021 ◽  
Vol 14 (5) ◽  
Author(s):  
Hamzeh Choobin ◽  
Sanaz Asiyabi ◽  
Khashayar Hesamizadeh ◽  
Taravat Bamdad
Keyword(s):  
Internal Control ◽  
Genomic Dna ◽  
Bioinformatics Analysis ◽  
False Negative ◽  
Rnase P ◽  
Rt Pcr ◽  
Specific Primers ◽  
Negative Results ◽  
False Negative Results ◽  
One Step

Background: Taqman one-step RT-PCR has special importance due to its high sensitivity and specificity in the diagnosis of infectious diseases such as viral infections. In the recent pandemic of SARS-CoV-2, diagnostic kits based on this method are commonly used for molecular detection. One of the main systematic errors that misinterpret the results is using inaccurate internal control in RT-PCR diagnostic kits. Designing primers and probes that span exon-exon junction (E-E-jn) will avoid genomic DNA amplification and lead to obtaining high specific results. Objectives: This study aimed to evaluate the endogenous internal control of primers and probe for RNase P RNA to reduce false-negative results in respiratory samples. Methods: In this study, 30 samples of patients who were negative for SARS-CoV-2, influenza A, and influenza B were re-evaluated for SARS-CoV-2 using newly designed primers and probes for RNase P RNA (ultra-specific primers and probe). We also performed bioinformatics analysis on CDC-approved primers and probes of RNase P endogenous internal control. Results: In this analysis, we specified the location of these newly designed primers and probe on target mRNA and genomic DNA. Then, the Taqman one-step RT-PCR method was performed using both CDC-approved primers and probes along with our ultra-specific primers and probe for RNase P RNA. Based on bioinformatics analysis, the attachment sites of the CDC-approved primers and probe for endogenous internal control of RNase P are located on the first exon of this gene. In addition to identifying the target gene sequence, these primers and probe also non-specifically detect similar sequences on the genomic DNA. Conclusions: The present study showed that the use of specific primers and probes introduced by CDC for SARS-CoV-2 and influenza virus may cause false results due to non-specific binding to the genomic DNA. Therefore, choosing the right internal control for RNase P RNA can be useful in achieving very accurate results.

Triplex us in the diagnosis of asymptomatic deep venous thrombosis

1997 ◽  
Vol 38 (2) ◽  
pp. 327-331 ◽  
Author(s):  
M. Mantoni ◽  
C. Strandberg ◽  
K. Neergaard ◽  
C. Sloth ◽  
P. Seest Jørgensen ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
False Negative ◽  
Cost Effective ◽  
Negative Results ◽  
Asymptomatic Patients ◽  
False Negative Results ◽  
Risk Patients ◽  
Sonographic Screening ◽  
Calf Vein

Purpose: to evaluate the accuracy of triplex ultrasound (TUS) compared with venography as a screening test for deep venous thrombosis (DVT), and to evaluate interobserver variation in the interpretation of the venographic studies. Material and Methods: A total of 133 postoperative hip fracture patients, asymptomatic of DVT, were prospectively examined with TUS and venography. All venograms were reviewed blindly and in case of disagreement a consensus was arrived at. Results: the incidence of DVT was 20%, with isolated calf vein thrombi in 63% of the cases. There were 7 false-negative and one false-positive result/s at TUS, with a sensitivity of 74%, specificity of 99% and accuracy of 97%. the kappa values ranged from 0.58 to 0.82. the false-negative results were all caused by missed calf vein thrombi in technically inadequate examinations. at sonography 2% of vein segments were noninterpretable, compared to 29% at venography. Conclusion: Venous US is less sensitive as a test for DVT in this study of asymptomatic patients than in earlier studies on symptomatic patients. Still, sonographic screening of high-risk patients would be both effective and cost effective. Fresh thrombi may cause a false-negative compression test.

scholarly journals Identification of Novel Mutations in the N Gene of SARS-CoV-2 That Adversely Affect the Detection of the Virus by Reverse Transcription-Quantitative PCR

2021 ◽  
Author(s):  
Rashedul Hasan ◽  
Mohammad Enayet Hossain ◽  
Mojnu Miah ◽  
Md Mahmudul Hasan ◽  
Mustafizur Rahman ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Binding Sites ◽  
False Negative ◽  
N Gene ◽  
Novel Mutations ◽  
Viral Spread ◽  
Negative Results ◽  
False Negative Results ◽  
Reverse Transcription Quantitative Pcr

Accurate and timely diagnosis of SARS-CoV-2 is a critical step toward controlling the viral spread, since it facilitates the identification and isolation of infected individuals. Mutations in the primer-/probe-binding sites may lead to false-negative results.

scholarly journals False-Negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

2020 ◽  
Author(s):  
Marcelo Fruehwirth ◽  
Açucena Veleh Rivas ◽  
Andressa Faria Rahyn Fitz ◽  
Aline Cristiane Cechinel Assing Batista ◽  
Cleypson Vinicius Silveira ◽  
...  
Keyword(s):  
Internal Control ◽  
False Negative ◽  
Rna Extraction ◽  
N Gene ◽  
Gold Standard Method ◽  
Negative Results ◽  
False Negative Results ◽  
Average Variation ◽  
Wrong Diagnosis ◽  
Inhibitor Compounds

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®) for extraction. rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Ct between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811Ct, 3.840Ct, and 3.842Ct, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.8% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

scholarly journals Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2474
Author(s):  
Jéssika Cristina Chagas Lesbon ◽  
Mirele Daiana Poleti ◽  
Elisângela Chicaroni de Mattos Oliveira ◽  
José Salvatore Leister Patané ◽  
Luan Gaspar Clemente ◽  
...  
Keyword(s):  
Real Time ◽  
False Negative ◽  
Gene Mutations ◽  
Sao Paulo ◽  
N Gene ◽  
São Paulo ◽  
Rt Pcr ◽  
Nucleocapsid Gene ◽  
Negative Results ◽  
False Negative Results

The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.

scholarly journals Assessment of sample pooling for clinical SARS-CoV-2 testing

2020 ◽  
Author(s):  
Sara B Griesemer ◽  
Greta Van Slyke ◽  
Kirsten St. George
Keyword(s):  
Data Transfer ◽  
False Negative ◽  
Automated Detection ◽  
Detection Rates ◽  
Detection Systems ◽  
Negative Results ◽  
False Negative Results ◽  
Sample Pooling ◽  
The Impact ◽  
Pooled Samples

AbstractAccommodating large increases in sample workloads has presented one of the biggest challenges to clinical laboratories during the COVID-19 pandemic. Despite the implementation of new automated detection systems, and previous efficiencies such as barcoding, electronic data transfer and extensive robotics, throughput capacities have struggled to meet the demand. Sample pooling has been suggested as an additional strategy to further address this need. The greatest concern with this approach in a clinical setting is the potential for reduced sensitivity, particularly the risk of false negative results when weak positive samples are pooled. To investigate this possibility, detection rates in pooled samples were evaluated, with extensive assessment of pools containing weak positive specimens. Additionally, the frequency of occurrence of weak positive samples across ten weeks of the pandemic were reviewed. Weak positive specimens were detected in all five-sample pools but failed to be detected in four of the 24 nine-sample pools tested. Weak positive samples comprised an average 16.5% of the positive specimens tested during the pandemic thus far, slightly increasing in frequency during later weeks. Other aspects of the testing process should be considered, however, such as accessioning and reporting, which are not streamlined and may be complicated by pooling procedures. Therefore, the impact on the entire laboratory process needs to be carefully assessed prior to implementing such a strategy.

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