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 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 Molecular detection of SARS-CoV-2 using a reagent-free approach

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243266
Author(s):  
Ronan Calvez ◽  
Andrew Taylor ◽  
Leonides Calvo-Bado ◽  
Donald Fraser ◽  
Colin G. Fink
Keyword(s):  
Internal Control ◽  
Molecular Detection ◽  
False Negative ◽  
Rna Extraction ◽  
Processing Method ◽  
Heat Processing ◽  
Negative Results ◽  
Treatment Conditions ◽  
False Negative Results ◽  
Different Temperatures

Shortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Many groups recently presented results using heat processing method of respiratory samples prior to RT-qPCR as an economical method enabling an extremely fast streamlining of the processes at virtually no cost. Here, we present our results using this method and highlight some major pitfalls that diagnostics laboratories should be aware of before proceeding with this methodology. We first investigated various treatments using different temperatures, incubation times and sample volumes to optimise the heat treatment conditions. Although the initial data confirmed results published elsewhere, further investigations revealed unexpected inhibitory properties of some commonly used universal transport media (UTMs) on some commercially available RT-qPCR mixes, leading to a risk of reporting false-negative results. This emphasises the critical importance of a thorough validation process to determine the most suitable reagents to use depending on the sample types to be tested. In conclusion, a heat processing method is effective with very consistent Ct values and a sensitivity of 96.2% when compared to a conventional RNA extraction method. It is also critical to include an internal control to check each sample for potential inhibition.

scholarly journals Fluorometric detection of HIV-1 genome through use of an internal control, inosine-substituted primers, and microtiter plate format

1996 ◽  
Vol 42 (5) ◽  
pp. 696-703 ◽  
Author(s):  
B Gérard ◽  
C Peponnet ◽  
G Brunie ◽  
H Cavé ◽  
E Denamur ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Internal Control ◽  
False Negative ◽  
Enzymatic Reaction ◽  
Pcr Amplification ◽  
Microtiter Plate ◽  
Fluorometric Detection ◽  
Negative Results ◽  
False Negative Results ◽  
Hiv 1

Abstract We describe a PCR-based fluorometric assay for the detection of the HIV-1 genome. This technique consists of a reverse hybridization with oligonucleotide probes covalently coated onto a microtiter plate as a solid support. Several improvements to the PCR amplification and detection steps gave greater sensitivity and specificity for HIV-1 screening and resulted in a convenient and rapid technique. False-positive results were avoided by using uracyl DNA glycosylase. False-negative results from the presence of PCR inhibitors were detected by coamplifying an internal control with the viral sequence. False-negative results from viral genome variability were limited by using two pairs of primers and by incorporating inosine at the primer positions corresponding to viral polymorphic nucleotides. Furthermore, the hybridization buffer and enzymatic reaction were optimized to increase the assay's sensitivity. The sensitivity and specificity of the fluorometric detection were similar to those of radioisotopic oligonucleotide solution hybridization; however, hands-on time was reduced, and the use of radioactivity was eliminated. We have used this technique routinely on 115 samples and obtained 100% specificity and high sensitivity (only one false-negative result) according to viral culture and (or) serological status of the patients.

scholarly journals Improving of the Nested PCR for Detection of Bovine Leukemia Virus

2021 ◽  
Vol 83 (3) ◽  
pp. 56-65
Author(s):  
L.M. Ishchenko ◽  
◽  
V.V. Nedosekov ◽  
V.D. Ishchenko ◽  
O.Yu. Kepple ◽  
...  
Keyword(s):  
Internal Control ◽  
Nested Pcr ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
False Negative ◽  
Envelope Gene ◽  
Rt Pcr ◽  
Second Stage ◽  
Negative Results ◽  
False Negative Results

Enzootic bovine leukosis caused by a bovine leukemia virus has a significant economic impact and is reported in World Organization for Animal Health(OIE). Aim. The purpose of our work was to improve the nested polymerase chain reaction (PCR) recommended by the OIE conducting it second-stage in real-time (RT) PCR. Such modification does not require the stage of gel electrophoresis and consequently reduces contamination risks and prevents false positive results. Methods. Primers that are recommended by the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE) were used for the first amplification stage. For the second stage of the proposed modification of nested PCR, the primers and probe were designed based on the alignment of the sequences envelope gene of different isolates of bovine leukemia virus including Ukrainian isolates. Amplification of the internal control was carried out for the second stage to prevent false negative results. Results. Comparative studies of 48 blood samples for bovine leukemia virus identification by a proposed nested RT-PCR, nested PCR recommended by the protocol of the OIE, and RT-PCR were conducted. The sample panel included both positive and negative samples. A 100% match of the results of the bovine leukemia virus presence in nested PCR proposed by the OIE and in our proposed nested RT-PCR was obtained. Comparative analysis of results that were obtained using the RT-PCR and the proposed nested RT-PCR showed that false-negative results in 5 samples and 3 doubtful results that require retesting were obtained by use of RT-PCR. The interpretation of the results using nested RT-PCR is more efficient than RT-PCR since the cycle threshold value of positive samples obtained using RT-PCR was in the range of 24–40 cycles, whereas in the case of nested RT-PCR using, the value of Ct was in the range of 4–20 cycles. Conclusions. Proposed nested PCR modification includes the combination of the OIE recommendation about nested PCR and the reduction of the risk of contamination by conducting the second stage in RT-PCR. Results of approbation of proposed nested RT-PCR give a reason to recommend it for the identification of bovine leukemia virus.

scholarly journals Patient DNA cross-reactivity of the CDC SARS-CoV-2 extraction control leads to an inherent potential for false negative results

2020 ◽  
Author(s):  
Adam P. Rosebrock
Keyword(s):  
Reverse Transcription ◽  
False Negative ◽  
Rna Extraction ◽  
Cross Reactivity ◽  
Sample Collection ◽  
Negative Results ◽  
False Negative Results ◽  
Important Diagnostic Tool ◽  
Control And Prevention ◽  
Careful Handling

AbstractTesting for RNA viruses such as SARS-CoV-2 requires careful handling of inherently labile RNA during sample collection, clinical processing, and molecular analysis. Tests must include fail-safe controls that affirmatively report the presence of intact RNA and demonstrate success of all steps of the assay. A result of “no virus signal” is insufficient for clinical interpretation: controls must also say “The reaction worked as intended and would have found virus if present.” Unfortunately, a widely used test specified by the US Centers for Disease Control and Prevention (CDC) incorporates a control that does not perform as intended and claimed. Detecting SARS-CoV-2 with this assay requires both intact RNA and successful reverse transcription. The CDC-specified control does not require either of these, due to its inability to differentiate human genomic DNA from reverse-transcribed RNA. Patient DNA is copurified from nasopharyngeal swabs during clinically-approved RNA extraction and is sufficient to return an “extraction control success” signal using the CDC design. As such, this assay fails-unsafe: truly positive patient samples return a false-negative result of “no virus detected, control succeeded” following any of several readily-encountered mishaps. This problem affects tens-of-millions of patients worth of shipped assays, but many of these flawed reagents have not yet been used. There is an opportunity to improve this important diagnostic tool. As demonstrated here, a re-designed transcript-specific control correctly monitors sample collection, extraction, reverse transcription, and qPCR detection. This approach can be rapidly implemented and will help reduce truly positive patients from being incorrectly given the all-clear.One Sentence SummaryA widely-used COVID-19 diagnostic is mis-designed and generates false-negative results, dangerously confusing “No” with “Don’t know” – but it’s fixable

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 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.

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 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.

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