scholarly journals Mutations in Animal SARS-CoV-2 Induce Mismatches with the Diagnostic PCR Assays

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 371
Author(s):  
Ahmed Elaswad ◽  
Mohamed Fawzy
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Animal Species ◽  
False Negative ◽  
Bioinformatic Analysis ◽  
Diagnostic Pcr ◽  
Animal Host ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Assays

Recently, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was detected in several animal species. After transmission to animals, the virus accumulates mutations in its genome as adaptation to the new animal host progresses. Therefore, we investigated whether these mutations result in mismatches with the diagnostic PCR assays and suggested proper modifications to the oligo sequences accordingly. A comprehensive bioinformatic analysis was conducted using 28 diagnostic PCR assays and 793 publicly available SARS-CoV-2 genomes isolated from animals. Sixteen out of the investigated 28 PCR assays displayed at least one mismatch with their targets at the 0.5% threshold. Mismatches were detected in seven, two, two, and six assays targeting the ORF1ab, spike, envelope, and nucleocapsid genes, respectively. Several of these mismatches, such as the deletions and mismatches at the 3’ end of the primer or probe, are expected to negatively affect the diagnostic PCR assays resulting in false-negative results. The modifications to the oligo sequences should result in stronger template binding by the oligos, better sensitivity of the assays, and higher confidence in the result. It is necessary to monitor the targets of diagnostic PCR assays for any future mutations that may occur as the virus continues to evolve in animals.

scholarly journals False-Negative Nasopharyngeal Swab RT-PCR Assays in Typical COVID-19: Role of Ultra-low-dose Chest CT and Bronchoscopy in Diagnosis

2020 ◽  
Author(s):  
Marco Marando ◽  
Adriana Tamburello ◽  
Pietro Gianella
Keyword(s):  
Low Dose ◽  
False Negative ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Oral Swab ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain ◽  
Pcr Assays

On 11 March 2020, the WHO declared COVID-19 a pandemic and global health emergency. We describe the clinical features and role of ultra-low-dose chest computed tomography (CT) and bronchoscopy in the diagnosis of coronavirus disease (COVID-19). In our patient, who was highly suggestive clinically and radiologically for COVID-19, we had two false-negative results for nasopharyngeal and oral swab reverse-transcriptase polymerase chain reaction (RT-PCR) assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Eventually, we confirmed the diagnosis using bronchoscopy and bronchoalveolar lavage (BAL).

scholarly journals Development and application of a canine endogenous internal positive control for use in real-time PCR assays

2018 ◽  
Vol 30 (5) ◽  
pp. 789-792 ◽  
Author(s):  
Joseph J. Modarelli ◽  
Pamela J. Ferro ◽  
Maria D. Esteve-Gasent
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Positive Control ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Negative Results ◽  
False Negative Results ◽  
Internal Positive Control ◽  
Pcr Assays

Real-time PCR (rtPCR) tests have become a method of choice in many diagnostic settings, both animal and human. A concern remains, however, regarding rtPCR assay inhibition during nucleic acid extraction and/or rtPCR reaction process that may result in false-negative results. The use of an internal positive control, either endogenous or exogenous, to mitigate this issue has become more commonplace. We identified and standardized an endogenous internal positive control that can be utilized in rtPCR assays targeting canine-specific pathogens in either a singleplex or multiplex format. The target chosen for the endogenous internal positive control (EIPC-K9) was a highly conserved region in canine mitochondrial DNA. Samples from 240 dogs and 11 other species were screened with EIPC-K9; all canine samples were detected, and no cross-amplification with other species tested was observed. Additionally, no inhibition was noted when comparing singleplex to multiplex rtPCR formats.

scholarly journals Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS-CoV-2

2020 ◽  
Author(s):  
Xiumei Hu ◽  
Taixue An ◽  
Bo Situ ◽  
Yuhai Hu ◽  
Zihao Ou ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
False Negative ◽  
Average Level ◽  
Heat Inactivation ◽  
Serum Antibodies ◽  
Serum Samples ◽  
Negative Results ◽  
False Negative Results ◽  
Before And After ◽  
Quantitative Fluorescence

AbstractThe detection of serum antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a new tool for the coronavirus disease-2019 (COVID-19) diagnosis. Since many coronaviruses are sensitive to heat, heating inactivation of samples at 56 °C prior to testing is considered a possible method to reduce the risk of transmission, but the effect of heating on the measurement of SARS-CoV-2 antibodies is still unclear. By comparing the levels of SARS-CoV-2 antibodies before and after heat inactivation of serum at 56 °C for 30 minutes using a quantitative fluorescence immunochromatographic assay, we shown that heat inactivation significantly interferes with the levels of antibodies to SARS-CoV-2. The IgM levels of all the 34 serum samples (100%) from COVID-19 patients decreased by an average level of 53.56%. The IgG levels were decreased in 22 of 34 samples (64.71%) by an average level of 49.54%. Similar changes can also be observed in the non-COVID-19 diseases group (n=9). Of note, 44.12% of the detected IgM levels were dropped below the cut-off value after heating, suggesting heat inactivation can lead to false-negative results of these samples. Our results indicate that heat inactivation of serum at 56 °C for 30 minutes interferes with the immunoanalysis of antibodies to SARS-CoV-2. Heat inactivation prior to immunoanalysis is not recommended and the possibility of false-negative results should be considered if the sample was pre-inactivated by heating.

scholarly journals Exogenous Controls Increase Negative Call Veracity in Multiplexed, Quantitative PCR Assays for Phakopsora pachyrhizi

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 343-352 ◽  
Author(s):  
James S. Haudenshield ◽  
Glen L. Hartman
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Arbitrary Sequence ◽  
Phakopsora Pachyrhizi ◽  
Synthetic Dna ◽  
Negative Results ◽  
Detection And Identification ◽  
False Negative Results ◽  
Pcr Assays ◽  
Rust Pathogens

Quantitative polymerase chain reaction (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5′-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi and P. meibomiae, two rust pathogens of soybean. Because of the extreme sensitivity of Q-PCR, the DNA of single urediniospores of these fungi can be detected from total DNA extracts of environmental samples. However, some DNA preparations unpredictably contain PCR inhibitors that increase the frequency of false negatives indistinguishable from true negatives. Three synthetic DNA molecules of arbitrary sequence were constructed as multiplexed internal controls (ICs) to cull false-negative results by producing a positive signal to validate the PCR process within each individual reaction. The first two, PpaIC and PmeIC, are a single-stranded oligonucleotide flanked by sequences complementary to the primers of either the P. pachyrhizi or P. meibomiae primary assay but hybridizing to a unique fluorogenic probe; the third contains unique primer- and probe-binding sequences, and was prepared as a cloned DNA fragment in a linearized plasmid. These ICs neither qualitatively nor quantitatively affected their primary assays. PpaIC and PmeIC were shown to successfully identify false-negative reactions resulting from endogenous or exogenous inhibitors, and can be readily adapted to function in a variety of diagnostic Q-PCR assays; the plasmid was found to successfully validate true negatives in similar Q-PCR assays for other soybean pathogens, as well as to function as a tracer molecule during DNA extraction and recovery.

scholarly journals Inhibition of Polymerase Chain Reaction: Pathogen-Specific Controls are better than Human Gene Amplification

2019 ◽  
Author(s):  
Guillaume Roux ◽  
Christophe Ravel ◽  
Emmanuelle Varlet-Marie ◽  
Rachel Jendrowiak ◽  
Patrick Bastien ◽  
...  
Keyword(s):  
Human Gene ◽  
False Negative ◽  
Routine Practice ◽  
Pcr Inhibition ◽  
Specific Sequence ◽  
Negative Results ◽  
Albumin Gene ◽  
False Negative Results ◽  
Pcr Inhibitor ◽  
Pcr Assays

AbstractPCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0 %), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden’s index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.

scholarly journals Multicentre comparison of quantitative PCR-based assays to detect SARS-CoV-2, Germany, March 2020

2020 ◽  
Vol 25 (24) ◽  
Author(s):  
Maximilian Muenchhoff ◽  
Helga Mairhofer ◽  
Hans Nitschko ◽  
Natascha Grzimek-Koschewa ◽  
Dieter Hoffmann ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Quantitative Pcr ◽  
Clinical Management ◽  
False Negative ◽  
Test Results ◽  
Rt Pcr ◽  
Diagnostic Pcr ◽  
Pcr Assays ◽  
Pcr Test ◽  
False Negative Test

Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.

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