False-negative results of a validated real-time PCR protocol for diagnosis of newcastle disease due to genetic variability of the matrix gene.
Ontology highlight
ABSTRACT:
SUBMITTER: Cattoli G
PROVIDER: S-EPMC2772626 | biostudies-literature | 2009 Nov
REPOSITORIES: biostudies-literature
Publications
False-negative results of a validated real-time PCR protocol for diagnosis of newcastle disease due to genetic variability of the matrix gene.
Cattoli G G De Battisti C C Marciano S S Ormelli S S Monne I I Terregino C C Capua I I
Journal of clinical microbiology 20090916 11
Similar Datasets
Project description:Real-time quantitative PCR is used routinely for the high-throughput diagnosis of viral pathogens, such as West Nile virus (WNV). Rapidly evolving RNA viruses present a challenge for diagnosis because they accumulate mutations that may render them undetectable. To explore the effect of sequence variations on assay performance, we generated every possible single point mutation within the target region of the widely used TaqMan assay for WNV and found that the TaqMan assay failed to detect 47% of possible single nucleotide variations in the probe-binding site and was unable to detect any targets with more than two mutations. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. The SYBR green-based assay was as sensitive as the TaqMan assay for WNV. Importantly, it detected 100% of possible WNV target region variants. The assay developed here adds an additional layer of protection to guard against false-negative results that result from natural variations or drug-directed selection and provides a rapid means to identify such variants for subsequent detailed analysis.
| S-EPMC387603 | biostudies-literature
Project description:Early treatment of meningococcal meningitis is mandatory but may negate the cerebrospinal fluid culture. Etiological diagnosis then mainly relies on PCR. Here, we report a case of false-negative results for real-time PCR for a Neisseria meningitidis serogroup B isolate with a polymorphism in the ctrA gene.
| S-EPMC3008480 | biostudies-literature
Project description:BackgroundMolecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite's DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite's DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.ResultsTwo primer pairs were designed to detect the plasmid pUC18 and a triplex PCR assay targeting the Leishmania braziliensis kinetoplast DNA, the external control and the internal control was standardized. The triplex PCR assay was assessed for its ability to detect the three target DNA fragments simultaneously. PCR products from pUC18 DNA resulted in bands of 368 (P1) and 316 (P2) base pairs (bp). The triplex PCR optimized with the chosen external control system (P1) allowed the simultaneous detection of the internal control (G3PD - 567 bp) as well as of small quantities (10 pg) of the target parasite's DNA, detected by amplification of a 138 bp product.ConclusionsThe new tool standardized herein enables a more reliable interpretation of PCR results, mainly by contributing to quality assurance of leishmaniasis diagnosis. Furthermore, after simple standardization steps, this protocol could be applied to the diagnosis of other infectious diseases in reference laboratories. This triplex PCR enables the assessment of small losses during the DNA extraction process, problems concerning DNA degradation (sample quality) and the detection of L. braziliensis kDNA.
| S-EPMC4012836 | biostudies-literature
Project description:Diphtheria surveillance depends on the rapid and reliable recognition of the toxin gene in Corynebacterium diphtheriae. Real-time PCR is a rapid tool to confirm the presence of the diphtheria toxin gene (tox) in an isolate or specimen. We report that some toxigenic Corynebacterium ulcerans strains show atypical results in a real-time PCR for tox.
| S-EPMC2224273 | biostudies-literature
Project description:Two strains of Neisseria meningitidis serogroup C with disparate sequences of ctrA were isolated. The nucleotide substitutions did not alter the corresponding protein sequences, but they impeded the detection of these meningococcal isolates by real-time PCR.
| S-EPMC2916631 | biostudies-literature
Project description:West Nile virus (WNV) causes significant morbidity and mortality worldwide. Transplant and transfusion recipients as well as the elderly are particularly at risk. WNV shows strain variation from season to season and from locale to locale. This poses a significant problem for diagnosis. Most assays use a single primer pair to detect WNV by QPCR, and can fail to detect novel stains. To overcome this limitation, a genome-wide, multiple primer-based real-time QPCR assay was developed for WNV. The same assay can be used for quantitation, viral variant discovery as well as for amplification of the entire viral genome using a single annealing temperature. It improves upon routine diagnosis as well as facilitates laboratory investigations of the pathology of WNV.
| S-EPMC3189403 | biostudies-literature
Project description: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.
| S-EPMC8707239 | biostudies-literature
Project description:Newcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (aMPV) can be similar making it critical to quickly differentiate them. Herein, we adapted pre-existing molecular-based diagnostic assays for NDV and ILTV, and developed new assays for aMPV A and B, for use under synchronized thermocycling conditions. All assays performed equivalently with linearity over a 5 log10 dynamic range, a reproducible (R² > 0.99) limit of detection of ≥ 10 target copies, and amplification efficiencies between 86.8%-98.2%. Using biological specimens for NDV and ILTV showed 100% specificity. Identical amplification conditions will simplify procedures for detection in diagnostic laboratories.
| S-EPMC8977536 | biostudies-literature
Project description:BackgroundCOVID-19 is diagnosed via detection of SARS-CoV-2 RNA using real time reverse-transcriptase polymerase chain reaction (rtRT-PCR). Performance of many SARS-CoV-2 rtRT-PCR assays is not entirely known due to the lack of a gold standard. We sought to evaluate the false negative rate (FNR) and sensitivity of our laboratory-developed SARS-CoV-2 rtRT-PCR targeting the envelope (E) and RNA-dependent RNA-polymerase (RdRp) genes.MethodsSARS-CoV-2 rtRT-PCR results at the Public Health Laboratory (Alberta, Canada) from January 21 to April 18, 2020 were reviewed to identify patients with an initial negative rtRT-PCR followed by a positive result on repeat testing within 14 days (defined as discordant results). Negative samples from these discordant specimens were re-tested using three alternate rtRT-PCR assays (targeting the E gene and N1/N2 regions of the nucleocapsid genes) to assess for false negative (FN) results.ResultsDuring the time period specified, 95,919 patients (100,001 samples) were tested for SARS-CoV-2. Of these, 49 patients were found to have discordant results including 49 positive and 52 negative swabs. Repeat testing of 52 negative swabs found five FNs (from five separate patients). Assuming 100% specificity of the diagnostic assay, the FNR and sensitivity in this group of patients with discordant testing was 9.3% (95% CI 1.5-17.0%) and 90.7% (95% CI 82.6-98.9%) respectively.ConclusionsStudies to understand the FNR of routinely used assays are important to confirm adequate clinical performance. In this study, most FN results were due to low amounts of SARS-CoV-2 virus concentrations in patients with multiple specimens collected during different stages of infection. Post-test clinical evaluation of each patient is advised to ensure that rtRT-PCR results are not the only factor in excluding COVID-19.
| S-EPMC7794619 | biostudies-literature
Project description:BackgroundReverse-transcription PCR (RT-PCR) assays are used to test for infection with the SARS-CoV-2 virus. RT-PCR tests are highly specific and the probability of false positives is low, but false negatives are possible depending on swab type and time since symptom onset.AimTo determine how the probability of obtaining a false-negative test in infected patients is affected by time since symptom onset and swab type.MethodsWe used generalised additive mixed models to analyse publicly available data from patients who received multiple RT-PCR tests and were identified as SARS-CoV-2 positive at least once.ResultsThe probability of a positive test decreased with time since symptom onset, with oropharyngeal (OP) samples less likely to yield a positive result than nasopharyngeal (NP) samples. The probability of incorrectly identifying an uninfected individual due to a false-negative test was considerably reduced if negative tests were repeated 24 hours later. For a small false-positive test probability (<0.5%), the true number of infected individuals was larger than the number of positive tests. For a higher false-positive test probability, the true number of infected individuals was smaller than the number of positive tests.ConclusionNP samples are more sensitive than OP samples. The later an infected individual is tested after symptom onset, the less likely they are to test positive. This has implications for identifying infected patients, contact tracing and discharging convalescing patients who are potentially still infectious.
| S-EPMC7812420 | biostudies-literature