scholarly journals One-Step Detection of Monilinia fructicola, M. fructigena, and M. laxa on Prunus and Malus by a Multiplex Real-Time PCR Assay

Plant Disease ◽  
2016 ◽  
Vol 100 (12) ◽  
pp. 2465-2474 ◽  
Author(s):  
C. Guinet ◽  
C. Fourrier-Jeandel ◽  
I. Cerf-Wendling ◽  
R. Ioos
Keyword(s):  
Real Time ◽  
High Performance ◽  
False Negative ◽  
Brown Rot ◽  
Pathogenic Fungi ◽  
Monilinia Fructicola ◽  
Pome Fruit ◽  
Universal Primer ◽  
Negative Results ◽  
Fruit Orchards

Brown rot is an economically important fungal disease affecting stone and pome fruit orchards, as well as harvested fruit during storage and on the market. Monilinia fructicola, M. laxa, and M. fructigena are the main causal agents of this disease and each have a different regulatory status depending on regional regulations. In this study, a new multiplex tool based on real-time polymerase chain reaction was developed to detect the three pathogenic fungi in a single reaction on fruit, twigs, and flowers of Prunus and Malus spp. Species-specific primer-hydrolysis probe combinations were designed to amplify a region located in a previously described MO368 sequenced characterized amplified region marker, and used in a quadruplex format coupled with the 18S Uni universal primer-probe test in order to check the quality of the DNA template. The assay was designed and optimized with the objective to provide high performance values. Experimental data supported its sensitivity, specificity, reproducibility, and robustness. In addition, a set of quality controls was implemented to minimize the risk of false-positive and false-negative results, thus making this new test fit for use in serial analyses and reliable in the framework of official controls.

Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

2020 ◽  
Vol 18 ◽  
Author(s):  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Streptococcus Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Cross Sectional Study ◽  
Laboratory Culture ◽  
Cross Sectional ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

scholarly journals Pooling of Upper Respiratory Specimens Using a SARS-CoV-2 Real-time RT-PCR Assay Authorized for Emergency Use in Low-Prevalence Populations for High-Throughput Testing

2020 ◽  
Vol 7 (11) ◽  
Author(s):  
Gwynngelle A Borillo ◽  
Ron M Kagan ◽  
Russell E Baumann ◽  
Boris M Fainstein ◽  
Lamela Umaru ◽  
...  
Keyword(s):  
Real Time ◽  
False Negative ◽  
False Negative Rate ◽  
Nucleic Acid Amplification ◽  
Rt Pcr ◽  
Pooled Testing ◽  
Negative Results ◽  
Single Positive ◽  
Upper Respiratory ◽  
Respiratory Specimens

Abstract Background Nucleic acid amplification testing is a critical tool for addressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Specimen pooling can increase throughput and conserve testing resources but requires validation to ensure that reduced sensitivity does not increase the false-negative rate. We evaluated the performance of a real-time reverse transcription polymerase chain reaction (RT-PCR) test authorized by the US Food and Drug Administration (FDA) for emergency use for pooled testing of upper respiratory specimens. Methods Positive specimens were selected from 3 prevalence groups, 1%–3%, >3%–6%, and >6%–10%. Positive percent agreement (PPA) was assessed by pooling single-positive specimens with 3 negative specimens; performance was assessed using Passing-Bablok regression. Additionally, we assessed the distributions of RT-PCR cycle threshold (Ct) values for 3091 positive specimens. Results PPA was 100% for the 101 pooled specimens. There was a linear relationship between Ct values for pooled and single-tested specimens (r = 0.96–0.99; slope ≈ 1). The mean pooled Ct shifts at 40 cycles were 2.38 and 1.90, respectively, for the N1 and N3 targets. The median Cts for 3091 positive specimens were 25.9 (N1) and 24.7 (N3). The percentage of positive specimens with Cts between 40 and the shifted Ct was 1.42% (N1) and 0.0% (N3). Conclusions Pooled and individual testing of specimens positive for SARS-CoV-2 demonstrated 100% agreement, which demonstrates the viability of pooled specimens for SARS-COV-2 testing using a dual-target RT-PCR system. Pooled specimen testing can help increase testing capacity for SARS-CoV-2 with a low risk of false-negative results.

scholarly journals False Negative Results in High Viremia Parvovirus B19-Samples Tested with Real-Time PCR

2010 ◽  
Vol 59 (2) ◽  
pp. 129-132 ◽  
Author(s):  
PIOTR GRABARCZYK ◽  
ALEKSANDRA KALIŃSKA ◽  
EWA SULKOWSKA ◽  
EWA BROJER
Keyword(s):  
Positive Result ◽  
Real Time ◽  
Real Time Pcr ◽  
Parvovirus B19 ◽  
False Negative ◽  
Immunocompromised Patients ◽  
Negative Results ◽  
False Negative Results ◽  
Parvovirus B 19 ◽  
The Way

Extremely high viremia is observed during some viruses infection, especialy in immunocompromised patients. False negative results of Parvovirus B 19 DNA tests performed with real-time PCR in high viremic samples are reported. The way of fluorescence diagrams analysis and algorithm of positive result confirmation to exclude such phenomenon are proposed.

scholarly journals Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

2003 ◽  
Vol 69 (12) ◽  
pp. 7145-7152 ◽  
Author(s):  
Zhonghua Ma ◽  
Michael A. Yoshimura ◽  
Themis J. Michailides
Keyword(s):  
Dna Sequences ◽  
Point Mutations ◽  
Brown Rot ◽  
Stone Fruit ◽  
Monilinia Fructicola ◽  
Tubulin Gene ◽  
Benzimidazole Fungicides ◽  
Specific Pcr ◽  
Allele Specific ◽  
Fruit Orchards

ABSTRACT Low and high levels of resistance to the benzimidazole fungicides benomyl and thiophanate-methyl were observed in field isolates of Monilinia fructicola, which is the causative agent of brown rot of stone fruit. Isolates that had low levels of resistance (hereafter referred to as LR isolates) and high levels of resistance (hereafter referred to as HR isolates) were also cold and heat sensitive, respectively. Results from microsatellite DNA fingerprints showed that genetic identities among the populations of sensitive (S), LR, and HR isolates were very high (>0.96). Analysis of DNA sequences of theβ -tubulin gene showed that the LR isolates had a point mutation at codon 6, causing a replacement of the amino acid histidine by tyrosine. Codon 198, which encodes a glutamic acid in S and LR isolates, was converted to a codon for alanine in HR isolates. Based on these point mutations in the β-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit.

scholarly journals Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control

2017 ◽  
Vol 29 (3) ◽  
pp. 351-356
Author(s):  
Amaresh Das ◽  
Ming Y. Deng ◽  
Shawn Babiuk ◽  
Michael T. McIntosh
Keyword(s):  
Real Time ◽  
False Negative ◽  
Positive Control ◽  
Lumpy Skin Disease ◽  
Outbreak Management ◽  
Negative Results ◽  
Beta Actin ◽  
False Negative Results ◽  
Internal Positive Control ◽  
Analytical Sensitivities

Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

scholarly journals Establishment and Validation of RNA-Based Predictive Models for Understanding Survival of Vibrio parahaemolyticus in Oysters Stored at Low Temperatures

2017 ◽  
Vol 83 (6) ◽  
Author(s):  
Chao Liao ◽  
Yong Zhao ◽  
Luxin Wang
Keyword(s):  
Real Time ◽  
Predictive Models ◽  
Vibrio Parahaemolyticus ◽  
False Negative ◽  
Bacterial Inactivation ◽  
Rt Pcr ◽  
Content Type ◽  
Low Temperature Storage ◽  
Reverse Transcription Pcr ◽  
Negative Results

ABSTRACT This study developed RNA-based predictive models describing the survival of Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) during storage at 0, 4, and 10°C. Postharvested oysters were inoculated with a cocktail of five V. parahaemolyticus strains and were then stored at 0, 4, and 10°C for 21 or 11 days. A real-time reverse transcription-PCR (RT-PCR) assay targeting expression of the tlh gene was used to evaluate the number of surviving V. parahaemolyticus cells, which was then used to establish primary molecular models (MMs). Before construction of the MMs, consistent expression levels of the tlh gene at 0, 4, and 10°C were confirmed, and this gene was used to monitor the survival of the total V. parahaemolyticus cells. In addition, the tdh and trh genes were used for monitoring the survival of virulent V. parahaemolyticus. Traditional models (TMs) were built based on data collected using a plate counting method. From the MMs, V. parahaemolyticus populations had decreased 0.493, 0.362, and 0.238 log10 CFU/g by the end of storage at 0, 4, and 10°C, respectively. Rates of reduction of V. parahaemolyticus shown in the TMs were 2.109, 1.579, and 0.894 log10 CFU/g for storage at 0, 4, and 10°C, respectively. Bacterial inactivation rates (IRs) estimated with the TMs (−0.245, −0.152, and −0.121 log10 CFU/day, respectively) were higher than those estimated with the MMs (−0.134, −0.0887, and −0.0732 log10 CFU/day, respectively) for storage at 0, 4, and 10°C. Higher viable V. parahaemolyticus numbers were predicted using the MMs than using the TMs. On the basis of this study, RNA-based predictive MMs are the more accurate and reliable models and can prevent false-negative results compared to TMs. IMPORTANCE One important method for validating postharvest techniques and for monitoring the behavior of V. parahaemolyticus is to establish predictive models. Unfortunately, previous predictive models established based on plate counting methods or on DNA-based PCR can underestimate or overestimate the number of surviving cells. This study developed and validated RNA-based molecular predictive models to describe the survival of V. parahaemolyticus in oysters during low-temperature storage (0, 4, and 10°C). The RNA-based predictive models show the advantage of being able to count all of the culturable, nonculturable, and stressed cells. By using primers targeting the tlh gene and pathogenesis-associated genes (tdh and trh), real-time RT-PCR can evaluate the total surviving V. parahaemolyticus population as well as differentiate the pathogenic ones from the total population. Reliable and accurate predictive models are very important for conducting risk assessment and management of pathogens in food.

scholarly journals Reducing False Negative PCR Test for COVID-19

2020 ◽  
Vol 9 (3) ◽  
pp. 408-410
Author(s):  
Fatemeh Bahreini ◽  
Rezvan Najafi ◽  
Razieh Amini ◽  
Salman Khazaei ◽  
Saeid Bashirian
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
False Negative ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Creative Commons ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain ◽  
Pcr Test

As the SARS-CoV-2 (COVID-19) pandemic spreads rapidly, there is need for a diagnostic test with high accuracy to detect infected individuals especially those without symptoms. Real-time polymerase chain reaction (RT-PCR) is a common molecular test for diagnosing SARS-CoV-2. If some factors are not taken into consideration when performing this test, it can have a relatively large number of false negative results. In this article, we discuss important considerations that could lead to false negative test reduction. Key words: • SARS-CoV-2 • COVID-19 • Real time polymerase chain reaction • RT-PCR test • Diagnosis • False negatives • Genetics • Emerging disease   Copyright © 2020 Bahreini et al. Published by Global Health and Education Projects, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0)which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in this journal, is properly cited.

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