scholarly journals Contrast of Real-Time Fluorescent PCR Methods for Detection of Escherichia coli O157:H7 and of Introducing an Internal Amplification Control

2019 ◽  
Vol 7 (8) ◽  
pp. 230 ◽  
Author(s):  
Zhao ◽  
Xia ◽  
Liu
Keyword(s):  
Real Time ◽  
Water Samples ◽  
False Negative ◽  
Internal Amplification Control ◽  
Pcr Assay ◽  
The Real ◽  
E Coli ◽  
Negative Results ◽  
False Negative Results ◽  
Fluorescent Pcr

Various constituents in food specimens can inhibit the PCR assay and lead to false-negative results. An internal amplification control was employed to monitor the presence of false-negative results in PCR amplification. In this study, the objectives were to compare the real-time PCR-based method by introducing a competitive internal amplification control (IAC) for the detection of Escherichia O157:H7 with respect to the specificity of the primers and probes, analytical sensitivity, and detection limits of contamination-simulated drinking water. Additionally, we optimized the real-time fluorescent PCR detection system for E. coli O157:H7. The specificity of primers and probes designed for the rfbE gene was evaluated using four kinds of bacterial strains, including E. coli O157:H7, Staphylococcus aureus, Salmonella and Listeria monocytogenes strains. The real time PCR assay unambiguously distinguished the E. coli O157:H7 strains after 16 cycles. Simultaneously, the lowest detection limit for E. coli O157:H7 in water samples introducing the IAC was 104 CFU/mL. The analytical sensitivity in water samples had no influence on the detection limit compared with that of pure cultures. The inclusion of an internal amplification control in the real-time PCR assay presented a positive IAC amplification signal in artificially simulated water samples. These results indicated that real-time fluorescent PCR combined with the IAC possessed good characteristics of stability, sensitivity, and specificity. Consequently, the adjusted methods have the potential to support the fast and sensitive detection of E. coli O157:H7, enabling accurate quantification and preventing false negative results in E. coli O157:H7 contaminated samples.

Scoring System for the Diagnosis of COVID-19

2020 ◽  
Vol 13 (1) ◽  
pp. 413-414 ◽  
Author(s):  
Mohamed Farouk Allam
Keyword(s):  
Scoring System ◽  
Clinical Symptoms ◽  
False Negative ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Negative Results ◽  
False Negative Results ◽  
Symptoms And Signs

Due to the international spread of COVID-19, the difficulty of collecting nasopharyngeal swab specimen from all suspected patients, the costs of RT-PCR and CT, and the false negative results of RT-PCR assay in 41% of COVID-19 patients, a scoring system is needed to classify the suspected patients in order to determine the need for follow-up, home isolation, quarantine or the conduction of further investigations. A scoring system is proposed as a diagnostic tool for suspected patients. It includes Epidemiological Evidence of Exposure, Clinical Symptoms and Signs, and Investigations (if available). This scoring system is simple, could be calculated in a few minutes, and incorporates the main possible data/findings of any patient.

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 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.

Development of a recombinase polymerase amplification assay forVibrio parahaemolyticusdetection with an internal amplification control

2018 ◽  
Vol 64 (4) ◽  
pp. 223-230 ◽  
Author(s):  
Huan-Lan Yang ◽  
Shuang Wei ◽  
Ravi Gooneratne ◽  
Anthony N. Mutukumira ◽  
Xue-Jun Ma ◽  
...  
Keyword(s):  
Bacterial Species ◽  
False Negative ◽  
Recombinase Polymerase Amplification ◽  
Internal Amplification Control ◽  
Target Sequence ◽  
Negative Results ◽  
False Negative Results ◽  
Amplification Assay ◽  
Pcr Method ◽  
Recombinase Polymerase Amplification Assay

A novel RPA–IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 103CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA–IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

scholarly journals PCR and Blood Culture for Detection ofEscherichia coli Bacteremia in Rats

1999 ◽  
Vol 37 (8) ◽  
pp. 2479-2482 ◽  
Author(s):  
Alexandra Heininger ◽  
Marlies Binder ◽  
Sibylle Schmidt ◽  
Klaus Unertl ◽  
Konrad Botzenhart ◽  
...  
Keyword(s):  
Blood Culture ◽  
Detection Rate ◽  
False Negative ◽  
Diagnostic Procedures ◽  
Microbial Pathogens ◽  
Gene Encoding ◽  
E Coli ◽  
Negative Results ◽  
False Negative Results ◽  
Male Wistar Rats

Critically ill patients often develop symptoms of sepsis and therefore require microbiological tests for bacteremia that use conventional blood culture (BC) techniques. However, since these patients frequently receive early empirical antibiotic therapy before diagnostic procedures are completed, examination by BC can return false-negative results. We therefore hypothesized that PCR could improve the rate of detection of microbial pathogens over that of BC. To test this hypothesis, male Wistar rats were challenged intravenously with 106 CFU of Escherichia coli. Blood was then taken at several time points for detection of E. coliby BC and by PCR with E. coli-specific primers derived from the uidA gene, encoding β-glucuronidase. In further experiments, cefotaxime (100 or 50 mg/kg of body weight) was administered intravenously to rats 10 min after E. colichallenge. Without this chemotherapy, the E. coli detection rate decreased at 15 min and at 210 min after challenge from 100% to 62% of the animals with PCR and from 100% to 54% of the animals with BC (P, >0.05). Chemotherapy decreased the E. coli detection rate at 25 min and at 55 min after challenge from 100% to 50% with PCR and from 100% to 0% with BC (P, <0.05). Thus, at clinically relevant serum antibiotic levels, PCR affords a significantly higher detection rate than BC in this rat model. The results suggest that PCR could be a useful adjunct tool supplementing conventional BC techniques in diagnosing bacteremia.

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.

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