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.

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 Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes

2021 ◽  
Author(s):  
Enos C Kline ◽  
Nuttada Panpradist ◽  
Ian T Hull ◽  
Qin Wang ◽  
Amy K Oreskovic ◽  
...  
Keyword(s):  
Molecular Diagnostics ◽  
Detection System ◽  
Point Of Care ◽  
False Negative ◽  
Cost Effective ◽  
Internal Amplification Control ◽  
Robust Detection ◽  
Negative Results ◽  
False Negative Results ◽  
Target Redundancy

AbstractThe increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multi-target redundancy and an internal amplification control. A convenient and cost-effective target specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-COV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multi-target redundancy. When challenged with extracted human samples the multiplexed assay showed 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction, and 100% specificity. These results are further evidence that conventional laboratory methodologies can be leveraged at the point-of-care for robust performance and diagnostic stability over time.

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.

scholarly journals High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

2020 ◽  
Author(s):  
Sanchita Bhadra ◽  
Timothy E. Riedel ◽  
Simren Lakhotia ◽  
Nicholas D. Tran ◽  
Andrew D. Ellington
Keyword(s):  
Point Of Care ◽  
False Negative ◽  
Human Saliva ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Target Sequence ◽  
One Pot ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

ABSTRACTIsothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (PCR)-based amplification assays, especially for point-of-care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva.

Different Subclones of Adult Acute Lymphoblastic Leukemia Detected by Clonal Igh and TCR Markers Behave Differently during Remission and at Relapse

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4860-4860
Author(s):  
Yulya Davidyan ◽  
Elena Parovichnikova ◽  
Vadim Surin ◽  
Valeryi Savchenko
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Gene Rearrangement ◽  
Lymphoblastic Leukemia ◽  
False Negative ◽  
Patient Specific ◽  
Gene Rearrangements ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Abstract INTRODUCTION. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient–specific targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). However they might be unstable during the disease course. False-negative results due to clonal evolution are the major disadvantage of using Ig/TCR gene rearrangements as PCR targets for MRD detection. In order to minimize false-negative results, it is necessary to use more PCR targets for MRD monitoring. PATIENTS AND METHODS. Bone marrow samples of 34 ALL patients (27 B-ALL, 7 T-ALL) were analyzed for IgH and TCR gene rearrangement by means of PCR method at diagnosis, after induction and consolidation of remission, before and after stem cell transplantation and during maintenance therapy. RESULTS. Clonal IgH and TCR gene targets for MRD detection were identified in 97% of the patients (33/34). In 70% of the patients (23/33) two or more specific markers for MRD monitoring were detected. Three clonal IgH gene rearrangements were discovered in 2 patients. We wished to investigate whether it was important to monitor two or more targets, because of the dynamics of various markers (subclones) may be different). Prolonged MRD monitoring with 2–3 markers was carried out in 9 patients on different stages of treatment. In 4 of 9 patients (3 precursor B-ALL, 1 pre-T-ALL) the results of MRD detection by 2–3 clonal markers at different time points were similar. And in 5 of 9 patients (3 precursor-B-ALL, 1 precursor T-ALL, 1 B-mature ALL) marker detectability during treatment was dissimilar and not parallel. Of note, 4 of those 5 patients with varying dynamics of different subclones relapsed. Relapse was not observed in the B-mature ALL patient. No relapses were detected in 4 patients with similar dynamics of MRD markers. Bone marrow samples of 8 adult ALL patients were subjected to a detailed analysis of IgH and TCR gene rearrangements by PCR method at diagnosis and relapse. In most patients (7/8) rearrangements identified at diagnosis were present also at relapse. In one patient with precursor-B-ALL one clonal incomplete IgH gene rearrangement (DH-JH) detected at diagnosis was lost at relapse and another one DH-JH rearrangement was preserved. Of note, acute myeloid leukemia was diagnosed at relapse in this patient. CONCLUSIONS. Comparative PCR analyses of IgH and TCR gene rearrangements did not detect differences between newly diagnosed and relapsed ALL although in a small group of adult patients (unlike childhood ALL). Our data indicate the necessity to monitor MRD in ALL patients with more than one PCR target in order to minimize false-negative results and to predict the course of the disease. The detectability of various MRD markers (subclones) during treatment may differ and this fact could predict a higher probability of relapse.

scholarly journals Quantitation of Viral DNA by Real-Time PCR Applying Duplex Amplification, Internal Standardization, and Two-Color Fluorescence Detection

2001 ◽  
Vol 67 (6) ◽  
pp. 2837-2839 ◽  
Author(s):  
Franz Gruber ◽  
Falko G. Falkner ◽  
Friedrich Dorner ◽  
Thomas Hämmerle
Keyword(s):  
Real Time ◽  
Fluorescence Detection ◽  
Real Time Pcr ◽  
Parvovirus B19 ◽  
False Negative ◽  
Viral Dna ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method ◽  
Internal Standardization

ABSTRACT A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 102 to at least 5 × 106 copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.

Critical Evaluation of Impedance Phlebography for the Diagnosis of Deep Vein Thrombosis

1974 ◽  
Vol 31 (02) ◽  
pp. 273-278
Author(s):  
Kenneth K Wu ◽  
John C Hoak ◽  
Robert W Barnes ◽  
Stuart L Frankel
Keyword(s):  
Deep Vein Thrombosis ◽  
False Positive ◽  
False Negative ◽  
Critical Evaluation ◽  
Original Method ◽  
Vein Thrombosis ◽  
Negative Results ◽  
False Negative Results ◽  
Deep Vein ◽  
Positive Results

SummaryIn order to evaluate its daily variability and reliability, impedance phlebography was performed daily or on alternate days on 61 patients with deep vein thrombosis, of whom 47 also had 125I-fibrinogen uptake tests and 22 had radiographic venography. The results showed that impedance phlebography was highly variable and poorly reliable. False positive results were noted in 8 limbs (18%) and false negative results in 3 limbs (7%). Despite its being simple, rapid and noninvasive, its clinical usefulness is doubtful when performed according to the original method.

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.

Sign in / Sign up

Export Citation Format

Share Document