scholarly journals Performance verification of pre-PCR and real-time PCR step in the molecular diagnosis of pneumococcal meningitis cases

2021 ◽  
Vol 319 ◽  
pp. 01063
Author(s):  
Soumaya Chaiboub ◽  
Réda Charof ◽  
Aicha Qasmaoui ◽  
Jamila Hamamouchi ◽  
El Hassan Berny
Keyword(s):  
Real Time ◽  
Molecular Diagnosis ◽  
Real Time Pcr ◽  
Performance Criteria ◽  
Contamination Rate ◽  
Standard Curve ◽  
Sample Contamination ◽  
A Value ◽  
Genes Encoding ◽  
Sensitivity Specificity

Isolation and determination of s.pneumoniae by culture and serological methods can be time consuming or indeterminate. Molecular diagnosis by real-time PCR is independent of the growth of the pathogen causing meningitis, and is not diminished with non-viable organisms. The aim of this study was to evaluate the performance criteria of pre-PCR-TR DNA extraction step and PCR-TR step by targeting two genes encoding s.pneumoniae. In this study we evaluated the inter-sample contamination of the pre-PCR-TR step, the intermediate fidelity and the repeatability of the DNA essay. PCR-TR verification was performed by two genes targeting s. pneumoniae the Lyt A and SP 2038 gene; sensitivity, specificity and LLD were determined. Contamination rate had a value of less than 0%, which is in agreement with an absence of inter-sample contamination; the repeatability and intermediate fidelity have a cv˂7%. The evaluation of the sensitivity and specificity of the RT-PCR assays targeted 100% the Lyt A gene and the SP 2038 gene. The standard curve generated detected less than 10copies for the Lyt A gene and less than 100copies for the SP 2038 gene. This study showed that the pre-PCR and PCR-TR assays met the performance criteria targeted in this study.

Molecular diagnosis of α-thalassemia by combining real-time PCR with SYBR Green1 and dissociation curve analysis

2006 ◽  
Vol 148 (1) ◽  
pp. 6-12 ◽  
Author(s):  
Jingzhong Liu ◽  
Mei Yan ◽  
Zhangyong Wang ◽  
Lirong Wang ◽  
Yan Zhou ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Diagnosis ◽  
Real Time Pcr ◽  
Curve Analysis ◽  
Dissociation Curve

scholarly journals Multiplex Molecular Diagnosis of MEFV Mutations in Patients with Familial Mediterranean Fever by LightCycler Real-Time PCR

2005 ◽  
Vol 51 (9) ◽  
pp. 1725-1727 ◽  
Author(s):  
Elena Rossou ◽  
Anastasia Kouvatsi ◽  
Charalampos Aslanidis ◽  
Constantinos Deltas
Keyword(s):  
Familial Mediterranean Fever ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Real Time Pcr ◽  
Mefv Mutations ◽  
Mediterranean Fever

scholarly journals A Set of Conventional and Multiplex Real-Time PCR Assays for Direct Detection of Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis in Citrus Fruits

Plant Disease ◽  
2019 ◽  
Vol 103 (2) ◽  
pp. 345-356 ◽  
Author(s):  
Yosra Ahmed ◽  
Jacqueline Hubert ◽  
Céline Fourrier-Jeandel ◽  
Megan M. Dewdney ◽  
Jaime Aguayo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Media ◽  
Direct Detection ◽  
Elongation Factor ◽  
Single Copy ◽  
The United States ◽  
Performance Criteria ◽  
In Planta ◽  
Conventional Pcr

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg μl−1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) μl−1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

Molecular Diagnosis of African Swine Fever by a New Real-Time PCR Using Universal Probe Library

2012 ◽  
Vol 60 (1) ◽  
pp. 48-58 ◽  
Author(s):  
J. Fernández-Pinero ◽  
C. Gallardo ◽  
M. Elizalde ◽  
A. Robles ◽  
C. Gómez ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Diagnosis ◽  
Real Time Pcr ◽  
African Swine Fever ◽  
Universal Probe Library

Comparison of Quantitative Analysis of Methylated Alleles Real-Time PCR and Methylation-Specific MLPA for Molecular Diagnosis of Beckwith-Wiedemann Syndrome

Pathobiology ◽  
2019 ◽  
Vol 86 (4) ◽  
pp. 217-224
Author(s):  
Massimiliano Bergallo ◽  
Ilaria Galliano ◽  
Paola Montanari ◽  
Cristina Calvi ◽  
Valentina Daprà ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Real Time Pcr

scholarly journals A SYBR green I real-time polymerase chain reaction (PCR) assay for detection and quantification of Trichomonas gallinae

2020 ◽  
Vol 119 (11) ◽  
pp. 3909-3913
Author(s):  
Zaida Rentería-Solís ◽  
Tran Nguyen-Ho-Bao ◽  
Shahinaz Taha ◽  
Arwid Daugschies
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Small Subunit ◽  
Sybr Green ◽  
Pcr Assay ◽  
Standard Curve ◽  
Trichomonas Gallinae ◽  
Host Interaction

Abstract Trichomonas gallinae are parasitic flagellates of importance in wild and domestic birds. The parasite is worldwide distributed, and Columbine birds are its main host. Current research focuses mostly on epidemiological and phylogenetic studies. However, there is still a lack of knowledge regarding parasite-host interaction or therapy development. Real-time PCR is a useful tool for diagnostic and quantification of gene copies in a determined sample. By amplification of a 113-bp region of the 18S small subunit ribosomal RNA gene, a SYBR green-based real-time PCR assay was developed. A standard curve was prepared for quantification analysis. Assay efficiency, linearity, and dissociation analysis were successfully performed. Specificity, sensibility, and reproducibility analysis were tested. This assay could be a useful tool not only for diagnostic purposes but also for future in vivo and in vitro T. gallinae studies.

Proline accumulation and real time PCR expression analysis of genes encoding enzymes of proline metabolism in relation to drought tolerance in Andean potato

2006 ◽  
Vol 29 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Roland Schafleitner ◽  
Amélie Gaudin ◽  
Raymundo Oscar Gutierrez Rosales ◽  
Carlos Alberto Alvarado Aliaga ◽  
Merideth Bonierbale
Keyword(s):  
Drought Tolerance ◽  
Real Time ◽  
Expression Analysis ◽  
Real Time Pcr ◽  
Proline Accumulation ◽  
Proline Metabolism ◽  
Genes Encoding ◽  
Andean Potato

scholarly journals Using the TxtAB Operon to Quantify Pathogenic Streptomyces in Potato Tubers and Soil

2008 ◽  
Vol 98 (4) ◽  
pp. 405-412 ◽  
Author(s):  
Xinshun Qu ◽  
Leslie A. Wanner ◽  
Barbara J. Christ
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Common Scab ◽  
Cost Effective ◽  
Potato Tubers ◽  
Pcr Assay ◽  
Streptomyces Species ◽  
Standard Curve ◽  
Target Dna

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R2 = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 101 to 106 pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 103 to 106 per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.

scholarly journals Comparison between Two Amplification Sets for Molecular Diagnosis of Toxoplasmosis by Real-Time PCR

2006 ◽  
Vol 44 (11) ◽  
pp. 4295-4295 ◽  
Author(s):  
S. Cassaing ◽  
M. H. Bessieres ◽  
A. Berry ◽  
A. Berrebi ◽  
R. Fabre ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Diagnosis ◽  
Real Time Pcr

Development of a LUX real-time PCR for the detection and quantification of human herpesvirus 7

2009 ◽  
Vol 55 (3) ◽  
pp. 319-325 ◽  
Author(s):  
Massimiliano Bergallo ◽  
Cristina Costa ◽  
Maria Elena Terlizzi ◽  
Francesca Sidoti ◽  
Samuela Margio ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Primary Infection ◽  
Latent Infection ◽  
Dynamic Range ◽  
Human Herpesvirus ◽  
Clinical Samples ◽  
House Light ◽  
Pcr Assay ◽  
Sensitivity Specificity

Human herpesvirus 7 is a highly seroprevalent β-herpesvirus that, following primary infection, remains latent in CD4+ T cells and determines a persistent rather than a latent infection in various tissues and organs, including the lung and skin. This paper describes the development of an in-house light upon extension real-time PCR assay for quantification of human herpesvirus 7 DNA in clinical samples. The efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated by evaluating the human herpesvirus 7 load in bronchoalveolar lavages and skin specimens, chosen as 2 persistency sites, from healthy and pathological individuals. The real-time PCR assay developed in this study could be a useful tool to detect and quantify human herpesvirus 7 DNA in different clinical specimens to elucidate its epidemiological and pathogenic roles.

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