scholarly journals Comparison of multiplexed-tandem real-time PCR panel with reference real-time PCR molecular diagnostic assays for detection of Giardia intestinalis and Tritrichomonas foetus in cats

2019 ◽  
Vol 266 ◽  
pp. 12-17 ◽  
Author(s):  
Maira N. Meggiolaro ◽  
Florian Roeber ◽  
Victoria Kobylski ◽  
Damien P. Higgins ◽  
Jan Šlapeta
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Giardia Intestinalis ◽  
Molecular Diagnostic ◽  
Tritrichomonas Foetus ◽  
Diagnostic Assays

scholarly journals Serology- and Blood-PCR-Based Screening for Schistosomiasis in Pregnant Women in Madagascar—A Cross-Sectional Study and Test Comparison Approach

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 722
Author(s):  
Tanja Hoffmann ◽  
Imke Carsjens ◽  
Raphaël Rakotozandrindrainy ◽  
Mirko Girmann ◽  
Njary Randriamampionona ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Real Time ◽  
Real Time Pcr ◽  
Disease Burden ◽  
Cross Sectional Study ◽  
Molecular Diagnostic ◽  
Sectional Study ◽  
Cross Sectional ◽  
Diagnostic Assays ◽  
Edta Plasma

This work was conducted as a cross sectional study to define the disease burden of schistosomiasis in pregnant Madagascan women and to evaluate serological and molecular diagnostic assays. A total of 1154 residual EDTA blood samples from pregnant Madagascan women were assessed. The nucleic acid extractions were subjected to in-house real-time PCRs specifically targeting S. mansoni complex, S. haematobium complex, and African Schistosoma spp. on genus level, while the EDTA plasma samples were analyzed using Schistosoma-specific IgG and IgM commercial ELISA and immunofluorescence assays. The analyses indicated an overall prevalence of schistosomiasis in Madagascan pregnant women of 40.4%, with only minor regional differences and differences between serology- and blood PCR-based surveillance. The S. mansoni specific real-time PCR showed superior sensitivity of 74% (specificity 80%) compared with the genus-specific real-time PCR (sensitivity 13%, specificity 100%) in blood. The laborious immunofluorescence (sensitivity IgM 49%, IgG 87%, specificity IgM 85%, IgG 96%) scored only slightly better than the automatable ELISA (sensitivity IgM 38%, IgG 88%, specificity IgM 78%, IgG 91%). Infections with S. mansoni were detected only. The high prevalence of schistosomiasis recorded here among pregnant women in Madagascar calls for actions in order to reduce the disease burden.

scholarly journals Can Testing Predict SARS-CoV-2 Infectivity? The Potential for Certain Methods to be a Surrogate for Replication-Competent Virus

2021 ◽  
Author(s):  
Matthew J. Binnicker
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Viral Rna ◽  
Molecular Diagnostic ◽  
Public Health Agencies ◽  
Diagnostic Assays ◽  
Molecular Tests ◽  
Primary Means ◽  
Clinical Illness ◽  
Antigen Tests

Since the beginning of the COVID-19 pandemic, molecular methods (e.g., real-time PCR) have been the primary means of diagnosing the disease. It is now well-established that molecular tests can continue to detect SARS-CoV-2 genomic RNA for weeks or months following the resolution of clinical illness. This has prompted public health agencies to recommend a symptom and/or time-based strategy for discontinuation of isolation precautions, which for hospitalized patients, results in significant use of personal protective equipment. Due to the inability of current molecular diagnostic assays to differentiate between the presence of remnant viral RNA (i.e., non-infectious) and replication-competent (i.e., infectious) virus, there has been interest in determining whether laboratory tests can be used to predict an individual’s likelihood of transmitting the virus to others. This review will highlight what is currently known about the potential for existing assays, such as real-time PCR and antigen tests, to predict active viral infection. In addition, data on the performance of new methods, such as molecular tests targeting viral RNA intermediates (e.g., subgenomic RNA), will be discussed.

scholarly journals A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e89395 ◽  
Author(s):  
Jiyoun Yeo ◽  
Erin L. Crawford ◽  
Thomas M. Blomquist ◽  
Lauren M. Stanoszek ◽  
Rachel E. Dannemiller ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Testing ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Testing ◽  
Ffpe Samples ◽  
Pcr Method

scholarly journals Variola Virus-Specific Diagnostic Assays: Characterization, Sensitivity, and Specificity

2015 ◽  
Vol 53 (4) ◽  
pp. 1406-1410 ◽  
Author(s):  
Ashley V. Kondas ◽  
Victoria A. Olson ◽  
Yu Li ◽  
Jason Abel ◽  
Miriam Laker ◽  
...  
Keyword(s):  
Public Health ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Variola Virus ◽  
Public Health Response ◽  
Diagnostic Assays ◽  
Health Response ◽  
Pcr Assays

A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified.

Parasitological versus molecular diagnosis of strongyloidiasis in serial stool samples: how many?

2017 ◽  
Vol 92 (1) ◽  
pp. 12-16 ◽  
Author(s):  
E. Dacal ◽  
J.M. Saugar ◽  
T. Soler ◽  
J.M. Azcárate ◽  
M.S. Jiménez ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Correct Diagnosis ◽  
Molecular Techniques ◽  
Maximum Sensitivity ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Techniques ◽  
Molecular Technique ◽  
Asymptomatic Patients ◽  
Stool Samples

AbstractStrongyloidiasis is usually an asymptomatic disease in immunocompetent patients, caused by Strongyloides stercoralis. However, in immunocompromised patients it can produce a severe clinical profile. Therefore, a correct diagnosis is necessary in these cases and in those chronic asymptomatic patients. The low sensitivity of classical parasitological techniques requires the analysis of multiple serial stool samples. Molecular diagnostic techniques represent an improvement in the detection of the parasite. The objective of this study was to evaluate the minimum number of samples necessary to achieve maximum sensitivity by real-time polymerase chain reaction (PCR). A total of 116 stool samples from 39 patients were analysed by direct microscopic observation, agar culture, Harada–Mori and real-time PCR, in one, two, three and four or more consecutive samples. After two serial samples, 6 out of 39 patients were positive by parasitological and molecular techniques, while 16 of them were real-time PCR positive, and all the patients detected by parasitology were also detected by the molecular technique, reaching 100.00% sensitivity versus 83.00% when analysing a single sample. These data also reflect apparently low specificity (51.52%) and positive predictive value (PPV) (27.27 %) values, due to the high number of cases detected by real-time PCR and not by parasitological techniques. These cases were confirmed as true positives when analysing three, four or more samples from the same patient. In conclusion, the application of molecular techniques decreases the number of serial stool samples necessary to give a diagnosis with the maximum sensitivity.

scholarly journals A critical assessment of two real-time PCR assays targeting the (SSU) rRNA and gdh genes for the molecular identification of Giardia intestinalis in a clinical laboratory

2014 ◽  
Vol 67 (9) ◽  
pp. 811-816 ◽  
Author(s):  
Samuel Boadi ◽  
Spencer D Polley ◽  
Sally Kilburn ◽  
Graham A Mills ◽  
Peter L Chiodini
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Laboratory ◽  
Protozoan Parasite ◽  
Molecular Techniques ◽  
Giardia Intestinalis ◽  
Diagnostic Sensitivity ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Pcr Assays

IntroductionGiardiasis is an intestinal diarrhoeal illness caused by the flagellate protozoan parasite Giardia intestinalis. Molecular techniques for the identification of G. intestinalis have generally been shown to offer a better detection rate of the parasite than the traditional faecal concentration and microscopy techniques.AimThe aim of this study was to critically assess the performance of a commercial and a published real-time PCR assay for their potential use as frontline tests for the diagnosis of giardiasis.MethodsA composite reference standard of enzyme immunoassay and rapid membrane test was used in a diagnostic accuracy study to assess the performance of Primerdesign's, and Verweij et alG. intestinalis real-time PCR assays, comparing them with the traditional ova, cysts and parasite microscopy test (OCP-M).ResultsThe Verweij real-time PCR used primers for the (SSU) rRNA gene, and produced a diagnostic sensitivity of 93.4% (95% CI 88.30% to 98.50%) and an efficiency of 100%. Primerdesign's real-time PCR used primers for the glutamate dehydrogenase gene and produced a diagnostic sensitivity of 61.5% (95% CI 51.50% to 71.50%) and an efficiency of 203%. The OCP-M sensitivity was 83.5% (95% CI 75.87% to 91.13%).ConclusionsThe Verweij real-time PCR was robust and the most sensitive assay suited for use as a first-line diagnostic test for giardiasis.

scholarly journals Conventional PCR Detection and Real-Time PCR Quantification of Reniform Nematodes

Plant Disease ◽  
2012 ◽  
Vol 96 (12) ◽  
pp. 1757-1762 ◽  
Author(s):  
Ronald J. Sayler ◽  
Courtney Walker ◽  
Fiona Goggin ◽  
Paula Agudelo ◽  
Terrence Kirkpatrick
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Its Region ◽  
Taqman Probe ◽  
Reniform Nematode ◽  
Rotylenchulus Reniformis ◽  
Conventional Pcr ◽  
Diagnostic Assays ◽  
Reniform Nematodes

Reniform nematode (Rotylenchulus reniformis) is a relatively recent introduction into the continental United States that can cause major yield losses on a variety of important crops including cotton and soybeans. DNA sequences from the internal transcribed spacer (ITS) region of this nematode were used to design primers for conventional and real-time PCR, as well as a TaqMan probe. These primers amplified DNA of reniform nematode isolates from a wide geographic range but did not detect genetically related species or other pathogenic nematodes found in production fields including Meloidogyne incognita and Heterodera glycines. Both SYBR green and TaqMan assays reliably quantified as little as 100 fg of reniform nematode DNA, and could be used to quantify as few as five reniform nematodes. An inexpensive and rapid DNA extraction protocol for high throughput diagnostic assays is described.

scholarly journals Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera

2019 ◽  
Author(s):  
Barbara Wong ◽  
Isabel Leal ◽  
Nicolas Feau ◽  
Angela Dale ◽  
Adnan Uzunovic ◽  
...  
Keyword(s):  
Real Time ◽  
Plant Pathogens ◽  
Treated Wood ◽  
Forest Products ◽  
Phytophthora Ramorum ◽  
Molecular Diagnostic ◽  
Diagnostic Assays ◽  
Non Timber Forest Products ◽  
Grosmannia Clavigera ◽  
Living Organisms

Abstract/KeywordsTo determine if living microorganisms of phytosanitary concern are present in wood after eradication treatment and to evaluate the efficacy of such treatments, the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can therefore only be produced by living organisms, providing a proxy for viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of wood infected with the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated wood samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and test the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade.

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