P181 Development of a new diagnostic method for histoplasmosis using a cycling probe-based real-time PCR to detect Histoplasma capsulatum

The ectoparasitic stubby root nematode,Paratrichodorus allius, transmits tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. A diagnostic method for direct quantification ofP. alliusfrom soil DNA using TaqMan probe and SYBR Green real-time PCR assays was developed to assist the potato industry in management of this important vector. Specificity of primers/probe designed from the internal transcribed spacer of ribosomal DNA ofP. alliuswas demonstrated byin silicoanalysis and experimental PCR tests with no cross reactions using non-target nematode species and nematode communities. The SYBR Green method was more sensitive than the TaqMan probe method during detection using serial diluted DNA templates. Standard curves were generated from serial dilutions of DNA extracted from autoclaved soil with artificially inoculatedP. alliusindividuals and were validated by high correlations between the numbers of target nematodes quantified by the assays and added to the soil. Moreover, the numbers ofP. alliusdetermined by the real-time PCR assays and estimated by the microscopic method in 17 field soil samples presented positive correlation relationships (). Although the quantification using TaqMan probe overestimated the target nematodes compared to using SYBR Green in eight out of ten field soil samples, results of the two methods correlated well (). This is the first report ofP. alliusquantification from soil DNA extracts using real-time PCR, providing a rapid and sensitive diagnostic method obviating time-consuming manual nematode extraction from soil and microscopic identification and quantification.

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Applying a Real-Time PCR Assay for Histoplasma capsulatum to Clinically Relevant Formalin-Fixed Paraffin-Embedded Human Tissue

Journal of Clinical Microbiology ◽

10.1128/jcm.01705-12 ◽

2012 ◽

Vol 50 (10) ◽

pp. 3395-3397 ◽

Cited By ~ 32

Author(s):

S. A. Koepsell ◽

S. H. Hinrichs ◽

P. C. Iwen

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Human Tissue ◽

Histoplasma Capsulatum ◽

Pcr Assay ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Detection of Blastomyces dermatitidis and Histoplasma capsulatum from Culture Isolates and Clinical Specimens by Use of Real-Time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.00673-11 ◽

2011 ◽

Vol 49 (9) ◽

pp. 3204-3208 ◽

Cited By ~ 72

Author(s):

N. Esther Babady ◽

Seanne P. Buckwalter ◽

Leslie Hall ◽

Kara M. Le Febre ◽

Matthew J. Binnicker ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Histoplasma Capsulatum ◽

Blastomyces Dermatitidis ◽

Clinical Specimens

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Developing a real-time PCR diagnostic method for a potential threat to chrysanthemum, Paratylenchus dianthus

Journal of Nematology ◽

10.21307/jofnem-2019-043 ◽

2019 ◽

Vol 51 ◽

pp. 1-11

Author(s):

Masanori Kawanobe ◽

Koki Toyota ◽

Hidehito Uchihara ◽

Mikoto Takae

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Diagnostic Method ◽

Potential Threat

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Comparative assessment of conventional PCR with multiplex real-time PCR using SYBR Green I detection for the molecular diagnosis of imported malaria

Parasitology ◽

10.1017/s0031182003004219 ◽

2004 ◽

Vol 128 (1) ◽

pp. 15-21 ◽

Cited By ~ 22

Author(s):

R. FABRE ◽

A. BERRY ◽

B. MORASSIN ◽

J. F. MAGNAVAL

Keyword(s):

Plasmodium Falciparum ◽

Real Time ◽

Real Time Pcr ◽

Diagnostic Method ◽

Comparative Assessment ◽

Falciparum Infection ◽

Imported Malaria ◽

Conventional Pcr ◽

University Hospitals ◽

Conventional Microscopy

For the diagnosis of imported malaria, optical or immunochromatographic methods are known to be less sensitive and less specific than PCR-based methods, which are conversely more complicated and time-consuming. An original strategy, based upon the sequential use of a multiplex competitive real-time PCR detectingPlasmodium falciparumorPlasmodiumspp. infection, followed by, if necessary, a single real-time PCR for species identification, was therefore performed and then tested versus conventional PCR in routine conditions. Conventional PCR has been used since October 1999 in the Department of Parasitology, University Hospitals in Toulouse, as a 2nd line diagnostic method. Out of 183 patients tested, 48 were found to be harbouring a falciparum infection by conventional microscopy, 60 by conventional PCR and 60 by multiplex competitive real-time PCR. Nine further patients had a non-falciparum infection, and concordant species identifications were obtained by both conventional PCR and single real-time PCR. The major value of PCR-based methods, when compared to microscopical techniques, was to ascertain the negativity of a suspect sample. Moreover, real-time PCR allows simplification of the operating procedure, with a diagnosis being made within 2 h.

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Differential Diagnosis of Fungal Pneumonias vs. Tuberculosis in AIDS Patients by Using Two New Molecular Methods

Journal of Fungi ◽

10.3390/jof7050336 ◽

2021 ◽

Vol 7 (5) ◽

pp. 336

Author(s):

Leticia Bernal-Martínez ◽

Laura Herrera ◽

Clara Valero ◽

Paula de la Cruz ◽

Larisa Ghimpu ◽

...

Keyword(s):

Differential Diagnosis ◽

Real Time ◽

Real Time Pcr ◽

Histoplasma Capsulatum ◽

Cost Effective ◽

Diagnostic Tools ◽

Clinical Samples ◽

Pcr Assay ◽

Aids Patients

Opportunistic fungal pneumonias (OFP) are the main cause of death in AIDS patients worldwide. Diagnosis of these infections is often late as tuberculosis (TB) is frequently the first suspicion. In addition, diagnostic tools have limitations and are unavailable in disadvantaged regions. To perform the differential diagnosis of the main fungi causing OFP in AIDS patients (Histoplasma capsulatum, Cryptococcus neoformans/C. gattii and Pneumocystis jirovecii) vs. the Mycobacterium tuberculosis complex (MTBC), two new assays were developed: (i) a multiplex real-time PCR (MRT-PCR) and (ii) a simple and cost-effective method based on real-time PCR and the analysis of melting curves after amplification (MC-PCR). Both of the techniques were optimized and standardized “in vitro”, showing a suitable reproducibility (CV ranged between 1.84 and 3.81% and 1.41 and 4.83%, respectively), a 100% specificity and detection limits between 20 and 2 fg of genomic DNA per 20 µL of reaction. A validation study was performed by retrospectively using 42 clinical samples from 37 patients with proven fungal infection or TB, and 33 controls. The overall sensitivity for the MRT-PCR assay and the MC-PCR assay was 88% and 90.4%, respectively. Both techniques were fast, sensitive and reproducible, allowing for the detection of these pathogens and the performance of a differential diagnosis.

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