scholarly journals Evaluation of the Multiplex Real-Time PCR DermaGenius® Assay for the Detection of Dermatophytes in Hair Samples from Senegal

2021 ◽  
Vol 8 (1) ◽  
pp. 11
Author(s):  
Mouhamadou Ndiaye ◽  
Rosalie Sacheli ◽  
Khadim Diongue ◽  
Caroline Adjetey ◽  
Rajae Darfouf ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Diagnostic Methods ◽  
Melting Curve Analysis ◽  
Microsporum Canis ◽  
Molecular Tools ◽  
Pcr Assay ◽  
Epidermophyton Floccosum ◽  
Hair Samples

For the successful treatment of dermatophytoses, especially tinea capitis, there is a need for accurate and rapid diagnostic methods. A lot of recent literature has focused on the detection of dermatophytes directly on sample material such as nails, hair and skin scrapings. Molecular tools offer the ability to rapidly diagnose dermatophytosis within 48 h. This study aimed to compare the results of a commercial real-time PCR (real-time PCR) assay DermaGenius®(DG) 2.0 complete multiplex kit with those of conventional diagnostic methods (direct microscopy and culture). A total of 129 hair samples were collected in Dakar (Senegal) from patients suspected of dermatophytosis. DG was applied for the molecular detection of Candida albicans, Trichophyton rubrum/soudanense, T. interdigitale, T. tonsurans, T. mentagrophytes, T. violaceum, Microsporum canis, M. audouinii, Epidermophyton floccosum, T. benhamiae and T. verrucosum. Dermatophytes species and C. albicans were differentiated by melting curve analysis. The sensitivity and specificity of the PCR assay were 89.3% and 75.3%, respectively. DG PCR was significantly more sensitive than culture (p < 0.001). DG PCR is fast and robust to contamination. In this paper, the main questions discussed were the replacement of culture by a broad-spectrum fungal real-time PCR and the implementation of DG PCR into a routine laboratory in Senegal.

scholarly journals Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples

2005 ◽  
Vol 11 (9) ◽  
pp. 713-718 ◽  
Author(s):  
M.I. Queipo-Ortuño ◽  
J.D. Colmenero ◽  
J.M. Reguera ◽  
M.A. García-Ordoñez ◽  
M.E. Pachón ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Rapid Diagnosis ◽  
Curve Analysis ◽  
Sybr Green I ◽  
Melting Curve Analysis ◽  
Human Brucellosis ◽  
Pcr Assay ◽  
Serum Samples

scholarly journals Rapid detection and grouping of porcine bocaviruses by an EvaGreen ® based multiplex real-time PCR assay using melting curve analysis

2016 ◽  
Vol 30 (4) ◽  
pp. 195-204 ◽  
Author(s):  
Xiaowen Zheng ◽  
Gaopeng Liu ◽  
Tanja Opriessnig ◽  
Zining Wang ◽  
Zongqi Yang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Pcr Assay

scholarly journals Rapid and Accurate Identification of Candida albicans and Candida dubliniensis by Real-Time PCR and Melting Curve Analysis

2018 ◽  
Vol 27 (6) ◽  
pp. 543-548 ◽  
Author(s):  
Mohammad Asadzadeh ◽  
Suhail Ahmad ◽  
Noura Al-Sweih ◽  
Ziauddin Khan
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gel Electrophoresis ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Pcr Assay ◽  
Candida Spp ◽  
Accurate Identification

Objective: Candida albicans and Candida dubliniensis are germ tube-positive pathogenic yeast species. Accurate identification of these two species is warranted since C. albicans is a highly pathogenic species while C. dubliniensis exhibits increased adherence to buccal epithelial cells, reduced susceptibility to azoles and resistance to flucytosine. We have developed a duplex real-time PCR assay for rapid detection and differentiation between clinical C. albicans and C. dubliniensis isolates. Materials and Methods: A duplex real-time PCR assay was developed by using two species-specific primer pairs and SYBR Green dye to differentiate C. albicans and C. dubliniensis isolates via melting curve analysis of real-time PCR amplicons. Amplification products were also analyzed by agarose gel electrophoresis to confirm real-time PCR results. Results: Melting temperatures (Tm) for reference strains of C. albicans and C. dubliniensis were 86.55 and 82.75°C, respectively. No amplicon was obtained with DNA from reference strains of 8 other common Candida spp. When real-time PCR was applied on 226 clinical isolates previously identified by the Vitek 2 system and/or PCR sequencing of rDNA, Tm values for C. albicans (n = 113) and C. dubliniensis (n = 98) were 86.68 ± 0.529 and 82.616 ± 0.535°C, respectively. The results were confirmed by agarose gel electrophoresis. No amplicon was obtained from 15 isolates belonging to 9 other Candida spp. Conclusions: The real-time PCR assay described here does not require prior identification of clinical yeast isolates as C. albicans/C. dubliniensis by germ tube formation and accurately reports results within 2 h. Detection of amplicons by agarose gel electrophoresis is also suitable for resource-poor settings devoid of real-time PCR facilities.

Development of a real-time PCR assay with fluorophore-labelled hybridization probes for detection of Schistosoma mekongi in infected snails and rat feces

Parasitology ◽  
2012 ◽  
Vol 139 (10) ◽  
pp. 1266-1272 ◽  
Author(s):  
O. SANPOOL ◽  
P. M. INTAPAN ◽  
T. THANCHOMNANG ◽  
P. SRI-AROON ◽  
V. LULITANOND ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
High Sensitivity ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Intermediate Hosts ◽  
Pcr Assay ◽  
Fecal Samples ◽  
Schistosoma Mekongi

SUMMARYSchistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts.

Optimization of duplex real-time PCR with melting-curve analysis for detecting the microsporidian parasites Nosema apis and Nosema ceranae in Apis mellifera

2010 ◽  
Vol 142 (3) ◽  
pp. 271-283 ◽  
Author(s):  
Karen L. Burgher-MacLellan ◽  
Geoffrey R. Williams ◽  
Dave Shutler ◽  
Kenna MacKenzie ◽  
Richard E.L. Rogers
Keyword(s):  
Apis Mellifera ◽  
Real Time ◽  
Real Time Pcr ◽  
Honey Bees ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Nosema Apis ◽  
Conventional Pcr ◽  
Pcr Assay

AbstractHoney bees, Apis mellifera (L.) (Hymenoptera: Apidae), are parasitized by the microsporidians Nosema apis (Zander) and Nosema ceranae (Fries). Molecular techniques are commonly used to differentiate between these parasites because light microscopy is inadequate. Our objectives were to (i) adapt the previously published duplex polymerase chain reaction (PCR) targeting the 16S rRNA gene of N. apis (321APIS-FOR, 321APIS-REV) and N. ceranae (218MITOC-FOR, 218MITOC-REV) using qualitative real-time PCR assay with SYBR® Green I dye (R-T PCR) and DNA melting-curve analysis, and (ii) determine whether the two Nosema species can be detected simultaneously in honey bees. Total spore counts and purified total genomic DNA were obtained from 37 bee samples (19 individual workers and 18 pooled samples of 15 workers) collected in Nova Scotia, Prince Edward Island, and Newfoundland, Canada. Overall, the prevalence of Nosema species was 86.5% (32/37 samples of bee DNA), based on conventional PCR and the optimized R-T PCR assay. The melting-curve analysis showed three groups of curve profiles that could determine the prevalence of N. apis, N. ceranae, and co-infection (21.9%, 56.2%, and 21.9%, respectively). The duplex R-T PCR assay was efficient, specific, and more sensitive than duplex conventional PCR because co-infection was identified in 5.4% (n = 2) more samples. Sequencing of R-T PCR products confirmed the results of the melting-curve analysis. Duplex R-T PCR with melting-curve analysis is a sensitive and rapid method of detecting N. apis, N. ceranae, and co-infection in honey bees.

scholarly journals New Panfungal Real-Time PCR Assay for Diagnosis of Invasive Fungal Infections

2016 ◽  
Vol 54 (12) ◽  
pp. 2910-2918 ◽  
Author(s):  
Clara Valero ◽  
Laura de la Cruz-Villar ◽  
Óscar Zaragoza ◽  
María José Buitrago
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fungal Infections ◽  
Melting Curve ◽  
Fungal Species ◽  
Invasive Fungal Infections ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Pcr Assay ◽  
Suitable Alternative

The diagnosis of invasive fungal infections (IFIs) is usually based on the isolation of the fungus in culture and histopathological techniques. However, these methods have many limitations often delaying the definitive diagnosis. In recent years, molecular diagnostics methods have emerged as a suitable alternative for IFI diagnosis. When there is not a clear suspicion of the fungus involved in the IFI, panfungal real-time PCR assays have been used, allowing amplification of any fungal DNA. However, this approach requires subsequent amplicon sequencing to identify the fungal species involved, increasing response time. In this work, a new panfungal real-time PCR assay using the combination of an intercalating dye and sequence-specific probes was developed. After DNA amplification, a melting curve analysis was also performed. The technique was standardized by using 11 different fungal species and validated in 60 clinical samples from patients with proven and probable IFI. A melting curve database was constructed by collecting those melting curves obtained from fungal species included in the standardization assay. Results showed high reproducibility (coefficient of variation [CV] < 5%; r > 0.95) and specificity (100%). The overall sensitivity of the technique was 83.3%, with the group of fungi involved in the infection detected in 77.8% of the positive samples with IFIs covered by molecular beacon probes. Moreover, sequencing was avoided in 67.8% of these “probe-positive” results, enabling report of a positive result in 24 h. This technique is fast, sensitive, and specific and promises to be useful for improving early diagnosis of IFIs.

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