Identification of opportunistic and nonopportunistic Exophiala species using high resolution melting analysis

2020 ◽  
Vol 58 (7) ◽  
pp. 938-945
Author(s):  
Tanaz Bahadori ◽  
Mojtaba Didehdar ◽  
Behzad Khansarinezhad ◽  
Tahereh Shokohi
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Its Region ◽  
Clinical Samples ◽  
Phenotypic Differences ◽  
Physiological And Biochemical Characteristics ◽  
Environmental Reference ◽  
Melting Analysis ◽  
Primer Sets ◽  
Exophiala Species

Abstract Exophiala is a genus comprising several species of opportunistic black yeasts. Exophiala species identification by morphological, physiological, and biochemical characteristics is challenging because of the low degree of phenotypic differences between species and its polyphyletic nature. We aimed to develop a high-resolution melting (HRM) assay based on the internal transcribed spacer (ITS) region to differentiate between pairs of clinical and environmental Exophiala species. HRM primers were designed based on the conserved ITS region of five Exophiala species (E. dermatitidis, E. phaeomuriformis, E. heteromorpha, E. xenobiotica, and E. crusticola). Environmental and clinical Exophiala isolates representing these five species (n = 109) were analyzed. The HRM assay was optimized using clinical and environmental reference isolates (n = 22), and then the results were compared with those obtained with nonreference isolates of Exophiala (n = 87) using two designed primer sets. The designed HRM assay was based on the normalized melting peak approach and two primer sets, and successfully distinguished between the five Exophiala species. The HRM1 primer set provided sufficient resolution, with a melting temperature (Tm) difference of approximately 2.5°C among the analyzed species and of approximately 1°C between E. dermatitidis and E. phaeomuriformis. HRM typing results were in agreement with those of ITS-sequence typing (100% sensitivity and specificity). The developed HRM assay can be used to ascertain the identity of Exophiala species, which may differ in clinical significance, with high accuracy. Its application to identify species directly in clinical samples and/or environmental niches may be possible in the future.

scholarly journals High-resolution melting analysis of gyrA codon 84 and grlA codon 80 mutations conferring resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples

2017 ◽  
Vol 29 (5) ◽  
pp. 711-715 ◽  
Author(s):  
Monica Loiacono ◽  
Piera A. Martino ◽  
Francesca Albonico ◽  
Francesca Dell’Orco ◽  
Manuela Ferretti ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Opportunistic Pathogen ◽  
High Resolution Melting Analysis ◽  
Clinical Samples ◽  
Mutation Site ◽  
Staphylococcus Pseudintermedius ◽  
Melting Analysis ◽  
Resistant Strains ◽  
Susceptibility Profiles

Staphylococcus pseudintermedius is an opportunistic pathogen of dogs and cats. A high-resolution melting analysis (HRMA) protocol was designed and tested on 42 clinical isolates with known fluoroquinolone (FQ) susceptibility and gyrA codon 84 and grlA codon 80 mutation status. The HRMA approach was able to discriminate between FQ-sensitive and FQ-resistant strains and confirmed previous reports that the main mutation site associated with FQ resistance in S. pseudintermedius is located at position 251 (Ser84Leu) of gyrA. Routine, HRMA-based FQ susceptibility profiles may be a valuable tool to guide therapy. The FQ resistance-predictive power of the assay should be tested in a significantly larger number of isolates.

scholarly journals Genotype Characteristics of Giardia duodenalis in Patients Using High Resolution Melting Analysis Technique in Khorramabad, Iran

2020 ◽  
Author(s):  
Akram SEPAHVAND ◽  
Ahmad HOSSEINI-SAFA ◽  
Hossein Ali YOUSOFI ◽  
Mohammad Hassan TAJEDINI ◽  
Reza PAHLAVAN GHAREHBABAH ◽  
...  
Keyword(s):  
High Resolution ◽  
Triosephosphate Isomerase ◽  
High Resolution Melting ◽  
Sucrose Solution ◽  
Giardia Duodenalis ◽  
Pcr Amplification ◽  
Clinical Samples ◽  
Analysis Technique ◽  
Melting Analysis ◽  
Comparison Of The Results

Background: We aimed at genotyping and evaluating the predominance of G. duodenalis assemblages isolated from patients referred to medical laboratories in Khorramabad, Iran from Nov 2015 to Sep 2016. Hence, the development of a costeffective HRM approach to determine genotypes of G. duodenalis based on the triosephosphate isomerase (tpi) gene was examined and the genotyping results with and without diarrhea was compared. Methods: Seventy G. duodenalis positive fecal samples were collected. A microscopic confirmation for the presence of Giardia spp. was performed, cysts of 70 Giardia spp. positive specimens were concentrated using sucrose flotation technique and sucrose solution PCR amplification was performed on 69 of 70 (98.5%) samples, and High Resolution Melting (HRM) analysis was performed using a software. Results: The results showed two distinct genotypes (assemblages A and B) of G. duodenalis but infections with mixture of both assemblages were not detected. The genotypes of G. duodenalis showed that the sub assemblage AI, BIII and BIV were present in a proportion of 68.1%, 20.3% and 11.6% respectively in samples. Assemblage AI was significantly (P<0.05) more frequently found in patients with diarrhea. Conclusion: The sub-assemblage AI, BIII, and BIV are more zoonotic potential. According to the comparison of the results of this study with the results of previous studies in this area and around of it, as well as the way people live and keep pets. This pattern established in Khorramabad city. HRM can be an ideal technique to detect and genotyping of G. duodenalis in clinical samples.

scholarly journals Exploring the Use of High-Resolution Melting Analysis and Helicase-Dependent Amplification for Discrimination of Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species and Trialeurodes vaporariorum

2020 ◽  
Vol 113 (5) ◽  
pp. 2511-2520
Author(s):  
Sharon A Andreason ◽  
Mohammad Arif ◽  
Judith K Brown ◽  
Francisco Ochoa-Corona ◽  
Astri Wayadande
Keyword(s):  
High Resolution ◽  
Bemisia Tabaci ◽  
High Resolution Melting ◽  
Trialeurodes Vaporariorum ◽  
High Resolution Melting Analysis ◽  
Molecular Techniques ◽  
Greenhouse Whitefly ◽  
End Point ◽  
Melting Analysis ◽  
Primer Sets

Abstract The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera; Aleyrodidae), and greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), are highly problematic plant pests and virus vectors with worldwide distributions. Identification of whitefly species is typically accomplished by observation of distinct morphological characters; however, because of morphological inconsistency and indistinguishability, the discrimination of B. tabaci species variants is dependent on molecular techniques based on genetic differences. New assays were designed for the detection of B. tabaci A, B, and Q mitotype groups, and T. vaporariorum. Specific primer sets were designed for amplification of the mitochondrial cytochrome c oxidase I gene of the four targets to perform in end-point PCR, real-time PCR coupled to high-resolution melting analysis (HRM), and the isothermal helicase-dependent amplification (HDA). Primer specificities were validated using end-point PCR, then tested in HRM and HDA. Bemisia tabaci A, B, and Q mitotypes, and T. vaporariorum-targeted primer sets discriminately amplified specimens of different populations within their target whitefly group. These tests provide three novel discrimination assays for the high-consequence, exotic B. tabaci B and Q groups, along with the native B. tabaci A group and T. vaporariorum.

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