High-resolution melting-curve (HRM) analysis for C. meleagridis identification in stool samples

Chelbi, Hanène; Jelassi, Refka; Bouzekri, Nesrine; Zidi, Inès; Salah, Hamza Ben; Mrad, Ilhem; Sghaier, Ikram; Abdelmalek, R.; Aissa, Sameh; Bouratbine, A.; Karim, Abdul

doi:10.1016/j.micpath.2017.12.070

Cited by 7 publications

(6 citation statements)

References 20 publications

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“…In this research, Cyt b and ITS-rDNA genes were targeted, and HRM technique was modified to be more trustworthy, strong, slight, and obvious, to allow the correct genotyping of Leishmania species 22 , 32 . In this method, new design of dedicated primers has been one of the most important parts of the work.…”

Section: Discussionmentioning

confidence: 99%

Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

AlaeeNovin

Parvizi

Ghafari

2022

Rev. Soc. Bras. Med. Trop.

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Background: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification.Methods: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High-Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis.Results: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol.Conclusions: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

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Section: Discussionmentioning

confidence: 99%

Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

AlaeeNovin

Parvizi

Ghafari

2022

Rev. Soc. Bras. Med. Trop.

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“…Previous studies showed that HRM method could diagnose different subtypes of influenza A 31 , astroviruses 32 , C. meleagridis 23 , and Yersinia pseudotuberculosis 33 . Besides, melting curve alteration in HRM method discriminated Iranian Leishmania parasites of L. major , L. tropica and mixed infection in the study of Ghafari SM et al 34 .…”

Section: Discussionmentioning

confidence: 99%

“…The advantage of HRM method is the amplification of short DNA fragments with high resolution and accuracy. However, some limitations, such as the mismatch of intercalated dyes to ambiguous sequences, like dimer primers and nonspecific products, should not be ignored 23 . This can lead to missing fragments that have an identical melting point 20 .…”

Section: Introductionmentioning

confidence: 99%

Discrimination of human papillomavirus genotypes using innovative technique nested-high resolution melting

Alirezaei

Mosawi²,

Afgar

et al. 2022

Sci Rep

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The prompt detection of human papillomavirus and discrimination of its genotypes by combining conventional methods in new molecular laboratories is essential to achieve the global call of eliminating cervical cancer. After predicting the melting temperature of an approximately 221 bp region of the L1 gene from different HPV genotypes by bioinformatics software, an innovative technique based on the nested- high resolution melting was designed with three approaches and using conventional PCR, qPCR, and diagnostic standards. HPV-positive samples identified by microarray along with diagnostic standards were evaluated by qPCR-HRM and discordant results were subjected to sequencing and analyzed in silico using reference types. In addition to screening for human papillomavirus, nested-qPCR-HRM is one of the modified HRM techniques which can discriminate some genotypes, including 6, 16, 18, 52, 59, 68 and 89. Despite the differences in diagnostic capabilities among HRM, microarray and sequencing, a number of similarities between HRM, and sequencing were diagnostically identified as the gold standard method. However, the bioinformatics analysis and melting temperature studies of the selected region in different HPV genotypes showed that it could be predicted. With numerous HPV genotypes and significant genetic diversity among them, determining the virus genotype is important. Therefore, our goal in this design was to use the specific molecular techniques with several specific primers to increase sensitivity and specificity for discriminating a wide range of HPV genotypes. This approach led to new findings to evaluate the ability of different approaches and procedures in accordance with bioinformatics.

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“…HRM analysis is sensitive enough to resolve a single base pair difference between amplicons, and has been applied to single nucleotide polymorphism (SNP) and tandem-repeat number based genotyping and determination of DNA methylation status [19] . Recently HRM analysis has been applied to the differentiation of Cryptosporidium species from other apicomplexan parasites, Toxoplasma gondii, Sarcocystis species, Neospora species, and to differentiate between Cryptosporidium species [20] , [21] , [22] . HRM analysis has also been successfully applied to the intra-species differentiation of Cryptosporidium cuniculus gp60 subtypes Va and Vb [23] .…”

Section: Methods Detailsmentioning

confidence: 99%

Development of novel methodology for the molecular differentiation of Cryptosporidium parvum gp60 subtypes via high resolution melting analysis

et al. 2020

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High-resolution melting-curve (HRM) analysis for C. meleagridis identification in stool samples

Cited by 7 publications

References 20 publications

Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

Discrimination of human papillomavirus genotypes using innovative technique nested-high resolution melting

Development of novel methodology for the molecular differentiation of Cryptosporidium parvum gp60 subtypes via high resolution melting analysis

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