High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans

Pangasa, Aradhana; Jex, Aaron R.; Campbell, Bronwyn E.; Bott, Nathan J.; Whipp, Margaret; Hogg, Geoff; Stevens, Melita; Gasser, Robin B.

doi:10.1016/j.mcp.2008.10.003

“…Therefore, the setup developed by Hadfield et al does not circumvent this potential problem, since this genus-specific PCR is based on SSU rRNA gene amplification. Interestingly, detection and differentiation of C. hominis, C. parvum, and C. meleagridis in human fecal samples can be performed by using high-resolution melting curve analysis of amplicons of the ITS2 region (165). Using this assay, C. hominis, C. parvum, and C. meleagridis were detected in 97, 44, and 2 samples, respectively, of 143 Cryptosporidium oocyst DNA samples originating from Australians with clinical cryptosporidiosis, and the results were in agreement with results previously obtained by single-strand conformation polymorphism analysis.…”

Section: Cryptosporidiummentioning

confidence: 99%

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

Verweij

¹

,

Stensvold

²

2014

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SUMMARY Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies.

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“…Until now high-resolution analysis of melting curves seems unparalleled (High Resolution Melting, HRM) [32]. This method enables highperformance mutation sequencing of single-nucleotide variants (SNPs, single nucleotide polymorphisms) smaller deletions and insertions, genotyping [33] or methylation analysis [34]. HRM is fast, simple, cost effective and at the same time high-performance sensitive method.…”

Section: Benefits Of High Resolution Melting Analysismentioning

confidence: 99%

“…It is a closed-tube method; i. e. amplification and analysis are done in the same tube without the need of further post-PCR manipulations, which lowers the risk of contamination [35]. This method was used in bacteriology [36], virology [Steer], mycology [37] and protozoology [33]. This type of DNA analysis has a potential for fast identification and simultaneous genotyping of medically important bacteria, viruses, protozoa or fungi in culture or in clinical or environmental samples.…”

Section: Benefits Of High Resolution Melting Analysismentioning

confidence: 99%

“…HRM should become a practical method for diagnostics, for detection of infection caused by Cryptosporidium, Microsporidia and Blastocystis in small geographic area and for monitoring outbreaks of these pathogens. This method was used for diagnostics of Cryptosporidium infection by authors Pangasa et al [33] where species C. hominis, C. parvum and C. meleagridis were identified. The use of this methods is also promising for identification and genotyping of other Cryptosporidium spp.…”

Section: Benefits Of High Resolution Melting Analysismentioning

confidence: 99%

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Untitled

2015

JDVAR

0

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Microsporidiosis, cryptosporidiosis, blastocystosis, etc., of humans and animals are an intestinal diseases caused predominantly by infection with zoonotic species and genotypes of these pathogens. These diseases are transmitted mainly via the faecal-oral route. Generally, these deseases are occurs worldwide, but in places with poor sanitation and crowded living conditions is endemic and is associated with source of water and food supply, age, and socioeconomic status [1]. The diagnosis and genetic characterization of the main species and population variants of Microsporidia, Cryptosporidium and Blastocystis infecting humans and animals are central to the prevention, surveillance and control of these diseases, particularly as there is presently no cost effective chemotherapeutic regimen or vaccine available. In this Minireview we want to draw attention to the possibility of using HRM analysis for species and genotypic differentiation of these pathogens in clinical samples of humans and animals. This approach is well suited for the rapid screening of large numbers of Cryptosporidium oocyst, Blastocystis cyst and Microsporidia spores DNA samples and, although qualitative, is significantly less time-consuming to carry out than electrophoretic analysis. This method provides a useful tool for investigating the epidemiology and outbreaks of cryptosporidiosis, microsporidiosis and blastocystosis and could be applicable to identification of species of these pathogens.

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“…31 Until now high-resolution analysis of melting curves seems unparalleled (High Resolution Melting, HRM). 32 This method enables high-performance mutation sequencing of single-nucleotide variants (SNPs, single nucleotide polymorphisms) smaller deletions and insertions, genotyping 33 or methylation analysis. 34 HRM is fast, simple, cost effective and at the same time high-performance sensitive method.…”

Section: Benefits Of High Resolution Melting Analysismentioning

confidence: 99%

Benefits of High Resolution Melting Analysis for Rapid Detection and Genotyping of Selected Protozoal and Fungal Parasites

Valenčáková¹

2015

JDVAR

0

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Microsporidiosis, cryptosporidiosis, blastocystosis, etc., of humans and animals are an intestinal diseases caused predominantly by infection with zoonotic species and genotypes of these pathogens. These diseasesare transmitted mainly via the faecaloral route.Generally, these deseasesare occurs worldwide, but in places with poor sanitation and crowded living conditions is endemic and is associated with source of water and food supply, age, and socioeconomic status. 1 The diagnosis and genetic characterization of the main species and population variants of Microsporidia, Cryptosporidium and Blastocystis infecting humans andanimalsare central to the prevention, surveillance and control of these diseases, particularly as there is presently no cost effective chemotherapeutic regimen or vaccine available. In this Minireview we want to draw attention to the possibility of using HRM analysis for species and genotypic differentiation of these pathogens in clinical samples of humans and animals. This approach is well suited for the rapid screening of large numbers of Cryptosporidium oocyst, Blastocystis cyst and Microsporidia spores DNA samples and, although qualitative, is significantly less time-consuming to carry out than electrophoretic analysis. This method provides a useful tool for investigating the epidemiology and outbreaks of cryptosporidiosis, microsporidiosis and blastocystosis and could be applicable to identification of species of these pathogens. Keywords Benefits of high resolution melting analysisEarly and correct diagnosis of disease is necessary for the beginning of rapid and effective actions that are essential for managing epidemiologic and epizootologic situation. Molecular identification and genotyping of pathogens is important part of this process, because response speed is decisive for minimization of final impacts from emerging outbreaks.27 Therefore, methods used for molecular identification and typing must be fast, credible and easily reproducible. Currently, various DNA analysis methods for detection of causal gene mutation or polymorphism associated with disease are used. Even though Sanger sequencing is still considered as a "golden standard" for detection of unknown mutations, there is a need to develop and use new methods which can reduce cost and time needed for individual examinations. There are many methods that are capable of detecting changes in nucleotide sequence of DNA such as: SSCP (single-strand conformation polymorphism analysis), 28 Conflict of interestAuthor declares that there is no conflict of interest. References

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High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans

Cited by 61 publications

References 68 publications

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

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Benefits of High Resolution Melting Analysis for Rapid Detection and Genotyping of Selected Protozoal and Fungal Parasites

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