Detection and Genotyping of Oocysts of
            <i>Cryptosporidium parvum</i>
            by Real-Time PCR and Melting Curve Analysis

Tanriverdi, Sultan; Tanyeli, Atila; Başlamışlı, Fikri; Köksal, Fatih; Kılınç, Yurdanur; Feng, Xin; Batzer, Glenda; Tzipori, Saul; Widmer, Giovanni

doi:10.1128/jcm.40.9.3237-3244.2002

Cited by 75 publications

(48 citation statements)

References 37 publications

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“…SYBR Green I was first used in melting analysis to distinguish PCR products that differed in T m by Ն2°C (9 ). Subsequently, SYBR Green I was used to identify deletions (23 ), genotype dinucleotide repeats (24 ), and various sequence alterations (10,(25)(26)(27). However, the T m difference between genotypes can be small and may challenge the resolution of current instruments.…”

Section: Discussionmentioning

confidence: 99%

Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes

et al. 2003

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Background:Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR. Methods: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (T m ). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required ϳ1 min and no sample processing was needed after PCR. Results: Polymorphisms in the HTR2A (T102C), ␤-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2°C/s) of denatured products, followed by rapid heating during melting analysis (0.2-0.4°C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in T m by <0.2°C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5 tail of identical sequence was added to one of the two unlabeled primers.

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Section: Discussionmentioning

confidence: 99%

Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes

et al. 2003

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“…Realtime PCR methods have been utilized in several areas of clinical parasitology, including the detection of fecal parasites. These real-time PCR assays have been shown to be more sensitive and specific than conventional methods (2,18,20,21). This study represents the first report of the application of a real-time PCR assay for the detection of D. fragilis in human fecal specimens.…”

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confidence: 97%

Evaluation of Three Diagnostic Methods, Including Real-Time PCR, for Detection of Dientamoeba fragilis in Stool Specimens

et al. 2006

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Dientamoeba fragilis is a protozoan parasite of humans that infects the mucosa of the large intestine and is associated with gastrointestinal disease. We developed a 5 nuclease (TaqMan)-based real-time PCR assay, targeting the small subunit rRNA gene, for the detection of D. fragilis in human stool specimens and compared its sensitivity and specificity to conventional PCR and microscopic examination by a traditional modified iron-hematoxylin staining procedure. Real-time PCR exhibited 100% sensitivity and specificity.Dientamoeba fragilis is a pathogenic protozoan parasite, often more prevalent than Giardia intestinalis, which causes gastrointestinal disease in humans (5,7,9,17). Due to the propensity of this organism to cause chronic infection, it is essential that correct diagnosis occur promptly (3,4,7,9,13,17). Two genotypes of D. fragilis have been described by analysis of the small subunit rRNA gene (10,12,17,22), with only one predominant in Australia (17).Diagnosis of D. fragilis relies on direct visualization of the trophozoites in stained fixed fecal smears by light microscopy, as demonstration of the characteristic nuclear structure cannot be achieved in unstained fecal specimens (4). D. fragilis may be difficult to distinguish from nonpathogenic protozoa (9).The aim of this study was to develop a real-time PCR method that is rapid, highly sensitive, and specific for the detection of D. fragilis in fecal specimens. Results from the real-time assay were compared to those derived by a conventional PCR and microscopic examination using a traditional modified iron-hematoxylin staining procedure in order to determine the usefulness and practicality of this real-time PCR test.(This research was performed by Damien Stark in partial fulfillment for the degree of Ph.D. at University of Technology Sydney.)Entamoeba histolytica HM-1:IMSS (ATCC strain 30459) and Trichomonas vaginalis (ATCC strain F1623) were passaged in TYI-S-33 broth: genomic DNA was extracted from them using a QIAamp DNA minikit (QIAGEN, Hilden, Germany).Stool specimens used in this study were those submitted to St. Vincent's Hospital Department of Microbiology, Sydney, for investigation of diarrhea. Portions of all stool samples were fixed in sodium acetate-acetic acid-formalin and permanently stained using a modified iron-hematoxylin stain (Fronine, Australia) according to the manufacturer's recommendations. DNA was extracted from fresh fecal specimens (Ͻ24 h old) using a QIAamp DNA stool minikit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. To ensure we were using "best practice" for the preparation of DNA from stool specimens using this kit, we also evaluated a modification of the manufacturer's instructions (8) using four samples positive for D. fragilis by microscopy. As a control, these same samples were also extracted following the manufacturer's instructions, and the two sets of DNA were then tested with conventional and real-time PCR.The small subunit (SSU) rRNA genes from D. fragilis was amplified using the prime...

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“…Molecular detection methods, primarily PCR based, have become increasingly common for the identification of viral and bacterial pathogens and appear to be especially attractive for the detection of protozoan parasites that are difficult to culture, morphologically similar, and capable of causing disease in low infectious doses. Thus, the sensitivity and specificity afforded by molecular detection methods such as PCR, restriction fragment length polymorphism, and real-time PCR have resulted in the development of rapid approaches for the detection and genotyping of protozoa in clinical and environmental samples (4,16,40).…”

Section: Discussionmentioning

confidence: 99%

“…A number of PCR-based assays, including gene amplification with specific primers (17,24,33), multiplex PCR (12,32), restriction fragment length polymorphism (3,5,10,46), and real-time PCR (2,4,16,23,40), have been developed for the identification of protozoan infections. However, the shortcomings of PCRbased assays become apparent during practical applications.…”

mentioning

confidence: 99%

Detection and Genotyping of Entamoeba histolytica , Entamoeba dispar , Giardia lamblia , and Cryptosporidium parvum by Oligonucleotide Microarray

2004

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Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.

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Detection and Genotyping of Oocysts of Cryptosporidium parvum by Real-Time PCR and Melting Curve Analysis

Cited by 75 publications

References 37 publications

Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes

Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes

Evaluation of Three Diagnostic Methods, Including Real-Time PCR, for Detection of Dientamoeba fragilis in Stool Specimens

Detection and Genotyping of Entamoeba histolytica , Entamoeba dispar , Giardia lamblia , and Cryptosporidium parvum by Oligonucleotide Microarray

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