Journal article
Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
Journal of clinical microbiology, Vol.49(11), pp.3892-3904
11/2011
DOI: 10.1128/JCM.r00764-11
PMCID: PMC3209110
PMID: 22042830
Abstract
The
Leishmania
species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate
Leishmania
species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all
Leishmania
species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect
Leishmania
parasites, make species determinations, and quantify
Leishmania
spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between
Leishmania
species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between
Leishmania
species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same
Leishmania
species.
Details
- Title: Subtitle
- Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples
- Creators
- Jason L Weirather - Interdisciplinary Graduate Program in Genetics and Departments of Internal Medicine, Microbiology, Biochemistry, and Epidemiology, University of IowaSelma M. B Jeronimo - Iowa City, Iowa; Department of Biochemistry, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, BrazilShalini Gautam - Banaras Hindu University, Varanasi, IndiaShyam Sundar - Banaras Hindu University, Varanasi, IndiaMitchell Kang - Interdisciplinary Graduate Program in Genetics and Departments of Internal Medicine, Microbiology, Biochemistry, and Epidemiology, University of IowaMelissa A Kurtz - Interdisciplinary Graduate Program in Genetics and Departments of Internal Medicine, Microbiology, Biochemistry, and Epidemiology, University of IowaRashidul Haque - International Center for Diarrheal Disease Research, Bangladesh (ICDDR,B), Dhaka, BangladeshAlbert Schriefer - Immunology Service, Federal University of Bahia, Salvador, BrazilSinésio Talhari - Tropical Medicine Foundation of the Amazon, Manaus, BrazilEdgar M Carvalho - Immunology Service, Federal University of Bahia, Salvador, BrazilJohn E Donelson - Interdisciplinary Graduate Program in Genetics and Departments of Internal Medicine, Microbiology, Biochemistry, and Epidemiology, University of IowaMary E Wilson - Interdisciplinary Graduate Program in Genetics and Departments of Internal Medicine, Microbiology, Biochemistry, and Epidemiology, University of Iowa
- Resource Type
- Journal article
- Publication Details
- Journal of clinical microbiology, Vol.49(11), pp.3892-3904
- Publisher
- American Society for Microbiology; 1752 N St., N.W., Washington, DC
- DOI
- 10.1128/JCM.r00764-11
- PMID
- 22042830
- PMCID
- PMC3209110
- ISSN
- 0095-1137
- eISSN
- 1098-660X
- Language
- English
- Date published
- 11/2011
- Academic Unit
- Microbiology and Immunology; International Programs; Epidemiology; Biochemistry and Molecular Biology; Internal Medicine
- Record Identifier
- 9984001134402771
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