A real-time quantitative polymerase chain reaction for the specific detection of Hammondia hammondi and its differentiation from Toxoplasma gondii
| Autor: | Mareen Tuschy, Maike Joeres, Majda Globokar Vrhovec, Pavlo Maksimov, Andrea Bärwald, Bretislav Koudela, Gereon Schares, Franz Josef Conraths, Jitender P. Dubey |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
In silico 030231 tropical medicine ved/biology.organism_classification_rank.species Genes Protozoan Toxoplasma gondii TOXOSOURCES Real-Time Polymerase Chain Reaction Genome lcsh:Infectious and parasitic diseases law.invention Hammondia hammondi Diagnosis Differential 03 medical and health sciences Feces Mice Oocyst 0302 clinical medicine law Quantitative polymerase chain reaction TaqMan Animals lcsh:RC109-216 Pathology Molecular Polymerase chain reaction Repetitive Sequences Nucleic Acid biology ved/biology Coccidiosis Research Oocysts biology.organism_classification Molecular biology 3. Good health 030104 developmental biology Infectious Diseases Real-time polymerase chain reaction Toxoplasmosis Animal Faecal examination Sarcocystidae TaqMan polymerase chain reaction Cats Parasitology Primer (molecular biology) Toxoplasma |
| Zdroj: | Parasites & Vectors Parasites & Vectors, Vol 14, Iss 1, Pp 1-11 (2021) |
| Popis: | Introduction Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites, but only T. gondii is zoonotic. Both species use felids as definitive hosts and cannot be differentiated by oocyst morphology. In T. gondii, a 529-base pair (bp) repetitive element (TgREP-529) is of utmost diagnostic importance for polymerase chain reaction (PCR) diagnostic tests. We identified a similar repetitive region in the H. hammondi genome (HhamREP-529). Methods Based on reported sequences, primers and probes were selected in silico and optimal primer probe combinations were explored, also by including previously published primers. The analytical sensitivity was tested using serial dilutions of oocyst DNA. For testing analytical specificity, DNA isolated from several related species was used as controls. The newly established TaqMan PCR (Hham-qPCR1) was applied to tissues collected from H. hammondi-infected gamma-interferon gene knockout (GKO) mice at varying time points post-infection. Results Ten forward and six reverse primers were tested in varying combinations. Four potentially suitable dual-labelled probes were selected. One set based on the primer pair (Hham275F, Hham81R) and the probe (Hham222P) yielded optimal results. In addition to excellent analytic specificity, the assay revealed an analytical sensitivity of genome equivalents of less than one oocyst. Investigation of the tissue distribution in GKO mice revealed the presence of parasite DNA in all examined organs, but to a varying extent, suggesting 100- to 10,000-fold differences in parasitic loads between tissues in the chronic state of infection, 42 days post-infection. Discussion The use of the 529-bp repeat of H. hammondi is suitable for establishing a quantitative real-time PCR assay, because this repeat probably exists about 200 times in the genome of a single organism, like its counterpart in T. gondii. Although there were enough sequence data available, only a few of the primers predicted in silico revealed sufficient amplification; the identification of a suitable probe was also difficult. This is in accord with our previous observations on considerable variability in the 529-bp repetitive element of H. hammondi. Conclusions The H. hammondi real-time PCR represents an important novel diagnostic tool for epidemiological and cell biological studies on H. hammondi and related parasites. Graphical Abstract |
| Databáze: | OpenAIRE |
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