New Molecular Approach for the Detection of Kinetoplastida Parasites of Medical and Veterinary Interest
Kinetoplastids are protozoa containing a range of ubiquitous free_living species-pathogens of invertebrates, vertebrates and even some plants. Some of them are causative agents of canine vector-borne diseases. Their diagnosis is often missing in a gold standard. Here, we proposed a molecular approac...
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Veröffentlicht in: | Microorganisms (Basel) 2020-03, Vol.8 (3), p.356 |
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Zusammenfassung: | Kinetoplastids are protozoa containing a range of ubiquitous free_living species-pathogens of invertebrates, vertebrates and even some plants. Some of them are causative agents of canine vector-borne diseases. Their diagnosis is often missing in a gold standard. Here, we proposed a molecular approach for the diagnosis and study of Kinetoplastida. The TaqMan qPCR assays target the following genes:
of Kinetoplastida,
of
spp.,
rRNA of
spp.,
of
spp., kinetoplast minicircle DNA
of
complex and
of
, were designed, validated for their sensitivity (Se) and specificity (Sp) in silico and in vitro using a panel of known DNAs. They were then used to screen 369 blood samples (358 dogs, 2 equids, 9 monkeys). In addition, new
primer sets are presented to use for Kinetoplastida's identification by PCR/sequencing. All qPCRs showed consistently high analytical sensitivities and reproducibility. They detect approximately 0.01 parasite/ mL blood for the
based- qPCRs and at least a single cell-equivalent of rDNA for the other systems. Based on the sequencing results, after screening, Se and Sp were: 0. 919 and 0.971, 0.853 and 0.979, 1.00 and 0.987, 0.826 and 0.995 for all of Kinetoplastida,
,
,
spp. specific qPCRs, respectively. kDNA based qPCRs were more sensitive and specific (Se: 1.00; Sp: 0.997). PCR/sequencing allowed the detection of Kinetoplastids in animal blood samples such as
and
spp. The molecular approach proposed here is useful for epidemiological studies, fundamental research such as screening for new Kinetoplastida species, diagnosis and therapeutic follow-up. In addition, researchers are free to choose the molecular tools adapted to their aims. |
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ISSN: | 2076-2607 2076-2607 |
DOI: | 10.3390/microorganisms8030356 |