Real-time qPCR coupled with high-resolution melting curve analysis for the detection of the internal transcribed spacer 1 of Angiostrongylus costaricensis.
Abdominal angiostrongyliasis
Molecular diagnosis
Nematode
Zoonosis
Journal
Parasitology research
ISSN: 1432-1955
Titre abrégé: Parasitol Res
Pays: Germany
ID NLM: 8703571
Informations de publication
Date de publication:
02 Sep 2024
02 Sep 2024
Historique:
received:
06
05
2024
accepted:
14
08
2024
medline:
2
9
2024
pubmed:
2
9
2024
entrez:
1
9
2024
Statut:
epublish
Résumé
Abdominal angiostrongyliasis (AA) is a zoonotic and severe parasitic infection caused by Angiostrongylus costaricensis. AA is currently diagnosed by the observation of A. costaricensis-compatible structures in biopsies or the detection of antibodies in serological tests. However, molecular methods targeting homologous sequences of A. costaricensis have not been designed before, and therefore, an HRM-coupled qPCR was developed to detect the internal transcribed spacer 1 (ITS1) of the parasite. The present assay successfully amplified DNA of A. costaricensis obtained from different hosts and identified slight sequence differences through the HRM analysis. The detection limit of the HRM-qPCR was 0.00036 ng/µL, 1.0 ng/µL, and 0.1 ng/µL when A. costaricensis DNA was diluted in nuclease-free water, whole blood, and sera, respectively, which highlights its potential use for cell-free DNA detection. Moreover, the reaction did not cross-amplify DNA of Angiostrongylus cantonensis, Strongyloides stercoralis, and other nematodes, thus emphasizing its specificity. Additionally, the assay tested positive in formalin-fixed paraffin embedded biopsies with visible A. costaricensis adults or eggs, but not in samples without evident parasites or a low number of larvae, which suggests that the reaction is useful for confirming the presence of the nematode in clinical samples. Finally, DNA of sera from patients with AA was evaluated with the HRM-qPCR but none tested positive, possibly due to long storage periods of the samples which could have led to cfDNA degradation. These results indicate that this assay may be useful in the confirmation of AA and its prospection for cell-free DNA detection protocols.
Identifiants
pubmed: 39218957
doi: 10.1007/s00436-024-08327-6
pii: 10.1007/s00436-024-08327-6
doi:
Substances chimiques
DNA, Ribosomal Spacer
0
DNA, Helminth
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
312Subventions
Organisme : Vicerrectoría de Investigación, Universidad de Costa Rica
ID : C2064
Informations de copyright
© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
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