Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene

Autor(es)

Pinhanelli, V. C. ^[1] ; Costa, P. N. M. ^[1] ; Silva, G. ^[1] ; Aguiar, D. M. ^[2] ; Silva, C. M. L. ^[1] ; Fachin, A. L. ^[1] ; Marins, M. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Ribeirao Preto, Unidade Biotecnol, Ribeirao Preto, SP - Brazil

^[2] Univ Fed Mato Grosso, Fac Agron Med Vet & Zootecnia, Cuiaba, MT - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Genetics and Molecular Research; v. 14, n. 4, p. 17885-17892, 2015.
• Citações Web of Science: 0

Resumo

Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME. (AU)

• Processo FAPESP: 12/15940-6 - Desenvolvimento e avaliação de ensaio baseado na técnica de amplificação isotérmica de DNA (LAMP) para a detecção de Ehrlichia canis em cães naturalmente infectados
• Beneficiário: Vitor Caressato Pinhanelli
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 12/01235-9 - Desenvolvimento e avaliação de ensaio baseado na técnica de amplificação isotérmica de DNA (LAMP) para a detecção de Ehrlichia canis em cães naturalmente infectados
• Beneficiário: Mozart de Azevedo Marins
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 13/08295-0 - Desenvolvimento e avaliação de ensaio de LAMP para detecção de E. canis em amostras de sangue canino
• Beneficiário: Péricles Natan Mendes da Costa
• Modalidade de apoio: Bolsas no Brasil - Programa Capacitação - Treinamento Técnico