Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR.
Detection of Fasciola hepatica infection in Lymnaea viatrix through analysis of histological cuts is based upon morphological characters of the parasite during the intra-mollusk phase of parasitism. At this stage, trematode forms are very similar and, thus, very difficult to differentiate. Specific detection may also be impaired by the presence of other helminthes in the mollusk. Histological samples are usually fixed in formalin, embedded in paraffin, sectioned and HE stained. In the current study, a method for the extraction of DNA from formalin-fixed, paraffin-embedded tissues was standardized by means of deparaffinizing with xylol and digesting with proteinase K. Extracted DNA was amplified in a multiplex-PCR, by using simultaneous primers in a single reaction under high stringency conditions. Results showed specific amplification of DNA from the trematode and the snails. The technique was sensitive enough to detect F. hepatica infections in L. viatrix, in histological sections in which the presence of larval stages could not be observed through brightfield microscopy. The profiles generated were: stair bands referring to F. hepatica DNAmt amplification; a band of 1200 bp referring to L. viatrix ITS and another of 1300 bp referring to F. hepatica ITS and other trematodes. Multiplex-PCR has shown to be a fast, safe, highly sensitive and specific method, which is able to amplify DNA from fixed tissues, despite a low DNA quantity and its degradation caused by fixation processes. Such methodology may be useful in studies on fascioliasis epidemiology, enabling the use of material from histological collections.
Magalhães
Kelly Grace
KG
Laboratório de Helmintoses Intestinais-Centro de Pesquisas René Rachou da Fundação Oswaldo Cruz, Av. Augusto de Lima 1715, Belo Horizonte, MG, Brazil. kellygrace@ioc.fiocruz.br
Jannotti-Passos
Liana K
LK
Caldeira
Roberta L
RL
Berne
Maria Elisabeth Aires
ME
Muller
Gertrude
G
Carvalho
Omar S
OS
Lenzi
Henrique Leonel
HL
D000818:Animals; D017931:DNA Primers; D018509:DNA, Helminth / Q000032:analysis*; D004197:Disease Reservoirs / Q000469:parasitology / Q000662:veterinary; D004199:Disease Vectors; D005210:Fasciola hepatica / Q000235:genetics / Q000302:isolation & purification*; D005784:Gene Amplification; D008195:Lymnaea / Q000469:parasitology*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D017931:DNA Primers; D018509:DNA, Helminth