Molecular identification of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) from fish and cetacean in Japanese waters

Umehara, Azusa; Kawakami, Yasushi; Matsui, Toshihiro; Araki, Jun; Uchida, Akihiko

doi:10.1016/j.parint.2006.07.001

Cited by 104 publications

(86 citation statements)

References 11 publications

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“…18 This region does not encode any product and has less impact on organism viability, which permits it to evolve at a faster rate than the ribosomal coding regions. 19 Recently, the evaluation of the 7SL rRNA gene for the identification of Leishmania spp. has produced satisfactory results.…”

Section: Discussionmentioning

confidence: 99%

The Use of Fluorescent Fragment Length Analysis (PCR-FFL) in the Direct Diagnosis and Identification of Cutaneous Leishmania Species

Tomás-Pérez

Fisa

Riera

2013

The American Journal of Tropical Medicine and Hygiene

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Abstract. Leishmaniasis is a disease caused by different species belonging to the genus Leishmania. It presents different epidemiological and clinical features and requires the development of rapid, sensitive techniques to improve specific diagnosis. In this study, we compared the traditional technique of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with PCR-fluorescent fragment length analysis (PCR-FFL). Fluorescently tagged primers, designed in the rRNA fragment ITS-1 and 7SL region, were used to amplify fragments, which were later digested and whose sizes were accurately determined using an automated DNA sequencer. We validated the technique using 19 Leishmania strains from five cutaneous Leishmania species before testing 36 clinical samples: 23 skin biopsies and 13 skin scrapings/lesion exudates on filter paper. In real diagnostic, PCR-FFL has proved to be quick, accurate, and more sensitive (83.3% testing the ITS-1 fragment and 94.4% testing the 7SL) than PCR-RFLP analysis (75% and 80.6%). Filter papers improved the specific diagnosis in both techniques using non-invasive samples.

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Section: Discussionmentioning

confidence: 99%

The Use of Fluorescent Fragment Length Analysis (PCR-FFL) in the Direct Diagnosis and Identification of Cutaneous Leishmania Species

Tomás-Pérez

Fisa

Riera

2013

The American Journal of Tropical Medicine and Hygiene

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“…According to this study, 84 out of 85 patients were infected with A. simplex s.s., and one was infected with A. pegreffii. Same researchers reported these two parasites and a hybrid genotype from fish and cetacean in Japanese waters 7 . Lee et al 16 15,23 .…”

Section: Umehara Et Almentioning

confidence: 95%

“…Universal primers A (5′-gtcgaattcgtaggtgaacctgcggaa ggatca-3′) and B (5′-gccggatccgaatcctggttagtttcttttcct-3′) were used for the amplification of rDNA (ITS1, 5.8 subunit rRNA and ITS2) 5,7,[14][15][16] . PCR was carried out in a final volume of 50 μl, containing 23.75 μl DNase, RNase free steril distilled water (Biobasic, Inc), 5 μl 10X PCR buffer, 6 μl 25 mM MgCl 2 , 5 μl 1 mM dNTP mix, 2.5 μl of each primer (50 pmol), 5 μl of template DNA, and 0.25 μl of TaqDNA polymerase (1.25 IU) (MBI, Fermentas).…”

Section: Pcr-rflp Analysismentioning

confidence: 99%

“…In PCR-RFLP analysis, primer pairs A-B and NC2-NC5 were used for the amplification of ITS region, AniF-Ani1R for 5.8 rRNA and JB3-JB4.5 for mt-CO1 genes 7,8,[14][15][16][22][23][24][25] . The most used restriction enzymes were HinfI, HhaI and TagI for the analysis of both ribosomal and mitochondrial genes 7,8,[14][15][16][22][23][24][25] 7,[14][15][16]22,25 .…”

Section: Umehara Et Almentioning

confidence: 99%

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Türkiye’nin Erzurum İlinde İnsan Tüketimi İçin Satılan İstavrit Balıklarında (Trachurus trachurus) Anisakis pegreffii’nin Moleküler Tanısı

Ütük¹,

Pişkin²,

Balkaya³

2009

Kafkas Univ Vet Fak Derg

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SummaryThis study was planned to identify six parasites detected in horse mackerels (Trachurus trachurus) sold for human consumption in Erzurum province. DNA extraction was performed on six parasites diagnosed as nematode. Species identification was done by PCR and PCR-RFLP analysis of ITS (ITS-1, 5.8S subunit rRNA gene and ITS-2) region of ribosomal DNA (rDNA) and partial sequencing of mitochondrial cytochrome c oxidase subunit 1 (mt-CO1) and mt-CO2 genes. According to the PCR and PCR-RFLP results, all parasites were identified as Anisakis pegreffii. Partial sequence of mt-CO1 gene of one randomly selected parasite was corresponding with A. simplex and mt-CO2 genes of two parasites were corresponding with A. pegreffii. With this study, A. pegreffii was molecularly detected for the first time in Turkey. Keywords: Anisakis pegreffii, Fish, PCR, PCR-RFLP, Sequencing Türkiye'nin Erzurum İlinde İnsan Tüketimi İçin Satılan İstavrit Balıklarında (Trachurus trachurus) Anisakis pegreffii'nin Moleküler Tanısı ÖzetBu çalışma, Erzurum ilinde insan tüketimi için satılan istavrit balıklarında (Trachurus trachurus) tespit edilen altı parazitin tür tanısını yapmak amacıyla planlanmıştır. Nematod olarak belirlenen altı parazitten DNA ekstraksiyonu yapılmıştır. Tür tanısı, rDNA'nın ITS bölgelerinin (ITS1, 5.8 alt ünite rRNA ve ITS2) PCR ve PCR-RFLP analizleri ve mitokondrial sitokrom c oksidaz alt ünite 1 (mt-CO1) ve mt-CO2 genlerinin kısmi olarak sekanslanması ile yapılmıştır. PCR ve PCR-RFLP sonuçlarına göre tüm parazitlerin Anisakis pegreffii olduğu belirlenmiştir. Rastgele seçilen bir parazitin mt-CO1 geninin kısmi sekans sonucu A. simplex, iki parazitin mt-CO2 geninin kısmi sekans sonuçları ise A. pegreffii ile uyuşmuştur. Bu çalışma ile A. pegreffii Türkiye'de ilk kez moleküler olarak tespit edilmiştir.

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“…The precise identification of Anisakis larvae up to species level is very difficult because no obvious morphological differences have been reported especially in differentiating A. simplex s.s. from A. pegreffii. The PCR-based approaches have been recently used for accurate diagnosis and the study of the systematic evolution of anisakid nematodes (Umehara et al, 2006;Mattiucci & Nascetti, 2008;Cavallero et al, 2011;2012). PCR-RFLP analysis and sequencing of the internal transcribed spacers of the ribosomal DNA have demonstrated that the ITS containing region is a specific and suitable marker for the identification of Anisakis members.…”

Section: Introductionmentioning

confidence: 99%

A polyphasic approach with molecular phylogeny for the characterization of Anisakis pegreffii (Anisakidae: Nematoda) in fishes from Adriatic Sea

Luca¹,

Vovlas

Troccoli³

et al. 2013

Helminthologia

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SummaryIn this study we investigated the morphometric and molecular characterization of a liver encapsulated third-stage larval population of Anisakis spp. infecting Merluccius merluccius and Lophius piscatorius caught in the Adriatic Sea waters (southern Italy). A polyphasic approach based on PCR-RFLP profiles of the ITS region, mitochondrial COI (cytochrome c oxidase subunit 1), sequencing and molecular phylogeny of ITS and mitochondrial COI was used to identify Anisakis larvae collected from fish samples. PCR-RFLP analysis showed three banding pattern corresponding to the peculiar pattern of A. pegreffii. Sequence data from ribosomal ITS and mitochondrial COI were analysed by Neighbour Joining, Minimum Evolution and Maximum Parsimony methods to evaluate the phylogenetic relationships among A. simplex sensu lato. The phylogenetic trees obtained for both ITS and COI revealed the existence of three distinct clades for A. simplex sensu sricto, A. simplex C and A. pegreffii and the sequences obtained in this study clearly clustered together with A. pegreffii sequences present in the database. Histopathological observations of anisakid nematode specimens detected on the liver surface of M. merluccius are illustrated. Encapsulated specimens of the L3 stage of the nematode were similar in size and morphometry to those found into the peritoneal cavity. Anisakis larvae encapsulated on the liver surface within dense and pearl coloured envelops caused host hepatic tissue necrosis, large cavities and oedematous liver spots to the host.

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Molecular identification of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) from fish and cetacean in Japanese waters

Cited by 104 publications

References 11 publications

The Use of Fluorescent Fragment Length Analysis (PCR-FFL) in the Direct Diagnosis and Identification of Cutaneous Leishmania Species

The Use of Fluorescent Fragment Length Analysis (PCR-FFL) in the Direct Diagnosis and Identification of Cutaneous Leishmania Species

Türkiye’nin Erzurum İlinde İnsan Tüketimi İçin Satılan İstavrit Balıklarında (Trachurus trachurus) Anisakis pegreffii’nin Moleküler Tanısı

A polyphasic approach with molecular phylogeny for the characterization of Anisakis pegreffii (Anisakidae: Nematoda) in fishes from Adriatic Sea

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