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2014 | 17 | 1 |

Tytuł artykułu

Detection of Echinococcus multilocularis in faeces by nested PCR with the use of diluted DNA samples

Autorzy

Treść / Zawartość

Warianty tytułu

Języki publikacji

EN

Abstrakty

EN
The aim of this study was to choose the optimal variant of PCR examination of faeces to detect Echinococcus multilocularis infection which would allow to reduce the influence of different inhibitors in faeces. The investigation was carried out by comparison of 3 different methods of DNA isolation from faeces and different DNA dilutions used in PCR. Thirty five intestines of red foxes were used. Small intestines were examined by the sedimentation and counting technique (SCT). Faeces were collected from the rectum for PCR and flotation. DNA were isolated with the use of 3 different methods. Two methods were dedicated for faeces: method 1 (M1) - for larger samples and method 2 (M2) - for standard samples. The third method, method 3 (M3), was not dedicated for faeces. DNA samples were tested by nested PCR in 6 variants: not diluted (1/1) and 5 diluted (1/2.5, 1/5, 1/10. 1/20, 1/40). E. multilocularis was found by SCT in 18 from 35 (51.4%) intestines. Taenia-type eggs were detected only in 20.0% of faecal samples. In PCR the highest number of positive results (45.7%) were obtained during examination of DNA isolated by M1 method, and then 40.0% and 34.3%, respectively, for M2 and M3. In some samples positive results in PCR were obtained only in diluted DNA. For example, 8 from 12 positive samples isolated by M3 method gave the PCR negative results in non-diluted DNA and positive only after dilution 1:2.5, 1:10 or 1:20. Also 3 samples isolated by methods dedicated for stool gave positive results only after DNA dilution. The investigation has revealed that in copro-PCR for detection of E. multilocularis infection additional using of diluted DNA (besides non diluted) can avoid false negative results causing by PCR inhibition. In the best method of DNA isolation (M1), the use of non diluted DNA sample together with diluted in proportion 1:10 seems to be optimal.

Słowa kluczowe

Wydawca

-

Rocznik

Tom

17

Numer

1

Opis fizyczny

p.79-83,fig.,ref.

Twórcy

autor
  • Department of Parasitology and Invasive Diseases, National Veterinary Research Institute, Al.Partyzantow 57, 24-100 Pulawy, Poland

Bibliografia

  • Al-Sabi MN, Kapel CM, Deplazes P, Mathis A (2007) Comparative copro-diagnosis of Echinococcus multilocularis in experimentally infected foxes. Parasitol Res 101: 731-736.
  • Casulli A, Manfredi MT, La Rosa G, Di Cerbo AR, Dinkel A, Romig T, Deplazes P, Genchi C, Pozio E (2005) Echinococcus multilocularis in red foxes (Vulpes vulpes) of the Italian Alpine region: is there a focus of autochthonous transmission? Int J Parasitol 35: 1079-1083.
  • Craig PS, Gasser RB, Parada L, Cabrera P, Parietti S, Borgues C, Acuttis A, Agulla J, Snowden K, Paolillo E (1995) Diagnosis of canine echinococcosis: comparison of coproantigen and serum antibody tests with arecoline purgation in Uruguay. Vet Parasitol 56: 293-301.
  • Deplazes P, Eckert J (1996) Diagnosis of the Echinococcus multilocularis infection in final hosts. Appl Parasitol 37: 245-252
  • Dinkel A, von Nickisch-Rosenegk M, Bilger B, Merli M, Lucius R, Romig T (1998) Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy. J Clin Microbiol 36: 1871-1876.
  • Dinkel A, Kern S, Brinker A, Oehme R, Vaniscotte A, Giraudoux P, Mackenstedt U, Romig T (2011) A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species. Parasitol Res 109: 493-498.
  • Karamon J, Sroka J, Cencek T (2010) Limit of detection of sedimentation and counting technique (SCT) for Echinococcus multilocularis diagnosis, estimated under experimental conditions. Exp Parasitol 124: 244-246.
  • Karamon J, Sroka J, Cencek T (2012) The first detection of Echinococcus multilocularis in slaughtered pigs in Poland. Vet Parasitol 185: 327- 329.
  • Lahmar S, Lahmar S, Boufana B, Bradshaw H, Craig PS (2007) Screening for Echinococcus granulosus in dogs: Comparison between arecoline purgation, coproELISA and coproPCR with necropsy in pre-patent infections. Vet Parasitol 144: 287-292.
  • Machnicka B, Dziemian E, Rocki B, Kołodziej-Sobocinska M (2003) Detection of Echinococcus multilocularis antigens in faeces by ELISA. Parasitol Res 91: 491-496.
  • Mobedi I, Zare-Bidaki M, Siavashi M, Naddaf S, Kia E, Mahmoudi M (2013) Differential detection of Echinococcus spp. copro-DNA by nested-PCR in domestic and wild definitive hosts in Moghan Plain, Iran. Iran J Parasitol 8: 107-113.
  • OIE (Office International des Epizooties) 2010 Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. World Organisation for Animal Health, Paris, pp. 179-180.
  • Raynaud JP (1970) Etude de l’efficacite d;une technique de coproscopie quantitative pour le diagnostic de routine et le controle des infestations parasitaires des bovins, ovins, equins et porcins. Ann Parasitol Hum Comp 45: 321-342.
  • Reiterová K, Miterpáková M, Turčeková L, Antolová D, Dubinský P (2005) Field evaluation of an intravital diagnostic test of Echinococcus multilocularis infection in red foxes. Vet Parasitol 128: 65-71.
  • Van der Giessen JW, Rombout YB, Franchimont JH, Limper LP, Homan WL (1999) Detection of Echinococcus multilocularis in foxes in The Netherlands. Vet Parasitol 82: 49-57.

Typ dokumentu

Bibliografia

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