Comparison of Two DNA Extraction Methods and Two PCRs for Detection of in the Stool Samples of Naturally Infected Red Foxes

Overview

Journal Animals (Basel)

Date 2020 Dec 16

PMID 33322623

Citations 4

Authors

Katarzyna Skrzypek

Jacek Karamon

Malgorzata Samorek-Pierog

Joanna Dabrowska

Maciej Kochanowski

Jacek Sroka

Ewa Bilska-Zajac

Tomasz Cencek

Affiliations

Soon will be listed here.

Abstract

(1) Background: Due to the increasing distribution of infections in final hosts, epidemiological investigations are important for recognizing the spreading pattern of this parasite and also to estimate risk infection for humans. (2) Methods: Investigations were conducted with two commercial kits dedicated for DNA extraction from feces: ZR Fecal DNA Mini Prep (Zymo Research, Freiburg, Germany) and QIAamp FAST DNA Stool Mini Kit (Qiagen, Hilden, Germany) (marked as Z and Q), together with two common PCR protocols (nested PCR and multiplex PCR). The goal was to compare their efficiency in detecting the genetic material of in the samples of feces. Stool samples from red foxes were collected in a highly endemic area in Poland. Sedimentation and counting technique (SCT) was used as a reference method. (3) Results: From 48 samples, 35 were positive in SCT. Further investigations showed that 40.0% of samples (from those with SCT positive result) after Z-DNA extraction and 45.7% after Q-DNA extraction gave positive results in nested PCR. In multiplex PCR, positive results were obtained in 54.3% of samples after Z isolation and 48.6% of samples after Q. Additionally, one sample that resulted in being negative in SCT gave a positive result in PCR. The number of worms detected in the intestines had no influence on PCR results. (4) Conclusions: Both of the extraction methods showed similar efficiency in DNA isolation and dealing with inhibitors; however, they showed relatively low sensitivity. This was probably caused by degradation of genetic material in the field-collected samples.

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References

Maksimov P, Schares G, Press S, Frohlich A, Basso W, Herzig M . Comparison of different commercial DNA extraction kits and PCR protocols for the detection of Echinococcus multilocularis eggs in faecal samples from foxes. Vet Parasitol. 2017; 237:83-93. DOI: 10.1016/j.vetpar.2017.02.015. View

Irie T, Ito T, Kouguchi H, Yamano K, Uraguchi K, Yagi K . Diagnosis of canine Echinococcus multilocularis infections by copro-DNA tests: comparison of DNA extraction techniques and evaluation of diagnostic deworming. Parasitol Res. 2017; 116(8):2139-2144. DOI: 10.1007/s00436-017-5514-y. View

Hofer S, Gloor S, Muller U, Mathis A, Hegglin D, Deplazes P . High prevalence of Echinococcus multilocularis in urban red foxes (Vulpes vulpes) and voles (Arvicola terrestris) in the city of Zürich, Switzerland. Parasitology. 2000; 120 ( Pt 2):135-42. DOI: 10.1017/s0031182099005351. View

Karamon J . Detection of Echinococcus multilocularis in faeces by nested PCR with the use of diluted DNA samples. Pol J Vet Sci. 2014; 17(1):79-83. DOI: 10.2478/pjvs-2014-0010. View

Maas M, van Roon A, Dam-Deisz C, Opsteegh M, Massolo A, Deksne G . Evaluation by latent class analysis of a magnetic capture based DNA extraction followed by real-time qPCR as a new diagnostic method for detection of Echinococcus multilocularis in definitive hosts. Vet Parasitol. 2016; 230:20-24. DOI: 10.1016/j.vetpar.2016.10.016. View

Trachsel D, Deplazes P, Mathis A . Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA. Parasitology. 2007; 134(Pt 6):911-20. DOI: 10.1017/S0031182007002235. View

Kapel C, Torgerson P, Thompson R, Deplazes P . Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats. Int J Parasitol. 2005; 36(1):79-86. DOI: 10.1016/j.ijpara.2005.08.012. View

Oksanen A, Siles-Lucas M, Karamon J, Possenti A, Conraths F, Romig T . The geographical distribution and prevalence of Echinococcus multilocularis in animals in the European Union and adjacent countries: a systematic review and meta-analysis. Parasit Vectors. 2016; 9(1):519. PMC: 5039905. DOI: 10.1186/s13071-016-1746-4. View

Hegglin D, Deplazes P . Control of Echinococcus multilocularis: strategies, feasibility and cost-benefit analyses. Int J Parasitol. 2013; 43(5):327-37. DOI: 10.1016/j.ijpara.2012.11.013. View

10.

Dinkel A, von Nickisch-Rosenegk M, Bilger B, Merli M, Lucius R, Romig T . Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy. J Clin Microbiol. 1998; 36(7):1871-6. PMC: 104943. DOI: 10.1128/JCM.36.7.1871-1876.1998. View

11.

Oines O, Isaksson M, Hagstrom A, Tavornpanich S, Davidson R . Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces. Parasit Vectors. 2014; 7:246. PMC: 4060867. DOI: 10.1186/1756-3305-7-246. View

12.

Knapp J, Umhang G, Poulle M, Millon L . Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis. Appl Environ Microbiol. 2016; 82(10):2950-2958. PMC: 4959075. DOI: 10.1128/AEM.03467-15. View

13.

Klein C, Liccioli S, Massolo A . Egg intensity and freeze-thawing of fecal samples affect sensitivity of Echinococcus multilocularis detection by PCR. Parasitol Res. 2014; 113(10):3867-73. DOI: 10.1007/s00436-014-4055-x. View

14.

Deplazes P, Alther P, Tanner I, Thompson R, Eckert J . Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations. J Parasitol. 1999; 85(1):115-21. View

15.

Nakao M, Xiao N, Okamoto M, Yanagida T, Sako Y, Ito A . Geographic pattern of genetic variation in the fox tapeworm Echinococcus multilocularis. Parasitol Int. 2009; 58(4):384-9. DOI: 10.1016/j.parint.2009.07.010. View

16.

Al-Sabi M, Kapel C, Deplazes P, Mathis A . Comparative copro-diagnosis of Echinococcus multilocularis in experimentally infected foxes. Parasitol Res. 2007; 101(3):731-6. DOI: 10.1007/s00436-007-0537-4. View

17.

Dinkel A, Kern S, Brinker A, Oehme R, Vaniscotte A, Giraudoux P . A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species. Parasitol Res. 2011; 109(2):493-8. DOI: 10.1007/s00436-011-2272-0. View

18.

Isaksson M, Hagstrom A, Armua-Fernandez M, Wahlstrom H, Agren E, Miller A . A semi-automated magnetic capture probe based DNA extraction and real-time PCR method applied in the Swedish surveillance of Echinococcus multilocularis in red fox (Vulpes vulpes) faecal samples. Parasit Vectors. 2014; 7:583. PMC: 4282741. DOI: 10.1186/s13071-014-0583-6. View

19.

Mathis A, Deplazes P, Eckert J . An improved test system for PCR-based specific detection of Echinococcus multilocularis eggs. J Helminthol. 1996; 70(3):219-22. DOI: 10.1017/s0022149x00015443. View

20.

Romig T . Echinococcus multilocularis in Europe--state of the art. Vet Res Commun. 2009; 33 Suppl 1:31-4. DOI: 10.1007/s11259-009-9244-1. View