Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Acta Parasitologica

4 Issues per year


IMPACT FACTOR 2016: 1.160
5-year IMPACT FACTOR: 1.185

CiteScore 2016: 1.24

SCImago Journal Rank (SJR) 2016: 0.532
Source Normalized Impact per Paper (SNIP) 2016: 0.721

Online
ISSN
1896-1851
See all formats and pricing
More options …
Volume 63, Issue 2

Issues

Evaluation of four commercial DNA extraction kits for the detection of Microsporidia and the importance of pretreatments in DNA isolation

Ülfet Çetinkaya / Arzuv Charyyeva
  • Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic
  • Department of Parasitology, Faculty of Medicine, Erciyes University, Kayseri, Turkey
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Eda Sivcan / Esra Gürbüz
Published Online: 2018-04-13 | DOI: https://doi.org/10.1515/ap-2018-0044

Abstract

Microsporidia are obligate intracellular parasitic protozoa infecting the wide variety of hosts and are commonly known as a cause of chronic diarrhea particularly in immunocompromised individuals. Molecular-based tests have high sensitivity and specificity in disease diagnosis. However, these tests’ performance relies on the isolation of DNA in a good concentration. The standard procedures of commercial DNA extraction kits are usually insufficient for this purpose due to the tough walls of spores. This study aimed to test the significance of pretreatments by glass beads and freeze-thawing processes in DNA isolation from microsporidia spores. The parasite was cultured in growing Vero cells and seven serial dilutions were prepared from the collected spores. DNA purification was performed according to different tissue kits and stool kit procedures with and without any pretreatment. Concentration of isolated DNA samples were evaluated by real-time PCR. As a result of this study, the detectable amount of spores is minimum 10 spores in each 100 μ! sample according to the different tissue kits’ standard protocols. However, according to the DNA stool mini kit, the detectable amount of spores was found to be 1,000 spores/100 μl of stool sample when pretreated with both the freeze-thawing and glass beads methods.In conclusion, the current study demonstrated that further pretreatments are an essential process for DNA extraction from the stool specimens in order to avoid possible false negativity in the diagnosis of microsporidiosis.

Keywords: Microsporidia; DNA extraction; freeze-thawing; glass beads; pretreatment

References

  • Ariefdjohan M.W., Savaiano D.A., Nakatsu C.H. 2010. Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens. Nutrition Journal, 9, 23. CrossrefWeb of SciencePubMedGoogle Scholar

  • Babaei Z., Oormazdi H., Rezaie S., Rezaeian M., Razmjou E. 2011. Giardia intestinalis: DNA extraction approaches to improve PCR results. Experimental Parasitology, 128, 159–162. CrossrefWeb of SciencePubMedGoogle Scholar

  • Cetinkaya U., Yazar S., Kuk S., Sivcan E., Kaynar L., Arslan D., Sahin I. 2016. The high prevalence of Encephalitozoon intestinalis in patients receiving chemotherapy and children with growth retardation and the validity of real-time PCR in its diagnosis. Turkish Journal Of Medical Sciences, 46, 1050–1058. CrossrefWeb of SciencePubMedGoogle Scholar

  • Curry A. 2005. Microsporidiosis. In: Cox FEG, Wakelin D, Gillespie SH, Despommier DD. (Eds Topley and Wilson’s) Microbiology and Microbial Infections. Washington, DC: ASM Press, 529–555Google Scholar

  • Franzen C., Muller A. 2001. Microsporidiosis: human diseases and diagnosis. Microbes and Infection, 3, 389–400CrossrefPubMedGoogle Scholar

  • Garcia L.S. (Ed.) 2007. Diagnostic medical parasitology. Washington DC: ASM Press, pp. 33–46Google Scholar

  • Ghosh K., Weiss L.M. 2009. Molecular diagnostic tests for microsporidia. Interdisciplinary Perspectives on Infectious Diseases, 2009, 926521. CrossrefPubMedGoogle Scholar

  • Hawash Y. 2014. DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR. Korean Journal of Parasitology, 52, 263–271. CrossrefWeb of ScienceGoogle Scholar

  • Joseph J., Sharma S. 2009. In vitro culture of various species of microsporidia causing keratitis: evaluation of three immortalized cell lines. Indian journal of medical microbiology, 27, 35–39PubMedGoogle Scholar

  • Lelu M., Gilot-Fromont E., Aubert D., Richaume A., Afonso E., Dupuis E., et al. 2011. Development of a sensitive method for Toxoplasma gondii oocyst extraction in soil. Veterinary Parasitology, 183, 59–67. CrossrefWeb of SciencePubMedGoogle Scholar

  • Mirjalali H., Mohebali M., Mirhendi H., Gholami R., Keshavarz H., Meamar A.R., Rezaeian M. 2014. Emerging Intestinal Microsporidia Infection in HIV(+)/AIDS Patients in Iran: Microscopic and Molecular Detection. Iranian Journal of Parasitology, 9, 149–154PubMedGoogle Scholar

  • Paulos S., Mateo M., de Lucio A., Hernandez-de Mingo M., Bailo B., Saugar J.M., et al. 2016. Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp. Journal of Microbiological Methods, 127, 68–73. CrossrefPubMedWeb of ScienceGoogle Scholar

  • Pessoa E.S.R., Mendonca Trajano-Silva L.A., Lopes da Silva M.A., da Cunha Goncalves-de-Albuquerque S., de Goes T.C., Silva de Morais R.C., et al. 2016. Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR. Journal of Microbiological Methods, 131, 34–41. CrossrefWeb of SciencePubMedGoogle Scholar

  • Pinar A., Akyon Y., Alp A., Erguven S. 2010. Adaptation of a sensitive DNA extraction method for detection of Entamoeba histolytica by real-time polymerase chain reaction. Mikrobiyoloji Bulteni, 44, 453–159PubMedGoogle Scholar

  • Polley S.D., Boadi S., Watson J., Curry A., Chiodini P.L. 2011. Detection and species identification of microsporidial infections using SYBR Green real-time PCR. Journal of Medical Microbiology, 60, 459–466. CrossrefWeb of SciencePubMedGoogle Scholar

  • Procop G.W. 2007. Molecular diagnostics for the detection and characterization of microbial pathogens. Clinical Infectious Diseases, 45, 99–111Web of ScienceCrossrefGoogle Scholar

  • Qi M., Wang H., Jing B., Wang R., Jian F., Ning C., Zhang L. 2016. Prevalence and multilocus genotyping of Giardia duodenalis in dairy calves in Xinjiang, Northwestern China. Parasites & Vectors, 9, 546. CrossrefWeb of SciencePubMedGoogle Scholar

  • Subrungruang I., Mungthin M., Chavalitshewinkoon-Petmitr P., Rangsin R., Naaglor T., Leelayoova S. 2004. Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens. Journal of Clinical Microbiology, 42, 3490–3494PubMedCrossrefGoogle Scholar

  • Weber R., Bryan R.T., Schwartz D.A., Owen R.L. 1994. Human microsporidial infections. Clinical Microbiology Reviews, 7, 426–461CrossrefWeb of SciencePubMedGoogle Scholar

  • Weiss L.M., Vossbrinck C.R. 1998. Microsporidiosis: molecular and diagnostic aspects. Advances in Parasitology, 40, 351–395CrossrefPubMedGoogle Scholar

  • Yazar S., Koru O., Hamamci B., Cetinkaya U., Karaman U., Kuk S. 2013. Microsporidia and microsporidiosis. Turkiye Parazitoloji Dergisi, 37, 123–134. CrossrefGoogle Scholar

About the article

Received: 2017-10-06

Revised: 2018-02-15

Accepted: 2018-02-16

Published Online: 2018-04-13

Published in Print: 2018-06-26


This article: Was presented at the international DNA Day and Genome Congress, Kirşehir, Turkish 2017.

Ethical standards: This study was carried out after being approved by the Ethics Committee of the Erciyes University Medical School. (protocol no: 2017/165).


Citation Information: Acta Parasitologica, Volume 63, Issue 2, Pages 386–392, ISSN (Online) 1896-1851, ISSN (Print) 1230-2821, DOI: https://doi.org/10.1515/ap-2018-0044.

Export Citation

© 2018 W. Stefański Institute of Parasitology, PAS. Copyright Clearance Center

Comments (0)

Please log in or register to comment.
Log in