Suitability of different tissue fixatives for subsequent PCR analysis of Cysticercus ovis

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Abstract

PCR amplification of specific DNA regions is a powerful tool for retrospective studies, but not all preservation or fixation methods render DNA that is suitable for subsequent amplification. Several factors affect sensitivity of polymerase chain reaction (PCR) amplification. There were reported the effects of commonly used fixation solutions — 10 % neutral buffered formalin, 20 % neutral buffered formalin and Carnoy’s solution and the efficiency of PCR amplification in fresh tissue and paraffin (or wax) embedded samples of Cysticercus ovis. DNA from samples was isolated and PCR product of 1300 bp was amplified. Results indicated that the samples fixed in Carnoy’s solution produced reliable amplification of desired fragments. The samples that were fixed in 10 % and 20 % neutral buffered formalin brought negative results.

[1] Foss, R. D., Guha-Thakurta, N., Canran, R. M., Gutman, P. (1994): Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue: Comparison of two housekeeping gene mRNA controls. Diagn. Mol. Pathol., 51: 148–155 http://dx.doi.org/10.1097/00019606-199409000-00003

[2] Goldová, M., Tóth, Š., Letková, V., Mojžišová, J., Kožárová, I., Pomfy, M. (2008): Comparison of the histological methods in the diagnostic of deer cysticercosis. Helminthologia, 3: 121–125. DOI: 10.2478/s11687-008-0023-2 http://dx.doi.org/10.2478/s11687-008-0023-2

[3] Gonzalez, L. M., Montero, E., Puente, S., Lopezvelez, R., Hernandez, M., Sciutto, E., Harrison, L. J. S., Parkhouse, R. M. E., Garate, T. (2002): PCR tools for the differential diagnosis of Taenia saginata and Taenia solium taeniasis/cysticercosis from different geographical regions. Diagn. Microbiol. Infect. Dis., 1: 243–249. DOI: 10.1016/S0732-8893(01)00356-X http://dx.doi.org/10.1016/S0732-8893(01)00356-X

[4] Greer, C. E., Wheeler, C. M., Manos, M. M. (1994): Sample preparation and PCR amplification from paraffinembedded tissues. Genome Res., 10: 113–122 http://dx.doi.org/10.1101/gr.3.6.S113

[5] Ke, W. Z., Yu, D. W., Gu, B. P., Zhuang, Z. W. (2001): Raman spectroscopic study of microscopic damage on the space structure of DNA with acetic acid. Biochemistry, 21: 790–793

[6] Lloyd, S. (1998): Cysticercosis and taeniosis Taenia saginata, Taenia solium and Asian Taenia. In: Zoonoses. Biology, Clinical Practice, and Public Health Control. Oxford University Press, Oxford, UK, 635–649

[7] Mayta, H., Talley, A., Gilman, R. H., Jimenez, J., Verastegui, M., Ruiz, M., Garcia, H. H., Gonzalez, E. (2000): Differentiating Taenia solium and Taenia saginata infections by simple hematoxylin-eosin staining and PCR-restriction enzyme analysis. J. Clin. Microbiol., 1: 133–137

[8] Mcpherson, M. J., Graham, R., Taylor, P. Q. (1991): PCR: a practical approach. Oxford University Press Inc., New York, 33–37

[9] Miething, F., Hering, S., Hanschke, B., Dressler, J. (2006): Effect of fixation to the degradation of nuclear and mitochondrial DNA in different tissues. J. Histochem. Cytochem., 3: 371–374. DOI: 10.1369/jhc.5B6726.2005

[10] Mitchell, D., Ibrahim, S., Gusterson, B. A. (1985): Improved immunohistochemical localization of tissue antigens using modified methacarn fixation. J. Histochem. Cytochem., 2: 491–495 http://dx.doi.org/10.1177/33.5.3921605

[11] Ogunremi, O., Macdonald, G., Geerts, S., Brandt, J. (2004): Diagnosis of Taenia saginata cysticercosis by immunohistochemical test on formalin-fixed and paraffinembedded bovine lesions. J. Vet. Diagn. Invest., 22: 438–441. DOI: 10.1177/104063870401600513 http://dx.doi.org/10.1177/104063870401600513

[12] Takagi, H., Shibutani, M., Kato, N., Fujita, H., Lee, K.Y., Takigami, S., Mitsumori, K., Hirose, M. (2004): Microdissected region-specific gene expression analysis with methacarn-fixed, paraffin-embedded tissues by RTPCR. J. Histochem. Cytochem., 4: 903–913. DOI: 10.1369/jhc.3A6215.2004 http://dx.doi.org/10.1369/jhc.3A6215.2004

[13] Turčeková, Ľ., Šnábel, V., Dudiňák, V., Gašpar, V., Dubinský, P.: Prevalence of cystic echinoccocosis in pig from Slovakia, with evaluation of size, fertility and number of hydatid cysts. Helminthologia, 2009, 46(3): 151–158. DOI: 10.2478/s11687-009-0029-4 http://dx.doi.org/10.2478/s11687-009-0029-4

[14] Wiegand, P., Domho, ver J., Brinkmann, B. (1996): DNA-Degradation in formalinfixiertem Gewebe. Pathologe, 8: 451–454 http://dx.doi.org/10.1007/s002920050185

[15] Yamasaki, H., Nakaya, K., Nakao, M., Sako, Y., Ito A. (2007): Mitochondrial DNA diagnosis for cestode zoonoses: application to formalin-fixed paraffin-embedded tissue specimens. Southeast Asian J. Trop. Med. Public Health, 38(Suppl 1): 166–174

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