Molecular Detection and Identification of Candida Species Isolates from Oral by RFLP-PCR

Zena W Al-jader; Jassim M Ado

doi:10.55544/jrasb.2.6.20

Authors

Zena W. Al-jader Department of Biology, College of Education for Pure Science, University of Mosul, IRAQ. https://orcid.org/0000-0002-0407-0546
Jassim M, Ado Department of Basic Science, College of Pharmacy, University of Duhok, IRAQ.

DOI:

https://doi.org/10.55544/jrasb.2.6.20

Keywords:

Candida, Genetic variation, Restriction enzyme, RFLP-PCR

Abstract

In this study, 10 local isolates from a total of 50 samples of Candida sp. were collected from oral swabs of patients with oral infections in Mosul hospitals. The isolates were diagnosed based on culturing, microscopic and biochemical characteristics, and then molecular methods. The first diagnosis by culturing, microscopic and biochemical tests found the isolates were identified as Candida sp. The ITS region was amplified using universal primers (ITS4-ITS5), The PCR product was size (510-721) bp. Performing RFLP-PCR using MspI, HhaI,and EcoRI, restriction enzyme to detect and identify Candida species, the results showed the presence of the cutting sequence of MspI and HhaI enzymes in the genomic DNA content of local isolate and the absence of the sequences for the EcoRI restriction enzyme. Two Candida species were identified (C. krusei and C. the basis of size and fragment sequences then compared with sequences of standard strains from the gene bank in previous studies. Therefore, it can be observed that there is a genetic variation between the local isolates and that there are different genotypes of rDNA 5.8S have been diagnosed in 10 isolates after the cutting process with three restriction enzymes. We conclude from this study that the RFLP-PCR technique was the best in diagnosing and identifying Candida species compared with traditional methods. and we are d the genetic variation between local isolates.

Downloads

Download data is not yet available.

Metrics

Metrics Loading ...

References

Warren, N. G. M. and Hazen, R. C. (1995).Candida, Cryptococcus and other yeast of Medical Importance In: Manual of Clinical Microbiology. R. P. Murray, E. J, Baron , M. A. P. Faller, F. C. Tenover and K. H. Yolen (Eds.), 6th Edn, Washington DC. ASM.

Neppelenbroek, K.H., Campanha, N.H., Spolidorio, D.M. (2006). Molecular fingerprinting methods for discrimination between C. albicans and C. dubliniensis. Oral Dis,V. 12, pp. 217-218.https://doi.org/10.1111/j.1601-0825.2006.01237.x

Dota, K.F.D., Consolaro M.E.L., Svidzinski, T.I.E.x, Bruschi ,M.L.(2011). Antifungal activity of Brazillian propolis microparticles against yeasts isolated from vulvo vaginal candidiasis. Brazil. J. EvidenceBased Complementary and Alternative Medicine. pp. 8. https://www.hindawi.com/journals/ecam/2011/201953/.

Mahmoudabadi, A.Z., Zarrin, M., Fard, M.B. (2013). Antifungal susceptibility of Candida species isolated from candidura. Iran, Ahwas. Jundishapur J. of Microbiol. V. 6, No. 1, pp.24-28. https://sites.kowsarpub.com/jjm/articles/18504.html.

Chu, J.H., Feudtner, C., Heydom ,K., Walsh, T.J., Zaoutis, T.E. (2006). Hospitalizations for endemic mycoses: apopulation-based national study. In United State. J.Clinical Infectious Diseases.,V. 42, No. 6, pp.822-825. https://academic.oup.com/cid/article/42/6/822/286617.

Liu, X., Ma, Z., Zhang J., Yang, L.(2017). Antifungal compounds against Candida infections from traditional chinese medicine. In China. Changchum. J. BioMed Research International. pp.12. https://www.hindawi.com/journals/bmri/2017/4614183/.

Lipperheide, V., Bikandi, J., Garcia-Femadez, J.F., Quindos, G., Ponton, J.(2002). Colony variation in Candida glabrata isolates from patients with vaginitis, Spain . Ibiza. J. Revista Iberoamericana de Micologia. V. 19, No. 3, pp. 161-164. http://www.reviberoammicol.com/2002-19/161164.pdf.

Timmermans, B., De las penas, A., Castano, I., Van Dijck, P.(2018). Adhesins in Candida glabrata, J.of fungi, V. 4, No. 2, pp. 160. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023314/.

Risan, M.H. (2016). Molecular identification of yeast Candida glabrata from candidemia patients in Iraq, Iraqi Journal of Science. V. 57, No. 2 A, pp. 808-813. https://www.iasj.net/iasj/article/119244.

Cirak, M. Y., Kalkanci, A., and Kustimur, S.(2003).Use of molecular methods in identification of Candida species and evolution of fluconazole resistance, Ankara, Turkey, Mem. Inst. Oswaldo Cruz.,V. 98, No.8, pp.1027-1032. https://doi.org/10.1590/S0074-02762003000800009.

Consuelo, F. ;Consuelo, F., Colom, F. and Frases, S. (2001).Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections, Spain, J. Clin. Microbiol., V. 39, No.8, pp. 2873-2879. https://jcm.asm.org/content/jcm/39/8/2873.full.pdf.

Mousavi, S. A.; Khalesi, E.; Bonjar, G. H.; Aghighi S.; Sharifi, F. and Aram, F. (2007). Rapid Molecular diagnosis for Candida Species Using PCR-RFLP, Mahan, Iran .J. Biotechnology, V. 6, No.4,pp.583-587. https://researchrepository.murdoch.edu.au/id/eprint/13990/1/rapid_molecular_diagnosis.pdf. 1

Kreger-Van Rji, N. J. (1984). The yeasts: a taxonomic study. 3rd ed Elsevier science publisher, Amsterdam, the Netherlands. https://www.elsevier.com/books/the-yeasts/kreger-van-rij/978-0-444-80421-1.

Mac Faddin, J.F. (1980). Biochemical Test for Identification of Medical Bacteria. Macro- and Micronutrient in Alkaline Soil Common. Soil Sci. J. Plant Anal.,V. 8 , pp.195-207.

Barnett, J. A.; Payne, R. W. and Yarrow, D.(1990). Yeasts: Characteristics and identification. 2 nd, and Cambridge Univ. press, Cambridge, UK. https://doi.org/10.1046/j.1525-1470.2001.1862020a.x.

Kurtzman, C. P. and Fell, J. W.(2000). The yeasts, A taxonomic study. 4thed, Elsevier, Amsterdam, Netherlands.https://www.elsevier.com/books/the-yeasts-a-taxonomic-study/kurtzman/978-0-444-81312-1

Yan, L. J., Thangthaeng, N., Sumien, N., Forster, M.J.(2013). Serum dehydrogenase dihydrolipoamide is a labile enzyme. Journal of Pharmacological and Biochemical Research, V. 1,(1),pp.(30-42).

Leena Sankari, S. ; Mahalakshmi, K.; Kumar, V. N.(2019). Chromogenic medium versus PCR– RFLP in the speciation of Candida: a comparative study, BMC Res. Notes. 12 (1):681. DOI: 10.1186/s13104-019-4710-5

White, T. J.;Bruns ,T. and . Lee, S. (1990). Amplification and direct sequencing of fungal ribosomal RNA gens for phylogenetic. PCR Protocol:a guide to methods and application press, Inc. Sandi ego. Calif. https://www.scienceopen.com/document?vid=f4505cd7-6ec2-43f4-b1d4-ddee592ba145.

Singh, S. ; Kumar, A. and Kumar, A.(2013). Species identification, antifungal susceptibility testing and genetic Variability among Candida species isolated from clinical samples. J. of Drug Discovery and Therapeutic. V.1. pp.01-11.

Al-Sofe, A. S. (2019). Evaluation of the efficiency of some species of yeasts on the production of indole acetic acid hormone and its effect on the growth of wheat plant. PH.D. Thesis. University of Mosul. Colleg of Education, Department of Biology, Iraq.

Boon, P. H. ; Ismail, A. ; Ong, E. and Sreeivasan, S. (2013). Phynotyping identification of Candida albicans for the production of in house helicase for nucleic acid based detection for fast diagnosis 2th ed. Pulau pinang. Malysia.

Habib, R. A. ; Habib, A. H. and Jasim, N. O. (2015).Isolation and diagnosis of some types Candida spp. and study of their sensitivity to some antifungal agents J. of University of Babylon. Iraq,V. 23. pp. 955-964, https://www.iasj.net/iasj?func=article&aId=107992

Akortha, E. E ; Nawaugo, V. O. ; Chikwe,N. O. (2009). Antifungal resistance among Candida species from patient with genitourinary tract infection isolated in Benin city, Edo state, Nigeria. Afri j. Microbial, V.3, pp., 694-699 . http://www.academicjournals.org/ajmr.

Bayona, J.V.M.; García,C. S.; Palop,N.T.; Martín, A.V; Padrón, C. G; Rodríguez,G, C.; Pemán, J.; Cardona,C. G. (2022). Novel Chromogenic Medium CHROMagarTM Candida Plus for Detection of Candida auris and other Candida Species from Surveillance and Environmental Samples: A Multicenter Study. 8, 281.

Nassir, N. I. (2010).Incidence of C. albicanis isolates from oral and vaginal candidiasis, study of their susceptibility and cross resistance to some antifungal agents. Thesis, College of Medicine/ Al-Qadisiya University, Iraq.

Shokohi, T. ; Hashemi Soteh, MB. ; Pouri, ZS. ; Hedayati, MT. ; Mayahi, S. (2010). Identification of Candida species using PCR-RFLP in cancer patients in Iran. Indian. J. Med Microbiol.V. 28,No.2, .pp.147-51.https://pubmed.ncbi.nlm.nih.gov/20404462/.

Mirhendi,H. ; Makimura, K. ; Khoramizade, M. H.; Yamaguchi, (2006). A one-Enzyme PCR-RFLP assay for identification of six medically important Candida species, Tahran, Jpn J Med Mycol., V. 47 ,pp. 225-229. https://doi.org/10.3314/jjmm.47.225.

Bakhshi T, Salari S, Naseri A, Esfandiarpour I, Mohammadi MA, Ghasemi Nejad Almani P. (2016). Molecular Identification of Candida Species in Patients with Candidiasis in Birjand, Iran, Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay. J Isfahan Med Sch; 33(359): 1986-93.

Shokoohi ,G. ; Rasekh-Jahromi ,A. ; Solhjoo , K. A.; Zhad ,H. ; Sisakht ,S.; Ahmadi, B. ; Teymouri ,Y. ; Shirvani , N. ; Abtahi F. ; Pooransari, P. and Ansari, S. (2020) Molecular Characterization and Antifungal Susceptibility of Candida Species Isolated From Vulvovaginitis in Jahrom City, South of Iran. Jundishapur J Microbiol.; 13(10). doi: 10.5812/jjm.106825.

Ortiz, B. ; Pérez-Alemána ,E. ; Galoa ,C. ; Fontecha b,G. (2018) Molecular identification of Candida species from urinary infections in Honduras. Rev Iberoam Micol.;35(2):73–77.

Nadăş, G. C. ; Kalmár,Z. ; Taulescu,M. A.; Chirila, F. ; Bouari, C. M. ; Răpuntean, S. ; Bolfa, P. ; Fit, N. I. (2014).Comparative identification of Candida species isolated from animals using phenotypic and PCR-RFLP methods. Bull Vet Inst Pulawy , Vol. 58, pp. 219-222,.

Mirhendi H and Makimura K. (2005). Differentiation of C. albicans and C. dubliniensis using a single-enzyme PCR-RFLP method. Jpn J Infect Dis; Vol. 58: pp. 35-7.