Detection of Clinical Isolates of Vibrio cholerae by Dot Blot Hybridization

Document Type : Original Research

Authors
S. Bagheri-Josheghani
B. Bakhshi
Department of Medical Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran
Abstract
Introduction: Vibrio cholerae, the causative agent of cholera, has attracted a great deal of attention as one of the major causes of morbidity and mortality worldwide, especially in developing countries. In most laboratories, biochemical assays are primarily performed for possible detection of these strains, which are then followed by a PCR (polymerase chain reaction) test to verify their identity. This study aimed to optimize dot blot technique to detect Vibrio cholerae bacteria for V. cholera as an easy-to-use and beneficial method.

Methods: A dot blot hybridization test was developed in this study to identify V. cholerae isolates as well as to assess the sensitivity and specificity of this test compared whit biochemical and PCR tests routinely performed for V. cholerae screening and detection in clinical specimens.

Results: Herein, the dot blot hybridization test was optimized to detect V. cholerae. A combination of three biochemical assays and PCR test confirmed the results of dot blot hybridization test. This test was able to identify V. cholerae strains with a high sensitivity and specificity of 100%. Using the newly developed method, a set of 26 V. cholerae isolates collected from clinical samples were accurately identified.

Conclusion: This study optimized dot blot technique as a simple and useful assay that could be employed in V. cholerae monitoring programs and strategies to effectively detect V. cholerae strains in surface water and fecal specimens.

Keywords

Subjects

References
1. Alves, B., et al., Application of a dot blot hybridization assay for genotyping Streptococcus uberis from Brazilian dairy herds. Journal of Dairy Science, 2021. 104(3): p. 3418-3426.
2. Petersen, L.M., et al., Third-generation sequencing in the clinical laboratory: exploring the advantages and challenges of nanopore sequencing. Journal of Clinical Microbiology, 2019. 58(1): p. e01315-19.
3. Menghi, C., C. Gatta, and L. Arias, Human cystic and alveolar echinococcosis. Current Treatment Options in Infectious Diseases, 2017. 9(2): p. 210-222.
4. Shafiei, R., et al., The seroprevalence of human cystic echinococcosis in Iran: A systematic review and meta-analysis study. Journal of parasitology research, 2016. 2016.
5. Moro, P.L., Clinical manifestations and diagnosis of echinococcosis. Up to date, 2016. 1.
6. Cecchini, F., et al., Identification of the unculturable bacteria Candidatus arthromitus in the intestinal content of trouts using Dot blot and Southern blot techniques. Veterinary microbiology, 2012. 156(3-4): p. 389-394.
7. Mahmood, T. and P.-C. Yang, Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 2012. 4(9): p. 429.
8. Hnasko, T.S. and R.M. Hnasko, The western blot, in ELISA. 2015, Springer. p. 87-96.
9. Kurien, B.T. and R.H. Scofield, Western blotting. Methods, 2006. 38(4): p. 283-293.
10. Stott, D., Immunoblotting and dot blotting. Journal of immunological methods, 1989. 119(2): p. 153-187.
11. Hoffmann, E., S. Muetzel, and K. Becker, A modified dot-blot method of protein determination applied in the tannin-protein precipitation assay to facilitate the evaluation of tannin activity in animal feeds. British journal of nutrition, 2002. 87(5): p. 421-426.
12. Clement, G. and J. Benhattar, A methylation sensitive dot blot assay (MS-DBA) for the quantitative analysis of DNA methylation in clinical samples. Journal of clinical pathology, 2005. 58(2): p. 155-158.
13. Putra, S.E.D., et al., Dealing with large sample sizes: comparison of a new one spot dot blot method to western blot. Clinical laboratory, 2014. 60(11): p. 1-7.
14. Organization, W.H., Ending cholera a global roadmap to 2030, in Ending cholera a global roadmap to 2030. 2017. p. 32-32.
15. Choopun, N., et al., Simple procedure for rapid identification of Vibrio cholerae from the aquatic environment. Applied and environmental microbiology, 2002. 68(2): p. 995-998.
16. Marashi, S.M.A., et al., Quantitative expression of cholera toxin mRNA in Vibrio cholerae isolates with different CTX cassette arrangements. Journal of medical microbiology, 2012. 61(8): p. 1071-1073.
17. Bakhshi, B., et al., A molecular survey on virulence associated genotypes of non-O1 non-O139 Vibrio cholerae in aquatic environment of Tehran, Iran. Water research, 2009. 43(5): p. 1441-1447.
18. Ali, M., et al., The global burden of cholera. Bulletin of the World Health Organization, 2012. 90: p. 209-218.
19. Hoffmann, M., et al., PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species. BMC microbiology, 2010. 10(1): p. 1-14.
20. Roozbehani, A., et al., A rapid and reliable species-specific identification of clinical and environmental isolates of Vibrio cholerae using a three-test procedure and recA polymerase chain reaction. Indian journal of medical microbiology, 2012. 30(1): p. 39-43.
21. Kamikawa, C. and A. Vicentini, Dot-blot methodology for rapid diagnosis of Paracoccidioidomycosis caused by Paracoccidioides brasiliensis. Journal of Infectious Diseases & Therapy, 2015.
22. Oude Elferink, S., et al., Detection and quantification of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification. Journal of Applied Microbiology, 1997. 83(1): p. 102-110.
23. Yoshimasu, M.A. and J. Zawistowski, Application of rapid dot blot immunoassay for detection of Salmonella enterica serovar Enteritidis in eggs, poultry, and other foods. Applied and environmental microbiology, 2001. 67(1): p. 459-461.
24. Niu, C., S. Wang, and C. Lu, Development and evaluation of a dot blot assay for rapid determination of invasion-associated gene ibeA directly in fresh bacteria cultures of E. coli. Folia microbiologica, 2012. 57(6): p. 557-561.
25. Poltronieri, P., et al., DNA arrays and membrane hybridization methods for screening of six Lactobacillus species common in food products. Food Analytical Methods, 2008. 1(3): p. 171-180.
26. Rojas, M. and P.L. Conway, [31] A dot-blot assay for adhesive components relative to probiotics. Methods in enzymology, 2001. 336: p. 389-402.
27. Borchardt, S.M., et al., Comparison of DNA dot blot hybridization and Lancefield capillary precipitin methods for group B streptococcal capsular typing. Journal of Clinical Microbiology, 2004. 42(1): p. 146-150.
28. Zhang, P., et al., Simple and sensitive detection of HBsAg by using a quantum dots nanobeads based dot-blot immunoassay. Theranostics, 2014. 4(3): p. 307.
29. Nikolaeva, O., et al., Improved Dot‐Blot Hybridization Assay for Large‐Scale Detection of Potato Viruses in Crude Potato Tuber Extracts. Journal of Phytopathology, 1990. 129(4): p. 283-290.
30. Cappione, A., et al., Rapid screening of the epidermal growth factor receptor phosphosignaling pathway via microplate-based dot blot assays. International journal of proteomics, 2012. 2012.
Pathobiology Reserach
Volume 24, Issue 2
February 2021
Pages 73-80