1. Evaluation of molecular typing methods- ERIC PCR and REP PCR to reveal genetic heterogeneity of Vibrio cholerae in water from river Turag Jannatul Ferdous1, 2, Md. Tasminuzzaman1*, Suhella Tulsiani2,3, Sumaiya Zaman1, Humaira Akhter1, Peter Mackie Kjaer Jensen2,3, Anowara Begum1 1Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh 2Section for Global Health, Institute of Public Health, University of Copenhagen, Denmark Copenhagen Centre for Disaster Research, University of Copenhagen, Denmark 1014
Results
Discussion
Discussion
Introduction
From this research we found that with REP sequences, the fingerprint patterns are more complex but the clustering result showed that the ERIC sequences are better, because the REP result shown only two big clusters and a small number of other cluster. The ERIC sequence is better than the REP sequence for analysis of V. cholerae samples, because it is less complex but more discriminative
Vibrio cholerae, the causative agent of cholera remains a major public health concern in developing countries like Bangladesh due to its potential to lead epidemics and pandemics. For epidemiological investigation, current molecular subtyping methods such as pulsedfield gel electrophoresis (PFGE), ribotyping, Multilocus Sequence typing (MLST) are traditionally available to measure the genetic diversity and to trace the global spread of clones. However, these methods are labor-intensive, expensive and time consuming compared with other simpler, non-sequencing PCR-based DNA fingerprinting approaches.
(i)
(ii)
Figure 1: Diagram of amplification patterns of Vibrio cholerae isolates generated by (i) ERIC and (ii) REP-PCR
Table 2: Fragment patterns obtained during analysis of ERIC and REP PCR
Methods
No. of
Predominant fragment size (kb)
% of strains with pre-dominant patterns
strains
patterns
ERIC 38 22
1.2, 1, 0.6, 0.48, 0.3
76.32, 89.47, 86.8, 84.2, 73.7
REP 16
1.8, 1.4, 1.3, 0.8, 0.75, 0.5
84, 84.2, 74, 92, 86.8, 95
Acknowledgement
This study was supported by the combined efforts of funds from Danish International Development Agency (DANIDA) and University Grants Commission of Bangladesh .
Methods and Materials
In this study, 38 isolates of Vibrio cholerae recovered from two different locations of river Turag in Dhaka were examined to investigate the genetic diversity and relatedness using two PCR-based fingerprinting methods: Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR, and Repetitive Extragenic Palindromic (REP) PCR.
References
Waturangi, D. E., Joanito, I., Yogi, Y., & Thomas, S. (2012). Use of REP- and ERIC-PCR to reveal genetic heterogeneity of Vibrio cholerae from edible ice in Jakarta, Indonesia. Gut Pathog, 4, 2-2
Mishra, A., Taneja, N., Sharma, R. K., Kumar, R., Sharma, N. C., & Sharma, M. (2011). Amplified fragment length polymorphism of clinical and environmental Vibrio cholerae from a freshwater environment in a cholera-endemic area, India. BMC infectious diseases, 11(1), 249.
Wong, H.-C., & Lin, C.-H. (2001). Evaluation of typing of Vibrio parahaemolyticus by three PCR methods using specific primers. J Clin Microbiol, 39(12),
Table 1: Origin of samples and number of positive samples
Regional
Location of sampling
Number of Presumptive isolates
Number of Positive isolates
Turag river, nearby northwest of Dhaka, in the Tongi sub-district of Bangladesh
Sarker-bari road 33 24
Dulafikiria road 27 14
(i)
(ii)
Figure 2: Dendograms of 38 isolates of (i) ERIC-PCR and (ii) REP-PCR. Dendograms were constructed using Gel 2K software based on Jackard co-efficient obtained after complete link cluster type analysis