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International Journal of Current Microbiology and Applied Sciences (IJCMAS)
IJCMAS is now DOI (CrossRef) registered Research Journal. The DOIs are assigned to all published IJCMAS Articles.
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National Academy of Agricultural Sciences (NAAS)
NAAS Score: *5.38 (2020)
[Effective from January 1, 2020]
For more details click here

ICV 2019: 96.39
Index Copernicus ICI Journals Master List 2019 - IJCMAS--ICV 2019: 96.39
For more details click here

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Original Research Articles

PRINT ISSN : 2319-7692
Online ISSN : 2319-7706
Issues : 12 per year
Publisher : Excellent Publishers
Email : editorijcmas@gmail.com / submit@ijcmas.com
Editor-in-chief: Dr.M.Prakash
Index Copernicus ICV 2018: 95.39
NAAS RATING 2020: 5.38

Int.J.Curr.Microbiol.App.Sci.2016.5(1): 430-442
DOI: http://dx.doi.org/10.20546/ijcmas.2016.501.043


Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Bacillus cereus Toxin Genes
Iychettira Machaiah Mandappa, Prasanna Joglekar, Munna Singh Thakur and Haravey Krishnan Manonmani
Food Protectants and Infestation Control Department, CSIR-Central Food Technological Research Institute, Mysore 570 020, Karnataka, India
*Corresponding author
Abstract:

In this communication we describe a Loop mediated isothermal amplification (LAMP) based method for quick and effective detection of specific Bacillus cereus enterotoxin genes. Primers for the enterotoxin genes (hemolysin-A (hblA), Enterotoxin-T (bceT), Enterotoxin-FM (entFM/cwpFM) and Cytotoxin-K (CytK) of Bacillus cereus were designed using Primer explorer 4. Hydroxynaphthol blue (HNB), SYBR Green I and Calcein were used to enable visual detection. An attempt was made to quantify the DNA using Varioskan Flash microplate reader. The primers showed negligible cross reactivity with other food borne pathogens studied. The lowest concentration of standard DNA detected corresponded to 10 2 , 102 and 10 3 copies of DNA respectively with Calcein, SYBR Green I and HNB dyes. The DNA isolated directly from the food matrix was used to validate the applicability of the developed LAMP assay. Negligible food matrix interference was observed in most cases. The method is quick, accurate and economical for onsite analysis. HNB was suitable for visual detection of B. cereus in some spiked food samples. Calcein proved to be a better dye for detection of spiked food samples when read in a Varioskan flash reader. This is one of the first reports on LAMP based detection of Bacillus cereus. The method is suitable for directly testing of food samples without any enrichment techniques and therefore could find its application in food safety analysis onsite. Such a detection system will ensure better food safety and can also be extended to clinical diagnosis of pathogenic organisms.


Keywords: Bacillus cereus LAMP, Isothermal amplification Food matrix Detection, Toxin genes
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How to cite this article:

Int.J.Curr.Microbiol.App.Sci. 5(1): 430-442. doi: http://dx.doi.org/10.20546/ijcmas.2016.501.043