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Different tolerance of loop-mediated isothermal amplification and polymerase chain reaction to inhibitors in chicken carcass rinse and feces for detecting Campylobacter jejuni
Poster Title: Different tolerance of loop-mediated isothermal amplification and polymerase chain reaction to inhibitors in chicken carcass rinse and feces for detecting Campylobacter jejuni
Submitted on 02 Nov 2017
Author(s): Hongsheng Huang, Beverley Phipps-Todd, Courtney Chew Leung and Dina Elleithy
Affiliations: Canadian Food Inspection Agency, Ottawa Laboratory – Fallowfield
Poster Views: 666
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Poster Information
Abstract: Campylobacter spp, particularly C. jejuni, are a leading cause of human foodborne gastroenteritis worldwide, with poultry being the major reservoir. There is a need for developing rapid methods to detect this organism in poultry for baseline studies and the reduction of this pathogen in poultry. Polymerase chain reaction (PCR) is a commonly used molecular procedure to detect pathogens including foodborne campylobacters. However, inhibition of PCR due to inhibitors in various types of samples has been problematic. Loop-mediated isothermal amplification (LAMP) procedure has been shown to be equivalent to or more sensitive than PCR and be less affected by the inhibitory substances compared with PCR. This study investigated the relative tolerance of LAMP and real-time PCR detecting C. jejuni to inhibitors in chicken carcass rinse and feces. Both procedures showed a similar sensitivity (10-100 and 100-1,000 colony forming unit (CFU)/mL for PCR and LAMP respectively) using purified DNA extracts from chicken carcass rinses and fecal homogenates experimentally contaminated with C. jejuni. The PCR and LAMP showed detection limit of 10,000 and 100,000 CFU/mL respectively using crude DNA extracts from chicken carcass rinses. When testing crude DNA extracts from fecal homogenates, LAMP demonstrated a detection limit at 100,00 CFU/mL, however, PCR showed negative results for all concentrations. This study demonstrates that LAMP is more tolerant than PCR to the inhibitor(s) in chicken feces, and could be complementary to PCR when different types of inhibitors are present. The LAMP assay could also be an alternative, simple and less expensive test amenable for use under various testing conditions.Summary: Loop-mediated isothermal amplification (LAMP) and real-time polymerase chain reaction (PCR) procedures for Campylobacter jejuni showed similar detection limits for samples with purified DNA extracts. The substances in chicken feces showed significantly more inhibition effect on real-time PCR than on LAMP when using crude DNA from chicken feces contaminated with C. jejuni. The LAMP using crude fecal DNA preparation could potentially be a less expensive and simple method.References: 1. Bacteriological Analytical Manual, US Food and Drug Administration.
2. MFLP-46,Compendium of Analytical Methods, Health Canada.
3. Yamazaki et al., 2008. J Med Microbial. 57:444-451.
4. Lund et al., 2004. J. Clin. Microbiol. 42:5125–5132.
5. Yamazaki et al., 2009. Appl Environ Microbial. 75:1597-1603.
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