Establishment of LAMP Assay for Detection of Bacillus cereus
Establishment of LAMP Assay for Detection of Bacillus cereus
Xu Yun-Ming1*, Sun Zhi-Yuan1, Yang Jian-Bo1, Bian Rong-Rong2, Ren Hong-Lin3, Zhong Si-Yuan1, Cai Yu-Hong1, Peng Jing1 and Bao Hua-Xia1
ABSTRACT
Bacillus cereus is a common foodborne and opportunistic pathogen. It is widely distributed in soil, water and animal intestines. Its special spore structure easily causes foodborne infection to humans or animals. In order to monitor and prevent the spread of B. cereus, a rapid and sensitive loop-mediated isothermal amplification assay has been developed to detect it in uniformly items. According to the entFM gene of B. cereus, four primers were designed, and reaction components and conditions optimized. Sensitivity was compared between optimized LAMP assay and the conventional PCR. The optimal reaction conditions of LAMP assay (25uL each reaction) comprised 1.0 mol/L betaine, 6 m mol/L Mg2+, 1.4 mmol/L dNTPs, 1.6 μmol/L inner primers (1:1), 0.2 μmol/L outer primers (1:1), 2.5 μL 10×thermpol reaction buffer, 1 μL Bst DNA Polymerase (8U/μL), 1 μL template DNA, at 65 ℃ incubating 60 min. Two strains of B. cereus out of 18 strains were positive result by LAMP assay. The detection limit of B. cereus genomic DNA(gDNA) by LAMP was 0.755 pg/μL which was 100 times more sensitive than conventional PCR assay. The CFU limit of LAMP assay was 14×103 CFU/mL. The artificial polluted samples (chicken meat) can be detected by LAMP, when at least 45 min must be needed to enrich bacteria. Visual dye hydroxynaphthol (HNB) successfully used in LAMP assay was established for detection of B. cereus in meat.
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