2019 年 36 巻 1 号 p. 47-52
We developed a multiplex loop-mediated isothermal amplification (mLAMP) assay to discriminate between Campylobacter jejuni and C. coli. The mLAMP assay is based on the fluorescence intensity of a fluorescent intercalator, which indicates whether DNA amplification has occurred. After the mLAMP reaction, two specific DNA products are discriminated based on differences in melting temperatures (Tm values). The mean Tm value obtained for C. jejuni by this assay was 84.26±0.19 and 88.34±0.30℃ for C. coli. The assay correctly discriminated between 34 C. jejuni strains and seven C. coli strains, and other bacteria associated with food poisoning were not detected. In sensitivity tests, the mLAMP assay effectively detected C. jejuni at levels of 7.3 cfu/test and C. coli at 13 cfu/test. In an assessment of viable bacterial counts and detection times, correlations in the range 10–104 cfu/test were obtained for both C. jejuni and C. coli (R2>0.9). In an analysis of five chicken samples using the mLAMP assay, C. jejuni was detected in three samples and C. coli was detected in one sample. These results corroborated those obtained based on bacterial cultures, but were obtained more rapidly. In conclusion, the developed mLAMP assay is a highly specific, sensitive and rapid tool for discriminating between C. jejuni and C. coli.
