Abstract:

The purpose of this study was to develop a real-time helicase-dependent isothermal DNA amplification (HDA)
method for the rapid detection of Listeria monocytogenes. Based on the platform of real-time PCR, pairs of primers targeting
the hemolysin gene (hly) of Listeria monocytogenes were designed, and genomic DNA was extracted from a standard strain
of L. monocytogenes for use as the template. The reaction temperature, primers and template DNA concentration were
optimized. Compared with real-time PCR method, the specificity and sensitivity of the real-time HDA method were
evaluated with L. monocytogenes and 10 bacteria control strains, and then this developed method was used to detect
L. monocytogenes in real samples. The results showed that the optimal primer concentration, reaction temperature and
time for real-time HDA system were 0.075 mol/L, 65 ℃ and 80 min (40 cycles), respectively. This system showed a
high specificity and sensitivity. Thus a real-time HDA method for rapid and specific detection of L. monocytogenes has
been successfully established.

Key words: Listeria monocytogenes, hemolysin gene, helicase-dependent isothermal amplification, real-time fluorescenceHDA, rapid detection