Evaluation of loop mediated isothermal amplification (LAMP) assay for rapid detection of Listeria monocytogenes from fish

Abstract

 A loop mediated isothermal amplification (LAMP) assay was optimised for rapid detection
of Listeria monocytogenes from fish by targeting haemolysin gene and compared with
conventional PCR and real time PCR (qPCR). All the assays were carried out using different
DNA extraction methods like commercial kit, phenol-chloroform-isoamyl alcohol method
and heat shock method. The analytical sensitivity of LAMP and qPCR was comparable and
the detection limit was found to be 9.6×101 CFU ml-1 from broth and 8×10² CFU ml-1 from
spiked fish whereas the detection limit of conventional PCR was found to be 9.8×102 CFU ml-1
and 8×10⁴ CFU ml-1 from broth and fish respectively, when commercial kit was used for
DNA extraction. The specificity of all these methods was 100% when compared with related
bacterial species. The optimised LAMP assay when applied directly on 204 field fish samples
gave an accuracy of 70.59% when compared to the gold standard while conventional PCR
showed a lower accuracy of 52.94%. However, enrichment of LAMP negative samples for 6 h
enhanced the sensitivity of detection to 100%. The optimised assay detected all negative
fish samples by culture as negative hence giving detection specificity of 100%. Moreover,
LAMP assay took the least detection time as compared to conventional PCR and qPCR.
Thus, the optimised LAMP assay developed can be used as a sensitive, rapid and simple
detection tool for the reliable detection of L. monocytogenes from fish.

Keywords:
Haemolysin gene (hlyA gene), Listeria monocytogenes, LAMP, PCR, qPCR