Single primer isothermal amplification coupled with SYBR Green II: Real-time and rapid visual method for detection of Listeria monocytogenes in raw chicken

Abstract

As a notorious foodborne pathogen worldwide, Listeria monocytogenes (L. monocytogenes) cause severe listeriosis with high mortality rate presenting a significant public health risk. Coupled with SYBR Green II, herein, a real-time and rapid visual single primer isothermal amplification (SPIA)method has been established for rapid detection of L. monocytogenes in raw chicken targeting the hlyA gene. The developed SPIA method enabled the resultscan be observed through the real-time amplification fluorescence curve, visual fluorescence or precipitate by naked eye. The method exhibited satisfied sensitivityof 1.4 × 101 CFU/mL by precipitate and 1.4 × 100 CFU/mL by fluorescence, respectively. With regard to the artificially inoculated raw chicken, the detection limits of SPIA were 1.4 × 101 CFU/g by precipitate and 1.4 × 100 CFU/g by fluorescence, respectively. In comparison with the conventional PCR method, the sensitivity of SPIA method was 10-fold or 100-fold better than that of PCR, while the detection limit was 10-fold or 100-fold lower than that of PCR. Compared with the ISO method, the relative sensitivity, specificity and accuracy of SPIA method were calculated as 100%, 97.47% and 97.65%, respectively. The results indicated that the developed SPIA method has the potential for rapid visual and on-site detection of L. monocytogenes in various food samples.