Recently, a research paper entitled "Rapid and visual detection of Listeria monocytogenes based on polymerase spiral reaction in fresh-cut fruits" has been published online in LWT - Food Science and Technology by Prof. Moran Zhang (first author), Prof. Yingjian Lu (corresponding author), and Jing Sun (corresponding author) of School of Food Science and Engineering, Nanjing University of Finance and Economics. Rapid and visual detection of Listeria monocytogenes based on polymerase spiral reaction in fresh-cut fruit".

Widespread foodborne pathogen Listeria monocytogenes exhibits a pronounced fatality rate among individuals with compromised immune systems, the elderly, and pregnant women. A simple and user-friendly approach is required to identify L. monocytogenes. A distinctive thermostatic nucleic acid amplification technology called polymerase spiral reaction (PSR) has been extensively employed in the identification of foodborne pathogens. In the study, a straightforward and intuitive approach for identifying L. monocytogenes in fresh-cut fruit was established using PSR in conjunction with hydroxy naphthol blue (HNB). Following optimization, the system's primary constitutes-betaine, dNTP, and MgSO4 were found to be 0.5 M, 1.0 mM, and 6 mM, respectively. Six strains of non- L. monocytogenes were used to test the assay's specificity. The PSR assay performed at 65 °C for 50 min demonstrated the capability to detect L. monocytogenes at level as low as 1 × 10−4 ng/μL DNA per tube and 5.1 × 101 CFU/g of freshly cut fruit. Notably, the sensitivity of this approach surpassed that of PCR by10-fold (1 × 10−3 ng/μL). In order to detect L. monocytogenes, the PSR assay was designed to be rapid, accurate, effective, and apparatus free. This innovative assay has provided researchers a fresh perspective on identification harmful microorganisms.

Fig. 1. a. PSR optimization of betaine; b. PSR optimization of dNTP; c. PSR optimization of MgSO4; d. PSR optimization of primers proportion (main primers: accelerating primers).

Fig. 2. a. Analytical sensitivity of PSR by fluorescence curve; b. Analytical sensitivity of PSR by 2% agarose gel electrophoresis. (Lane 1-6:100 ng/μL, 10−1 ng/μL, 10−2 ng/μL, 10−3 ng/μL, 10−4 ng/μL, 10−5 ng/μL; Lane M: 2000 bp Marker; Lane N: Negative control); c. Analytical sensitivity of PSR by HNB. (Tube 1–6: 100 ng/μL, 10−1 ng/μL, 10−2 ng/μL, 10−3 ng/μL, 10−4 ng/μL, 10−5 ng/μL); d. Analytical sensitivity of PCR by 2% agarose gel electrophoresis (Lane 1–6: 100 ng/μL, 10−1 ng/μL, 10−2 ng/μL, 10−3 ng/μL, 10−4 ng/μL, 10−5 ng/μL; Lane M: 2000 bp Marker; Lane N: Negative control).

Fig. 3. a. Analytical specificity of PSR by fluorescence curve; b. Analytical specificity of PSR by 2% agarose gel electrophoresis (Lane 1–6: L. monocytogenes, S. aureus, S. typhimurium, S. enteritidis, B. cereus, S. flexneri; Lane M: 2000 bp Marker; Lane N: Negative control); c. Analytical specificity of PSR by HNB (Tube 1–6: L. monocytogenes, S. aureus, S. typhimurium, S. enteritidis, B. cereus, S. flexneri; Tube N: Negative control).

Fig. 4. a. PSR detection of L. monocytogenes from artificial contamination samples by fluorescence curve; b. PSR detection of L. monocytogenes from artificial contamination samples by 2% agarose gel electrophoresis (Lane 1–6: 5.1 × 106 CFU/g, 5.1 × 105 CFU/g, 5.1 × 104 CFU/g, 5.1 × 103 CFU/g, 5.1 × 102 CFU/g, 5.1 × 101 CFU/g, 5.1 × 100 CFU/g; Lane M: 2000 bp Marker; Lane N: Negative control); c. PSR detection of L. monocytogenes from artificial contamination samples by HNB (Tube 1–6: 5.1 × 106 CFU/g, 5.1 × 105 CFU/g, 5.1 × 104 CFU/g, 5.1 × 103 CFU/g, 5.1 × 102 CFU/g, 5.1 × 101 CFU/g, 5.1 × 100 CFU/g; Tube N: Negative control).