Saltatory rolling circle amplification assay for simple and visual detection of Listeria monocytogenes in milk and milk products

Abstract

A novel saltatory rolling circle amplification (SRCA) assay was established for prompt identification of Listeria monocytogenes in milk and Indian milk cake (kalakhand). The SRCA and conventional PCR were independently able to detect 63 fg and 630 fg L. monocytogenes DNA per reaction, respectively. The detection limit of SRCA was evaluated in artificially contaminated milk and kalakhand. In milk, without any enrichment, SRCA was able to detect 4.4 × 102 cfu mL−1 of milk, indicating 100-fold greater sensitivity than PCR (4.4 × 104 cfu mL−1). After enrichment up to 3 h and 6 h, detection in SRCA improved to 4.4 cfu mL−1 of milk. Spiking studies in kalakhand showed 10-times better results for SRCA than PCR. The study illustrates that the developed SRCA-based test is sensitive, cost-effective, rapid, and most importantly simple for the detection of L. monocytogenes and it can be adopted for on-site detection of L. monocytogenes in a variety of milk and milk products.