Development of novel visual detection methodology for Salmonella in meat using saltatory rolling circle amplification

• Published in: Journal of applied microbiology 2021-11, Vol.131 (5), p.2361-2371
• Main Authors: Milton, A.A.P., Momin, K.M., Priya, G.B., Ghatak, S., Das, S., Gandhale, P.N., Angappan, M., Sen, A.
• Format: Article
• Language: English
• Subjects: Amplification; Assaying; Deoxyribonucleic acid; Detection limits; DNA; Enrichment; Food; Hydrogen enrichment; invA; isothermal assay; Meat; Polymerase chain reaction; Pork; Salmonella; Salmonella enterica; saltatory rolling circle amplification; Screening
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.15099

Aim The aim of this study was to develop a saltatory rolling circle amplification (SRCA) assay for rapid, simple and visual detection of Salmonella in meat. Methods and Results Saltatory rolling circle amplification assay was established using simple PCR primers targeting the invA gene of Salmonella enterica. The specificity of the SRCA assay was determined using 28 Salmonella and 15 non‐Salmonella strains. The analytical sensitivity of the developed SRCA, conventional and real‐time PCR assays were 70 fg, 7 pg and 700 fg S. enterica DNA per tube, respectively. The limit of detection (LoD) of the SRCA assay was 40 CFU per gram of meat without enrichment and 4 CFU per gram after including 6 h brief enrichment step. The detection limits of 40 CFU per gram and 4 CFU per gram of meat were achieved within 165 min and 9 h, respectively (including DNA extraction). To assess the real‐world relevance of the SRCA assay, it was used to screen Salmonella from the field pork samples (n = 82). The same samples were also tested with culture (ISO 6579: 2002) method, conventional and real‐time PCR assays. Using the developed assay with 6‐h enrichment step, it could give accurate results as that of the culture method. Conclusions The results of this study showed that the SRCA assay is a rapid, simple, sophisticated equipment‐free and user‐friendly method for accurate detection of Salmonella in meat foods. To our information, this is the first study to deploy SRCA assay for screening foods for Salmonella. Significance and Impact of the Study The developed SRCA assay is cost‐effective, easy‐to‐perform and equipment‐free; therefore, it has the potential to replace other molecular detection methods for regular screening of Salmonella in foods in field laboratories.