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

Abstract

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 costeffective, 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.

Rapid detection methods for foodborne pathogens based on nucleic acid amplification: Recent advances, remaining challenges, and possible opportunities

“…Therefore, more effort is needed to improve the performance of RCA in conjunction with biosensors in detecting pathogens in food samples. Nevertheless, a study reported that RCA technology could detect pathogens in pork better than conventional PCR and qPCR ( Milton et al, 2021b ).…”

“…SRCA follows an initial amplification mechanism similar to RCA, using a circular DNA template and a DNA polymerase with strand displacement activity. However, during the SRCA process, specific recognition sites within the template sequence are encountered ( Milton et al, 2021b , Milton et al, 2021a , Milton et al, 2021c , Zhang et al, 2019a ). At these recognition sites, DNA-cutting enzymes, such as restriction endonucleases or nicking enzymes, introduce breaks in the circular DNA.…”

“…As a result, SRCA produces shorter, non-contiguous DNA segments, referred to as saltatory products. In recent studies, this method has been used to detect numerous pathogens in various types of food samples, including PIF, milk, pork, chicken meat, and oysters ( Huang et al, 2023b , Milton et al, 2021b , Milton et al, 2021a , Milton et al, 2021c , Prasad et al, 2023 , Yang et al, 2019 , Yuan et al, 2022 , Zhang et al, 2019a ).

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Recent advances in biological detection with rolling circle amplification: design strategy, biosensing mechanism, and practical applications

“…Up to now, SRCA assays have been exploited in Salmonella, 34 Staphylococcus aureus, 113 Shigella, 112 Cronobacter sp., 114 Clostridium perfringens 115 and Alicyclobacillus acidoterrestris. 100 As a signal amplification technology, RCA can be employed in combination with numerous biosensor technologies to detect pathogenic bacteria. Chen et al combined LAMP with RCA technology and adopted molecular beacons to pair the amplification products to emit fluorescence.…”

“…Up to now, SRCA assays have been exploited in Salmonella , 34 Staphylococcus aureus , 113 Shigella , 112 Cronobacter sp., 114 Clostridium perfringens 115 and Alicyclobacillus acidoterrestris . 100…”