Detection of Salmonella Enterica in Egg Yolk by PCR on a Microfluidic Disc Device Using Immunomagnetic Beads

Abstract

Salmonella enterica is a pathogenic bacterium that causes foodborne illness. One of the vehicle foods of S. enterica are chicken eggs. Efficient collection of the bacterium is necessary to detect it specifically. We developed a method to detect S. enterica by PCR on a microfluidic disc device using a fluorescent probe. Salmonella enterica cells were isolated in the microchambers on the device, followed by thermal lysis and PCR targeting with the invA gene, a gene specific to S. enterica, were observed by measurement of the fluorescent signal that resulted from gene amplification. However, the developed method was unable to discriminate viable cells from dead cells. Consequently, in this study, magnetic beads modified with anti-Salmonella antibody were utilized to detect viable Salmonella cells from egg yolk prior to PCR on the device. While using the antibody-modified beads, egg yolk components, which inhibit PCR, were removed. The collected cells were subsequently detected by PCR of the invA gene on a microfluidic disc device. This method enabled the detection of viable cells without the inhibition of PCR by any egg component. S. enterica was detected at 5.0×104 cells mL−1 or at a higher concentration of egg yolk within 6 h including the sampling time.

Recent advancements in microfluidic chip biosensor detection of foodborne pathogenic bacteria: a review

“…This convenient experimental device enables the development of a real-time diagnostic system while maintaining appropriate sensitivity. Kubol et al [ 29 ] developed a method for detecting intestinal Salmonella by PCR using fluorescent probes on microfluidic disc devices. The cells of Salmonella enterica were isolated in the microchamber of the apparatus and then thermally lysed, and the polymerase chain reaction targeting the invA gene specific to S. enterica was observed by measuring the fluorescence signal generated by gene amplification.…”

Application of Lab-on-Chip for Detection of Microbial Nucleic Acid in Food and Environment

“…After washing and cell lysis, DNA/RNA is obtained for nucleic acid amplification ( Kant et al, 2018 ). Kubo et al (2020) designed a LOC device to detect Salmonella in egg yolks using magnetic beads modified with anti- Salmonella antibodies. After thermal lysis, the target genes were amplified by PCR, and then detected by fluorescence probe.…”

Rapid label‐free detection of Salmonella enterica with biolayer interferometry

“…Culture‐based conventional methods are considered to be the “gold standard” and are commonly used for S. enterica detection. Coupled with limited dilution analysis and selective medium, these methods are highly sensitive and specific, as even few colony‐forming units (CFU)/ml of S. enterica can be identified in food (Kubo, Kajiya, Aramaki, & Furutani, 2020). However, because of complicated procedures, they are arduous and time‐consuming, taking at least 3–4 days for confirmation of contamination by S. enterica (Lee, Runyon, Herrman, Phillips, & Hsieh, 2015).…”

“…However, because of complicated procedures, they are arduous and time‐consuming, taking at least 3–4 days for confirmation of contamination by S. enterica (Lee, Runyon, Herrman, Phillips, & Hsieh, 2015). Over the last few years, several typical methods have been applied for the rapid detection of S. enterica , including enzyme‐linked immunosorbent assay (ELISA; Jay, 1996; Li et al, 2019) and polymerase chain reaction (PCR) techniques (Bhandari, Chen, Hamal, & Bridgman, 2019; Kubo et al, 2020; Vinayaka et al, 2019). Although ELISA is simple to use, it has two limitations: low sensitivity for detecting the causal pathogen (typically around 10 5 CFU/ml) and long assay time (4 hr ~).…”