A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on label-free amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction

Authors: BAI, YALONG - Shanghai Jiaotong University; SONG, MINGHUI - Shanghai Jiaotong University; CUI, YAN - Shanghai Jiaotong University; SHI, CHUNLEI - Shanghai Jiaotong University; WANG, DAPENG - Shanghai Jiaotong University; Paoli, George; SHI, XIANMING - Shanghai Jiaotong University

Submitted to: Analytica Chimica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/29/2013
Publication Date: 6/2/2013
Publication URL: http://handle.nal.usda.gov/10113/57268
Citation: Bai, Y., Song, M., Cui, Y., Shi, C., Wang, D., Paoli, G., Shi, X. 2013. A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on label-free amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction. Analytica Chimica Acta. 787:93-101.

Interpretive Summary: This study was conducted to develop an improved method for the detection of harmful bacteria (pathogens) from food. To protect consumer health, food producers and government regulators require rapid and specific methods to detect pathogens in food. Most foods do not contain harmful bacteria, and when they are present, they are present in very small amounts. Thus, the detection methods must also be very sensitive. A common method to detect foodborne pathogens, the polymerase chain reaction (or PCR), is based on the detection of specific genes associated with the particular pathogenic bacterium. Most procedures that use PCR to detect pathogenic bacteria from food require the food to be incubated in a microbial growth medium for several hours to overnight in order to increase the concentration the of pathogens to a level that can be detected by PCR. This growth step greatly increases the time to result for foodborne pathogen testing and prevents the enumeration of the pathogens in the food. In the present study, rather than using a lengthy microbial growth step to increase the concentration of the bacteria in the food, microscopic magnetic particles (nanoparticles) were used to capture and concentrate the DNA directly from the bacteria in the food prior to the detection of the pathogen genes by PCR. Using artificially contaminated raw milk, the nanoparticles proved very effective in capturing the DNA, allowing for very rapid and sensitive detection of pathogens (both Salmonella Enteritidis and Listeria monocytogenes) by PCR without the need for the lengthy microbial growth step. This method has the potential to improve public health by providing food producers and regulators a quicker time to result for foodborne pathogen testing as well as allow for the enumeration of pathogens in food.

Technical Abstract: A method based on amino-modified silica-coated magnetic nanoparticles (ASMNPs) and polymerase chain reaction (PCR) was developed to rapidly and sensitively detect foodborne pathogens in raw milk. After optimizing parameters such as pH, temperature, and time, a trace amount of genomic DNA of pathogens could be extracted directly from complex matrices such as raw milk using ASMNPs. The magnetically separated complexes of genomic DNA and ASMNPs were directly subjected to single PCR (S-PCR) or multiplex PCR (M-PCR) to detect single or multiple pathogens from raw milk samples. Salmonella Enteritidis (Gram-negative) and Listeria monocytogenes (Gram-positive) were used as model organisms to artificially contaminate raw milk samples. After magnetic separation and S-PCR, the detection sensitivities were 8 CFU/ml and 13 CFU/ml, respectively. Furthermore, this method was successfully used to detect multiple pathogens (S. Enteritidis and L. monocytogenes) from artificially contaminated raw milk using M-PCR at sensitivities of 15 CFU/ml and 25 CFU/ml, respectively. This method has great potential to rapidly and sensitively detect pathogens in raw milk or other complex food matrixes.