Optimization of a rapid method for detection of Listeria monocytogenes in food matrices

Abstract

Detection of Listeria monocytogenes by the microbiological standard method (ISO 11290-1) is time consuming and requires several days to get results, thus a more rapid detection method is required. The aim of this study is to continue the optimization of a rapid method that is based on quantitative polymerase chain reaction (qPCR) to detect L. monocytogenes in food matrices.
The method is based on a shorter enrichment step in 50 mL non-selective medium, followed by DNA extraction with EZ1 robot and analysis with qPCR. The extraction method and qPCR analysis have been chosen and optimized to handle larger sample volumes without interference from the background matrices. In this study, sausages from HKScan, green frozen peas and swabs from HKScans food production line were used as models for different background matrices. Different sample volumes from the enrichment step with sausages and green frozen peas (2, 5 and 10 mL) and with swab matrices (1, 2 and 5 mL) were tested to determine which volume gave the highest L. monocytogenes DNA yield. For food matrices, 2 mL volume gave the highest yield, while 1 mL gave the highest yield for swab matrices. Larger volumes led to decreased yield, most likely due to interference from the background flora and inhibitors from the matrices. The temperature of cell culturing incubation was also optimized for L. monocytogenes. It was found that incubation at 35 °C and 37 °C gave the same L. monocytogenes growth, but that incubation at 37 °C promoted faster growth of the unwanted background flora.
The qPCR step was optimized for two detection gene assays: Listeria gene mix and prfA. After optimizing the assays in regard to annealing temperature and magnesium concentration, a comparison between the two qPCR assays was done. It was shown that Listeria gene mix was more specific than prfA assay for detection of L. monocytogenes DNA, and was thus chosen for this method.

Popular Abstract

Listeriosis is a foodborne illness that is caused by ingestion of Listeria monocytogenes. The mortality rate is high, between 20-30%. People at risk are older adults, people with weakened immune systems, pregnant women and newborns. According to World Health Organization estimation in 2010, there were 23,150 reported cases of listeriosis with 5,463 deaths in the U.S., while the European Union in 2009 accounted for 1,645 reported cases of listeriosis with 270 deaths. Human listeriosis is commonly associated with outbreaks that are often caused by consumption of ready-to-eat products.
According to the U.S. Food and Drug Administration (FDA), the estimated total cost of listeriosis is $2.6 billion which makes it the third most costly foodborne disease. This includes expenses of healthcare and decreased productivity. In addition to these expenses, there are high expenses to the producer of the contamination source in case of outbreaks.
The conventional method for detection of Listeria monocytogenes requires the use of enrichment cultures followed by selective plating and it takes up to a week to get a result. Although new and faster methods based on molecular detection using quantitative polymerase chain reaction (qPCR) or ELISA have been developed, these methods often require selective enrichment before analysis and it often takes more than 24 hours to get reliable results. Most producers of ready-to-eat food cannot wait as long as 24 hours to release their products, thus even faster detection methods of Listeria monocytogenes are highly desirable. A more rapid method would reduce the expenses of listeriosis by preventing its outbreak and result in faster release of finished food products which is a clear benefit for food producers.
This study has been part of the development of a more rapid method for detection of Listeria monocytogenes. This method includes a single and short non-selective enrichment step, followed by DNA extraction and qPCR analysis. The approach is based on the pre-PCR processing concept, where the aim is to maximize the amount of target molecules and at the same time minimize the effect of inhibitory substances. This is achieved by optimizing all steps of the analysis chain, i.e. enrichment, nucleic acid extraction and amplification and detection of PCR products, to the sample and to be compatible to each other.
This work included selection and optimization of a multiplex real-time PCR assay for Listeria species and for Listeria monocytogenes, and optimization of a DNA extraction method for Listeria.

• author: Mikhael, Lara
• supervisor: Linda Jansson; Sebastian Jankowski
• organization: Applied Microbiology
• course: KMBM05 20181
• year: 2018
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: extraction, EZ1, genus Listeria, hlyA, Listeria monocytogenes, peas, pre-lysis, prfA, prs, qPCR, sausages, swab, applied microbiology, teknisk mikrobiologi
• language: English
• id: 8940245
• date added to LUP: 2018-05-25 10:48:58
• date last changed: 2018-05-25 10:48:58