Abstract
Pathogene: A portable, low-cost, microfluidic lab-on-a-chip based device for rapid detection of multiple foodborne pathogens
Despite regulations in place to ensure the distribution of safe food, foodborne diseases (FBDs) remain a global concern. To address the worldwide challenge of FBDs, we have devised a customizable device for the simultaneous detection of multiple food-borne pathogens (FBPs). The device detects specific DNA sequences associated with four FBPs: Campylobacter, Listeria monocytogenes, Salmonella, and Vibrio cholerae using the isothermal amplification techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The use of isothermal techniques allows the device to be more portable and cost-effective compared to conventional PCR systems, while the use of microfluidics allows for multiplexing and rapid high-throughput screening. The parameters of the device such as the number of pathogens, and amplification and detection methods can be customized as required. This novel lab-on-a-chip based device is rapid, portable, affordable, sensitive, specific, and customizable, making it ideal for resource-limited settings and point-of-care testing.
Background
Food safety and prevention of foodborne illness remains an important plight of members of the international community and more specifically the scientific community. Even though most countries have food safety regulations and laws, it is still a challenge to ensure that all food companies and manufacturers, and not just the large-scale ones, are compliant to these rules and sell food that is safe to consume. Often, the challenges that the small-scale food producers or everyday consumers face in deciding whether food is safe to consume are challenges of accessibility, affordability, and portability of the methods of detection. Thus, this project aims to build on and improve on last year’s NYU Abu Dhabi iGEM project that detected the presence of STEC in food. They created a device that was a cost-effective and portable. This year’s team hopes to build on it by developing a service of customizable devices to suit anyone’s need. The team started by analysing different target groups and realized that there is a huge need for a quick detection of food pathogens in many different areas (See more in target group info under ideation). We focused on making our service wide by providing different techniques (LAMP and RPA), waiting time, different visualization methods (fluorescence and color change) and detection of various different pathogens that cause foodborne illnesses. We made sure that the device is efficient and portable and guided this year’s project based on WHO’s ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid, Robust, Equipment free, and Delivered to those who need it) ideal features for diagnostic tests (1).
The team has selected four pathogens to be investigated: Campylobacter, Listeria monocytogenes, Salmonella, and Vibrio cholerae. All of these pathogens are most widely spread and are significant threats to human health around the world. Detailed information about each pathogen, why they are a significant concern, their mode of action, geographical locations most affected, the symptoms of infection, and a bacteria-specific gene that was selected by the team can be found below:
![](https://static.igem.org/mediawiki/2018/6/61/T--NYU_Abu_Dhabi--CB.png)
What is it?
Campylobacter is a bacteria genus with 17 known species and 6 subspecies. The two species C.jejuni and C.coli are the most commonly associated with human diseases. Their structure is mainly spiral and "S" shaped, or curved and rod shaped (1). Campylobacter Jejuni functions by arresting the cell cycle in G2 phase using Cytolethal Distending Toxin (4).
Why are they a major concern? Which parts of the world are the most affected?
Yearly, 550 million people are infected with diarrheal diseases (1). Campylobacter is one of the four main causes of diarrheal diseases and is the most common cause of gastroenteritis, in this case called Campylobacteriosis, an infection in the stomach and small intestine (1). Campylobacteriosis is a zoonosis, meaning the source of its transmittance to humans is animals.
What are the symptoms of the infection?
The infection has symptoms that last 3 to 6 days such as diarrhea, nausea, vomiting, headaches, and abdominal pain that appear 2 to 4 days after an infection (1). Infections by Campylobacter are usually caused by injected contaminated undercooked poultry, or contaminated water. Such infections can be fatal in young children, elderly and immunosuppressed patients, such as those with diseases like AIDs. In developing countries there is a high incidence of Campylobacter infections in children under two and are sometimes fatal (1). In rare cases, a Campylobacter infection can lead to other diseases and disorders such as hepatitis, pancreatitis, miscarriages and Guillain-Barré syndrome (2). Guillain-Barré syndrome is a neurodegenerative disorder that occurs due to the immune system attacking the peripheral nervous system, including the cranial nerves, the spinal nerves and the autonomic nervous system (3).
Gene of choice. Why?
hipO gene codes for the hippurate hydrolase in Campylobacter jejuni. It was chosen due its specificity to the organism, the lack of toxicity and previous research projects that have used this gene to detect the pathogen by PCR (5, 6).
References:
1. Campylobacter (2018) World Health Organization. Available at: http://www.who.int/mediacentre/factsheets/fs255/en/ [Accessed April 18, 2018].
2. Hofreuter D (2006) Unique Features of a Highly Pathogenic Campylobacter jejuni Strain. American Society for Microbiology 74 no. 8 4694-4707.
3. Yuki N & Hartung H-P (2012) Guillain–Barré Syndrome. New England Journal of Medicine 366(24):2294-2304.
4. Whitehouse CA (1998) Campylobacter jejuni Cytolethal Distending Toxin Causes a G2-Phase Cell Cycle Block. American Society for Microbiology 66 no. 5 1934-1940.
5. Ayaz, N. D., Goncuoglu, M., Cakmak, O., & Erol, I. (2016). Comparison of hipO and ceuE gene based PCR assays for the detection of Campylobacter jejuni. J Clin Microbiol Biochem Technol 2 (1): 006, 8(006).
6. Caner, V., Cokal, Y., Cetin, C., Sen, A., & Karagenc, N. (2008). The detection of hipO gene by real-time PCR in thermophilic Campylobacter spp. with very weak and negative reaction of hippurate hydrolysis. Antonie Van Leeuwenhoek, 94(4), 527.
![](https://static.igem.org/mediawiki/2018/4/41/T--NYU_Abu_Dhabi--Listeria.png)
What is it?
Listeria Monocytogenes is a rod-shaped, food-borne, pathogenic species of gram-positive bacteria that ultimately infects both animals and humans, culminating in the disease Listeriosis. L. monocytogenes are characterised by their outstanding ability not only to survive in harsh conditions but also proliferate, these conditions include pH environments of 4.3-9.6, temperatures between 1-45°C and saline environments (less than 10%) (1-2). Three out of the total 13 identified serotypes of the bacterium, 1/2a, 1/2b and 4b are responsible for most of the cases of listeriosis, albeit all 13 serovars have the pathogenicity to cause listeriosis (3).
Why are they a major concern? Which parts of the world are the most affected?
L. Monocytogenes infections do not usually lead to serious listeriosis in healthy adults, however of the susceptible population of infants, elderly and pregnant women who contract invasive listeriosis, there is a significantly high mortality rate of 20-30% (4). This high mortality rate is especially concerning considering that the bacterium persists in a diverse range of environments and foods thus triggering sporadic outbreaks all over the world. According to the World Health Organisation, as of March 2018, the largest listeria outbreak is occurring in South Africa and has seen 978 cases of laboratory-confirmed listeriosis, of which 183 have resulted in a fatal outcome (5). Due to the bacterium’s robust nature and commonness, outbreaks are a common global occurrence and are non-region specific thus making them a serious international public health threat.
What are the symptoms of the infection?
The motility of L. Monocytogenes is reliant on their flagella, active at relatively low temperatures and so they are able to travel from food sources such as raw or unpasteurised milk and milk products, raw and processed vegetables, fruit and meat to food-contact surfaces, especially in food-processing and handling environments. From the source, humans ingest the bacteria orally after which the bacterium travels directly from cell to cell through the intestinal mucosa where dendritic cells under Peyer’s patches are infected and promote bacterial spread to blood and lymph (2-3). The majority vulnerable population of neonates and foetuses (~40%) are infected at birth or in-utero from the mother, prompting abortions and stillbirths (6).
Two different forms of the disease manifest after infection by L. Monocytogenes depending on the general health and immune strength of the infected. Healthy adults who contract the disease may suffer a mild non-invasive gastrointestinal illness including symptoms of fever, diarrhoea and vomiting with an incubation period between 18 and 20 hours and is usually self-resolving. Infected elderly, pregnant women, cancer patients and others affected by immunosuppression are more likely to contract an invasive septicaemic or neuropathic form of the disease resulting in a combination of the following set of symptoms: fever, malaise, fatigue, abdominal pain, ataxia, seizures, meningitis. This invasive form has an incubation period of 3-90 days (2).
Gene of choice. Why?
Lmo0773 gene codes for a lmo0773 protein, which is involved in the transcription regulation. The gene was chosen as it is not involved in any toxic activities in the bacteria and is specific for Listeria monocytogenes (7, 8).
References:
1. Bowman JP, Bittencourt CR, & Ross T (2008) Differential gene expression of Listeria monocytogenes during high hydrostatic pressure processing. Microbiology 154(2):462-475.
2. Roberts A & Wiedmann M (2003) Pathogen, host and environmental factors contributing to the pathogenesis of listeriosis. Cellular and Molecular Life Sciences CMLS 60(5):904-918.
3. World Health Organisation (2012) Foodborne Pathogenic Microorganisms and Natural Toxins. Food and Drug Administration.
4. Anonymous (2004) Risk assessment of Listeria monocytogenes in ready-to-eat foods. in Technical Report (World Health Organisation, Rome, Italy).
5. Anonymous (2018) Listeriosis- South Africa. Emergencies preparedness, response.
6. Farber J & Peterkin P (1991) Listeria monocytogenes, a food-borne pathogen. Microbiological reviews 55(3):476-511.
7. Chatterjee, S. S., Hossain, H., Otten, S., Kuenne, C., Kuchmina, K., Machata, S., ... & Hain, T. (2006). Intracellular gene expression profile of Listeria monocytogenes. Infection and immunity, 74(2), 1323-1338.
8. Glaser, P., Frangeul, L., Buchrieser, C., Rusniok, C., Amend, A., Baquero, F., ... & Charbit, A. (2001). Comparative genomics of Listeria species. Science, 294(5543), 849-852.
![](https://static.igem.org/mediawiki/2018/7/7f/T--NYU_Abu_Dhabi--Salmonella.png)
What is it?
Salmonella is a bacteria that causes diarrheal diseases in organisms. It is a gram negative rods and belongs to the Enterobacteriaceae family. There are two major species, Salmonella bongori and Salmonella enterica, and over 2500 different serotypes have been identified within the two species. Of the various serotypes, Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Typhimurium are two of the most important and prevalent types that affect human around the world. Salmonella is a hardy bacteria that can survive under unfavorable circumstances: it can survive several weeks in a dry environment and can survive several months in water (1).
Why are they a major concern? Which parts of the world are the most affected?
According to WHO, 1 in 10 people fall ill each year because of foodborne diseases, and salmonella is one of the four major causes of diarrheal diseases throughout the world (1). Salmonella infection is usually caused by consuming raw or undercooked meat, poultry, eggs, egg products or contaminated raw fruits and vegetables. Salmonella can be exterminated by cooking and pasteurization (2).
What are the symptoms of the infection?
Depending on the serotype of Salmonella infected, two types of illnesses might result, either nontyphoidal salmonellosis or typhoid fever. For nontyphoidal salmonellosis, the illness is developed through the penetration and passage of Salmonella organisms from gut lumen into epithelium of small intestine. Evidence have shown that enterotoxin (toxin affecting the intestines) may be produced within enterocytes (a cell of the intestinal lining). For typhoid fever, the illness is developed through the penetration and passage of typhoid Salmonella organisms from gut lumen into epithelium of small intestine and further into the bloodstream. Once the bacteria enter the bloodstream, it may be carried to other sites of the body. There is also evidence that enterotoxin may be produced within the cells of the intestinal lining (3).
The disease caused by salmonella is called Salmonellosis. It is usually characterized by acute fever, abdominal pain, diarrhea, nausea, and vomiting. The symptoms of salmonellosis is relatively mild: some people infected don’t exhibit any signs of infection while others can recover without specific medical treatment (2). However, for those with weaker immune system, especially in children or the elderly, the associated dehydration can become severe and life-threatening. Symptoms of the disease occurs 6–72 hours after the ingestion of Salmonella, and the illness usually lasts 2–7 days.
Gene of choice. Why?
The invA gene codes for the invasion gene involved in the invasion of the intestinal epithelium cells. This gene is specific to all Salmonella serotypes and alone does not cause toxicity. Previous studies have also utilized this gene for detection of Salmonella by PCR and LAMP (4-6).
References:
1. Salmonella (non-typhoidal) (2018) World Health Organization. Available at: http://www.who.int/mediacentre/factsheets/fs139/en/ [Accessed April 18, 2018].
2. Salmonella infection - Symptoms and causes (2018) Mayo Clinic. Available at: https://www.mayoclinic.org/diseases-conditions/salmonella/symptoms-causes/syc-20355329 [Accessed April 18, 2018].
3. Center for Food S & Applied N, The bad bug book.
4. Rahn K, et al. (1992) Original article: Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Molecular and Cellular Probes 6:271-279.
5. Chiu CH & Ou JT (1996) Rapid Identification of Salmonella Serovars in Feces by Specific Detection of Virulence Genes, invA and spvC, by an Enrichment Broth Culture-Multiplex PCR Combination Assay. (AMERICAN SOCIETY FOR MICROBIOLOGY, United States), p 2619.
6. Hara-Kudo, Y., Yoshino, M., Kojima, T., & Ikedo, M. (2005). Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS microbiology letters, 253(1), 155-161.
![](https://static.igem.org/mediawiki/2018/6/6b/T--NYU_Abu_Dhabi--Cholera.png)