Rapid methods for the detection of pathogens have been gaining acceptance in the food industry. Recent advances in technology can result in faster detection of pathogens, more convenient, more sensitive methods. We have seen many new alternative methods being proposed in the past couple of years. Below is an example of such a novel method.According to a press release from University of Massachusetts Amherst (https://www.umass.edu/newsoffice/article/umass-amherst-food-scientists-are ) a team of scientists (including Lily He, Lynne McLandsborough, and Brooke Pearson) developed a low cost, rapid method for the detection of bacteria in food samples.The assay steps include rinsing the fruit or vegetable, collecting the rinse water sample, placing the water to the chemical-based microchip that captures the harmful bacteria, and detecting the bacteria using a smartphone with a light microscope adaptor. He said:” If there are harmful bacteria, it will be shown as visible dots, to indicate that you may have, for example, salmonella or Listeria.”
The Chip includes 3-mercaptophenylboronic acid (3-MBPA) that attracts and binds to any bacteria. Food particles, sugars, fat, and proteins can be washed away with a high-pH buffer.There are two detection methods: (i) surface-enhanced Raman spectroscopy” (SERS) that relies on silver nanoparticles and the optical microscopy method. Both the optical method and the SERS mapping methods have a sensitivity of detection as few as 100 CFU/mL according to the publication (http://pubs.rsc.org/en/content/articlelanding/2017/ay/c7ay01596h#!divAbstract). The total assay time for the optical method is 1.25 hours and for SERS imaging 3 hours.The method seems very attractive at first glance. It is straightforward and inexpensive. The use of a smartphone instead of a microscope is eye-catching. A lay person can use it without the need for a laboratory.However, it seems to me that the method lack the desired selectivity as it detects all bacteria and not necessarily pathogenic bacteria. For pathogens, it lacks the desired sensitivity. While sensitivity of 100 CFU/mL is impressive, a system for pathogens needs to be 10,000 folds more sensitive.There is a danger of prematurely promoting such rapid methods; it might take many years of research to obtain the desired specificity and sensitivity.
Dr. Timothy Lu, an associate professor in biological engineering at the Massachusetts Institute of Technology, found a new potential way to kill superbugs with a DNA editor called CRISPR-Cas9. The Wall Street Journal reported that Dr. Lu said: “is is basically a molecular scissor” that can snip bacterial genes that make bacteria drug-resistant, killing the bug in the process. The technology combines bacteriophages and CRISPR-Cas9 to target drug-resistant genes.
What is CRISPR?
CRISPRis used to edit or delete genes from living cells. “CRISPR” means Clustered Regularly Interspaced Short Palindromic Repeats. They are the characteristic of a bacterial defense system thatformsthe basis for CRISPR-Cas9 genome editing technology.CRISPR-Cas9 can be programmed to target portions of genetic code and edit DNA at exact locations. It allows researchers to modify genes in living organisms permanently. This technology can be used to remove the genes that make the bacteria drug-resistant, and in the process, it can also kill the bacteria.
The New Technology to Eliminate Drug-Resistant Bacteria
Dr. Lu is studying ways to eliminate superbugs with CRISPR-Cas9. He is contemplating combining the CRISPR-Cas9 technology with bacteriophages, and engineering the bacteriophages to attack only bacteria with drug-resistant genes. They were successful in including the CRISPR-Cas9 into a bacteriophage that was designed to attack a drug-resistant E. coli (Nature Biotechnology, 2014 Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases, R. J. Citorik, M. Mmee, and T. K. Lu). The new technology has the advantage of being a more targeted approach.
Numerous hurdles need to be overcome before this technology can be used against superbugs, including the demonstration that in humans, bacteriophages are safe and effective to use. Another concern is that the CRISPR can deviate from the target, thereby slicing the wrong genes. However, there is a race among scientists to find new applications for this novel technology.One major concern is that CRISPER can veer off target, slicing away the wrong genes with potentially harmful effects, scientists say. There are also fears of unknown effects due to the use of CRISPER to modify bacteria. Regardless of the promise of this technology, any potential therapy is years away. Nevertheless, many other scientists are trying to harness this novel technology for a variety of applications.
Can a similar technology be used in food plants to eliminate pathogenic bacteria from the environment?
Bacteriophages have been recommended for rapid detection of food-borne pathogens as well as a natural food preservative (Front Microbiol. 2016; 7: 474). Phage cocktails were created for the treatment of foods contaminated with various pathogens (Campylobacter jejuni, Cronobacter sakazakii, E. coli O157:H7, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus, and Vibrio spp). Numerous other studies report that phages may be useful for controlling specific food pathogen. However, there is no widespread use of bacteriophages to control pathogens.Several of bacteriophage-based applications have been approved for pre-harvest control of food pathogens in livestock and poultry. Another application is the decontamination of surfaces in food-processing facilities (Neha Bhardwaj, Sanjeev K. Bhardwaj, Akash Deep, Swati Dahiya and Sanjay Kapoor, 2015. Lytic Bacteriophages as Biocontrol Agents of Foodborne Pathogens. Asian Journal of Animal and Veterinary Advances, 10: 708-723.) Using the new advanced technology described above might improve the stability of the bacteriophages and improve their ability to attack the bacteria. As a result, it might gain more traction in the food industry.
Sample6 has two main products: DETECT, the pathogen detection system for the detection of Listeria monocytogenes in environmental samples, and CONTROL a food safety software package. Sample6 businesses were split into two. The pathogen detection system (DETECT) was acquired by IEH while Sample6 will continue with its CONTROL software.PRNews reported that Sample6 and IEH had jointly announced that IEH is acquiring the DETECT platform while Sample6 will continue to pursue the CONTROL software.
An engineered phage designed to interact with the target pathogens (i.e., Listeria monocytogenes, or Salmonella), makes the bacteria produce a large amount of the reporter enzyme. After a few hours, the bacterial cells go through a lysis step, and the reported enzyme is detected. The enzyme introduced by the phage makes the bacteria produce a biolumination compound that glows. The Biolumination signal is detected by the system. It is an enrichment free system capable of detecting one cfu/swab in 4 hours.The CEO of Sample 6, Dr. Michael Koeris said: “IEH’s resources and reach will allow for a more rapid deployment of the groundbreaking in-shift, on-site technology, as well as the successful launch of the high-throughput platform into the central and 3rd party laboratory market worldwide.”
Sample6 CONTROL is environmental monitoring software, allowing to schedule, monitor and report environmental program data. It allows gaining an insight into the effectiveness of the environmental monitoring system.Automated scheduling can be obtained by the system and results from any test method can be easily entered into the CONTROL software. In the event of a nonconforming or presumptive positive test result, a corrective action is generated.The system provides the reporting tools necessary to evaluate the performance of the environmental monitoring plan and to make adjustments based on historical and real-time data.
IEH Laboratory and Consulting Group
The company owns more than 95 laboratories, combining consulting with accredited testing laboratory. IEH is serving the food and pharmaceutical industries, providing services in a variety of disciplines. The company is lead by Dr. Mansour Samadpour.
Naturepublished in July 2017, an article byGwangseong Kim, Horatiu Vinerean & Angelo Gaitason a simple novel device to concentrate and detect food pathogens (immunocapturing method). The technique has the potential of being used for both clinical applications and food testing.
The technique employs polymer (polydimethylsiloxane) tubes (1.02 mm in diameter) coated with an antibody. The test sample is circulating through the antibody coated tubes. The re-circulating of liquid media containing the bacteria through the antibody conjugated tubes result in the capturing of the pathogens by the conjugated antibodies.Several tubes can be used with different antibodies in each, thereby allowing the capture of different pathogens. Alternatively, several identical tubes can be used to increase the efficiency of the capturing.As a result, the pathogens present in the sample are concentrated and accumulated in the tubes. This concentration step results in a higher concentration of the pathogens in a small volume of liquid.
The results show that in larger volumes of 100-250 mL and small starting bacterial numbers of anywhere from 1 to 10 CFU anywhere from 55%-91% of bacteria were captured inside the tubes within 6-7 hours.Ground chicken and ground beef were used as matrices to demonstrate the ability of the immuno-capturing method. 25 CFU of Salmonellatyphimurium in 25 grams of ground meat was used to show the systems ability to work with real foods. The product was diluted 1:10 in 225 ml of buffered peptone water (BPW) or Romer Labs Primary enrichment media supplemented with phage. After 5-7 hours Salmonella was detected from these samples, representing significant time savings over the traditional methodology.The two food matrices tested did not clog the 1mm tubes. To test larger volumes of samples required in food pathogens, long (120 cm) antibody coated tube was split into four 30 mm tubes. The 250 ml sample was circulated approximately 10 times in the 7-hour experiment.Use of Molecular Methods: The S. Typhimurium DNA was directly extracted from the concentration tubes by inserting DI water in the tube and heating to 100 °C for 10 minutes. Other methods for DNA extraction were also tested. Detection of the presence of the pathogens was done using either microscope fluorescence imaging or RT PCR. 10 µm from the content can be directly used for RT PCR without further purification steps.Use of Lateral flow devices: have a higher limit of detection than PCR, and therefore requires longer enrichment time. However, they are low cost and easy to use. Therefore they also were tested with the immunocapturing method.As shown below, 25 cfu of S. typhimurium in 25 gram of ground meat could detect in 14 hours with traditional enrichment, and in 9 hours when using the Romer Primary enrichment medium with phage. These time frames are significantly lower than the traditional methodology (36-44 hours).
(b) Positive results using Neogen Reveal 2.0 Salmonella strip in 14 hours in non-selective media.(c) Positive result using Romer Labs RapidChek SELECT Salmonella strip in 14 hours in non-selective media, (d) Positive result using Romer Labs RapidChek SELECT Salmonella strip in 9 hours in selective media
There is certainly a need for a faster method to find food pathogens because it allows for faster intervention and faster corrective action. It allows to link pathogen strains to specific cases and can be useful in preventing outbreaks and illnesses.This novel method can allow for results from food matrices in less than a single shift. However, the technology is currently in prototype stage and will need to be developed to a full commercial product.The inventors of the technology are currently seeking funding to finish the commercialization of the product. They expect the product to be commercially available in the next two years.
The Journal of Dairy Sciences reports that a team of researchers from China (Zhao et al. J. Dairy Sci. 100:3480–3496 May 2107) developed a new method for the detection of Salmonella in infant powdered milk.The developed method is claimed to be rapid, specific, and sensitive. It is is based upon loop-mediated isothermal amplification technique combined with a lateral flow dipstick (LAMP-LFD) as the detection step.
LAMPis a powerful new nucleic acid amplification method that detects very low levels of DNA. The method amplifies a few copies of target DNA with high specificity, efficiency and rapidity. The method uses a set of 4 specifically designed primers that recognize 6 distinct sequences of target DNA, and a DNA polymerase.The cycling reaction can result in the accumulation of 109 fold of copies in less than 1 hour. The method is claimed to be more specific and less susceptible to interference than PCR, it is very fast, without the need of denaturing step.
The target gene invA encodes a Salmonella invasion protein and is thus considered a virulence gene located on Salmonella pathogenicity island (SPI), and is used frequently for the detection of Salmonella. The SPI4 region includes genes from siiA to siiF that are important for adhesion to polarized epithelial cells, and plays an important role in Salmonella pathogenicity.The authors claim that this is the first attempt to use LAMP and the siiA gene to detect Salmonella.
Lateral Flow Dipstick (LFD)
Lateral flow immunoassays dipsticks are used routinely to detect pathogens in food. Lateral flow dipstick use a sandwich type ELISA and the majority use polyclonal antibody as a capture antibody and a monoclonal antibody as the detection antibody. The antibodies are fixed on a hydrophobic membrane in immobilized in lines. Their role is to react with the analyte bound to the conjugated antibody. Recognition of the sample analyte results in an appropriate response on the test line, while a response on the control line indicates the proper liquid flow through the strip.In the LAMP-LFD assay LFD strip is inserted into a tube that allows the strip to be immersed in the amplified sample. The sample migrates through theconjugate pad, which contains antibodies specific to the target analyte and are conjugated to colloidal gold and latex microspheres. The sample, together with the conjugated antibody bound to the target analyte, migrates along the strip into the detection zone. A number of researchers have combined LAMP with LFD. In this combination the LFD is soaked in LAMP amplified sample and the liquid travels by capillary action across the membrane to react with the antibodies and provide a color band.
Elimination of carryover Contamination
The high sensitivity of LAMP can become its largest potential disadvantage because trace left over material can be amplified and detected, causing false positive results, after several times of detection in the same place. Therefore, there is a need to eliminate any contamination from previous LAMP reactions.To reduce incidence of LAMP contamination, the authors applied propidium monoazide (PMA) to eliminate carryover contamination of LAMP. The appropriate concentration of PMA diluted in water was applied to the working environment of any contaminated area and adequate light exposure conditions were used to complete the decontamination process.
A very specific and conserved Salmonella target gene siiA was used to establish the LAMP-LFD detection method for Salmonella in powdered Infant formula.In this study, the limit of detection of the LAMP-LFD for inoculated powdered infant formula, without enrichment was 2.2 cfu/g, which is 100x lower than the limit of detection for most PCR methods. A pure culture study of 21 Salmonella strains (with limited number of serotypes), and 60 inoculated samples of powdered infant formula yielded all positive results. 31 non-Salmonella strains (75% gram positive), including 20 non inoculated samples all yielded negative results. While more testing of this method is required, the reported method seems to be very rapid, specific, and sensitive for the detection of Salmonella in powdered infant formula. PMA needs to be used to eliminate the LAMP carryover contamination.
Rapid methods for pathogen testing have been gaining acceptance in the food industry. Recent advances in technology result in faster detection and identification of pathogens, more convenient, more sensitive, more reproducible, and more specific than conventional methods. Many new methods are available involving antibody-based assays, genetic amplification methods, and newer sensor development methods.However, the industry is always looking for faster, simpler and cost effective new methods. An article in ACS Central Science describes the work of researchers at Massachusetts Institute of Technology (MIT) that are developing a new method for pathogen detection, utilizing Janus emulsions. The team is lead by Timothy Swager and Qifan Zhangis the lead author.The test is based on the analysis of liquid droplets (Janus droplets, or Janus emulsion) that are powerful liquid phase sensing particles. These droplets are formed from two equally sized hemispheres. One half is composed of a fluorocarbon and the other from a hydrocarbon.The fluorocarbon is denser than the hydrocarbon when droplets sit on a surface, therefore the fluorocarbon orients to the bottom. When the different hemispheres are functionalized to have orthogonal physical and biochemical properties, they can be used as sensors. Consequently Janus particles with covalently modified surfaces have been used for sensing applications.From above the droplets are transparent but when viewed sideways they appear opaque. This property relates to the way that light passes through the droplet, and it is the path of light that can be adapted to make the sensor.Building on this the scientists at MIT developed a surfactant molecule that contains mannose sugar to form the top half of the droplet surface. These molecules are capable of binding to a protein called lectin. Lectin is a protein that can bind specifically to certain sugars and cause agglutination of particular cells, and it is found on the surface of strains of E. coli.The emulsion assay uses the carbohydrate surfactant molecule, which self-assembles at the droplet surfaces during the emulsification process. Therefore, no further element is required for bacterial recognition. These changes in the alignment of the Janus droplets are used for the detection of analytes. The droplets are capable of binding to specific bacterial proteins. The mannose surfactant functionalized emulsion assay described in this work was designed specifically for E. coli as a model system.
Whenever E. coli is present the droplets attach to the Lectin proteins. This causes the droplets to clump together causing light to scatter in many directions. The Janus emulsion assay enables detection of E. coli bacteria at a concentration of 104 cfu/mL.The figure below shows the effect of the agglutination process.
On the left, Janus droplets are viewed from above. After the droplets encounter their target, they clump together (right). Credit: Qifan ZhangThe intrinsic optical lensing behavior of the Janus droplets also enables both qualitative and quantitative detection of protein and E. coli bacteria. The qualitative assay is very simple and can be scanned with a Smartphone. To demonstrate the simplicity of the agglutination assay for qualitative results, the researchers placed inside a Petri dish QR barcode (Quick Response Code two-dimensional barcode)
As seen in the figure above when E. coli are present, the droplets clump together and the QR code can’t be read.( Credit: Qifan Zhang)To precisely quantify the degree of agglutination, the researchers implemented an image processing program to calculate the percentage of area covered by agglutinated Janus emulsions and to evaluate the differences in optical intensity of the images before and after exposure to ConA (concanavalin A, serves as a functional substitute for E. coli bacteria). The program uses the adaptive threshold algorithm to distinguish areas with higher transparency (pristine Janus emulsions) from the opaque regions (agglutinated Janus emulsions).The MIT teamplans to create droplets customized with more complex sugars that would bind to different bacterial proteins. In this paper the researchers used a sugar that binds to E. coli, but they expect that they could adapt the sensor to other pathogens.The researchers are now working on optimizing the food sample preparation so they can be placed into the wells with the droplets. They also plan to create droplets customized with more complex sugars that would bind to different bacterial proteins. The team leader, Savagatrup says “You could imagine making really selective droplets to catch different bacteria, based on the sugar we put on them”.The researchers are also trying to improve the sensitivity of the sensor, which currently is similar to existing techniques but has the potential to be much more sensitive, they believe. They hope to launch a company to commercialize the technology within the next year and a half.Explaining a clear advantage of the technology, one of the lead scientists, Professor Timothy Swager, said: “What we have here is something that can be massively cheaper, with low entry costs. The sensor has been tested out with multiple samples of the infective bacterium and the results are sufficiently successful for the sensor to be considered for commercialization”
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