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A Novel Concentration Device for the Detection of Food Pathogens

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Nature published in July 2017, an article by Gwangseong KimHoratiu Vinerean & Angelo Gaitas on 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.

 System Set-Up

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.

Results

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 Salmonella typhimurium 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 STyphimurium 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

Bottom-line

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.
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A New Method for the Detection of Salmonella in Powdered Dairy Products

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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.

Loop-Mediated Isothermal Amplification Technique (LAMP)

LAMP is 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.
 

Target Genes

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 the conjugate 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.
 

Results

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.
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MIT Developed Novel Pathogen System Based on Janus Emulsions

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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 Zhang
The 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 team plans 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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Comments about “Modified Polymerase Chain Reaction Distinguish between Live and Dead Bacteria”

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The blog about PCR and distinguishing between live and dead bacteria have drawn some comments as shown below:
Gerold Schwarz You might have a look for Reagent D – we established a protocol for Enterobacteriaceae in instant milk formula and Yeast and Mold in dairy products – it works similar utilizing a halogen light in addition source to eliminate dead cells before amplification.
 Gerold Schwarz Send the following additional Information:
 By: Dr. Gerold Schwarz, Produktmanager BIOTECON Diagnostics GmbH
 

Elimination of DNA form dead cells prior a PCR-Setup:

Reagent D is designed for the rapid elimination of DNA from dead cells to avoid false-positive PCR results. The reagent contains a light sensitive substance which can penetrate the cell membranes of dead cells, whereas the outer membrane compartments of living cells can actively protect their cytosolic compartments.
  1. After a brief incubation of a freshly prepared enrichment culture with reagent D.
            
  1. The complete Assay is exposed 5 minutes to a high-power halogen light source.
 
  1. Isolate/ extract DNA
  1. Run PCR
After incubation with light the DNA is irreversibly linked and amplification is blocked.  
 
  1. Final PCR Results and no or low false positive rates.
We have tested and validated the procedure for our foodproof Enterobacteriaceae plus Cronobacter Detection Kit and the foodproof Yeast & Mold Quantification LyoKit and third with the foodproof Vibrio Detection LyoKit.

Other Comments:

Relating to the original Blog:
Angela Aucoin This could be invaluable in reducing or eliminating triple re-examination costs of false positive environmental samples
Peter Ball Similar methods using propidium monoxide and ethidium monoxide have been published multiple times and are well known to most working with QPCR.
 
Francesc Codony Iglesias  The dynamic approach in vPCR has been suggested in the past , mainly by Dr Soejima working with different chemical compounds based on Pt and Pd. In the patent appointed by Ruth the authors also are following the dynamic approach, with a phenantridinium dimer. Although the dye interaction is quite strong, it’s not irreversible therefore during PCR denaturation this DNA will be available for amplification. For this purpose I opine that photo-reactive phenantridiniums are better reagents because their reagents can be activated by light and the binding to DNA is irreversible. Otherwise during sampling transport and handling, it’s quite critical in some clinical and food applications, the reagent will remain active affecting post sampling damaged cells. Regarding the patent, probably the inventive novelty can be refuted during future examinations.
 
John Mackay Use of PMA etc is patented – does this method vary sufficiently? Although doesn’t seem to need cross-linking. Patent describes a comparison among the dyes.
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Rapid Methods for Pathogen Detection in the FSMA Era

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Food online just published my guest column “Rapid Pathogen Detection Methods — What To Consider” Below are exerts from this blog:

Why Rapid Methods?

Rapid methods of 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. The main reasons for their adaptation are because faster results mean:
  • Faster intervention and corrective actions
  • Fewer lost lots or reduced amount of product in a contamination event
  • Faster reaction to a problem
  • Improved throughput and reduced warehouse space
  • Decreased manufacturing cycle through faster release of inventory
  • Ability to link strains of pathogens to a specific case
  • Accelerates root cause analysis
  • Rapid pathogen testing can be useful in preventing an outbreak of illness
For ready-to-eat products, the new FSMA proposal requires environmental pathogens to be controlled. As a result, environmental pathogen testing is required in some segments of the industry. In most cases, the cost of assay materials increases with new methods. However, the operational and financial benefits far outweigh the expense.

Available Methods

It is important to remember, in most foods, rapid methods still lack sufficient sensitivity and specificity for direct testing of the food. Therefore, the foods still need to be enriched in a culture media before the rapid method analysis. New methods include antibody-based assays, genetic amplification methods, and newer sensor development methods.  The Food OnLine article discusses in more details:
  • Growth-based methods
  • Immunological-based methods
  • Molecular detection methods
  • Biosensor devices
  • Whole Genome Sequencing (WGS)
 

What About FSMA?

FSMA requires food producers, processors, manufacturers, and service providers to certify their products are free from pathogens, such as Listeria, Salmonella, and pathogenic E. coli. The testing results obtained must use valid pathogen testing protocols.
Typically, all methods for pathogen testing are validated by vendors through an organization, such as AOAC International  , MicroVal, or AFNOR, either by an independent laboratory or a more stringent multi-laboratory study. However, according to FSMA, all microbiology methods utilized must be proven to be adequate for the particular products tested. Validation is required to demonstrate the method is equivalent to the reference method (for the matrices validated). Method verification will demonstrate it achieves an acceptable level of precision and accuracy, and there are no matrix effects or interference when utilized for the particular product(s) of the company.

Laboratory Accreditation

FSMA mandates laboratory accreditation with the objective to align commercial laboratories with government labs. This would assist the acceptance of analytical data, improve the efficiency of government labs, and support the testing of food imports. The FDA establishes an accredited third-party certification body that must be used in a laboratory accreditation. The accreditation is done to the ISO 17025 standard, or equivalent. These accredited labs will report results of public health concern directly to the FDA. The agency is required to establish a registry of accrediting bodies and accredited laboratories that includes laboratory contact information. Laboratories must be accredited for the particular sampling or analytical testing methodologies used to analyze their particular products. The new rules are placing greater emphasis on laboratory expectations, technical competence, and use of validated methods, and thereby, improved consistency across the industry in producing reliable data. According to Tom Wechler, lab accreditation is not inconsequential. A sizable initial investment is required in order to put systems in place and provide proper training for staff. The review fee for accreditation can run $15,000 or more and, once accredited, labs can expect additional ongoing costs for staffing, management and overall compliance.

Pathogen Testing Market Size

The demand for microbiological testing in the food industry is higher than ever before. Pathogen testing seems to account for over 50 percent of the entire microbiological testing market and its growth rate is three times greater than that of the total market. For more details go to Food OnLine article.
 

Difficult Choices

While the new methods offer advantages in technology, speed, and accuracy, they represent an incremental progress, rather than a revolution. It seems like every so often we hear about a new better methodology coming to market.
With the large number of competitors in the pathogen testing arena, it is hard to differentiate among the technologies and find the clear winners. Every small progress of one technology is soon followed by a competitor that makes it slightly better.
With over 40 different assays on the market it is increasingly more difficult for companies to choose a new rapid method and to decide to invest in it. This might be one of the reasons that laboratories. Small- and mid-sized food plant labs are closing, and looking to contract testing labs for their pathogen testing. The extensive validation requisite as part of FSMA, as well as the increased training and documentation requirement, is driving smaller manufacturers toward contract laboratories. Some of the difficult choices include:
  • Internal (In-House Testing Lab) Or External Testing Lab Internal laboratories can yield faster results. With faster results comes faster access to data. However, increasingly sophisticated and price competitive contract laboratories offer a good alternative. Furthermore, food company customers — including global food retail and food service companies — are requesting analytical results provided by an accredited third-party lab rather than the food plant itself.
     
  • Standard Methods Or New Methods? If New, Which New Method Should You Use? Newer and more rapid method are generally more sensitive, specific, time-efficient, labor-saving, and reliable than conventional methods and are currently more frequently used. Most of the U.S. market has moved to newer methods, while close to 50 percent of the European market is using the newer methods. Technology continues to evolve at a faster pace and the next generation assays are being developed.
Currently, numerous technologies are under development, pushing the limits of current methodologies. Some are trying to move current technologies forward, such as digital PCR, next generation sequencing. One essential step is the separation or concentration of the organisms from the sample prior to the utilization of rapid methods.
One technology attracting interest is Magnetic nanoparticles (MNPs) due to its super-paramagnetic property and large surface-to-volume ratio as excellent ligand attachment. There is a need for automation and the removal of manual steps that slow down the assay time. We expect constant progress in new method development over the next couple of years.
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Modified Polymerase Chain Reaction Distinguish Between Live and Dead Bacteria

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DNA-based diagnostic, such as Polymerase Chain Reaction (PCR) tends to overestimate the number of live cells because it will also measure the DNA from dead cells. This is because of the relatively long perseverance of DNA after cell death (e.g. up to 3 weeks). DNA extracted from a sample can originate from any cell, regardless of its metabolic state. Consequently, most DNA-based diagnostics cannot distinguish between live and dead bacteria, and this is a major drawback of the techniques.
After processes such as pasteurization or disinfections, dead bacteria might be present. While the dead bacteria present no hazard it can still be detected by PCR. Injured cells are virulent and may or may not be detected by standard procedures. PCR offers a more rapid and sensitive method than culture-based techniques, but the major limitation is the lack of differentiation of DNA from live or dead bacteria. In food matrices and the environment, DNA can be very stable and persist for extended periods of time, and therefore, it is desirable to have DNA-based assays that can identify only viable organisms.
A study by Tuskegee University researchers (Drs. Temesgen Samuel, Woubit S. Abdela, and Tsegaye Habtemariam with Yehualaeshet) allows the separation between live and dead bacteria. “PCR has been developed decades back, and the time spent for conventional PCR and our PCR — we call it viability PCR — protocol is the same,” Yehualaeshet said . “The novel aspect of our patent is that we developed a modified sample preparation, which enables the PCR to detect only viable, or live, bacteria.”
“During the sample preparation for PCR, we used a safe compound which will be ideal as a routine detection protocol for the presence of viable organisms. This invention will be mainly beneficial, but not limited, to the food industry to monitor biological decontamination, disinfection or the sanitization process.” That’s important, Dr. Yehualaeshet said, because the risk of contamination and disease comes from live bacteria. “If a detection method could not differentiate the dead from the live bacteria, then there is always a risk of false positive alarm”.
The research team from Tuskegee just got a United States Patent no. 9434976, for the rapid and more reliable detection of viable foodborne, pathogens and other infectious microbes using modified Polymerase Chain Reaction sample preparation. It provides a method of detecting the presence of a live microbe in a sample. The method comprises of:
  1. Isolating the microbe from the culture;
  2. Adding Gel Red™ dye to the isolated microbe from step (a);
  3. Extracting DNA from the microbe after step (b);
  4. Performing PCR on the DNA from step (c);
  5. Analyzing PCR results from step (d) for the presence or absence of amplified DNA using real time PCR and further gel electrophoresis confirmation; and
  6. Correlating the presence of amplified DNA from step (e) with the presence of live bacteria in the test sample. It may be desirable to further confirm that no viable bacteria were present by culturing on an appropriate media after heat and isopropyl alcohol inactivation of the culture.
The key ingredient used in the assay is Gel Red™ a DNA-intercalating chemical that is highly selective in penetrating only into dead bacteria (not live). The penetrating chemical binds with the DNA and blocks amplification of the targeted gene. It is claimed to be extremely stable and environmentally safe fluorescent nucleic acid dye.
“If there is no live organism after sterilization, it means the chemical used and the sterilization process works well. Therefore, the best detection technology should be able to detect only viable bacteria.”