The Pathogen DETECT SystemThe principle of the technology is described in an article entitled “Advancing bacteriophage-based microbial diagnostics with synthetic biology” by Lu et al. (Lu TK, Bowers J, Koeris MS.2013).
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.”
CONTROL SoftwareSample6 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 GroupThe 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.
System Set-UpThe 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.
ResultsThe 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 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
Bottom-lineThere 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.
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 GenesThe 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 ContaminationThe 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.
ResultsA 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.
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”
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.
- After a brief incubation of a freshly prepared enrichment culture with reagent D.
- The complete Assay is exposed 5 minutes to a high-power halogen light source.
- Isolate/ extract DNA
- Run PCR
- Final PCR Results and no or low false positive rates.