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.
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.
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
Available MethodsIt 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 AccreditationFSMA 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 SizeThe 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 ChoicesWhile 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.
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:
- Isolating the microbe from the culture;
- Adding Gel Red™ dye to the isolated microbe from step (a);
- Extracting DNA from the microbe after step (b);
- Performing PCR on the DNA from step (c);
- Analyzing PCR results from step (d) for the presence or absence of amplified DNA using real time PCR and further gel electrophoresis confirmation; and
- 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.