“Saving The Bees Is Burning Down The House” - Triplex qPCR Using Dual-Target Assays For The Highly Pathogenic Bacteria American Foulbrood, Using Novel eDNA Methods.
dnature diagnostics & research Ltd, Gisborne, New Zealand
American Foulbrood (AFB) is the most devastating pathogen of honeybee diseases. It is estimated that AFB has a minimum direct cost of more than US$7 million dollars per year to New Zealand beekeepers. Worse, the incidence of the disease is increasing at an estimated 15% per year. We have developed a multiplex qPCR for AFB and are using this to screen bee and honey samples, as well as testing new sampling strategies to predict the development of this disease and prevent further spread.
A snapback high resolution melting method for multiple SNP resolution in apiculture will be also be described.
A Portable, Accurate, and Cost-Effective Strategy for Rapid On-Site Authentication and Characterization of Commercially Important Species and Pathogens Using Bio Molecular Systems’ MIC qPCR Cycler
Thermagenix, Inc, Natick, Massachusetts, United States of America
PROBLEM: In the seafood industry, mislabeled products disguising lesser-value/lower-quality species unfairly compete for profits, harm brands/consumer trust, and prevent proper safety tests for species-specific hazards and pathogens. In agriculture, invasive/destructive species continuously threaten crops and farmers’ livelihoods. Routine large-scale species testing imperative for these and many other industries is currently not possible due to the high-cost and complexities of species DNA sequencing and the complications of using a different DNA test for each species. SOLUTION: In response, ThermaGenix developed a broadly-applicable strategy for rapid and cost-effective identification of up to hundreds of species and pathogens in a single-tube test. One set of reagents in the test identifies multiple species individually without sequencing. ThermaGenix’s universal species DNA tests run on the MIC qPCR Cycler, a highly accurate, portable, and affordable instrument from Bio Molecular Systems for field applications. TECHNOLOGY: ThermaGenix’s High Precision PCR (based on ThermaStop™, a proprietary reagent for error-free DNA amplification) coupled with paired sets of positive/negative Nielsen hybridization probes convert any species-specific DNA sequences into highly accurate fluorescent signatures. Sequence-specific fluorescent signatures are then automatically compared against a reference library for immediate species identification. APPLICATIONS: FASTFISH-ID™, ThermaGenix’s first product for the MIC qPCR Cycler, provides rapid on-site DNA authentication of >700 individual species in commercial fish products in a single-tube test in about two hours. Another ThermaGenix test identifies any of 17 bacterial and fungi pathogens associated with sepsis in a single-tube. SIGNIFICANCE: ThermaGenix’sHigh-Precision PCR reagents and platform technology together with the high accuracy, reproducibility, and portability of Bio Molecular Systems’MIC qPCR Cycler provides an innovative turnkey solution for rapid on-site identification of large numbers of species and pathogens in a single-tube using a single set of reagents. Application target areas include on-site food integrity and safety testing, detection of invasive pest species and their pathogens, environmental bioassessment programs; additional uses include point-of-care diagnostics for cancer, infectious diseases, and other fields.
Development Of An Event-Specific qPCR Method For Detection Of Genetically Modified Alfalfa
1Bavarian Health and Food Safety Authority, Germany; 2Federal Office of Consumer Protection and Food Safety, Germany; 3Lower Saxony State Office for Consumer Protection and Food Safety, Germany
Genetically modified (gm) plants (GMP) have gained importance since commercialization in 1996. Cultivation areas increased from 1.7 million hectares in 1996 to almost 190 million hectares in 2017. In Europe, GMPs need to be authorized before being placed on the market and food and feed products containing authorized GMPs need to be labeled above a gm content of 0.9 %. Non-authorized products must not be placed on the EU market.
One of the emerging GMP species is alfalfa (Medicago sativa), which is one of the most important forage crops worldwide. Modified gm alfalfa events J101 and J163 gained herbicide tolerance against glyphosate by incorporating a CTP2-CP4 epsps gene. In event KK179, the RNA interference technique was used to knock-out the caffeoyl-CoA-3-O-methyltransferase (CCOMT) translation. CCOMT is a key enzyme in the lignin pathway and a knock-out leads to an improved digestibility for ruminants. Gm alfalfa is commercially cultivated in the US and in Canada.
In order to develop a qPCR-based detection method, we designed plasmids for each gm alfalfa event, based on published patent sequences. Further, we designed primers and a hydrolysis probe targeting the junction sequence spanning the plant genome and the transgenic insert (=event-specific detection). Plasmids were quantified using ddPCR and used for optimization and in-house validation of the methods. An estimated LOD95% of 10 copies per PCR was observed and PCR efficiencies of 95 – 97 % were achieved. Different qPCR instruments and PCR conditions were applied to test for robustness. Certified reference material for different GMP was used to test for specificity. No unspecific amplification signal was observed for any of the developed methods.
An inter-laboratory comparison study with seven participating laboratories was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters. Our cooperation partner (Federal Office of Consumer Protection and Food Safety, Berlin) was able to procure ground seed material for all three gm alfalfa events, which could be used in this inter-laboratory comparison study. All participants reported qPCR efficiencies between 95.9 % and 106.9 % and all laboratories were able to detect 10 nominal copies in twelve replicates. All results underline the suitability of the methods for the specific detection of gm alfalfa events J101, J163 and KK179.
A full collaborative trial validation study of the developed methods is planned for 2019.
MyPOLS Biotec: Shaping DNA Polymerases For Your Needs
1myPOLS Biotec GmbH, Technologiezentrum Konstanz, Blarerstraße 56, 78462 Konstanz, Germany; 2Chair of Organic Chemistry / Cellular Chemistry, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany
Based on their experience in basic and applied research on DNA polymerases, Dr. Ramon Kranaster and Prof. Dr. Andreas Marx founded myPOLS Biotec GmbH in Konstanz, Germany, as a spin-off from the University of Konstanz in 2014. Since then, myPOLS Biotec’ business activities, focused on the development of innovative applications of DNA polymerases, turned out to be very successful.
Off-the shelf, myPOLS Biotec offers DNA polymerase-based products like DIRECT BLOOD GENOTYPING KITS that tolerate blood ingredients in real-time PCR allowing genotyping directly from blood specimen, thereby saving time and money by omitting the nucleic acid extraction step; HiDi DNA POLYMERASE – a DNA polymerase that provides significantly enhanced selectivity of matched versus mismatched primers during PCR extension steps, rendering it the first choice in mutation-detection assays via allele-specific PCRs; VOLCANO2G DNA POLYMERASE – an enzyme that is capable of performing reverse transcription PCR without the need of an isothermal reverse transcription step to promote “zero-step” RT-PCR; Kits for DIRECT PCR FROM PLASMA – are currently developed in collaboration with the University of Konstanz that will allow analysis directly from blood plasma e.g., for the detection of cancer mutations by real-time, liquid biopsy PCRs.
To provide solutions for customized in-vitro-diagnostics, myPOLS Biotec develops and produces tailored products for IVD applications. For instance, the PCR LYOBEADS and LYOCAKES product lines: As freeze-dried ready to use master mixes, they can be shipped and stored at room-temperature and contain all components necessary (i.e., enzymes, primers, and probes) for a rapid, sensitive and reproducible detection and quantification of nucleic acid targets.
In contract research projects, myPOLS Biotec offers its specialized knowledge as a highly reliable, and transparent partner in challenging research projects based on DNA polymerases and their tailoring for advanced applications.
In the presentation, Ramon Kranaster will introduce the company myPOLS Biotec and provide an overview about newest developments, applications and products.