OptiGene

Company Background

• Established January 2008

• Complementary expertise in instrument design and biochemistry

• Standard products and customised options

• Based in Horsham, West Sussex, in the South of England

• Privately owned with no external investment

Fields of Application

• Plant health

• Food safety

• Meat / fish / fruit / vegetable authentication

• Clinical diagnostics

• Veterinary

• Environmental monitoring

• Biosecurity

Isothermal Amplification of DNA & RNA

• Several Isothermal methods available but LAMP is our preferred method

• Single temperature – simple control

• Simple assay design

• Highly specific

– Simple sample preparation

– Robust assays

• Extremely fast

• Fluorescence or turbidity

• Product anneal - confirmation

• Most LAMP publications use Bst polymerase

• Assay time is normally LONG i.e.1 hour

• OptiGene has developed new enzymes, which are much faster and a thermostable, proof-reading, strand displacing enzyme capable of 95oC denaturation prior to LAMP

• Isothermal master mixes containing proprietary enzymes

• Highest speed available in a fluorescent LAMP reaction

• RT and thermostable master mixes available

• Lyophilised Reagents

• On-going development

Reagents

LAMP - 1x106 copies Aeropyrum pernix gDNA template

RT-LAMP - 1x109 copies MS2 RNA template

No amplification from Bst/GspM or Tin DNA polymerases

Sample matrices

Tested on Genie® platform include…

Blood, urine, tissue, leaves, wood, whole insects

Sample preparation is simple

Lyse and lamp (e.g., meat authenticity)

Thermally stable enzyme (e.g. Campylobacter) and shaking in a tube (e.g. ash dieback).

Plastic loops for transfer and no pipettes required

Campylobacter

• On-Farm detection of Campylobacter in poultry (turkeys prior to slaughter)

• TSB Funded project with Bernard Mathews, Cranberry Foods & Poultry Council

• Detection from faeces within 30 minutes

• 1st stage field trial successful, 2nd stage underway

Plant pathogens do not respect borders

Importing food & Pathogens

• Year round requirement for fruit & vegetables means we have to import

• Potatoes, The UK imported 1,500,000 metric tonnes of fresh, processed and seed potatoes in 2012 : 5 -6 diseases are important

• Tomatoes, UK imported from Southern EU - 400,000 tonnes of fresh tomatoes in 2012 (4 out of 5 sold are imports) 6 diseases present in EU crops

• Inspectors have to check, visual check, but maybe further testing is sent to the laboratory for analysis

Importing food & Pathogens

• Mango as an example

• Mangos imported under- ripe, ready for supermarket shelves

• 2 - 3 day turn around for analysis at the lab

• Disease OK, then shipment can be disposed of

• What if NO Disease?

• Rapid testing at ports of entry is essential tool for inspectorate services.

Notifiable DiseasesBacteriaXylella fastidiosaBrown rot of potato FireblightRing rot of potato

FungiKarnal bunt Phytophthora kernoviaePhytophthora lateralisPhytophthora ramorumPhytophthora ramorum and P. kernoviae diseases on bilberry Potato wart disease Strawberry black spot

Virus/ViroidsEmerging viroid threats to UK Tomato production Plum pox

Notifiable PestsBeetlesAnoplophora longhorn beetle Argentine stem weevil Listronotus bonariensisRed-necked longhorn Aromia bungiiCitrus Longhorn Beetle Colorado beetle Diabrotica species Lemon Tree Borer Pepper Weevil Potato Flea Beetle Red Palm Weevil Western corn rootworm

BugsBemisia tabaci - Tobacco whitefly Platanus Lace Bug Trialeurodes abutiloneus (Banded-winged whitefly)Wheat Bug White Peach Scale

CaterpillarsEggplant Borer Palm BorerOmnivorous leafrollerSouth American Tomato Moth Spodoptera species Tomato Pinworm

FliesLiriomyza leaf miners

ThripsThrips palmi

MitesFuchsia Gall Mite Goji Gall Mite Swamp Cypress Rust Mite

SnailsPomacea species Apple Snails

NematodesPotato Cyst Nematodes Stem nematode on Narcissus and Tulip

Zurich Airport

• The melon thrips, Thrips palmi, is a notifiable pest in the UK, EU & Switzerland.

• It can cause damage to a widerange of glasshouse ornamental and vegetable crops, particularly plants such as cucumber, aubergine, tomato and sweet pepper.

• It is imported on a wide range of vegetables, ornamental cuttings, pot plants and other cut flowers

1mm in size

Zurich Airport

• Cut flower shipments are examined and Carbon Dioxide is injected into the bag to cause the insect to lose its grip on the plant material.

• The bags are emptied into petri dishes and the insects are placed into the Genie strip directly.

• The assay is preloaded and the amplification proceeds in under 30 minutes.

• Confirmation is made via amplification and anneal curve. A very rapid confirmation can be carried out by the inspectorate services saving time and also the expense of laboratory analysis.

Heathrow Airport

• Guignardia citricarpa is a fungal pathogen which causes Citrus Black Spot disease.

• Visual inspection of symptoms can be difficult and ambiguous.

• Plant health inspectors at Heathrow have to check imports of all citrus fruit

Heathrow Airport• G. citricarpa lesions are excised from the surface of infected fruit using a sterile

scalpel and placed into extraction buffer.

• The material is lightly crushed, and the resulting crude extract is added directly to the LAMP reaction.

• Positive detection of G. citricarpa in crude extracts is observed in less than 20 minutes; a characteristic annealing temperature peak in the range 86-87oC of the amplification product serves as a confirmation for specific amplification of the correct pathogen.

• This rapid and easy to use G.citricarpa sampling and detection method in conjunction with the Genie® II makes it an invaluable diagnostic tool especially for importers and inspectorate services.

Real-time LAMP Genie®

• Real-time fluorescence detection

• 16 reactions per run

• Membrane touchpad

• 3” display

Genie®

• 1 Prototype

• 4 UK Government Agencies

• 4 Further prototypes

• Feedback

• 60+ instruments sold

• Requirement for use in low power /no power settings, Larger screen/touchpad, connectivity

• Technology Strategy Board funding

Genie® II

• Isothermal DNA amplification with fluorescence measurement

• Target confirmation by product anneal

• Low power with internal Li-Po battery

• Stand-alone operation via 7” touchscreen

• Internal data storage and upload via USB

• Accurate & precise thermal control

• High-quality optics

• Two blocks - 16 samples

• In full production since March 2011

Genie® StripsGenie® II & III use a proprietary tube strip that maximises optical and thermal efficiencies with a locking cap providing a closed-tube system. The strips have the following advantages:-

• Seal-and-lock mechanism to prevent contamination• Individually capped• Non-fluorescent and optically clear• Wings for ease of handling• Each strip has 8-tubes with a working volume of 20 - 150 µl

Agroscope Changins-Wädenswil, SwitzerlandOptiGene Limited, UKCentro Internacional de la Papa, PeruNational Institute of Biology, SloveniaUniversità Degli Studi di Padova, Italy Stichting Dienst Landbouwkundig Onderzoek, The NetherlandsUniversity of Nottingham, Sutton Bonnigton, UK

LAMP-based assays

Q-detect LAMP-based assaysPests Bursaphelenchus xylophilus, Monochamus galloprovincialis, Thrips palmi,

Liriomyza huidobrensis, Liriomyza sativae, Liriomyza trifolii, Bemisia tabaci, Ips acuminatus

Fungal Guignardia citricarpa, Ophiostoma brunneo-ciliatum, Ophiostoma clavatum, Fusarium graminearum, Phytophthora ramorum, Phytophthora kernoviae

Bacterial Ralstonia solanacearum, Clavibacter michiganensis subsp. sepedonicus, Xanthomonas arboricola pv. pruni, Erwinia amylovora, Pseudomonas syringae pv. actinidae, Pantoea ananatis, Curtobacterium flaccumfaciens pv. falccumfaciens

Viral Potato spindle tuber viroid, Cassava brown streak virus, Tomato chlorotic dwarf viroid, Chrysanthemum sten necrosis virus, Potato yellow vein virus, Tomato chlorosis virus, Tomato infectious chlorosis virus, Cucurbit yellow stunting disorder virus, Tomato yellow leaf curl virus, Cotton leaf curl virus, Sweet potato feathery mottle virus, Sweet potato chlorotic stunt virus, Potato virus Y

Phytoplasmas Bois Noir phytoplasma, Florescence doree phytoplasma, Candidatus liberibactersolanacearum, Aster yellows group 16SrI phytoplasmas, 16SrIII group phytoplasmas, Napier grass stunt and Bermuda grass whiteleaf phytoplasmas, 16SrXXII Coconut lethal disease type phytoplasmas, 16SrXII Stolbur group phytoplasmas

EU wide Standardised procedures to EPPO

UK Outbreak – Ash diebackChalara fraxinea

LAMP for detection of Chalara fraxinea

Primer design and initial test end of October 2012

Development of field extraction method for ash

November

Improved assay design mid-November

Finalised protocol for validation

Shake for 1 minute

Dilute(using plastic

loop)

Crude extraction using alkaline PEG buffer (based on Chomczynski et al. 2006)

LAMP for detection of Chalara fraxinea

Take sample from leading edge of lesion

Transfer approx. 5 µl per LAMP reaction

Place in tube with PEG buffer and shake for 1 minute

Transfer approx. 10 µl into tube containing 490 µl water

2-5 minutes per sample

using inoculating loop if in the field

1 minute up to 8 samples

2 minutes up to 14 samples

2 minutes up to 14 samples

Run LAMP on Genie II instrument

30 minutes up to 14 samples

Approx. 30 minutes hands-on time to test 14 samples (mostly

sampling)

LAMP for detection of Chalara fraxinea

LAMP for detection of Chalara fraxinea

sensitivity: approx. 7 pg C. fraxinea DNA/reaction

150 previously-tested samples were tested by LAMP end November: 34% infected, 66% C. fraxinea-negative, Since Jan routine Genie detection.

Chytrid fungus Batrachytrium dendrobatidsInstitute of Zoology, London; Czech University of Life Sciences, Prague; & University of Veterinary and Pharmaceutical Sciences, Brno

• Batrachytrium dendrobatids Bd is a chytrid fungus that causes the disease chytridiomycosis in amphibians.

• First described in 1998 following a number of mass mortality events in Australia and Central America. Initially thought to have originated in Africa chytrid has been transported throughout the world potentially as a result of the amphibian trade where it is believed to have caused the extinction of 200+ amphibian species

• qPCR detection has limitations. Firstly a number of B. d. lineages cannot be detected using the standard methodology; secondly, it is prone to phenolic compound inhibition, and thirdly assays have to be carried out in the laboratory by personnel trained in qPCR. This often takes place days to weeks after the field work has taken place enabling time for DNA degradation

Chytrid fungus Batrachytrium dendrobatids

• Assay designed for the detection of more lineages of B. d. than previously possible plus the detection of the recently discovered Batrachochytriumsalamadrivorans (B. s.) in the same test

• The method used a simple 0.3M KOH extraction whereby a swab of an amphibian’s skin was ‘twizzled’ in 50μl KOH before 5μl of extracted DNA/KOH solution was added to 20μl of a ready to use mastermix before being run on the Genie® II

• The entire process from start to finish taking 25 minutes per 8 samples

Chytrid fungus Batrachytrium dendrobatids

• Field work was conducted in the Czech Republic

• The site chosen was a known positive B. d. site

• Samples were run in 8 well strips consisting of 7 amphibian swab samples and one known positive control.

• The results of the run showed that practically all B.variegata cryptically carried B. d. with no visible signs of infection, while only a small number of Pelophylax produced a positive result. Although a reasonably small study, this represents the first time it has been possible to detect B. d. in the field.

• Further testing is still required, however, the implications of this method in terms of mitigation strategies and general disease identification are evident and it is hoped that this strategy will be adopted as a standard tool by field ecologists for disease control management.

Foot-and-mouth disease (FMD)

• Foot-and-mouth disease (FMD) is an economically devastating vesicular disease of cloven-hooved animals.

• FMD is endemic in many countries within Asia, Africa and South America, and while mostly eradicated in developed countries, outbreaks can occur due to the highly contagious nature of the causative virus.

• Clinical signs are indistinguishable from other vesicular diseases, therefore diagnosis is currently confirmed at national reference laboratories: a lengthy process which delays critical decision making.

• Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) provides a realistic option for rapid, sensitive, in situ detection.

• Having developed and validated a lyophilised RT-LAMP assay in the laboratory we took the Genie® II to Morogoro, Tanzania for field trials.

Foot-and-mouth disease (FMD)

• For the field trials: results were obtained in less than 30 minutes from sample collection to result calling

• Positive amplification detected in two cattle at 6:45min and 15:30min. • Melt curve analysis confirmed LAMP amplicons to be FMDV specific with

concordance to rRT-PCR.

Foot-and-mouth disease (FMD)

“The Genie® II proved to be a robust andportable platform for RT-LAMP.Furthermore, the compatibility ofRT-LAMP with the use of simple samplepreparation methods and lyophilisedreagents reduces difficulties associatedwith field deployment. Further trials areplanned for testing the assay on acutely

infected animals in situ”

Foot-and-mouth disease (FMD)

Animal Diseases

Swine vesicular disease (SVD): Time to result approx. 12 - 18 minsFoot and mouth disease (FMD): Time to result approx. 9 - 10 mins Vesicular Stomatitis Virus (VSV): Time to result approx. 10 - 14 minsAfrican Swine Fever Virus (ASFV): Time to result approx. 12 - 15 mins

Institute of Animal Health – Pirbright, UK

Genie® III

• Development of more portable system

• Technology Strategy Board Funding

• FMDV & ASF ‘in-field testing’

Genie® III

• Single block – 8 samples

• Two-colour fluorescence detection

• Low power with internal Li-Po battery

• Stand-alone operation via 4.3” touchscreen

• Wireless communications via Bluetooth

• Network connection via WiFi

• Positional information via GPS

• Sealed enclosure

• Simple user interface

• In full production since Jan 2014

• Food pathogen LAMP tests developed– E-coli, Listeria and Salmonella

• Development of LAMP test for Campylobacter in poultry underway– Assay development and sample extraction from faecal boot swab

• LAMP tests developed for meat speciation– Simple DNA extraction from pure and processed sample

– Rapid, sensitive and specific assays

– Meat targets horsemeat, pork, beef, lamb, chicken and turkey

– White fish targets cod, haddock, pollock, catfish and whiting

• Validation of horsemeat test complete– Validation undertaken by Fera

– Comparison against ‘gold standard’ qPCR assay

– Validation of other tests to follow

Food Safety and Authentication

Simple sample preparation – “Lyse & LAMP”– Small amount of sample collected (e.g. raw meat, processed product)

– Placed into tube with alkaline lysing solution

– Taken to 95°C and held for 5 minutes

– 5 µl of solution added to Genie tube that contains all other reagents

Meat Testing Protocol

Run in Genie® II– DNA amplification for 30 minutes (or less)

– Product anneal to confirm target (5 minutes)

– Straightforward result calling that is simple to automate

Test Results: Hot DogSample Brand 1

Sample Brand 2

Acknowledgements

• Fera: Neil Boonham, Jenny Tomlinson, Sioban Ostoja-

Starzewska, Catherine Harrison, Hez Hird

• FC: Ben Jones, Helen Carter, Barnaby Wylder

• IAH: Emma Howson, Veronica Fowler

• ZSL: Chris Durrant; CULS Prague: Milič Solský

• All at OptiGene

Contact

OptiGene LimitedUnit 5 Blatchford RoadHorshamWest SussexRH13 5QRUK

T: +44 (0)1403 274980F: +44 (0)1403 271017

E: info@optigene.co.ukW: www.optigene.co.uk