DNAble Field Test Kit for Rapid Detection of Clavibacter michiganensis subsp. michiganensis in Tomato Tissue

Transcription

1 DNAble Field Test Kit for Rapid Detection of Clavibacter michiganensis subsp. michiganensis in Tomato Tissue Tania R. Spenlinhauer, PhD Manager, Molecular Diagnostics Applications Development EnviroLogix Inc. October 1, 2011

2 Features of DNAble QuickStick Technology Easy to use Reagents are stable (20-2 o C) Simple field adaptable sample preparation Rapid qualitative results (< 20 minutes) Nucleic acid amplification platform NEAR Technology Lateral Flow Detection Specificity down to a single nucleotide base Sensitive detection of DNA from a crude matrix (no purification needed) Complete validated kits

3 NEAR Technology Genome Fragmentation N.E. Pol.

4 NEAR Technology Primer Anneal and Extension Target Pol. Pol. Complement Amplification Duplex

5 NEAR Technology Rapid Amplification of Duplex N.E. Pol. Target N.E.

6 Lateral Flow (DNA) Background Product (-) potato Specific Product Positive Capture Control Sample Pad QuickStix Absorbent

7 Crude Sample Matrices Sample direct from Leaf Tissue or Stem No DNA purification requirement

8 Tomato Tissue Analysis Sample Procedure Sample plant tissue Eject sample into extraction tube Macerate with pestle Add 0.mL extraction buffer Macerate with pestle Assay 10 ul Using the tube and cap interface of a 0.mL microcentrifuge tube to collect stem or leaf punch. Transfer to a 1. ml micro-centrifuge tube and grind for 20 sec. Add 1mL extraction buffer and grind for 20 sec. Demonstrated results in Laboratory Infected Samples and Naturally occurring Greenhouse Infected Samples

9 Assay Procedure Step 1 Step 2 Step Add Sample Equilibrium 6 C Transfer Re-cap Amplification 6 C Add vial to LFD cartridge Detection min min 10 min Reaction Buffer Lyophilized Master Mix Heat block Strip in closed cartridge; visual detection Traditional test and control lines on strip within 2 minutes

10 Laboratory Infected Tomato Tissue Testing 1 Site of infection 7 9 NTC Site of lesion 8 The plant was inoculated by injection of Cmm into the stem The plant became weakly symptomatic 16 days post infection Test results on this day were positive at a lesion (1 cm), Leaf Petiole ( cm) and Rachis (10 cm) as shown below 1 Primary sampling locations Tube1 Tube2 Tube Tube4 RFU NTC (-) Leaf Petiolule (-) Leaf 1.Terminal Leaflet 2. Leaf Petiolule. Leaf Petiolule 4. Rachis. Leaf Petiolule 6. Rachis 7. Petiole 8 Lesion 9 Apical Meristem 20 cm 1 cm 10 cm 10 cm cm cm 1 cm 1 cm 20 cm+ Tissue (distance from infection site)

11 Greenhouse Infected Tomato Tissue Testing Mature plant showing symptoms of Cmm infection was harvested Analysis of samples from a leaf (using leaf procedure) and from a lesion containing stem (using stem procedure) was conducted Test result of the stem was positive Row A Row B Row C mv NTC Leaf Stem

12 Cmm Assay- Sensitivity Analysis Sensitivity analysis achieved by a titration of bacterial suspensions in a tomato tissue matrix Able to detect consistently at a concentration of 1x10 CFU/ml in 1ml of crude sample matrix Testing 10ul of crude sample results in consistent detection of 1000 CFU/rxn 10 0 control 00 cfu/rxn (x10 4 cfu/ml) 1000 cfu/rxn (1x10 cfu/ml) 2000 cfu/rxn (2x10 cfu/ml) cfu/rxn x10 4 cfu/ml 1000 cfu/rxn 1x10 cfu/ml (LOD) 2000 cfu/rxn 2x10 cfu/ml

13 Cmm Assay- Specificity Analysis Genetically diverse panel of Clavibacter michiganensis subsp. michiganensis with international origin # Isolates 94 # Positive Detection 94 # Detection 0 Sample Pseudomonas corrugata Pectobacterium carotovorum subs. Carotaovorum Xanthamonas vesicatoria Xanthamonas vesicatoria strain SV 2 Xanthamonas axonopodis Microbacterium paraxydans Fusarium oxysporum Verticillium dahliae Ocrobactrum grignonense Ocrobacterum tritici Result Results show specific detection of Cmm isolates *Representative data of out-group panel

14 Summary DNAble Field Test Kit for Detection of Clavibacter michiganensis subsp. michiganensis in Tomato Tissue Amplification direct from a crude sample with no purification Exceptional Speed Portability Superior diagnostic tools for the industry Utilization of a new technology Give same-day results, on-site Currently being evaluated in a field setting to verify the product performance Following this verification of product specifications, product to be release for validation with our collaborating Life Science Companies

15 Robust assay system allowing for PCR quality results direct from the crude sample matrix and in a nonlaboratory environment For more information regarding DNAble Products Contact: dnable@envirologix.com