On-site detection of Potato spindle tuber viroid in a disease outbreak using loop-mediated isothermal amplification (LAMP)

Transcription

1 On-site detection of Potato spindle tuber viroid in a disease outbreak using loop-mediated isothermal amplification (LAMP) Linda Zheng, Fiona Constable, Angela Freeman, Brendna Rodoni Department of Economic Development, Jobs, Transport and Resources Plant Biosecurity Cooperative Research Centre

2 Why are diagnostics important? Accurate, rapid, low cost tools for detecting exotic pathogens are the foundation for: - secure border protection - rapid response to incursions - active surveillance programs Correct identification is critical - identification failures result in inappropriate responses - false negative, false positive

3 Sample collection Sample submission Sample preparation Diagnostic tests For most plant and animal pathogens, accurate, rapid, low cost tools for on-site detection are not currently available Mostly laboratory based techniques such as PCR and ELISA

4 Smart Surveillance Tools - Molecular diagnostics Field-deployable diagnostic tools - mobility (equipment) - simple and easy to use - easy sample preparation - robust amplification of target template - easy interpretation of results Tools currently available - LAMP loop mediated isothermal amplification - RPA recombinase polymerase amplification - Nanopore portable real time sequencing (MinIon)

5 Loop-mediated isothermal amplification 5

6 Visualisation of LAMP products Mg precipitation Colorimetric Electrophoresis Lateral flow dipstick DNA intercalating dye

7 Conventional PCR vs LAMP PCR LAMP 1 Requires temperature cycling Isothermal single temperature 2 Requires 2 primer sites Requires at least 6 primer sites 3 Slow: Typically >1hr Rapid: Typically <40 mins 4 Not amenable to visual detection Amenable to visual detection based on turbidity etc. 5 Sensitive to sample matrix inhibitors Tolerant to sample matrix inhibitors 6 Can be multiplexed Difficult to multiplex Desirable traits for field deployment less energy consumption no need for thermocyclers faster reaction time results in less than 40 minutes can tolerate crude nucleic acid extracts no need for lab-based template preparation

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9 Small circular RNA molecule Highly infectious can be spread by vectors, pollen, seed and mechanically Exotic plant pathogen to Australia Serious threat to tomato and potato crops Systematic infection Potato spindle tuber viroid (PSTVd) Causes fruit deformity in tomatoes reduces marketable yield up to 60% Sporadic outbreaks in greenhouse tomatoes in Australia possible from infected seed

10 Evaluation of LAMP assays Potato spindle tuber viroid Two PSTVd LAMP assays available - Lanarcic and Tsutsumi - Lanarcic more specific Sensitivity comparable to conventional RT-PCR Total RNA dilution Lanarcic RT-LAMP PSTVd RT-PCR RT-qPCR

11 Glasshouse (Victoria)

12 The 6 step LAMP procedure 1. Add plant tissue (approx. 0.2g/leaf) to lysis buffer 1. Close lid firmly and shake by hand for 1 minute 2. Transfer 1 loop (10 ul) of tissue extract to the dilution buffer tube 3. Transfer 5 ul of diluted extract to a LAMP test reaction tube 4. Record sample ID 1. Transfer tube to the LAMP machine and run the program (Push two buttons)

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14 Results Symptomatic and non-symptomatic plant sampled 10 red roma samples 5 were positive 10 yellow roma samples 6 were positive Results corroborated by anneal curve analysis Confirmed by conventional RT-PCR Well Name Peaks 1 well1-2 well2 08:30 3 well3 19:30 4 well4 28:45 5 well5 18:00 6 well6 20:15 7 well7 12:45 8 well8 16:45 Negative

15 Summary LAMP was successfully used for the rapid detection of PSTVd on-site It is rapid and yields results within 40 minutes, which can minimise - economic loss - environmental impact - social impact on farming communities Potential to partner up with suppliers to provide customised offthe-shelf tests Is not the ultimate diagnosis but confident we can confirm a negative

16 Acknowledgement Agriculture Victoria - Jim Moran - Elizabeth Minchinton - Dean Harapas La trobe University - Shivraj Braich (ex Master student)

17 Thank you For more information, please