Mod 5 - Advanced diagnostics

Question 1

Give examples of 3 types of advanced diagnostic methods for plant pests used regularly.

Answer 1

• biochemical tests - carbohydrate or oxygen utilisaiton assays (bacteria)
• Immunological tests - ELISAs (viruses)
• Molecular tests - PCR

Question 2

Why are biochemical tests good for bacteria? How do they work?

Answer 2

• They are based on the biochemistry or phisiology bacterial pathogens
• They ID the by-products of common metabolic pathways used by bacteria.

Question 3

What is an ELISA test - how does it work?

Answer 3

ELISA = Enzyme Linked ImmunoSorbent Assay
- It uses antibodies to detect and quantify the amount of antigen in a sample.
- anitbody linked enzyme changes colour to indicate a reaction occured - more colour = more antigen.
- Anti body = type of protein
- Antigen = susbtance that stimulates antibody production e.g. bacteria, toxin

Question 4

What are the advanatges and issues with ELISA tests.

Answer 4

Adv:
- fast, convenient and sensitive
Issues:
-antigens can be masked by host
- antigens may not be produced under certain enviro conditions

Question 5

Why is DNA used for diagnostics?

Answer 5

• Its the same in all cells in every organism
• It doesn’t change with development stage or enviro conditions.
• DNA sequences are sufficiently variable to differentiate genera, species, individuals etc.

Question 6

What is PCR? How is it done?

Answer 6

PCR = Polymerase Chain Reaction
- Very small amounts of DNA are amplified and detected very specifically in a machine
- 93 step process
- Denaturation –> Annealing –> Extension x40

Question 7

What are 3 different types of PCR?

Answer 7

• Nested = 2 primer pairs used therefore is more specific and sensitive.
• Real time = measures the product after each amplification round = can quantify the amount of DNA in a sample.
• Reverse transcriptase = Used RNA which is then converted to DNA using an enzyme. More complex more $$$

Question 8

Adv and Disadv of PCR for plant BS?

Answer 8

Adv:
- quick, highly sensitive, and specific
Disadv:
- False positives and negs
- inhibition of PCR due to DNA quality

Question 9

Why does PCR need controls?

Answer 9

• DNA quality and the release of inhibitors can cause false - and + results
• PCR controls are important to identify when an error has occurred.
• Pos control = amont of DNA we know will be amplified
• Neg control = DNA free test to make sure reagents arent contaminated.

Question 10

What is phylogenetics? How can it be used in plant BS?

Answer 10

• Phylogenetics = study of biological organisms and their classification based on evolutionary descen.
• It can ID plant pests based on their DNA sequence.

Question 11

How to differentiate a live pest from a dest pest using DNA based diagnostics?

Answer 11

• Measure the amount of DAN before and after a period of incubation to see if it has changed.
• Measure RNA as it is less stable than DNA - if RNA is present this means there is current or was recent viability.

Question 12

Whats the difference between biomarkers and biosensors?

Answer 12

• Biosensor = device that detects, collects and transmits information about a physical cahange or presence of chemical or biological material in an enivronment.
• Biomarker = a naturally occuring gene, molecule or characteristic by which an organism/disease/process can be indentified.

Question 13

What are some examples of new diagnostic methods that may become routine for plant BS in the future?

Answer 13

• Microarrays
• Biomarkers
• Biosensor
• Nanotechnology

Question 14

Briefly explain microarrays

Answer 14

• Allows genetic material to be spotted onto a microarray chip.
  Can be used for:
• genotyping - detect the presence of an organism’s DNA
• Resequencing - ID pathogenic strains or mutations.

Question 15

How can biosensors be applied in plant BS?

Answer 15

Detects a small number of invertebrate plant pests in large areas or volumes e.g. shipping containers or bulk storage.
- Extract and anlyse air in the grain dor chemical signs of insects.

Question 16

Briefly explain nanotechnology

Answer 16

substances e.g. antibodies can be bound to nanobeads –> used to detect pathogens in animal/plant cells extracts or enviro samples
- cheaper than microarray chips, with greater flexibility.

Question 17

Whats the “Omics” revolution?

Answer 17

• A shift in research that has looked into high throughput molecular techniques in this case to ID plant pests.
• It has the potential to provide new biomarkers - based on the proteome (stuidy of protein) and metabolome (study of metabolism) of plant pests.