Virus Diagnosis

Question 0

Antigen definition

Answer 0

Forgein substance that evokes production of specific proteins in host called immoglobulins(antibodies)

Question 1

Antigen

Answer 1

Antibody generator

Question 2

Antibodies recognize forgein substances

Answer 2

Antibodies recognize and stick to forgein substances- antigens
Structure of the recognition face mirrors that of the forgein substance precisely
Antibodies vary in shape of recognition face and stick to different forgein substances

Question 3

Immunoglobulins(antibodies)

Answer 3

Defensive proteins produced in response to presence of an antigen

Question 4

Basic structure of antibody

Answer 4

Rsemble Y
2 arms- Fab
Other end- Fc is the effector arm in immune system

Question 5

Serology

Answer 5

Use of antibodies

Need antisera for each virus to diagnose

Question 6

Make virus specific antiserum

Answer 6

Purify virus

Particles purified from infected plant

Question 7

Figure- ateps of purification

Answer 7

1

Question 8

Make virus specific antiserum

Answer 8

Inject purified proteins into mammal- rabbits/goats
Purify IgG from blood
Humoral response
Purified antibodies will bind same antigen/virus as injected
Enzyme antibody conjugate

Question 9

ELISA to detect antigens

Answer 9

Double antibody sandwich ELISA:
Bind
Wash
Label
Eash
Read

Question 10

Das ELISA advantage

Answer 10

Simple

Sensitive very specific

Question 11

Das ELISA disadvantage

Answer 11

Very specific- steric hinderance of enzyme

Need conjugate for each antigen

Question 12

Immuno- electron microscopy

Answer 12

E dense stain
Antibody
Virus(antigen)
Antibody

Question 13

Nucleic acid hybridization

Answer 13

Hybridization: nothern/southern blots,micro-array
Amplification: PCR,RT-PCR

Question 14

Nucleic acid hybridization

Labeked probe

Answer 14

Label:
Radioactive element
Digoxigenin
Avidin
Fluorescent compounds

Question 15

Nucleic acid hybridization

Dot blot

Answer 15

Take membrane and spot RNA onto spot- wash-
Cover membrane to prevent move nucleic avid bind
Put on probe

Question 16

Southern/northen blot

Answer 16

DNA
Seperate DNA on agrose gel
Transfer blot DNA fragments from gel to membrane
Membrane with DNA bands transferred to it
Radiolabelled probe incubated with membrane
Bound DNA bands exposed on film

Question 17

PCr- DNA amplification

Answer 17

Denturation
Annealing- FP RP
Extension-dNTP

Need virus specific primers and positive controls

Question 18

Multiplex PCR

Answer 18

Primer pairs combined detects numerous viruses in one reaction

Question 19

Alter specificity for PCR diagnosis

Answer 19

Design primers to conserved region can detect virus having to those primer sites- potyvirus(polyspecific PCR)

Primer in variable region(ID specific virus/strain of virus)

Only detect dominant virus

Question 20

PCR exponential

Answer 20

Quantitative during exponential phase
Reagents not limiting and amount OCR is proportional to quantity of input template

As cycles increase, reaction- plateau-
reagents limiting-
DNA pol loses activity
Inhibitors build up and PCR product accumulate- reannel of denatured compete primer annealing

Question 21

Real time PCR

Answer 21

Improved accuracy
Eliminating time effort

Based on recognition of PCR amplified DNA by fluorescent:

• inserted into the amplicon
• labelled probe hybridizinf with the amplicon
• dyes- false + and not diff between spec and non spec products

Question 22

DNA binding dye

Answer 22

Quencher labelled primers and SYBR green I dye
Become flurescent when bound dsDNA- fluoresence detected thermal cycler- graph
Qunecher bound to primer repress fluoresence of nearby SYBR green I dye

Melting curve analysis

Question 23

Direct seq

Answer 23

Clone and seq

Next generation seq