35.2 Laboratory Diagnostic Methods

Question 1

What are the two main ways a viral infection is detected?

Answer 1

Viraemia - detection of virus in the blood or CSF as an indication of current infection.
Serology - Existence of serum antibodies to virus indicates previous infection

Question 2

List the tests which can indicate viraemia

Answer 2

PCR
ELISA
RT-PCR
Plaque assay
Haemagglutination

Question 3

What are plaque assays used for?

Answer 3

Used to measure the number of viral particles present in a sample.

Question 4

How do plaque assays work?

Answer 4

Cells cultured with the virus at different serial dilutions (serum samples spread on a monolayer of cells)
Different densities of plaques (lysed infected cells which die and fall off the plate)
Plaque forming units can be calculated and cytopathic effects can be seen (changes in cell shape/size and fusion)

Question 5

How does ELISA work?

Answer 5

Blood sample taken -
Known antibody to the virus bound to the surface of wells
If virus present in blood then it will bind
Sample of antibody linked to an enzyme is added
Substrate normally broken down to become colour/colourless if the antibody binds

Question 6

How does haemagglutination inhibition assays detect the presence of antibodies made to a specific virus?

Answer 6

If the virus and antibody are homologous then the virus is blocked from attaching to the erythrocytes and causing a clump
No haemagglutination occurs in the presence of that antibodies
Degree of lattice development (virus binding to erythrocytes rather than antibodies) is inversely proportional to the amount of antibody present
Only works for influenza, parainfluenza and mumps

Question 7

What type of virus is generally tested for using haemagglutination?

Answer 7

Influenza virus

Question 8

How does PCR work?

Answer 8

(1) denaturation of the template into single strands;
(2) annealing of primers to each original strand for new strand synthesis;
(3) extension of the new DNA strands from the primers.
Amplifies DNA in the virus
Add DNA from known sample with fluorescent markers to see if they attach (SYBR green dye binds to dsDNA- binds to DNA in each round of PCR)

Question 9

Why does PCR have the highest resolution?

Answer 9

Can detect very small viral loads due to amplification step

Question 10

When is RT-PCR used instead of PCR?

Answer 10

Reverse transcriptase activity is first employed to transcribe DNA from RNA templates
Used for retroviruses (use RNA as genetic material) in Baltimore class VI and VIII

Question 11

How does the haemagglutinin test work?

Answer 11

Haemagglutin is a viral glycoprotein found on the envelope of influenza/mumps
Erythrocytes attach to the glycoprotein and form a lattice and solid clumps within a sample which changes the colour of the solution

Question 12

Which antibody detection can diagnose current infection?

Answer 12

Presence of IgM

Question 13

Why does the presence of IgG no diagnose current infection?

Answer 13

IgG may be due to an infection in the past (need to compare acute and convalescent)

Question 14

What do serology tests depend on?

Answer 14

Depends on the specificity of the immune response

Question 15

Which tests can be used to detect the presence of antibodies to a virus?

Answer 15

ELISA
inhibition of haemagglutination
plaque formation