W6L10 - Routine Diagnostic Testing by Nucleic Acid Methods

Question 1

Methods Used

Answer 1

PCR
Real time PCR
Multiplex PCR
PCR followed by:
- Restriction Fragment Length Polymorphism
- Probing
- Sequencing

Question 2

What is tested?

Answer 2

Viruses
Other microbes
- difficult or unusual bacteria 
- chlamydia, gonorrhoea
Genetic diseases
- mutation detection: CF, sickle cell anaemia
Cancer
- mutation detection 
Tissue typing
- HLA typing for organ transplantation

Question 3

What are the 4 sources of contamination for PCR reactions?

Answer 3

Carryover - products from previous amplifications
Cloned DNA
Sample to sample
Environmental DNA

Question 4

How to Avoid Contamination

Answer 4

Separation of individual areas
Lab practice
- separate lab coats for each area
- PPE
- aseptic technique 
- UV light

Question 5

Multiplex PCR

Answer 5

Several primers in one reaction to amplify different targets
Detection of different infectious agents in one tube
Detect oncogene mutations
Melting temperatures should be similar for each primer

Question 6

Nested PCR

Answer 6

Set of primers designed to amplify a target
Use amplification product of this PCR as target for a second PCR with primers designed internally
Second PCR run for a short internal PCR product
Increases sensitivity approx x100

Question 7

HLA-DR Typing

Answer 7

Multiplex PCR followed by DNA sequencing of PCR product
Two alleles expressed, if heterozygote two sequences overlayed over each other
If homozygous there will still be two sequences overlayed but they will be the same
Resolve HLA-DR types by analysis of polymorphisms in chromatogram

Question 8

Why has Real Time PCR replaced Nested PCR?

Answer 8

• closed system
• reduced possibility of contamination
• fast results
• can quantify
• melt curve analysis
• can multiplex
  Disadvantage is that the products can’t be used for sequencing

Question 9

Masstag PCR: Novel Multiplex PCR

Answer 9

Label primers with 'Masstag'
- each Masstag has a different molecular weight
- detect by mass spectrometry
To date up to 22 primer sets successfully used (44 tags)
Adapted for
- respiratory pathogens (22)
- viral haemorrhagic fevers (10)
- encephalitis (14)

Question 10

Advantages of Masstag PCR

Answer 10

Multiplexing
- not limited to dyes available (more Tags)
High throughput
- 20 plex assays in 96 well tray = 1920 tests
Reduced risk of false positives
- no second round of PCR
- no probes
Potential for detection of related sequences or virus discovery
However, it is less sensitive than RT-PCR

Question 11

Hybrid Capture Signal Amplification

Answer 11

1. Hybridising RNA probe mixed with target DNA to form DNA/RNA hybrid
2. RNA/DNA hybrid is captured onto a solid phase by antibodies specific for RNA/DNA hybrids
3. Anti RNA/DNA antibodies conjugated with alkaline phosphatase added to bind hybrid at multiple sites
   Much less sensitive than PCR
   Used for HPV detection with two sets of probes, one for HR-HRP and another for LR-HPV