PCR As Diagnostic Tool

Question 1

what is PCR

Answer 1

amplifies a specific region of nucleic acid (target)

generates millions-billions of target copies (amplicons)

detect/isolate nucleic acid copies

Question 2

how does amplification of target DNA

Answer 2

target nucleic acid amplified by polymerase in presence of short (~20nt) oligonucleotide primers (which bind to complementary sequence in the target and define the amplified sequence) and nucleotides in an appropriate environment

Question 3

what are the steps in amplification of target DNA

Answer 3

1. thermal cycling to allow denaturation of target
2. annealing of primers (which define the sequence amplified)
3. extension of DNA

Question 4

what are the ingredients of PCR

Answer 4

1. DNA target
2. primers 1, 2
3. Taq pol
4. dNTP
5. buffer

Question 5

what is process of PCR

Answer 5

https://www.youtube.com/watch?v=IaKXv1JqBtY

Question 6

how can product of PCR be detected

Answer 6

gel/capillary electrophoresis

Question 7

what is real time PCR - qPCR

Answer 7

based on PCR principles

but amplified product is detected in real time rather than at end of PCR

potential quantification of target sample

laballed with fluorescent dye

Question 8

what are the advantages vs. disadvantages of real time PCR

Answer 8

advantages

1. more sensitive
2. more quantitative
3. faster - no gel needed
4. safer - no ethidium bromide or radioactivity
5. possibly lowered risk of contamination - closed tube

disadvantages

1. more specialized equipment
2. more expensive
3. requires known sequence across primer and probe regions for Taqman PCR

Question 9

why is avoiding contamination important in PCR

Answer 9

PCR/RT-PCR is so sensitive it is imperative to avoid

clean rooms for setting up master mixes

Addition of target in rooms where product is not

amplified using equipment not used to handle product

• Wearing of protective gear (clothes/gloves)
• Use of UV hoods which irradiate (break up)

contaminating DNA/product

Question 10

what is primer non-specificity

Answer 10

primers can bind elsewhere in

target nucleic acid sequence then some products may not be

of desired sequence/target sequence may not be amplified

efficiently

• possible false positives/false negatives

Question 11

what is sequence variation across primer binding site

Answer 11

if there are mismatches across primer binding site then efficiency of reaction may be lessened/no product amplified

• NB when using PCR to detect pathogens in clinical samples

as far as possible primer should be designed to areas of

pathogen genome which are conserved across strains

• possible false negatives if primers do not bind to particular strain in

clinical sample

Question 12

what are the veterinary uses of qPCR

Answer 12

1. disease diagnosis: infectious disease detection of pathogen genomes in host
2. monitoring disease/infection progress
3. screening to prevent intro of infection (or genetic disease) into household/geographic area
4. research- gene expression and disease

Question 13

what useful clinical info is provided

Answer 13

Rapid (24-48hr report) , sensitive, specific diagnosis

Identify pathogens non-cultivable / grow slowly

Early infections (pre-antibodies)

Identification of carriers + shedders

Strain-specific identification

Vaccine vs field infections (sequence differences)

Quantitative: pathogen load

Question 14

what are diagnostic PCR issues

Answer 14

1. pre-lab issues (degradation of sample, cross-contamination)
2. inhibition: substances contained within clinical sample that interfere with PCR reaction –> false negative
3. contamination: laboratory contamination from amplicons –> false positive

Question 15

what are the limitations of diagnostic PCR

Answer 15

1. RNA viruses
2. no info on viability/infectivity
3. no data on antimicrobial susceptibility

Question 16

what are the key things for vets re PCR

Answer 16

1. Be clear about the purpose of your test (e.g. for

diagnosis, for screening) – is a PCR test REALLY the

best one to achieve your goal? [e.g. it detects

pathogen nucleic acid - does not tell you whether

infectious agent is replicating]

2. Consider where to send the sample : are there

scientific reports regarding the efficacy of tests by

different laboratories?

3. Check to make sure you submit the best sample

for your purposes, e.g. swab from correct location

and be aware of contamination issues

4. Be aware of limitations of PCR – e.g. genetic

variation in RNA viruses: false negatives possible

(e.g. for some genetically variant viruses better tests

may exist than PCR)

www. catvirus.com
5. Presence of organism does not always

mean that it is the cause of the disease