Routine diagnostic testing by nucleic acid methods Flashcards
What are the benefits of Nested PCR and what are the problems?
Benefit: inc sensitivity by 100x
Problems; contamination, can’t continue amplification cycles in single PCR rxn bc reagents run out & DNA pol. digest PCR product
Why would multiplexing be useful in a diagnostic lab?
- can use diff primers to detect diff targets
> detect diff. infectious agents
> detect oncogene mutations
How were the primers designed for the Multiplex nested PCR for HPV?
- designed manually
- 2 groups of PCR & each round had a set of primers = each group could detect a region on HPV
What are the two phylogenetic groups that contain the High-Risk HPV types?
Groups 1: 18 …
Groups 2: 16,33,35 …
What region was used to sequence for HLA DR (multiplex PCR then DNA sequ) typing and why was it used?
PCR relevant region
e.g. DR-B1 bc highly polymorphic = able to be identified)
What are the advantages and disadvantages of sequencing vs serological typing for HLA?
Sequ; √: reveals more HLA type than serology. X: slow
Serolo: √ quicker typing. X: reveals less types
Explain the principal of Mass-Tag PCR.
- label primers w/ “Masstag”
- each Masstag has a diff. molecular weight
- detect by mass spectro
What are the advantages of Mass-Tag PCR
- Multiplexing = more tags (not limited)
- High throughput: lots of tests in short time frame (6-8hrs)
- Reduce risk of False pos. bc no 2nd round of PCR or probes
- Potential to detect virus
Disadvantage of Mass-Tag PCR
less sensitive than RT-PCR (bc makes more copies than RT-PCR)
Explain the principal of the Hybrid Capture method.
- Hybridising RNA probe mixed w/ target DNA => RNA/DNA hybrid
- RNA/DNA hybrid captured onto solid phase by Ab - specific for the hybrids
- Anti-RNA/DNA Ab conjugate (couple) w/ alkaline phosphatase => bind hybrid @ multiple sites
Disadvantage of Hybrid Capture method.
less sensitive than PCR
Use of Hybrid Capture method.
HPV detection w/ 2 probe sets : 1 for HRHPV & LRHPV
