Diagnosis of viral infections pt 2

Question 1

PCR used for ?

Answer 1

amplification of viral genome/DNA

Question 2

PCR steps

Answer 2

denaturation –> annealing –> extension/elongation

Question 3

real time PCR aka ?
useful for ?
what can be used for it?
results mean?

Answer 3

quantitative PCR
advanced form of PCR, allows for monitoring and quantification of increasing accumulation of PCR products/nuclei acid load as the reaction progresses - useful to study virus load in patient

1. a target specific probe (labelled with a fluorescent dye) such as 5’ nuclease TaqMan probes, molecular beacons or FRET hybridization probes
2. OR intercalating dyes, such as SYBR green
   can be used in real time PCR
   visualize fluorescent

decrease CT value = stronger rxn = increase viral genome concentration in sample

Question 4

genome sequencing def

Answer 4

DNA sequencing is process by which sequence of bases in a DNA molecule is elucidated/obtained and read

Question 5

next gen sequencing

Answer 5

-NGS replacing conventional sequencing methods
-NGS platforms, such as Illumina (Solexa) sequencing, are significantly cheaper, quicker, needs less DNA, has high throughput, and is more accurate and reliable than Sanger sequencing

Question 6

metagenomics definition

Answer 6

study of collective set of microbial populations in a sample by analyzing the samples entire nucleotide sequence content, and is a method for random detection of existing and new pathogens

Question 7

metagenomics - method

Answer 7

amplify and sequence whole genome (DNA and/or RNA) content of a sample followed by filtering and analysis of obtained data by comparing genome databases
-NGS platforms due to high sequencing capacity are being used in metagenomics

Question 8

importance of genome sequencing (7)

Answer 8

1. pathogen detection
2. studies on genetic variation, such as genotyping, evolution and interspecies transmission of pathogens
3. ID of novel and unidentified strains
4. development of diagnostics, such as genotyping primers or probes
5. ID of genes associated with drug resistance
6. development of therapeutics
7. judging the efficacy of current vax and formulating new vax strategies

Question 9

phylogenetic analysis

Answer 9

use of virus genome sequence data to study evolution of viruses and genetic relationships among viruses

Question 10

microarrays -def/method
-advantage

Answer 10

several thousands of known DNAs (probes) are amplified by PCRs and spotted onto a glass or silicon chip
-target/sample DNA are fluorescently labelled and then added to chip containing DNA probes
-positive reactions between probe DNA and sample DNA (hybridization) generate a fluorescent signal from the spot where probe DNA is spotted in the chip
-advantage in detection of pathogens in surveillance studies is that hundreds of pathogens can be screened for simultaneously suing a single microarray chip