MGY378 24: NAAT and serology

NAAT

= nucleic acid amplification test

• ex: PCR

• Extract the nucleic acid and remove everything else

• RT: RNA > cDNA

  • If not an RNA virus, don't need to do this step

• Amp: primers and probes bind to target, amp, gen a signal. U can make ur test a multiplex -- test for many pathogens at once

  • Ex: test for Sars2, RSV, influenza

• Detect: if probe binds to target > fluorescence > amp curves

• U dk if it infected at other places or not, could just be dead virus there

Ct

= cycle threshold

• Ct = when signal goes above background = +ve result

• Higher Ct = the longer it took to go above threshold = lesser virus in sample

Non-specific probes

Sybr green

specific probes

Taqman

RT-PCR

= real-time polymerase chain reaction

• type of NAAT

• Design primers to bind to regions only your virus OI has so u don't get cross reactivity w another virus

• Host probes and primers target host DNA and shows amplification - control

  • Way of telling that the specimen is good quality - know the NP swab actually got some cells and they might have virus in it

• sensitivity and specificity: How likely is it for my test to test for the actual disease. Does it cross react with any other pathogen? You want the accuracy to be 98-99%

• Need a series of patient samples to make sure test actually works

Samples for covid

• nasopharyngeal = ideal

• saliva = alternate. 98% agreement, 91% sensitivity

metagenomic WGS

• Seq everything that is in the sample

  • Bacteria, virus, host DNA, RNA -- shotgun approach

• Computer analysis sorts out what everything is

• Do this when u have no idea what the virus is

• Did this in the beginning of Sars2

  • Had a cluster of pneumonia cases in Wuhan, didn't know what it was > metagenomic wgs > novel Sars

• High throughput

amplicon-based WGS

method where the entire genome (or nearly all of it) is covered by multiple overlapping PCR amplicons, which are then sequenced to reconstruct the full genome.

1. Design primers that target multiple regions across the genome (tiling method).

2. PCR amplify those regions — these are your amplicons.

3. Pool the amplicons together.

4. Sequence them using platforms like Illumina or Oxford Nanopore.

5. Assemble the full genome from these overlapping reads.

deep sequencing

Deep sequencing = ultra-high-throughput sequencing that gives very detailed information, including rare variants or low-frequency mutations.

coverage

how much of the genome has been sequenced

depth

how many times has a region been sequenced

steps of sequencing

1. library prep

2. amplification

3. determine the seq

4. bioinformatic analysis

library prep

• convert to DNA using RT (RNA>DNA) (unless starting with DNA)

• may shear DNA into smaller parts

• generate cDNA

• add barcodes (to ID specific virus in multiplexing)

Case study: persistently +ve Sars2 patient with HIV

• same alpha-Sars2 variant reinfecting

• found antiviral resistance to Nirmaltrevir (part of paxlovid that increases EC₅₀ in blood)

  • resistance mutation = E166

• used molnupiravir to inhibit virus

serology

• Detecting the immune response to the virus

• Look for abs specific to virus OI

  • Some look for viral ag too

• Look for abs during acute phase of infection

• Also convalescent sera -- abs from before

• ID which ag causes a humoral response

• Avidity - how well the abs bind to see if its new vs old infection

  • New - abs don’t bind as well bc BCs did not mature yet (not memory)

• Unable to differentiate from prev exposure vs active infectiom

significant ab titre

4-fold rise = new infection

systemic infections

• measles

• YFV

• mumps

• polio

• hep A

long-lived abs

mucosal infections

• RSV

• Sars2

short-lived abs

IgM

first ab generated = acute infection

IgG

generated late = infection resolving

direct ab detection

• Direct assay: add ab for it, things light up, the virus is present

  • CMV

indirect ab detection

• Indirect: coat a plate w ag, put patient serum, Abs bind to ag, add a 2ndary signal ab on top

capture ab detection

• Capture: put a capture antibody on the plate, add ag

  • Capture ab captures viral ag

  • Then add an ab against the viral pr - viral ag sandwiched btwn capture ab and primary ab

  • Add a secondary signal ab

  • Adds sensitivity

HIV serology and ab testing

• HIV

• Test for ab and p24 ag

• Incr sensitivity and works w course of disease in HIV

  • Initially, presence of viral RNA

  • 1st thing virus gen detectable levels of is p24 ag

  • P24 decreases over time

  • Then ab against HIV ag are gen

• Testing for p24 shows the infection earlier than testing just for ab