Cepheid

Question 1

describe the general principles of qPCR

Answer 1

generation of products from template is detected in real time through an increase in fluorescence generated by fluorophore labels

fluorescent signal intensity > set threshold

cycle number at which fluorescent signal crosses threshold known as Ct or Cp

Question 2

T or F. The Ct value is inversely proportional to the amount of starting material (template NA)

Answer 2

T

Question 3

qPCR amplification curve

Answer 3

a baseline that gradually transitions into (2) an exponential region, followed by (3) a plateau, which indicates that amplification is reducing

generated based on the quantitative relationship between the fluorescence signal accumulation and cycles

Question 4

explain the principles of Taqman probes aka 5’ nuclease probes

Answer 4

• have 5’ fluorophore and 3’ quencher
• probe intact = FRET occurs = no light detected
• DNA polymerase cleaves probe = separating fluorophore from quencher
• free fluorophore signal detected

Question 5

overview of 5’ nuclease assays

Answer 5

• primers and probe hybdridize; sequence- dependent to complementary DNA strand; quencher absorbs fluorescence by fluorophore
• polymerase extends from primers and begins DNA synthesis
• polymerase reaches probe; exonuclease activity of pol cleaves HYBRIDIZED probe; fluorophore = signal
• fluorescence measured by real-time instrument

repeated for each PCR cycle; if intercalating dyes used = primer-dimers and non-specific producTs will contribute to fluorescence BUT Taqman is specific and fluorescence will only be produced for DNA sequence probe and primers hybridize to

Question 6

explain principles of molecular beacon probes

Answer 6

single stranded molecule with a quencher and fluorophore at the ends

5’ end and 3’ end complementary = form hairpin so fluorophore and quencher are in close proximity and FRET occurs

when probe hybridizes to template, fluorophore and quencher separated = SIGNAL

NOTE: probe not destroyed by Taq

Question 7

steps of molecular beacon probes PCR

Answer 7

1. molecular beacon probes hybridize to target sequences = separates the Q and F (straighten from hairpin loop) = FLUORESCENCE
   probes that do not hybridize, reform hairpin = no fluorescence = not detected
2. polymerase extend from primers and begins DNA synthesis
3. polymerases displaces probe without being degraded; molecular beacons can participate in multiple rounds of annealing
4. pol continues extension of primers to complete synthesis of DNA strand

Question 8

explain scorpion probes (primers)

Answer 8

• bi-functional molecules in which a primer is covalently linked to the probe
• fluorophore and quencher
• absence of target = quencher absorbs fluorescence
• presence of target = fluorophore and quencher separate = increase in fluorescence
• fluorescence detected and measured in reaction tube
• between primer and probe is a blocker region which prevents elongation of the reverse strand

Question 9

two different methods of nucleic acid extraction used i the Cepheid cartridge

Answer 9

physical = sonication
chemical = lysis reagent

Question 10

briefly describe how the sample moves thorugh the Cepheid cartridge chambers

Answer 10

syringe motor raises and lowers through cartridge chambers

this action creates vacuum = pulls fluid through microvalves = moves sample and dissolved reagents from one chamber to the next

Question 11

identify the physical state of Cepheid amplification PCR reagents

Answer 11

• PCR reagents = solid beads
• DNA polymerase, buffer, cofactors, nucleotides = enzyme beads
• primers and probes = TSR (target specific reagents) beads
• some chambers contain liquid

Question 12

describe the function of SPC in Cepheid cartridge

Answer 12

• SPC = sample processing control: exogenous nucleic acid preloaded in cartridge that co-xtracts and co-amplifies along with the sample nucleic acids; positive control, internal control

Question 13

internal control

Answer 13

detects PCR inhibition

Question 14

four controls in Cepheid cartridge

Answer 14

SPC = sample processing control
PCC = probe check control (aka reagent control)
SVC = sample volume CONTROL
SAC = sample adequacy control (not in all assays)

NOTE: positive and negative external controls should be run regularly
QC batch each batch of cartridges; once per day the test is performed

Question 15

PCC

Answer 15

probe check control aka reagent control
- fluorescence reading in the rxn tube are compared to the default settings several times during procedure before PCR stage

done to ensure the fluorophores are properly emitting and haven’t spoiled in cartridge

Question 16

SVC

Answer 16

SAMPLE VOLUME CONTROL
ensures sample was correctly added to cartridge

Question 17

sample adequacy control

Answer 17

not in all assays
- reagents amplify and detect an endogenous single copy human gene

• this nucleic acid target is found in the sample and co-extracts with the target nucleic acid