5 - Integrated PCR Technologies

Advantages

• Small benchtop instruments, suitable for POC testing

• Minimal hands on operation, less training, faster TAT

• High sensitivity and specificity

• Closed systems reduce risk of contamination (FP)

BioFire FilmArray systems and panels

• Fully integrated, 1h TAT, assay specific pouches available, simultaneous detection of viruses, bacteria etc

• Respiratory 2.1 plus panel - pathogens commonly associated with respiratory infections, nasopharyngeal swab used as sample, 19 viruses (corona) and 4 bacteria (s. pneumoniae)

• Blood culture identification panel - positive blood culture grown first, targets gram positive and negative bacteria and yeasts

• GI panel - stool sample, 5 viruses, 13 bacteria, 4 parasites

• Meningitis / encephalitis panel - CSF sample, 7 viruses, 6 bacteria, 1 yeast

Assay principle

• Pouch inserted into loading station and injected with hydration solution

• Sample is combined with a buffer and inserted into pouch and assay is run

• During the run the system lyses the sample by bead agitation and extracts and purifies all nucleic acids

• Performs nested multiplex PCR with multiple outer primers

• Then multiple single second stage PCR reactions are performed with inner primers

• Melting analysis identifies multiple NA’s and generates the results, LCGreen Plus dye used (binds to dsDNA), releases as temperature increases

• Each target has a unique peak due to difference in lengths and sequences, 3 replicates analysed

Quality control

• Two controls included in pouch, both must be positive, external positive and negative controls ran regularly

• RNA process control which targets an RNA transcript from a yeast, indicates all steps were successful

• Second stage PCR control detects a DNA target dried onto the wells along with corresponding primers

• Interference - blood, mucus, genomic DNA, commensals, medications, disinfectants (bleach)

Interpretation of results

• Melting analysis performed after PCR2 is complete, detects fluorescence in each well

• Tm of product is compared to the Tm range for the specific assay, either detected or not detected

• Analysis of replicates - software evaluates three replicates for each assay, at least 2 melt curves must match and Tm values must be within one degree

GeneXpert (closed system)

• Automated and integrated sample purification, NA amplification and detection using RT q PCR 

• Base unit used with disposable cartridges preloaded with the necessary reagents for the assay e.g. respiratory

• Several can be run at one time, FilmArray can only do one pouch at a time :(

Cartridges

• Processing chambers - hold samples, reagents and waste

• Valve body - rotates to allow fluid to move to different cartridge chambers and the reaction tube, specimen are ultrasonically lysed and mixed with reagents, move into reaction tube

• Reaction tube - rapid thermal cycling and fluorescent excitation and detection

• Cycle threshold - first cycle in which there is significant increase in fluorescence above background fluorescence

Quality control

• Probe check control - before PCR the fluorescent signal of the probes is measured to ensure dye stability, reagent rehydration and tube filling

• Sample processing control - ensures sample was correctly processed, verifies lysis and detects PCR inhibition

• Sample adequacy control - normalises target and ensures there is sufficient sample used in the test

Result analysis

• MRSA/SA blood culture test - simultaneous detection of S. aureus and MRSA

• Fluorescent probes used for specific DNA targets e.g. Staphylococcal protein A (spa) or methicilin resistance gene (mecA)

Verigene system

• Automated multiplex system, rapid TAT (2h), uses cartridges with reagent pack and substrate holder

• Used for blood cultures, c.diff, respiratory pathogens

• Cell lysis by magnetic beads and isolation, NA fragmented and denatured to form ssDNA

• Binds to capture oligos specific for target of interest on microarray slide

• Mediator oligo binds to target DNA and a common oligo attached to a gold nanoparticle

• Silver coats the gold nanoparticles making the spots visible by light scatter, which can be detected

Cobas Liat System

• Multiplex real time PCR assay (20 min TAT) for covid, influenza, RSV

• Integrated silica magneic particle based purification, NA amplification and tetection using taqman probes

• Internal positive control included