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)