SPADA Draft Documents

increasing the concentration until at least 58 of the 60 replicates are quantified with a pre-defined 680 level of precision. The upper limit of quantification (ULOQ) can similarly be determined if 681 needed. While only quantitative results within this range can be reported, one can also report 682 qualitative results (positive or negative) that are outside this range (34).

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Table 2. Recommendations for PCR Experiments

Item

Recommendation

Reason

Gather Target Sequences

Database and literature research on target sequences

Learn about target sequence variation, design PCR to conserved regions

Enzyme

not HiFi, no 3'-exonuclease

3'-exonuclease causes off-target amplification, PCR failure

PCR Cycles

45

For 20 uL reaction, a single target molecule has Ct of about 38. Using 45 cycles ensures detection Reduce delayed onset due to template re- annealing, lower Ct value observed Test if primers work before ordering expensive fluorophore-labeled probes Often detect brackground amplification, causing false positives Determine if "primer dimers" and other false amplicons are formed. Verify that the amplicon product is indeed the correct target If a reaction doesn't work as singleplex, then it isn't going to work in multiplex either. Verify that PCR is working, so that a negative for the target analyte is meaningful Verify that primers are compatible with each other Keep enzyme activity high

Denaturation temperature

First 3 cycles use 95 °C and 20 seconds

Denaturation temperature

Cycles 4-50 use 94 °C for 5 seconds Good to evaluate if primers work

Dye based detection

Dye based detection

Do Not use for clinical testing

No Template Control

Run PCR without template DNA

Sanger Sequencing

Perform sequencing on PCR reaction product

Singleplex testing

Test all targets as singlplex before performing multiplex Add to your analyte target

Positive control

Multiplex testing

Combine validated singleplexes into larger multiplexes Use synthetic gBlocks for initial testing Use actual patient samples, known positives and negatives

Synthetic Target

Cheap, non-pathogenic

Testing with Patient samples

Determine sensitivity, specificity, and LOD

Gather Assay Information

Use MIQE standards

Ensure assay is reproducible and documented

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