Unit Tests for Covid-19 Polymerase Chain Reaction Software

The Francis Crick Institute has repurposed its laboratories as an emergency Covid-19 testing facility. The Crick is helping combat the spread of infection and allow key workers to perform lifesaving duties and remain safe.

One of the main technologies the Crick is using in this effort are Polymerase Chain Reaction machines. PCR machines test for the presence of a specific nucleic acid. The end to end process involves capturing molecules on a swab that are then broken down into genetic code, using special chemicals and liquid handling robots. The PCR (polymerase chain reaction) machine can then make billions of copies of DNA strands from the original swab. The PCR machine tests for the presence for the Covid-19 RNA. This is done on a series of 94 Wells (each containing an individual swab) on a Plate within the ThermoFisher PCR machines. The final step involves specialist clinicians making the decision on whether the sample contains sufficient RNA to justify the presence of Coronavirus.

The qPCR test produces a graph showing the exponential progress (or not) of the Cq (Ct) value as it traverses the threshold. The Cq value is the cycle quantification value of the PCR cycle number at which the sample’s reaction curve intersects the threshold line. This value tells how many cycles it took to detect a real signal from your samples. Real-Time PCR runs will have a reaction curve for each sample, and therefore many C_q values. Your cycler’s software calculates and charts the Cq value for each of your sample

To help support the clinician diagnostic phase we have written a series of complementary tests. These seven tests (github) test each Well, each Plate and a series of Plates. The test data comes from the the ThermoFisher PCR machines and QuantStudio software.

T#	Test Name	Possible Outcomes	Scope	Data Required
1	Ct Range	Ct value ? < Threshold : > Threshold	Per Well	Ct Value, Exponential Phase, Pre-Exponential Phase, Threshold
2	Amplification Effect	Measure of exponential phase approximation to 100%. If Slope <0.01 test failed	Per Well	Intercept, Plot, Slope
3	R^2	R2  is the Coefficient of Determination of a whole plate with a maximum and perfect value of 1 R Squared < X == failed test	Per Well & Per Plate-	R2 value
4	Plate Standard Curve	Standard curve for baseline wells (A1 and H8)	Per Plate	Test well positions and reference gene
5	DeltaDelta Ct	Very similar to ANCOVA. Requires Treatment, Control and Reference genes. Difference between Delta CT Values	Multiple Plates Multiple Samples	Compare DeltaCT across multiple Gene samples. Requires a Reference Gene and a Reference Group
6	ANCOVA	Covariance analysis – what is the Ct difference of target gene value between treatment and control sample after corrected by reference gene	Multiple Plates (need reference & target, treatement & control)	CT and Concentration.
7	Efficiency Model	Log score per Well between 90% & 110% Across multiple Plates a value of Rsquared greater than >0.99	Per Well & Multiple Plates	Per Well: Column R in Results Sheet provides efficiency score per WellMultiple Plates: Slope: ~ –3.3R2 >0.99

The outcome of these tests can then be used in conjunction by the clinician reviewing 94 individual wells (each representing a unique swab). The intent is that this helps reduce human error and can improve the clinical throughput.