dPCR - CB-Gene

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dPCR Testing Service

Service Principle

The full name of DdPCR is Droplet Digital PCR, which is described by bio-rad as Droplet Digital PCR technology is a digital PCR method utilizing a water-oil emulsion droplet system. Droplets are formed in a water-oil emulsion to form the partitions that separate the template DNA molecules The droplets serve essentially the same function as individual test tubes or wells in a plate in which the PCR reaction takes place, albeit in a much smaller format. The massive sample partitioning is a key aspect of the ddPCR technique.

From the basic principles, it is not difficult to see that the biggest advantage of DdPCR is absolute quantification, which is expressed as:

Absolute quantification

ddPCR technology can provide absolute counts of target DNA copies for each input sample without inputting a standard curve, making this technology ideal for target DNA measurement, viral load analysis and microbial quantification.

High precision

The large number of sample assignments provided by ddPCR can reliably measure small differences in the copy number of the target DNA sequence in the sample.

Improved signal-to-noise ratio

Dilute high-copy templates and background, effectively enrich the template concentration in the target-positive partition, so that rare targets can be ·sensitively detected, and quantitative accuracy of ±10% can be achieved, the sensitivity can reach a single nucleic acid molecule, and the detection limit As low as 0.001%.

Eliminate PCR bias

Eliminate qPCR amplification efficiency dependence, reduce the error rate, and detect small (1.2 times) differences, here it should be pointed out.

Reduced consumable costs

Reaction volumes in the picoliter to nanoliter range reduce reagent usage and sample volume per data point, especially for precious samples.

Excellent partitioning

ddPCR technology can generate 20,000 droplets per 20 µl sample, and nearly 2 million partitioned PCR reactions can be performed on a 96-well plate. The more partitions, the higher the precision.

Service Application

Combined with the field of tumors, we generally have 4 applications related to it:

Detection of Gene Expression, mainly quantitative analysis of mRNA detection.

Detect Mutation (SNV and Indel), can detect copy number, and can also detect ratio.

Detect Fusion, can detect the copy number, and can also detect the ratio.

Detection of CNV (copy number variation analysis), relative quantification.

Case Presentation

CB-Gene develops corresponding detection kits for the above applications, and uses CB-Gene's reference products to evaluate the performance of the kits. Let's take EGFR T790M as an example:

Experiment 1: LOB blank limit test

Samples to be tested: 20 negative samples and 1 NTC. The input amount was set to 10ng and 40ng respectively, and each sample was tested by Bio-Rad digital PCR in duplicate.

Sample 10ng 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 NTC

n=1 0 0 0 0 0 0 0 0 0 0 1.2 0 0 0 0 0 0 0 0 0 0

n=2 0 0 0 0 0 0 0 0.6 0 0 0 0 0 0.8 0 0 0 0 0 0 0

Sample 40ng 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 NTC

n=1 0 0 0 0 0 0 0 0 0 1.3 0 0 0 0 0 0 0 0.9 0 0 0

n=2 0 0 0 0.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

From the above data, it can be seen that whether it is 10ng or 40ng, LOB is controlled within 2 copies (generally the noise copies are within 5), and the noise does not show dose dependence and is a randomly generated noise signal.

Experiment 2: Accuracy test

Samples to be tested: 10 samples verified as positive by NGS (numbered 1-10); 10 samples verified as negative by NGS (numbered 11-20). Digital PCR was used to test the 20 samples.

Sample	1	2	3	4	5	6	7	8	9	10
Value	72.21%	66.63%	13.25%	5.29%	10.34%	43.01%	22.19%	3.27%	1.07%	13.29%
Results	Positive	Positive	Positive	Positive	Positive	Positive	Positive	Positive	Positive	Positive

Sample	11	12	13	14	15	16	17	18	19	20
Value	0%	0%	0%	0%	0%	0%	0%	0%	0%	0%
Results	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative

From the above data, we can see that:
① Test 10 NGS positive samples, the test results are all positive, the positive compliance rate is 100%, and the variation frequency is quantitatively detected.
② Test 10 NGS negative samples, the test results are all negative, the negative compliance rate is 100%, and the fixed variation frequency is 0%.

Experiment 3: Linear range test

1. Linear test between input amount and copy number and frequency
First, select the EGFR T790M standard (CBP10402) with a mutation frequency of 50%. This standard is a two-allele gene with CN=2 edited by gene editing. One is mutation and the other is WT. After theoretical value calculation, Sanger sequencing, and Bio-Rad DdPCR kit (Assay ID: dHsaCP2000019), it is confirmed to be 50% frequency.
1.1 Set 10 gradients between 0.1ng and 200ng input amount, and perform digital PCR detection (2 replicates) to consider whether the FAM copy number is linearly related to the input amount and whether the VIC copy number is linearly related to the input amount;
1.2 Set 10 gradients between 0.1ng and 200ng input amount, and perform digital PCR detection (2 replicates) to determine whether the fluctuation of %AF=50±10% is satisfied.

Input ng	200	100	50	20	10	5	2	1	0.5	0.1
FAM Copies(n=1)	18970	14970	7238	3327	1570	786	288	139	87	23
FAM Copies(n=2)	18367	15013	7139	3122	1430	722	290	151	67	21
VIC Copies(n=1)	20183	15180	7312	3290	1540	739	267	150	93	31
VIC Copies(n=2)	19783	14962	7016	3017	1510	697	281	143	80	30

Input ng	200	100	50	20	10	5	2	1	0.5	0.1
AF%(n=1)	48.45	49.65	49.75	50.28	50.48	51.54	51.89	48.10	48.33	42.59
AF%(n=2)	48.14	50.09	50.43	50.86	48.64	50.88	50.79	51.36	45.58	41.18

From the above data, we can see that:
①200 ng input is too large, the copy number is not linear, although it meets the range of 45-55%, it is not suitable for single channel copy number determination;
②0.1 ng input is too small, the copy number is not linear, and 42.59% is also lower than 45%, which is not suitable for single channel or dual channel;
Therefore: the range is 0.5-100ng

2. Linear test of theoretical values and detection values at different frequencies of linear dilution
Based on the above conclusions, we selected 10ng as a further exploration of the %AF linear range. First, we used the corresponding negative standard of EGFR T790M, performed limiting dilution on 50% of the standard, set 9 AF gradients between 0.1% and 50%, and performed digital PCR detection (2 replicates)

Expected %AF	50±10%	25±10%	10±10%	5±20%	3±20%	1±30%	0.5±30%	0.2±30%	0.1±50%
%AF(n=1)	49.14	24.82	9.82	4.52	2.94	0.95	0.55	0.16	0.13
%AF(n=2)	50.25	26.20	10.24	4.98	2.55	0.79	0.59	0.19	0.13

From the above data, we can see that at an input of 10 ng, AF% shows very good linear dilution, with some fluctuations, but all within the error range.

Experiment 4: Detection limit test

We can predict the possible detection limit range through theoretical calculation. When the control copy is within 15000 (corresponding to 50ng input), the LOB data is 2, 2/15000=0.0133%, and the predicted detection limit is 0.0200%.
In such a low frequency, the clinical application scenario is suitable for ctDNA. Generally speaking, in clinical practice, the median ctDNA of 10ml whole blood is 20-40ng, and the lowest value is 20ng. The theoretical value of 20ng is 6000 copies, 2/6000=0.0333%, and the predicted detection limit is 0.0500%.
The error range of 0.05% is ±50%, which is 0.025-0.075%, so we choose 3 points and explore LODs of 0.010%, 0.050%, and 0.100%, respectively, requiring 95% detection.

0.010%
Perform digital PCR test on 20 samples with 0.01% to see if 95% detection is achieved within 0.005-0.015%

0.05%
Digital PCR testing was performed on 20 0.05% samples to observe whether 95% detection was achieved within 0.025-0.075%.

0.1%
Digital PCR testing was performed on 20 0.1% samples to observe whether 95% detection was achieved within 0.05-0.15%.

Prediction frequency LOD 0.01% 0.05% 0.10%

Achievement rate 65% 95% 100%

From the above data, it can be seen that 0.05% is selected as the detection limit LOD of the detection kit.

Experiment 5: Precision test

Select 20 samples with strong positive rate of 2%, 20 samples with medium positive rate of 1%, and 20 samples with weak positive rate of 0.2% for digital PCR detection.

	High Positive2%	Medium Positive 1%	Low Positive 0.2%
AVG	1.9959%	1.0027%	0.1878%
SD	0.001039	0.001135	0.000295
CV%	5.2058	11.3177	15.7011
Measurement uncertainty	1.16%	2.53%	3.51%

From the above data, it can be seen that under the three detection systems of strong positive 2%, medium positive 1% and weak positive 0.2%, the errors are all within a reasonable range and the precision is good.

Experiment 6: Recovery test

First, the corresponding negative standard of EGFR T790M was used to perform limiting dilution on 50% of the standard, and each gradient was diluted twice with the negative sample, and then 11 samples were tested.

Sample 1 2 3 4 5 6 7 8 9 10 11

%AF 50.00 25.00 12.50 6.25 3.125 1.563 0.781 0.391 0.195 0.098 0.049

%Recovery rate 99.0 100.1 99.4 108.8 95.6 91.1
112.2
92.4 84.8 81.3 82.1

From the above data, it can be seen that when AF is in the range of 0.05%-50%, the corresponding recovery rate is in the range of 80-120%, which is in line with expectations.

Experiment 7: Repeatability test

Take 10 standard samples with different AF (1%~50%) and arrange Operator A and Operator B to perform four tests on the same day.

It can be seen from the above data that the EGFR T790M detection system is stable and reproducible.

Experiment 8: Reproducibility test

Take 10 standard samples with different AF (1%~50%) and arrange ① Operator A and Operator B to perform four experimental tests each on the same day; ② Operator A performs four experimental tests on the first day and the second day respectively; ③ Operator A performs four experimental tests on the first day and Operator B performs four experimental tests on the second day.

From the above data, it can be seen that the EGFR T790M detection system is stable and reproducible in different people and at different times.

CB-Gene has developed a variety of dPCR kit detection systems and used its own standards to verify performance. It can be used for the detection of gene mutations, fusions, copy number variations, etc. At the same time, we can provide scientific research services for dPCR detection, based on its strong advantages of absolute value and precise quantification, to provide high-quality services for customers' sample testing.

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