1

Ultrasensitive and quantitative detection of EGFR mutations in plasma samples from

patients with non-small-cell lung cancer using a dual PNA clamping-mediated LNA-

PNA PCR clamp †

Shichao Zhang,a,c,† Zhiyao Chen,a,† Chenrong Huang,a Cheng Ding,b Chang Li,b Jun Chen,b

Jun Zhao*b and Liyan Miao*a,c

aDepartment of Clinical Pharmacology, The First Affiliated Hospital of Soochow University,

Suzhou, 215006, PR China

bDepartment of Thoracic Surgery, The First Affiliated Hospital of Soochow University,

Suzhou, 215006, PR China

cCollege of Pharmaceutical Sciences, Soochow University, Suzhou, 215006, PR China

†These authors contributed equally to this work.

*Correspondence:

Liyan Miao

No. 188, Shi Zi Street, Suzhou, 215006, PR China. Tel.: (+86) 512-6778-0467; Fax: (+86)

512 67780040; E-mail address: miaolysuzhou@163.com.

Jun Zhao

No. 188, Shi Zi Street, Suzhou, 215006, PR China. Tel.: (+86) 512-6778-0507; Fax: (+86)

512 67780507; E-mail address: zhaojia0327@126.com.

Electronic Supplementary Material (ESI) for Analyst.This journal is © The Royal Society of Chemistry 2019

2

List of Electronic Supplementary Information (ESI)

Table S1 Sequences of the PCR primers and probes used for the LNA-dPNA PCR clamp.

Table S2 Clinical characteristics of the patients with NSCLC.Figure S1 Efficiency of the LNA-dPNA PCR clamp at various

concentrations of the M-LNA probe (A), cycle numbers (B), concentrations of IPs (C), and concentrations of OPs (D).

Figure S2 Mutation enrichment detected by the LNA-dPNA PCR clamp at various concentrations of the W-PNA probe using 103 M:103 W (A) and 103 M:106 W (B) as templates.

Figure S3 Mutation enrichment detected by the LNA-dPNA PCR clamp at annealing temperatures of 72 °C (A-1, B-1), 70 °C (A-2, B-2), 68 °C (A-3, B-3), and 66 °C (A-4, B-4) using OPs and IPs.

Figure S4 Mutation enrichment detected by the LNA-dPNA PCR clamp at annealing temperatures of 64 °C (A-1, B-1), 60 °C (A-2, B-2), and 56 °C (A-3, B-3) using OPs and IPs.

Figure S5 Evaluation of the effects of one-tube nested PCR (A) and dual PNA clamp (B) on the LNA-dPNA PCR clamp.

Figure S6 Evaluation of the sensitivity (A), specificity (B), and linearity (A) using ddPCR for EGFR L858R detection.

Figure S7 Evaluation of the reproducibility of LNA-dPNA PCR clamp in detection of EGFR L858R.

Figure S8 Typical results for EGFR L858R analysed using the LNA-dPNA PCR clamp (A, C, E, G) and ddPCR (B, D, F, H) from plasma (A, B, E, F) and tissue (C, D, G, H) samples.

Table S3 Comparison of ddPCR and the LNA-dPNA PCR clamp for detecting plasma EGFR mutations and tissue EGFR mutations.

Table S4 Comparison of EGFR mutations in tumour tissues and matched plasma samples for different tumour stages.

Table S5 Comparison of labour and cost between the LNA-dPNA PCR clamp and ddPCR for quantifying plasma mutations.

3

Table S1. Sequences of the PCR primers and probes used for the LNA-dPNA PCR clamp.

Primer name Sequence (5’-3’)Tm

(C)

Length of

amplicon

EGFR858-IP-F CAGCATGTCAAGATCACAGATT 61.0

EGFR858-IP-R CCTTACTTTGCCTCCTTCTG 60.087 bp

EGFR858-OP-F CTACTTGGAGGACCGTCGCTTGGTGC 69.5

EGFR858-OP-R CCTGGTCCCTGGTGTCAGGAAAATGCT 69.5200 bp

EGFR858-W-PNA TGGGCTGGCCA ~ 63.9

EGFR858-M-LNA 6-FAM-TTGGGCGGGCCAAA-TAMRA 65.8/

EGFR858-UP HEX-TCTTTCTCTTCCGCACCCAGC-BHQ1 67.3 /

EGFR19-2237-IF ATCCCAGAAGGTGAGAAAGT 60.7

EGFR19-2237-IR GGGCCTGAGGTTCAGAG 60.8121 bp

EGFR19-2237-OF TGGCACCATCTCACAATTGCCAGTT 66.9

EGFR19-2237-OR GCAGCTGCCAGACATGAGAAAAGGT 66.8207 bp

EGFR19-W-PNA AGGAATTAAGAGAAGCAACATCT ~ 64.5 /

EGFR19-2237-LNA 6-FAM-CAAGGCCGAAAGCC-IABkFQ 70.5 /

EGFR19-2240-UP HEX-ACAGCAAAGCAGAAACTCACATCGA-BHQ1 67.7

EGFR-T790M-IF ATCTGCCTCACCTCCAC 60.3

EGFR-T790M-IR GGAGCCAATATTGTCTTTGTGT 61.296 bp

EGFR-T790M-OF AGCCACACTGACGTGCCTCTCC 67.9

EGFR-T790M-OR ATCTGCACACACCAGTTGAGCAGGT 68.1211 bp

EGFR-T790M-PNA GCTCATCACGCAGCTCAT ~ 66.3 /

EGFR-T790M-LNA 6-FAM-TCATCATGCAGC-IABkFQ 65.4 /

EGFR-T790M-UP HEX-TCCCGGACATAGTCCAGGAG-BHQ1 66.3 /

EGFR797-2390-IF ATCTGCCTCACCTCCAC 60.3

EGFR797-2390-IR ACCAGTTGAGCAGGTACT 60.1115 bp

EGFR797-2390-OF CTCTCCCTCCCTCCAGGAAGCCTA 67.3

EGFR797-2390-OR TCCCTGATTACCTTTGCGATCTGCACAC 67.9213 bp

EGFR797-2390-PNA CTTCGGCTGCCTCCTGG ~ 69.7 /

EGFR797-2390-LNA 6-FAM-TTCGGCTCCCTCCT-IABkFQ 71.1 /

EGFR797-2390-UP HEX-AGCTGCGTGATGAGCTGC-BHQ1 66.0 /

Note: The Tm values of the primers and probes were calculated by OligoAnalyzer 3.1

(Integrated DNA Technologies, Inc., Iowa, USA). The LNA bases were marked in italic. The

Tm values of the PNA probes were 2-13 °C higher than the values calculated by

OligoAnalyzer 3.1. Because a base mismatch occurs between the W-PNA and the mutant

sequence, the Tm of W-PNA shifts 2-13 °C.

4

Table S2. Clinical characteristics of patients with NSCLC.

Patients with NSCLC

(N=212)

Age, median

(range)63 (32-83)

Sex (n, %)

Male 112 (52.8%)

Female 100 (47.2%)

Smoking history

(n, %)

Never smoked 149 (70.3%)

Smoker 63 (29.7%)

Histological

type

Adenocarcinoma 159(75.0%)

Squamous cell

carcinoma38(17.9%)

Others 15(7.1%)

Stage

Stage I 102 (48.1%)

Stage II 48 (22.7%)

Stage III 49 (23.1%)

Stage IV 13 (6.1%)

5

10 20 30 40

0.0

0.8

1.6

2.4

3.2

CTBlank=UD

CT0.4 µM=21.0

CT0.1 µM=24.1

CT0.2 µM=22.2

CT0.6 µM=20.7CT0.8 µM=20.0

CT1.0 µM=20.2

Del

ta R

n

PCR cycles10 20 30 40

0.0

0.8

1.6

2.4

3.2

CTBlank=UD

CT5 cycles=16.2CT10 cycles=11.4

CT15 cycles=6.3

Del

ta R

n

PCR cycles

10 20 30 40

0.0

0.8

1.6

2.4

3.2

CTBlank=UD

CT0.4 µM=11.6

CT0.2 µM=11.5

CT0.1 µM=11.6

Del

ta R

n

PCR cycles

CT0.6 µM=11.7

10 20 30 40

0.0

0.8

1.6

2.4

3.2

CTBlank=UD

CT0.2 µM=6.3

CT0.1 µM=6.9

CT0.02 µM=13.9

CT0.05 µM=8.6

Del

ta R

n

PCR cycles

A B

C D

Figure S1. Efficiency of the LNA-dPNA PCR clamp at various concentrations of M-LNA

probe (A), cycle numbers (B), concentrations of IPs (C), and concentrations of OPs (D). All

experiments were performed at the same concentration of template (106 mutant plasmid), and

each assessment was performed twice. The selected conditions are marked with red arrows.

Note: The efficiency of the LNA-dPNA PCR clamp was affected by the concentrations of the

M-LNA probe, IPs, and OPs as well as the cycle numbers. Therefore, we optimized these key

factors and found that a high efficiency was achieved when performing the LNA-dPNA PCR

clamp with 0.8 μM M-LNA probe (Figure S1A), 0.2 μM IPs (Figure S1B), and 15 cycles of

pre-amplification (Figure S1C). We also found that a higher efficiency was achieved at a

higher concentration of OPs (0.2 μM), but a smoother S-curve was achieved at a lower

concentration of OPs (0.05 μM). Therefore, we selected 0.05 μM as the optimized

concentration (Figure S1D).

6

10 20 30 40

0.0

0.8

1.6

2.4

0.2

CTW+10 µM PNA=UDCTW+8 µM PNA=UD

CTM+8 µM PNA=17.8

CTW+0 µM PNA=18.5

CTM+0 µM PNA=18.0

CTM+10 µM PNA=17.6D

elta

Rn

PCR cycles

CTW+4 µM PNA=UD

CTM+4 µM PNA=17.6

10 20 30 40

0.0

0.8

1.6

2.4

0.2

CTW+0 µM PNA=7.9

CTM+8 µM PNA=26.7

CTW+10 µM PNA=UD

CTM+0 µM PNA=UD

CTW+4 µM PNA=28.6CTM+10 µM PNA=30.0D

elta

Rn

PCR cycles

CTW+8 µM PNA=UD

CTM+4 µM PNA=29.7

A B

Figure S2. Mutation enrichment detected by the LNA-dPNA PCR clamp at various

concentrations of the W-PNA probe using 103 M:103 W (A) and 103 M:106 W (B) as

templates. “M” means mutant DNA; “W” means wild-type DNA; “UD” means “Undetected”.

Each assessment was performed twice, and the selected conditions are marked with red

arrows.

Note: The mutation enrichment detected by the LNA-dPNA PCR clamp was affected by the

W-PNA probe concentration and annealing temperature. Therefore, we first optimized the

PNA concentration and found that mutant DNA amplification was unaffected, and wild-type

DNA amplification was completely blocked when the concentration of W-PNA was changed

from 4 μM to 10 μM at a lower concentration of wild-type template (103 copies) (Figure

S2A). We selected 8 μM W-PNA as the optimal concentration because more efficient

blockade of wild-type DNA (UD vs. 29.7) and more efficient mutant DNA amplification

(26.7 vs. UD) were obtained when the W-PNA concentration was 8 μM vs. 4 μM at a higher

concentration of wild-type DNA (106 copies) (Figure S2B).

7

10 20 30 400.0

0.4

0.8

1.2

CTW+PNA=UDCTW=UD CTBlank=UDCTM+PNA=UD

Delta

Rn

PCR cycles

CTM=UD

B-1 PCR with IPs at 72 °C

10 20 30 400.0

0.4

0.8

1.2

CTW=19.8

CTBlank=UD

CTW+PNA=22.6

CTM+PNA=19.3

Delta

Rn

PCR cycles

CTM=19.1

1.8

A-1 PCR with OPs at 72 °C

10 20 30 400.0

0.4

0.8

1.2

CTW=23.4

CTBlank=UD

CTW+PNA=33.0

CTM+PNA=24.8

Delta

Rn

PCR cycles

CTM=24.3

9.6

B-3 PCR with IPs at 68 °C

10 20 30 400.0

0.4

0.8

1.2

CTW=19.3

CTBlank=UD

CTW+PNA=24.1

CTM+PNA=18.5

Delta

Rn

PCR cycles

CTM=19.24.8

A-4 PCR with OPs at 66 °C

10 20 30 400.0

0.4

0.8

1.2

CTW=19.4

CTBlank=UD

CTW+PNA=22.6

CTM+PNA=18.6

Delta

Rn

PCR cycles

CTM=19.0

3.1

A-2 PCR with OPs at 70 °C

10 20 30 400.0

0.4

0.8

1.2

CTW+PNA=UDCTW=UD CTBlank=UDCTM+PNA=UD

Delta

Rn

PCR cycles

CTM=UD

B-2 PCR with IPs at 70 °C

10 20 30 400.0

0.4

0.8

1.2

CTW=19.3

CTBlank=UD

CTW+PNA=23.7

CTM+PNA=18.9

Delta

Rn

PCR cycles

CTM=18.9

4.6

A-3 PCR with OPs at 68 °C

CT OPs blocking + IPs blocking=

1.8+0=1.8

CT OPs blocking + IPs blocking=

3.1+0=3.1

CT OPs blocking + IPs blocking=

4.6+9.6=14.2

CT OPs blocking + IPs blocking=

4.8+6.2=11.0

10 20 30 400.0

0.4

0.8

1.2

CTW=18.7

CTBlank=UD

CTW+PNA=24.9

CTM+PNA=19.1

Delta

Rn

PCR cycles

CTM=19.1

6.2

B-4 PCR with IPs at 66 °C

Figure S3. Mutation enrichment detected by the LNA-dPNA PCR clamp at annealing

temperatures of 72 °C (A-1, B-1), 70 °C (A-2, B-2), 68 °C (A-3, B-3), and 66 °C (A-4, B-4)

using OPs and IPs. “M” means mutant DNA; “W” means wild-type DNA; “PNA” means W-

PNA probe; “UD” means “Undetected”. Each assessment was performed twice, and the

selected conditions are marked with red arrows.

8

10 20 30 400.0

0.4

0.8

1.2

CTW=19.2

CTBlank=UD

CTW+PNA=24.9

CTM+PNA=18.8

Delta

Rn

PCR cycles

CTM=18.85.7

A-1 PCR with OPs at 64 °C10 20 30 40

0.0

0.4

0.8

1.2

CTW=18.2

CTBlank=UD

CTW+PNA=24.2

CTM+PNA=18.6

Delta

Rn

PCR cycles

CTM=18.66.0

B-1 PCR with IPs at 64°C

10 20 30 400.0

0.4

0.8

1.2

CTW=17.7

CTBlank=UD

CTPNA+W=24.3

CTPNA+M=18.1

Delta

Rn

PCR cycles

CTM=18.2

6.6

CT OPs blocking + IPs blocking=

5.7+6.0=11.7

CT OPs blocking + IPs blocking=

6.2+6.6=12.8

CT OPs blocking + IPs blocking=

6.5+7.1=13.6

10 20 30 400.0

0.4

0.8

1.2

CTW=19.0

CTBlank=UD

CTPNA+W=25.2

CTPNA+M=18.6

Delta

Rn

PCR cycles

CTM=18.76.2

A-2 PCR with OPs at 60 °C B-2 PCR with IPs at 60 °C

10 20 30 400.0

0.4

0.8

1.2

CTBlank=UD

CTPNA+W=24.7

CTW=18.2

CTPNA+M=18.3

Delta

Rn

PCR cycles

CTM=18.3

6.5

A-3 PCR with OPs at 56 °C

10 20 30 400.0

0.4

0.8

1.2

CTW=17.2

CTBlank=UD

CTPNA+W=24.3

CTPNA+M=17.9

Delta

Rn

PCR cycles

CTM=17.77.1

B-3 PCR with IPs at 56 °C

Figure S4. Mutation enrichment detected by the LNA-dPNA PCR clamp at annealing

temperatures of 64 °C (A-1, B-1), 60 °C (A-2, B-2), and 56 °C (A-3, B-3) using OPs or IPs.

“M” means mutant DNA; “W” means wild-type DNA; “PNA” means W-PNA probe; “UD”

means “Undetected”. Each assessment was performed twice, and the selected conditions are

marked with red arrows.

Note: We optimized the annealing temperatures, and 68 °C for the OPs and 56 °C for the IPs

were selected because we found that more efficient blockade was achieved when the OPs

were amplified at 68 °C (14.2) rather than 72 °C (1.8), 70 °C (3.2), or 66 °C (11.0) (Figure

S3). We further observed that more efficient blockade was achieved when the IPs were

amplified at 56 °C (13.6) rather than 60 °C (12.8) or 62 °C (11.7) (Figure S4). A better

specificity was achieved when the IPs were amplified at 60 °C rather than 56 °C when the

clinical samples were amplified (data not shown).

9

10 20 30 40

0.0

0.8

1.6

2.4

3.2

CTBlank=UD

CTwithout OPs=20.0Del

ta R

n

PCR cycles10 20 30 40

0.0

0.8

1.6

2.4

3.2

CTBlank=UD

CTwith OPs=8.6 CT without OPs=20.1

Del

ta R

n

PCR cycles

11.5

A-1 A-2

0 10 20 30 400.0

0.4

0.8

1.2

CTW=19.0

CTM=18.7

CTW+PNA=25.2

CTM+PNA=18.6

Del

ta R

n

PCR cycles

6.2

CTBlank=UD

10 20 30 400.0

0.4

0.8

1.2

CTW=17.7

CTM+PNA=18.2

CTW+PNA=24.3

CTM+PNA=18.1

Del

ta R

n

PCR cycles

6.6

CTBlank=UD

10 20 30 400.0

0.4

0.8

1.2

CTW+PNA=23.7

CTW=19.3

CTM=18.9

CTBlank=UD

CTM+PNA=18.9

Del

ta R

n

PCR cycles

4.6

10 20 30 400.0

0.4

0.8

1.2

CTW=23.4

CTW+PNA=33.0

CTM+PNA=24.8

CTM=24.3

Del

ta R

n

PCR cycles

9.6

CTBlank=UD

B-3

B-1 B-2

B-4

Conventional PCR One-tube nested PCR

PCR with OPs at 68 °C PCR with IPs at 68 °C

PCR with OPs at 60 °C PCR with IPs at 60 °C

Figure S5. Evaluation of the effects of one-tubed nested PCR (A) and dual PNA clamp (B) on

the LNA-dPNA PCR clamp. “M” means mutant DNA; “W” means wild-type DNA; “PNA”

means W-PNA probe. Each assessment was performed twice.

10

A-1

A-3

A-2

A-4

A-5

A-6

A-7

Theoretical Value: 0 M (105 W);

Measured Value: 0 M

Theoretical Value: 100 M;

Measured Value: UN

Theoretical Value: 101 M;

Measured Value: 0.6×101 M

Theoretical Value: 102 M;

Measured Value: 0.80×102 M

Theoretical Value: 103 M;

Measured Value: 0.98×103 M

Theoretical Value: 104 M;

Measured Value: 1.07×104 M

Theoretical Value:105 M;

Measured Value: 1.01×105 M

B-1

B-2

B-3

B-4

B-5

B-7

B-6

Theoretical Value: 0.001%(100 M:105 W);

Measured Value: UN

Theoretical Value: 0.01 %(101 M:105 W);

Measured Value: 0.0083%(1.16×101 M:1.40×105 W)

Theoretical Value: 0.05%(5×101 M:105 W);

Measured Value: 0.032%(4.60×101 M:1.42×105 W)

Theoretical Value: 0.1%(102 M:105 W);

Measured Value: 0.076%(1.04×102 M:1.36×105 W)

Theoretical Value: 1%(103 M:105 W);

Measured Value: 0.67%(0.94×103 M:1.40×105 W)

Theoretical Value: 10%(104 M:105 W);

Measured Value: 6.08%(0.90×104 M:1.39×105 W)

Theoretical Value: 50%(105 M:105 W);

Measured Value: 46.60%(0.89×105 M:1.02×105 W)

Figure S6. Evaluation of the sensitivity (A), specificity (B), and linearity (A) using ddPCR for EGFR L858R detection. “M” means mutant DNA; “W” means wild-type DNA; “UN” means undetermined. Each assessment was performed twice.

11

10 20 30 40

0.0

0.7

1.4

2.1

2.8

Delta

Rn

PCR cycles

0% Blank0.01%

0.2

50% 10% 1% 0.1%

10 20 30 40

0.0

0.7

1.4

2.1

2.8

3.5

Delta

Rn

PCR cycles

0.2 Blank105 104

102

103101 100

10 20 30 40

0.0

0.8

1.6

2.4

3.2

Delta

Rn

PCR cycles

0.2Blank

105104

102103 101 100

10 20 30 40

0.0

0.8

1.6

2.4

3.2

Delta

Rn

PCR cycles

0% Blank0.01%0.2

50% 10% 1% 0.1%

10 20 30 40

0.0

0.8

1.6

2.4

3.2

Delta

Rn

PCR cycles

0.2Blank

104 102103 101100

10 20 30 40

0.0

0.8

1.6

2.4

3.2

4.0

Delta

Rn

PCR cycles

0% Blank0.01%

0.250% 10% 1% 0.1%

A-1

A-2

A-3

B-1

B-2

B-3

y = 3.3506x+ 5.3655R² = 0.9942

y = 3.9568x+ 3.6407R² = 0.9867

y = 3.6471x+ 3.6985R² = 0.9967

y = 3.4613x+ 3.0756R² = 0.9947

y = 3.535x+ 5.5968R² = 0.9927

y = 3.3654x+ 5.9421R² = 0.9975

Figure S7. Evaluation of the reproducibility of LNA-dPNA PCR clamp in detection of EGFR

L858R. The amplification sensitivity (A) and limit of detection (LOD) (B) were repeatedly

detected in triplicate from sample dilution to real-time PCR. A-1: The amplification

sensitivity was tested first; A-2: the amplification sensitivity was tested second; A-3: the

amplification sensitivity was tested third. B-1: the LOD was tested first; B-2: the LOD was

tested second; B-3: the LOD was tested third. Each assessment was performed twice.

Note: 0.01% (101M:105W) was repeatedly detected in the six parallel tests. A 100

copy/reaction was detected four times in six parallel tests due to its extremely low content.

12

10 20 30 40

0.0

0.8

1.6

2.4

3.2

4.0

0.2

CTC=19.1

CTM=17.5

Del

ta R

n

PCR cycles

10 20 30 40

0.0

0.8

1.6

2.4

0.2

CTC=25.1

CTM=22.7D

elta

Rn

PCR cycles

C D

A B

10 20 30 40

0.0

0.4

0.8

1.2

1.6

0.2

CTC=28.2

CTM=UD

Delta

Rn

PCR cycles

10 20 30 40

0.0

0.4

0.8

1.2

1.6

0.2

CTC=21.3

CTM=UD

Del

ta R

n

PCR cycles

E F

G H

Figure S8. Typical results for EGFR L858R analysed using the LNA-dPNA PCR clamp (A,

C, E, G) and ddPCR (B, D, F, H) from plasma (A, B, E, F) and tissue (C, D, G, H) samples.

“M” means mutant DNA; “C” means universal control; “UD” means “Undetected.” Each

assessment was performed twice.

13

Table S3. Comparison of ddPCR and the LNA-dPNA PCR clamp for detecting plasma EGFR

mutations and tissue EGFR mutations.

LNA-dPNA PCR

clampddPCR

Positive Negative

TotalSensitivity

(%)

Specificity

(%)

Positive 46 0 46 100.0 /Tissues

Negative 0 86 86 / 100.0

Total 46 86 132 / /

Positive 12 5 17 70.6 /Plasmas

Negative 2 113 115 / 98.3

Total 14 118 132 / /

14

Table S4. Comparison of EGFR mutations in tumor tissues and matched plasma samples for

different tumour stages.

Matched plasma samplesTumor

stagesTissues

Positive NegativeSensitivity

I 29 2 27 6.9%

II 11 2 9 18.2%

III 8 1 7 12.5%

IV 3 3 0 100%

15

Table S5. Comparison of labour and cost between the LNA-dPNA PCR clamp and ddPCR

for quantifying plasma mutations.

LNA-dPNA PCR clamp ddPCR

StepsTime

(h)

Cost

($)Steps

Time

(h)Cost ($)

Standard curve

preparation 0.5 0.02

Reagent

preparation0.5 22.04

Reagent

preparation0.5 39.52

Droplet

preparation0.5 28.08

PCR 1.25 0 PCR 2.0 0

Data analysis 0.5 0 Droplet reading 0.5 5.92

Data analysis 0.5 0

Total 2.75 39.68a Total 4.0 56.04 b

a Eight reactions are needed to quantify one mutation site with the LNA-dPNA PCR clamp, including 5 reactions for the

standard curve, 1 reaction for the negative control, 1 reaction for the blank control, and 1 reaction for the sample. The cost of

one reaction is 4.96 $, and the cost for the reaction is as follows: EASY Dilution (for real time PCR): 0.02 $/reaction; PNA

probe: 2.49 $/reaction; TaKaRa Premix Ex TaqTM (probe qPCR): 0.69 $/reaction; PCR tube (Axygen): 0.09 $/reaction; LNA

Probe: 1.54 $/reaction; universal probe: 0.13 $/reaction.

b Four reactions are needed to quantify one mutation site by ddPCR, including 1 reaction for the positive control, 1 reaction

for the negative control, 1 reaction for the blank control, and 1 reaction for the sample. The cost of one reaction is 14.01 $,

and the cost for the reaction is as follows: PrimePCRTM ddPCRTM Mutation Assay (Bio-Rad): 3.52 $/reaction; probes: 1.83

$/reaction; PCR tube and seal foil: 0.16 $/reaction; droplet generation oil: 0.96 $/reaction; droplet generator: 4.76 $/reaction;

DG8 gaskets: 1.3 $/reaction; droplet-reading oil: 1.48 $/reaction.