S-1
Supporting Information
A Lyophilized Ready-to-use Mix for the Real-time Polymerase Chain Reaction 
Diagnosis
Siyu Yang 1; Weijia Wen 1, *. 
1 Department of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, 
Kowloon, Hong Kong
These authors contributed equally to this work.
Corresponding Author
*Wen Weijia – Department of Physics, The Hong Kong University of Science and Technology, Clear Water 
Bay, Sai Kung, New Territories, Hong Kong SAR (China) 999077. Email: phwen@ust.hk


S-2
Table S1 Sequence the template used in this work.
Template Type
Hepatitis B virus (HBV)
Sequence
TCTTTCGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAA
ATGCCCCTATCTTATCAACACTTCCGGAGACTACTGTTGTTAGACG
ACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGAC
GAAGGTCTCAATCGCCGCGTCGCAGAAGGTCTCAATCTCGGGAAT
CTCAATGTTAGTATACCTTG


S-3
Table S2 Compatibility test of all additives to be tested.
Additive(s)
Cq 
Compatibility *
/
21.62
N/A
Trehalose
21.78
compatible
BSA
21.36
compatible
Sorbitol
22.07
compatible
Gelatin
21.89
compatible
Raffinose
21.71
compatible
PVP
21.86
compatible
Ficoll 400
22.20
compatible
Dextran
22.05
compatible
Glucose
21.74
compatible
* Compatibility: The additive is compatible with the basic mixture if the Cq value 
has a difference of no more than 2 cycles


S-4
Table S3 Amplification result of lyophilized mix with a single additive.
Additive(s)
Dried cake
0-day Cq 
Positive Control
N/A
21.66
/
No
30.56
Trehalose
No
21.42
BSA
Yes
26.75
Sorbitol
No
28.60
Gelatin
Yes
26.41
Raffinose
No
27.97
PVP
No
28.76
Ficoll 400
No
21.75
Dextran
No
21.94
Glucose
No
29.92


S-5
Table S4 Shelf-life and sample condition of some selected formulations.
Additive(s)
Dried cake
Shelf-life *
Positive Control
N/A
Cq means: 21.43
5% ~ 15%Trehalose 
+ 0.5% ~ 3% BSA
Yes
120 days
5% ~ 15%Trehalose 
+ 0.1% ~ 3% Gelatin
Yes
180 days
5% ~ 15%Trehalose 
+2% ~ 10% Dextran 
+ 0.1% ~ 3% Gelatin
Yes
60 days
5% ~ 15%Trehalose 
+2% ~ 10% Ficoll 400 
+ 0.1% ~ 3% Gelatin
Yes
more than 300 days
* Shelf-life: The longest period that the material can provide an Cq result 
similar to positive control (no more than 2 cycles).


S-6
Table S5 The Cq result of all tested days with materials at room temperature and 
controlled temperatures.
Storage 
time
R. T. 1
4 ℃
30 ℃
35 ℃
40 ℃
45 ℃
0 day
21.77
21.71
21.75
21.80
22.31
21.43
30 days
21.65
22.21
21.89
22.09
22.00
21.91
60 days
21.45
21.77
21.55
22.35
22.04
21.79
90 days
21.90
21.55
21.25
21.39
21.24
21.10
120 days
21.07
21.35
22.14
21.94
22.05
21.78
150 days
21.44
20.76
21.13
21.32
22.66
21.94
180 days
21.64
21.50
21.32
20.76
21.05
21.89
210 days
21.86
22.15
21.93
21.94
21.89
22.00
300 days
22.04
21.73
21.62
21.56
21.68
21.45


S-7
0
5
10
15
20
25
30
35
40
45
0
500
1000
1500
2000
2500
3000
RFU
Cycles
Baseline
PC
5 months
6 months
7 months
Amplifications of the basic mixture stored at 4 ℃
Figure S1 The amplification result of the basic mixture liquid stored at 4 °C after 5, 6, 
and 7 months.


S-8
0
5
10
15
20
25
30
35
40
45
0
500
1000
1500
2000
2500
3000
RFU
Cycles
Baseline
 10-2 ng
 10-3 ng
 10-4 ng
 10-5 ng
 10-6 ng
 10-7 ng
 10-8 ng
 10-9 ng
Sensitivity of the freshly prepared mixture 
Figure S2 Sensitivity test of the freshly prepared mixture as a reference for 
comparison with the sensitivity of the final material stored at 45 ℃ for 300 days