Drug-Resistant Gene Detection Kit For Mycobacterium Tuberculosis (Real-Time PCR) Stability Study Data

Drug-Resistant Gene Detection Kit for
Mycobacterium Tuberculosis (Real-Time

PCR)
Stability Study Data
Applicant: CapitalBio Corporation

Address: No.18, Life Science Park Road, Changping District, Beijing
Contact Person: Jiang Di
Tel.: 010-61777461, 13520971176
1. Foreword
Drug-Resistant Gene Detection Kit for Mycobacterium Tuberculosis (Real-Time PCR) is
mainly used to qualitatively test the drug-resistance gene mutation of mycobacterium
tuberculosis, and to assist to the clinical diagnosis and epidemiological survey and other fields.
The kit composition is shown in table 1.
Table 1 Composition (24 tests/kit) of Drug-Resistant Gene Detection Kit for Mycobacterium
Tuberculosis (Real-Time PCR)
Component Name Spec. Qty.
Operating Instructions / 1 copy
Product Certificate / 1 copy
Amplification reaction mixture A 630 µL/tube 1 tube
Amplification reaction mixture B 630 µL/tube 1 tube
Amplification reaction mixture C 630 µL/tube 1 tube
Amplification reaction mixture D 630 µL/tube 1 tube
Amplification reaction mixture E 630 µL/tube 1 tube
Positive control 400 μL/tube 1 tube
Negative control 400 μL/tube 1 tube
During the R&D process of the Drug-Resistant Gene Detection Kit for Mycobacterium
Tuberculosis (Real-Time PCR), for considering the circumstances may occur under the real
condition, confirming the storage period of the kit under real storage and use condition and
ensuring the authentic and reliable test result, we conduct the stability study for the kit under
real storage condition, including:
Long-term Stability Study
Repeated Freeze-thaw Stability Study
2. Long-term Stability Study

The long-term stability refers to the period that the kit can keep its test performance under the
real storage condition. During the R&D process of the Drug-Resistant Gene Detection Kit for
Mycobacterium Tuberculosis (Real-Time PCR), we conduct the study on storage period of the
kit under real storage condition for making sure the long-term stability of the kit.
2.1 Experimental Design
In the long-tern stability study, we adopt the corporate references (including 11 positive
references and 10 negative references) of Drug-Resistant Gene Detection Kit for
Mycobacterium Tuberculosis (Real-Time PCR) for testing, the performance of the kits for
R&D and trial production from three lots under the real storage condition (stored at -20℃) is
studied within the ten-month period according to the performance test indexes of the corporate
reference of the kit in a way of monthly test.
2.2 Test Instrument
Real-time Fluorescent Quantitative PCR Instrument: ABI7500 realtime PCR system
The lot numbers of three lots of kits for R&D and trial production are: 20130401, 20130402,
20130403
The composition of corporate reference of the Drug-Resistant Gene Detection Kit for
Mycobacterium Tuberculosis (Real-Time PCR) is shown in table 2 with the lot number of:
20130301.
-1-
Table 2 Composition of Corporate Reference for Stability Study
Type No. Strain Name Genotype Concentration Purpose
P1 Tuberculosis drug-resistance strain T511C&G315A&A43G 105 cfu/mL
P2 Tuberculosis drug-resistance strain T533C&G315C&A43G 105 cfu/mL
A516T&G315C&A94G 105 cfu/mL
P3 Tuberculosis drug-resistance strain
&A306G&A88G
P4 Tuberculosis drug-resistance strain A526G&C-15T&A306G 105 cfu/mL
C531T&G315C&A94G 105 cfu/mL It's used to
Positive &G306A&A88G evaluate the
Reference C526G&G315C&C90T 105 cfu/mL accuracy,
(1mL/copy) &A88G LOD and
A526T&C-15T&A43G& 105 cfu/mL repeatability
A1401G
P8 Tuberculosis drug-resistance strain C526T 105 cfu/mL
P9 Tuberculosis drug-resistance strain A1401G 105 cfu/mL
P10 Tuberculosis drug-resistance strain C1402T 105 cfu/mL
P11 Tuberculosis drug-resistance strain G1484T 105 cfu/mL
Standard mycobacterium 105 cfu/mL
N1 Wild type
tuberculosis strain H37RV
Mycobacterium tuberculosis 105 cfu/mL
N2 Wild type
sensitive strain
N3 Wild type
sensitive strain
N4 Wild type
sensitive strain
Negative N5 Wild type It's used for
sensitive strain
Reference negative
(1mL/copy) N6 Wild type evaluation
sensitive strain
N7 Wild type
sensitive strain
N8 Wild type
sensitive strain
N9 Wild type
sensitive strain
N10 Wild type
sensitive strain
2.3 Test Standard

The judgment for validity of the kits from different lots and at different time is made according
to the positive cut-off value of the kit, study data of the analytical performance as well as the
technical requirement of the kit. The test results shall be consistent with the theoretical test
result in table 3.
Table 3 Test Positive Reference of the Kit
Reference
Reference No. Test Result of the Kit
Concentration (cfu/mL)
FAM(rpoB) Negative
A
VIC(katG) Positive
FAM(rpoB) Positive
B
VIC(inhA) Negative
P1 105
FAM(gyrA) Negative
C
VIC(embB) Negative
D FAM(rpsL) Positive
E FAM(rrs) Negative
-2-
FAM(rpoB) Negative
A
VIC(katG) Positive
FAM(rpoB) Positive
B
VIC(inhA) Negative
P2 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Positive
A
VIC(katG) Positive
FAM(rpoB) Negative
B
VIC(inhA) Negative
P3 105
FAM(gyrA) Positive
C
VIC(embB) Positive
E FAM(rrs) Negative
FAM(rpoB) Positive
A
VIC(katG) Negative
FAM(rpoB) Negative
B
5 VIC(inhA) Positive
P4 10
FAM(gyrA) Positive
C
VIC(embB) Negative
D FAM(rpsL) Negative
E FAM(rrs) Negative
FAM(rpoB) Positive
A
VIC(katG) Positive
FAM(rpoB) Negative
B
VIC(inhA) Negative
P5 105
FAM(gyrA) Positive
C
VIC(embB) Positive
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Positive
FAM(rpoB) Positive
B
VIC(inhA) Negative
P6 105
FAM(gyrA) Negative
C
VIC(embB) Positive
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Positive
B
VIC(inhA) Positive
P7 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
FAM(rpoB) Positive
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
P8 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
P9 105 A FAM(rpoB) Negative
-3-
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
P10 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
P11 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
Table 4 Test LOD Reference of the Kit

Reference Concentration
(cfu/mL)
FAM(rpoB) Positive
A
VIC(katG) Positive
FAM(rpoB) Negative
B
VIC(inhA) Negative
P3 104
FAM(gyrA) Positive
C
VIC(embB) Positive
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Positive
B
VIC(inhA) Positive
P7 104
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
P9 104
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
-4-
Table 5 Test Result of Negative Reference
(cfu/mL)
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N1 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N2 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
5 VIC(inhA) Negative
N3 10
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
N4 10
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N5 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N6 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
N7 105 FAM(rpoB) Negative
B
VIC(inhA) Negative
C FAM(gyrA) Negative
-5-
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N8 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N9 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N10 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
Table 6 Test Results of Repeatability Reference

(cfu/mL)
FAM(rpoB) Positive
A
VIC(katG) Positive
FAM(rpoB) Negative
B
VIC(inhA) Negative
P3 105
FAM(gyrA) Positive
C
VIC(embB) Positive
E FAM(rrs) Negative
Note: each repeatability reference is tested repeatedly for 10 times and 10 test results shall be
consistent.
2.4 Analysis and Conclusion
Through the ten-month testing (the test lasts from June 8, 2013 to March 8, 2014), three lots of
kits are tested by using the partial corporate references, and all indexes are consistent with the
theoretical value, and all three lots of the kit are qualified. The test results are shown in Table 7.
Viewing from the test data, the performance of the kit from three lots can reach the intended
standard in the long-term (10 months) storage test. Therefore, the kit can be stored at -20℃ for
10 months while the valid period of the kit is determined as 8 months according to the relevant
requirements for IVD reagent.
-6-
Table 7 Detailed Test Results of Three Lots of Trial Production Reagent for Long-term Stability
1st month
Kit Lot
Sample Genotype
20130401 20130402 20130403
FAM(rpoB) Negative Negative Negative
A
VIC(katG) Negative Negative Negative
B
VIC(inhA) Negative Negative Negative
Negative control
FAM(gyrA) Negative Negative Negative
C
VIC(embB) Negative Negative Negative
D FAM(rpsL) Negative Negative Negative
E FAM(rrs) Negative Negative Negative
FAM(rpoB) Positive Positive Positive
A
VIC(katG) Positive Positive Positive
B
VIC(inhA) Positive Positive Positive
Positive control
FAM(gyrA) Positive Positive Positive
C
VIC(embB) Positive Positive Positive
D FAM(rpsL) Positive Positive Positive
E FAM(rrs) Positive Positive Positive
A
B
P1
C
A
B
P2
C
A
Positive VIC(katG) Positive Positive Positive
Reference FAM(rpoB) Negative Negative Negative
B
P3
C
A
B
P4
C
A
P5
B
-7-
C
A
B
P6
C
A
B
P7
C
A
B
P8
C
A
B
P9
C
A
B
P10
C
A
B
P11
C
A
Negative
N1 FAM(rpoB) Negative Negative Negative
Reference B
C FAM(gyrA) Negative Negative Negative
-8-
A
B
N2
C
A
B
N3
C
FAM(rpoB) Nega tive Negative Negative
A
B
N4
C
A
B
N5
C
A
B
N6
C
A
B
N7
C
A
N8 B
C
-9-
A
B
N9
C
A
B
N10
C
A
B
Repeatability P3-1~P3-1 VIC(inhA) Negative Negative Negative
Reference 0 FAM(gyrA) Positive Positive Positive
C
A
B
P3
C
A
B
LOD VIC(inhA) Positive Positive Positive
P7
Reference FAM(gyrA) Negative Negative Negative
C
A
B
P9
C
2nd month
Kit Lot
Sample Genotype
20130401 20130402 20130403
A
Negative control
B
-10-
C
A
B
Positive control
C
A
B
P1
C
A
B
P2
C
A
B
P3
C
Positive
Reference
A
B
P4
C
A
B
P5
C
A
P6 FAM(rpoB) Positive Positive Positive
B
-11-
A
B
P7
C
A
B
P8
C
A
B
P9
C
A
B
P10
C
A
B
P11
C
A
B
N1
C
Negative D FAM(rpsL) Negative Negative Negative
Reference E FAM(rrs) Negative Negative Negative
A
N2 B
C
-12-
A
B
N3
C
A
B
N4
C
A
B
N5
C
A
B
N6
C
A
B
N7
C
A
B
N8
C
A
B
N9 VIC(inhA) Negative Negative Negative
C
-13-
A
B
N10
C
A
B
Repeatability P3-1~P3-1 VIC(inhA) Negative Negative Negative
Reference 0 FAM(gyrA) Positive Positive Positive
C
A
B
P3
C
A
B
P7
C
A
B
P9
C
3rd month
Kit Lot
Sample Genotype
20130401 20130402 20130403
A
B
Negative control
C
A
Positive control FAM(rpoB) Positive Positive Positive
B
C FAM(gyrA) Positive Positive Positive
-14-
A
B
P1
C
A
B
P2
C
A
B
P3
C
A
Positive FAM(rpoB) Negative Negative Negative
B
Reference VIC(inhA) Positive Positive Positive
P4
C
A
B
P5
C
A
B
P6
C
A
P7 B
C
-15-
A
B
P8
C
A
B
P9
C
A
B
P10
C
A
B
P11
C
A
B
N1
C
A
Negative B
Reference N2
C
A
B
N3 VIC(inhA) Negative Negative Negative
C
-16-
A
B
N4
C
A
B
N5
C
A
B
N6
C
A
B
N7
C
A
B
N8
C
A
B
N9
C
A
B
N10
C
-17-
A
B
Repeatability VIC(inhA) Negative Negative Negative
P3-1~P3-10
Reference FAM(gyrA) Positive Positive Positive
C
A
B
P3
C
A
B
P7
C
A
B
P9
C
4th month
Kit Lot
Sample Genotype
20130401 20130402 20130403
A
B
Negative control
C
A
B
Positive control
C
A
Positive FAM(rpoB) Positive Positive Positive
P1 B
Reference VIC(inhA) Negative Negative Negative
C
-18-
A
B
P2
C
A
B
P3
C
A
B
P4
C
A
B
P5
C
A
B
P6
C
A
B
P7
C
A
B
P8 VIC(inhA) Negative Negative Negative
C
-19-
A
B
P9
C
A
B
P10
C
A
B
P11
C
A
B
N1
C
A
B
N2
C
Negative E FAM(rrs) Negative Negative Negative
A
B
N3
C
A
B
N4
C
-20-
A
B
N5
C
A
B
N6
C
A
B
N7
C
A
B
N8
C
A
B
N9
C
A
B
N10
C
A
B
P3-1~P3-10
C
LOD P3 A FAM(rpoB) Positive Positive Positive
-21-
Reference VIC(katG) Positive Positive Positive
B
C
A
B
P7
C
A
B
P9
C
5th month
Kit Lot
Sample Genotype
20130401 20130402 20130403
A
B
Negative control
C
A
B
Positive control
C
A
B
P1
C
Positive
Reference
A
B
P2 VIC(inhA) Negative Negative Negative
C
-22-
A
B
P3
C
A
B
P4
C
A
B
P5
C
A
B
P6
C
A
B
P7
C
A
B
P8
C
A
B
P9
C
-23-
A
B
P10
C
A
B
P11
C
A
B
N1
C
A
B
N2
C
A
B
Negative N3
Reference C
A
B
N4
C
A
B
N5
C
N6 A FAM(rpoB) Negative Negative Negative
-24-
B
C
A
B
N7
C
A
B
N8
C
A
B
N9
C
A
B
N10
C
A
B
P3-1~P3-10
C
A
B
P3
LOD FAM(gyrA) Positive Positive Positive
C
Reference VIC(embB) Positive Positive Positive
P7 A
-25-
B
C
A
B
P9
C
6th month
Kit Lot
Sample Genotype
20130401 20130402 20130403
A
B
Negative control
C
A
B
Positive control
C
A
B
P1
C
A
B
Positive VIC(inhA) Negative Negative Negative
P2
C
A
B
P3
C
-26-
A
B
P4
C
A
B
P5
C
A
B
P6
C
A
B
P7
C
A
B
P8
C
A
B
P9
C
A
B
P10
C
P11 A FAM(rpoB) Negative Negative Negative
-27-
B
C
A
B
N1
C
A
B
N2
C
A
B
N3
C
Negative FAM(rpoB) Negative Negative Negative
A
Reference VIC(katG) Negative Negative Negative
B
N4
C
A
B
N5
C
A
B
N6
C
N7 A
-28-
B
C
A
B
N8
C
A
B
N9
C
A
B
N10
C
A
B
P3-1~P3-10
C
A
B
P3
C
LOD A
Reference
B
P7
C
A
P9 VIC(katG) Negative Negative Negative
B FAM(rpoB) Negative Negative Negative
-29-
C
7th month
Kit Lot
Sample Genotype
20130401 20130402 20130403
A
B
Negative control
C
A
B
Positive control
C
A
B
P1
C
A
B
P2
C
Positive
Reference A
B
P3
C
A
B
P4
C
P5 A FAM(rpoB) Positive Positive Positive
-30-
B
C
A
B
P6
C
A
B
P7
C
A
B
P8
C
A
B
P9
C
A
B
P10
C
A
B
P11
C
Negative FAM(rpoB) Negative Negative Negative
N1 A
Reference VIC(katG) Negative Negative Negative
-31-
B
C
A
B
N2
C
A
B
N3
C
A
B
N4
C
A
B
N5
C
A
B
N6
C
A
B
N7
C
A
N8 VIC(katG) Negative Negative Negative
-32-
C
A
B
N9
C
A
B
N10
C
A
B
P3-1~P3-10
C
A
B
P3
C
A
B
P7
C
A
B
P9
C
8th month
Kit Lot
Sample Genotype
20130401 20130402 20130403
Negative control A FAM(rpoB) Negative Negative Negative
-33-
B
C
A
B
Positive control
C
A
B
P1
C
A
B
P2
C
A
B
P3
Positive FAM(gyrA) Positive Positive Positive
C
Reference VIC(embB) Positive Positive Positive
A
B
P4
C
A
B
P5
C
P6 A
-34-
B
C
A
B
P7
C
A
B
P8
C
A
B
P9
C
A
B
P10
C
A
B
P11
C
A
B
N1
Negative C
Reference
A
N2 VIC(katG) Negative Negative Negative
-35-
C
A
B
N3
C
A
B
N4
C
A
B
N5
C
A
B
N6
C
A
B
N7
C
A
B
N8
C
A
N9
B
-36-
C
A
B
N10
C
A
B
P3-1~P3-10
C
A
B
P3
C
A
B
P7
C
A
B
P9
C
9th month
Kit Lot
Sample Genotype
20130401 20130402 20130403
A
B
Negative control
C
Positive control A
-37-
B
C
A
B
P1
C
A
B
P2
C
A
B
P3
C
Positive A
Reference
B
P4
C
A
B
P5
C
A
B
P6
C
A
P7 VIC(katG) Negative Negative Negative
B FAM(rpoB) Positive Positive Positive
-38-
C
A
B
P8
C
A
B
P9
C
A
B
P10
C
A
B
P11
C
A
B
N1
C
A
Negative VIC(katG) Negative Negative Negative
B
N2
C
A
N3
B
-39-
C
A
B
N4
C
A
B
N5
C
A
B
N6
C
A
B
N7
C
A
B
N8
C
A
B
N9
C
A
B
-40-
A
B
P3-1~P3-10
C
A
B
P3
C
A
B
P7
C
A
B
P9
C
10th month
Kit Lot
Sample Genotype
20130401 20130402 20130403
A
B
Negative control
C
A
B
Positive control
C
Positive A
P1 VIC(katG) Positive Positive Positive
Reference
B FAM(rpoB) Positive Positive Positive
-41-
C
A
B
P2
C
A
B
P3
C
A
B
P4
C
A
B
P5
C
A
B
P6
C
A
B
P7
C
A
P8
B
-42-
C
A
B
P9
C
A
B
P10
C
A
B
P11
C
A
B
N1
C
A
B
N2
C
Negative
Reference
A
B
N3
C
A
B
-43-
A
B
N5
C
A
B
N6
C
A
B
N7
C
A
B
N8
C
A
B
N9
C
A
B
N10
C
A
Repeatability FAM(rpoB) Negative Negative Negative
P3-1~P3-10 B
Reference VIC(inhA) Negative Negative Negative
C
-44-
A
B
P3
C
A
B
P7
C
A
B
P9
C
3. Repeated Freeze-thaw Stability Study

All reagents in the kit can be stored -20℃ and the repeated freeze-thaw may occur during the
actual use process. For making sure the kit stability during the use process and ensuring the
authentic and reliable test results, we test the stability of the kit under the repeated freeze-thaw
condition and provide the test basis for the stability during the actual test process.
3.1 Experimental Design
The specification of the kit is 24 tests/kit and one reagent can test it for 24 times maximally. For
covering all mutation types tested by the kit and testing the specificity, LOD, repeatability and
accuracy of the kit, totally 29 tests (including one negative and positive control respectively)
are needed at least for one test. Therefore, we provide 10 sets of kit from the third lot (20130403)
of R&D and trial production for repeated freeze-thaw stability study and those kits can be
repeatedly frozen and thawed for at least 8 times.
After completing freeze-thaw each time, we adopt the partial corporate references for testing.
3.2 Test Instrument
Real-time Fluorescent Quantitative PCR Instrument: ABI7500realtimePCRsystem
The lot numbers of three lots of kits for R&D and trial production are: 20130401, 20130402,
20130403
The composition of corporate reference of the Drug-Resistant Gene Detection Kit for
Mycobacterium Tuberculosis (Real-Time PCR) is shown in table 8 with the lot number of:
20130301.
Table 8 Composition of Corporate Reference for Repeated Freeze-thaw Stability Study
-45-
Type No. Strain Name Genotype Concentration Purpose
Tuberculosis
P1 drug-resistance T511C&G315A&A43G 105 cfu/mL
strain
Tuberculosis 105 cfu/mL
P2 drug-resistance T533C&G315C&A43G
strain
P3 drug-resistance A516T&G315C&A94G&A306G&A88G
strain
P4 drug-resistance A526G&C-15T&A306G
strain
P5 drug-resistance C531T&G315C&A94G&G306A&A88G
strain It's used to
Positive Tuberculosis 105 cfu/mL evaluate the
Reference P6 drug-resistance C526G&G315C&C90T&A88G accuracy,
(1mL/copy) strain LOD and
Tuberculosis 105 cfu/mL repeatability
P7 drug-resistance A526T&C-15T&A43G&A1401G
strain
P8 drug-resistance C526T
strain
P9 drug-resistance A1401G
strain
P10 drug-resistance C1402T
strain
P11 drug-resistance G1484T
strain
Standard 105 cfu/mL
mycobacterium
N1 Wild type
tuberculosis
strain H37RV
Mycobacterium 105 cfu/mL
N2 tuberculosis Wild type
sensitive strain
sensitive strain
Negative It's used for
Reference negative
sensitive strain
(1mL/copy) evaluation
sensitive strain
sensitive strain
sensitive strain
N8 Wild type
tuberculosis
-46-
sensitive strain
sensitive strain
sensitive strain
3.3 Test Standard

The judgment for validity of the kits from different lots and at different time is made according
to the positive cut-off value of the kit, study data of the analytical performance as well as the
technical requirement of the kit. The test results shall be consistent with the theoretical test
result in table 9.
Table 9 Test Positive Reference of the Kit
(cfu/mL)
FAM(rpoB) Negative
A
VIC(katG) Positive
FAM(rpoB) Positive
B
VIC(inhA) Negative
P1 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Positive
FAM(rpoB) Positive
B
VIC(inhA) Negative
P2 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Positive
A
VIC(katG) Positive
FAM(rpoB) Negative
B
P3 10
FAM(gyrA) Positive
C
VIC(embB) Positive
E FAM(rrs) Negative
FAM(rpoB) Positive
A
VIC(katG) Negative
FAM(rpoB) Negative
B
P4 10
FAM(gyrA) Positive
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Positive
A
VIC(katG) Positive
FAM(rpoB) Negative
B
P5 105 VIC(inhA) Negative
FAM(gyrA) Positive
C
VIC(embB) Positive
-47-
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Positive
FAM(rpoB) Positive
B
VIC(inhA) Negative
P6 105
FAM(gyrA) Negative
C
VIC(embB) Positive
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Positive
B
P7 10
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
FAM(rpoB) Positive
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
P8 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
P9 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
P10 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
P11 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
-48-
Table 10 Test LOD Reference of the Kit
(cfu/mL)
FAM(rpoB) Positive
A
VIC(katG) Positive
FAM(rpoB) Negative
B
VIC(inhA) Negative
P3 104
FAM(gyrA) Positive
C
VIC(embB) Positive
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Positive
B
VIC(inhA) Positive
P7 104
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
P9 10
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Positive
Table 11 Test Result of Negative Reference
(cfu/mL)
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N1 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N2 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N3 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
N4 105 A FAM(rpoB) Negative
-49-
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N5 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N6 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
N7 10
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N8 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N9 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
A
VIC(katG) Negative
FAM(rpoB) Negative
B
VIC(inhA) Negative
N10 105
FAM(gyrA) Negative
C
VIC(embB) Negative
E FAM(rrs) Negative
Table 12 Test Results of Repeatability Reference
-50-
(cfu/mL)
FAM(rpoB) Positive
A
VIC(katG) Positive
FAM(rpoB) Negative
B
VIC(inhA) Negative
P3 105
FAM(gyrA) Positive
C
VIC(embB) Positive
E FAM(rrs) Negative
Note: each repeatability reference is tested repeatedly for 10 times and 10 test results shall be
consistent.
3.4 Analysis and Conclusion
Three lots of the kit are tested by using the partial corporate references, all indexes are
consistent with the theoretical value. The test results of repeated freeze-thaw stability for R&D
and trial production are summarized in table 13.
Viewing from following data, the R&D and trial production reagents conform to the test
standard in each test under the condition of repeated freezing and thawing for 8 times.
Therefore, the kit performance will not be affected if repeated freezing and thawing not over 8
times under the normal use condition.
Table 13 Detailed Test Results of R&D and Trial Production Reagent for Repeated Freeze-thaw
Stability
Repeated Freeze-thaw Frequency
Sample Genotype
1st time 2nd time 3rd time 4th time
FAM(rpoB) Negative Negative Negative Negative
A
VIC(katG) Negative Negative Negative Negative
B
VIC(inhA) Negative Negative Negative Negative
Negative control
FAM(gyrA) Negative Negative Negative Negative
C
VIC(embB) Negative Negative Negative Negative
D FAM(rpsL) Negative Negative Negative Negative
E FAM(rrs) Negative Negative Negative Negative
FAM(rpoB) Positive Positive Positive Positive
A
VIC(katG) Positive Positive Positive Positive
B
VIC(inhA) Positive Positive Positive Positive
Positive control
FAM(gyrA) Positive Positive Positive Positive
C
VIC(embB) Positive Positive Positive Positive
D FAM(rpsL) Positive Positive Positive Positive
E FAM(rrs) Positive Positive Positive Positive
A
B
P1
Positive C
Reference
A
P2 VIC(katG) Positive Positive Positive Positive
B FAM(rpoB) Positive Positive Positive Positive
-51-
C
A
B
P3
C
A
B
P4
C
A
B
P5
C
A
B
P6
C
A
B
P7
C
A
B
P8
C
A
P9
B
-52-
C
A
B
N1
C
A
B
N2
C
A
B
N3
C
A
Negative
Reference B
N4
C
A
B
N5
C
A
B
N6
C
A
N7 FAM(rpoB) Negative Negative Negative Negative
B
C FAM(gyrA) Negative Negative Negative Negative
-53-
A
B
N8
C
A
B
N9
C
A
B
N10
C
A
B
Repeatability VIC(inhA) Negative Negative Negative Negative
P3-1~P3-10
Reference FAM(gyrA) Positive Positive Positive Positive
C
A
B
P3
C
A
LOD FAM(rpoB) Positive Positive Positive Positive
B
Reference VIC(inhA) Positive Positive Positive Positive
P7
C
A
P9 B
C
-54-
Repeated Freeze-thaw Frequency
Sample Genotype
5th time 6th time 7th time 8th time
A
B
Negative control
C
A
B
Positive control
C
A
B
P1
C
A
B
P2
C
A
Positive
Reference B
P3
C
A
B
P4
C
A
P5 FAM(rpoB) Negative Negative Negative Negative
B
C FAM(gyrA) Positive Positive Positive Positive
-55-
A
B
P6
C
A
B
P7
C
A
B
P8
C
A
B
P9
C
A
B
N1
C
A
Negative FAM(rpoB) Negative Negative Negative Negative
B
Reference VIC(inhA) Negative Negative Negative Negative
N2
C
A
N3 B
C
-56-
A
B
N4
C
A
B
N5
C
A
B
N6
C
A
B
N7
C
A
B
N8
C
A
B
N9
C
A
B
N10 VIC(inhA) Negative Negative Negative Negative
C
-57-
A
B
Repeatability VIC(inhA) Negative Negative Negative Negative
P3-1~P3-10
Reference FAM(gyrA) Positive Positive Positive Positive
C
A
B
P3
C
A
B
LOD VIC(inhA) Positive Positive Positive Positive
P7
Reference FAM(gyrA) Negative Negative Negative Negative
C
A
B
P9
C
4. Stability Study Summary

Through the above studies, we summarize the following conclusions:
The Drug-Resistant Gene Detection Kit for Mycobacterium Tuberculosis (Real-Time PCR) can
be stably stored for 10 months under the normal storage condition (-20℃) without significant
performance change. The valid period of the kit is determined as 8 months according to the
result of long-term stability study.
When repeatedly using the Drug-Resistant Gene Detection Kit for Mycobacterium
Tuberculosis (Real-Time PCR) within the valid period, the test performance of the kit will not
be affected after being repeatedly frozen and thawed for 8 times.
-58-

Drug-Resistant Gene Detection Kit For Mycobacterium Tuberculosis (Real-Time PCR) Stability Study Data

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Drug-Resistant Gene Detection Kit For Mycobacterium Tuberculosis (Real-Time PCR) Stability Study Data

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Drug-Resistant Gene Detection Kit for

Mycobacterium Tuberculosis (Real-Time

Applicant: CapitalBio Corporation

2. Long-term Stability Study

2.3 Test Standard

Table 4 Test LOD Reference of the Kit

Table 6 Test Results of Repeatability Reference

3. Repeated Freeze-thaw Stability Study

3.3 Test Standard

4. Stability Study Summary

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