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
P5 Tuberculosis drug-resistance strain
Positive &G306A&A88G evaluate the
Reference P6 Tuberculosis drug-resistance strain C526G&G315C&C90T 10 cfu/mL accuracy,
5
(1mL/copy) &A88G LOD and

A526T&C-15T&A43G& 105 cfu/mL repeatability
P7 Tuberculosis drug-resistance strain 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 tuberculosis strain Wild type
H37RV
Mycobacterium tuberculosis 105 cfu/mL
N2 sensitive strain Wild type
N3 Wild type
sensitive strain
N4 Wild type
sensitive strain
Negative Mycobacterium tuberculosis 105 cfu/mL It's used for
Reference N5 sensitive strain Wild type negative
(1mL/copy) Mycobacterium tuberculosis 10 cfu/mL evaluation
5
N7 Wild type
sensitive strain
N8 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
FAM(rpoB) Positive
E FAM(rrs) Negative
FAM(rpoB) Positive
FAM(rpoB) 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 VIC(inhA) Positive
D FAM(rpsL) Negative
E FAM(rrs) Negative
FAM(rpoB) Positive
FAM(rpoB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Positive
5
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Positive
5
E FAM(rrs) Positive
FAM(rpoB) Positive
FAM(rpoB) Negative
5
E FAM(rrs) Negative
P9 105 A FAM(rpoB) Negative
-3-
VIC(katG) Negative
FAM(rpoB) Negative
FAM(gyrA) Negative
E FAM(rrs) Positive
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Positive
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Positive
Table 4 Test LOD Reference of the Kit

Reference Concentration
(cfu/mL)
FAM(rpoB) Positive
FAM(rpoB) Negative
4
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Positive
4
E FAM(rrs) Positive
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Positive
-4-
Table 5 Test Result of Negative Reference
(cfu/mL)
FAM(rpoB) Negative
FAM(rpoB) Negative
N1 105 FAM(gyrA) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
N7 10 5 FAM(rpoB) Negative
C FAM(gyrA) Negative
-5-
VIC(embB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Negative
FAM(rpoB) Negative
FAM(rpoB) Negative
E FAM(rrs) Negative
Table 6 Test Results of Repeatability Reference

Reference No. (cfu/mL) Test Result of the Kit
FAM(rpoB) Positive
FAM(rpoB) Negative
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
P1 FAM(gyrA) Negative Negative Negative
Positive FAM(rpoB) Negative Negative Negative
Reference B VIC(inhA) Negative Negative Negative
P3 FAM(gyrA) Positive Positive Positive
P5 FAM(rpoB) Negative Negative Negative
-7-
FAM(gyrA) Positive Positive Positive
Negative FAM(rpoB) Negative Negative Negative
N1 B
Reference VIC(inhA) Negative Negative Negative
C FAM(gyrA) Negative Negative Negative
-8-
VIC(embB) Negative Negative Negative
N2 FAM(gyrA) Negative Negative Negative
FAM(rpoB) Nega tive Negative Negative
N8 B VIC(inhA) Negative Negative Negative
FAM(gyrA) Negative Negative Negative
-9-
Repeatability P3-1~P3-1
Reference 0 FAM(gyrA) Positive Positive Positive
LOD P7 FAM(gyrA) Negative Negative Negative
Reference C VIC(embB) Negative Negative Negative
2nd month
Kit Lot
Sample Genotype 20130401 20130402 20130403
Negative control FAM(rpoB) Negative Negative Negative
-10-
Positive
Reference
P6 FAM(rpoB) Positive Positive Positive
-11-
VIC(embB) Positive Positive Positive
Negative D FAM(rpsL) Negative Negative Negative
Reference E FAM(rrs) Negative Negative Negative
N2 B VIC(inhA) Negative Negative Negative
-12-
N9
-13-
Repeatability P3-1~P3-1
Reference 0 FAM(gyrA) Positive Positive Positive
3rd month
Kit Lot
Sample Genotype 20130401 20130402 20130403
Positive control FAM(rpoB) Positive Positive Positive
C FAM(gyrA) Positive Positive Positive
-14-
VIC(embB) Positive Positive Positive
Positive
P7 B VIC(inhA) Positive Positive Positive
-15-
Negative
Reference N2 FAM(gyrA) Negative Negative Negative
N3
-16-
-17-
Repeatability
P3-1~P3-10 FAM(gyrA) Positive Positive Positive
Reference C VIC(embB) Positive Positive Positive
4th month
Kit Lot
Sample Genotype 20130401 20130402 20130403
Positive FAM(rpoB) Positive Positive Positive
P1 B VIC(inhA) Negative Negative Negative
Reference
-18-
P8
-19-
Reference A VIC(katG) Negative Negative Negative
-20-
Repeatability
LOD P3 A FAM(rpoB) Positive Positive Positive
-21-
Reference VIC(katG) Positive Positive Positive
5th month
Kit Lot
Sample Genotype 20130401 20130402 20130403
Positive E FAM(rrs) Negative Negative Negative
Reference FAM(rpoB) Negative Negative Negative
P2
-22-
-23-
Negative C VIC(embB) Negative Negative Negative
Reference
N6 A FAM(rpoB) Negative Negative Negative
-24-
VIC(katG) Negative Negative Negative
Repeatability
LOD C
Reference VIC(embB) Positive Positive Positive
P7 A VIC(katG) Negative Negative Negative
-25-
6th month
Kit Lot
Sample Genotype 20130401 20130402 20130403
Positive P2 FAM(gyrA) Negative Negative Negative
-26-
P11 A FAM(rpoB) Negative Negative Negative
-27-
Negative
Reference FAM(rpoB) Negative Negative Negative
N7 A VIC(katG) Negative Negative Negative
-28-
Repeatability
LOD A VIC(katG) Negative Negative Negative
Reference FAM(rpoB) Positive Positive Positive
P9
B FAM(rpoB) Negative Negative Negative
-29-
VIC(inhA) Negative Negative Negative
7th month
Kit Lot
Sample Genotype 20130401 20130402 20130403
Positive FAM(rpoB) Positive Positive Positive
Reference
P5 A FAM(rpoB) Positive Positive Positive
-30-
VIC(katG) Positive Positive Positive
N1 A VIC(katG) Negative Negative Negative
Reference
-31-
N8
-32-
Repeatability
8th month
Kit Lot
Sample Genotype 20130401 20130402 20130403
Negative control A FAM(rpoB) Negative Negative Negative
-33-
Positive C VIC(embB) Positive Positive Positive
Reference D FAM(rpsL) Positive Positive Positive
P6 A VIC(katG) Positive Positive Positive
-34-
Negative C VIC(embB) Negative Negative Negative
Reference D FAM(rpsL) Negative Negative Negative
N2
-35-
N9 FAM(rpoB) Negative Negative Negative
-36-
Repeatability
9th month
Kit Lot
Sample Genotype 20130401 20130402 20130403
Positive control A VIC(katG) Positive Positive Positive
-37-
Positive FAM(rpoB) Negative Negative Negative
Reference B VIC(inhA) Positive Positive Positive
P7
B FAM(rpoB) Positive Positive Positive
-38-
VIC(inhA) Positive Positive Positive
Negative
Reference B VIC(inhA) Negative Negative Negative
-39-
-40-
Repeatability
10th month
Kit Lot
Sample Genotype 20130401 20130402 20130403
Positive A VIC(katG) Positive Positive Positive
P1
Reference B FAM(rpoB) Positive Positive Positive
-41-
P8 FAM(rpoB) Negative Negative Negative
-42-
Negative D FAM(rpsL) Negative Negative Negative
Reference E FAM(rrs) Negative Negative Negative
-43-
Repeatability FAM(rpoB) Negative Negative Negative
P3-1~P3-10 B VIC(inhA) Negative Negative Negative
Reference
-44-
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-
Tipe No. Nama Strain Genotip Konsentrasi Tujuan
Obat tuberculosis
P1 -Strain resistensi T511C&G315A&A43G 105 cfu/mL
Obat tuberculosis 105 cfu/mL

-Strain resistensi T533C&G315C&A43G
P2
Obat tuberculosis A516T&G315C&A94G&A306G&A 105 cfu/mL
P3 -Strain resistensi 88G
-Strain resistensi A526G&C-15T&A306G
P4
P5 -Strain resistensi C531T&G315C&A94G&G306A&A
88G
P6 -Strain resistensi C526G&G315C&C90T&A88G It's used to
Positi evaluate the
ve
Obat tuberculosis 105 cfu/mL accuracy,
Refer P7 -Strain resistensi A526T&C-15T&A43G&A1401G LOD and
ence repeatability
(1mL Obat tuberculosis 105 cfu/mL
/copy P8 -Strain resistensi C526T
)
P9 -Strain resistensi A1401G

P10 -Strain resistensi C1402T
Obat tuberculosis G1484T 105 cfu/mL

P11 -Strain resistensi
Standar 105 cfu/mL
N1 mycobacterium Tipe Liar
tuberculosis
saring H37RV
Strain sensitif 105 cfu/mL
N2 Mycobacterium Tipe Liar
tuberculosis
tuberculosis
Negat N4 Mycobacterium Tipe Liar It's used for
ive tuberculosis negative
Refer Strain sensitif 105 cfu/mL evaluation
ence Mycobacterium
N5 Tipe Liar
(1mL tuberculosis
/copy Strain sensitif 105 cfu/mL
) Mycobacterium
N6 Tipe Liar
tuberculosis
tuberculosis
N8 Tipe Liar
Mycobacterium
tuberculosis
-46-
tuberculosis
N10 Mycobacteriu Tipe Liar
m tuberculosis
3.3 Standar Pengujian

Penilaian keabsahan kit dari lot yang berbeda dan pada waktu yang berbeda dibuat
berdasarkan nilai batas positif kit, mempelajari data kinerja analitis serta persyaratan teknis
kit. Hasil pengujian harus konsisten dengan hasil pengujian teoritis pada Tabel 9.
Tabel 9 Tes Referensi Positif Kit
Konsentrasi Referensi
Nomor Referensi. (cfu/mL) Hasil Tes Kit
FAM(rpoB) Negatif
A VIC(katG) Positif
FAM(rpoB) Positif
B VIC(inhA) Negatif
5
P1 10 FAM(gyrA) Negatif
C VIC(embB) Negatif
D FAM(rpsL) Positif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A VIC(katG) Positif
FAM(rpoB) Positif
B VIC(inhA) Negatif
C VIC(embB) Negatif
D FAM(rpsL) Positif
E FAM(rrs) Negatif
FAM(rpoB) Positif
A VIC(katG) Positif
FAM(rpoB) Negatif
B VIC(inhA) Negatif
P3 105 FAM(gyrA) Positif
C VIC(embB) Positif
D FAM(rpsL) Positif
E FAM(rrs) Negatif
FAM(rpoB) Positif
A VIC(katG) Negatif
FAM(rpoB) Negatif
B VIC(inhA) Positif
C VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
FAM(rpoB) Positif
A VIC(katG) Positif
FAM(rpoB) Negatif
B VIC(inhA) Negatif
P5 105
FAM(gyrA) Positif
C VIC(embB) Positif
D FAM(rpsL) Positif
-47-
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A VIC(katG) Positif
FAM(rpoB) Positif
B VIC(inhA) Negatif
C VIC(embB) Positif
D FAM(rpsL) Positif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A VIC(katG) Negatif
FAM(rpoB) Positif
B VIC(inhA) Positif
C VIC(embB) Negatif
D FAM(rpsL) Positif
E FAM(rrs) Positif
FAM(rpoB) Positif
A VIC(katG) Negatif
FAM(rpoB) Negatif
B VIC(inhA) Negatif
C VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A VIC(katG) Negatif
FAM(rpoB) Negatif
B VIC(inhA) Negatif
C VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Positif
FAM(rpoB) Negatif
A VIC(katG) Negatif
FAM(rpoB) Negatif
B VIC(inhA) Negatif
5
C VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Positif
FAM(rpoB) Negatif
A VIC(katG) Negatif
FAM(rpoB) Negatif
B VIC(inhA) Negatif
5
C VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Positif
-48-
Tabel 10 Uji Referensi LOD Kit
Nomor Referensi. Hasil Tes Kit
(cfu/mL)
FAM(rpoB) Positif
A VIC(katG) Positif
FAM(rpoB) Negatif
B VIC(inhA) Negatif
4
C VIC(embB) Positif
D FAM(rpsL) Positif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A VIC(katG) Negatif
FAM(rpoB) Positif
B VIC(inhA) Positif
4
C VIC(embB) Negatif
D FAM(rpsL) Positif
E FAM(rrs) Positif
FAM(rpoB) Negatif
A VIC(katG) Negatif
FAM(rpoB) Negatif
B VIC(inhA) Negatif
C VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Positif
Tabel 11 Hasil Uji Referensi Negatif

Nomor Referensi (cfu/mL) Hasil Tes Kit
FAM(rpoB) Negatif
A VIC(katG) Negatif
FAM(rpoB) Negatif
B VIC(inhA) Negatif
5
N1 10 FAM(gyrA) Negatif
C VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A
VIC(katG) Negatif
FAM(rpoB) Negatif
B
VIC(inhA) Negatif
N2 105
FAM(gyrA) Negatif
C
VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A
VIC(katG) Negatif
FAM(rpoB) Negatif
B
VIC(inhA) Negatif
N3 105
FAM(gyrA) Negatif
C
VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
N4 105 A FAM(rpoB) Negatif
-49-
VIC(katG) Negatif
FAM(rpoB) Negatif
B
VIC(inhA) Negatif
FAM(gyrA) Negatif
C
VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A
VIC(katG) Negatif
FAM(rpoB) Negatif
B
VIC(inhA) Negatif
N5 105
FAM(gyrA) Negatif
C
VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A
VIC(katG) Negatif
FAM(rpoB) Negatif
B
VIC(inhA) Negatif
N6 105
FAM(gyrA) Negatif
C
VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A
VIC(katG) Negatif
FAM(rpoB) Negatif
B
VIC(inhA) Negatif
N7 105
FAM(gyrA) Negatif
C
VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A
VIC(katG) Negatif
FAM(rpoB) Negatif
B
VIC(inhA) Negatif
N8 105
FAM(gyrA) Negatif
C
VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A
VIC(katG) Negatif
FAM(rpoB) Negatif
B
VIC(inhA) Negatif
N9 105
FAM(gyrA) Negatif
C
VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
FAM(rpoB) Negatif
A
VIC(katG) Negatif
FAM(rpoB) Negatif
B
VIC(inhA) Negatif
N10 105
FAM(gyrA) Negatif
C
VIC(embB) Negatif
D FAM(rpsL) Negatif
E FAM(rrs) Negatif
Tabel 12 Hasil Uji Pengulangan Referensi
-50-
Nomor Referensi (cfu/mL) Hasil Tes Kit
FAM(rpoB) Positif
A VIC(katG) Positif
FAM(rpoB) Negatif
B VIC(inhA) Negatif
C VIC(embB) Positif
D FAM(rpsL) Positif
E FAM(rrs) Negatif
Catatan: setiap referensi pengulangan diuji berulang kali sebanyak 10 kali dan 10 hasil uji
harus konsisten.
3.4 Analisis dan Kesimpulan
Tiga lot kit diuji dengan menggunakan referensi perusahaan parsial, semua indeks konsisten dengan
nilai teoretis. Hasil pengujian stabilitas pencairan beku berulang untuk R&D dan produksi percobaan
dirangkum dalam Tabel 13.
Melihat dari data berikut, reagen R&D dan percobaan produksi sesuai dengan standar pengujian di
setiap pengujian dalam kondisi pembekuan dan pencairan berulang selama 8 kali.
Oleh karena itu, kinerja kit tidak akan terpengaruh jika pembekuan dan pencairan berulang kali tidak
lebih dari 8 kali dalam kondisi penggunaan normal.
Tabel 13 Hasil Uji Detil R&D Dan Percobaan Produksi Reagen Untuk Stabilitas Pencairan-
Pembekuan Berulang
Frekuensi Pencairan-Pembekuan Berulang
Sampel Genotip 1 kali 2 kali 3 kali 4 kali
FAM(rpoB) Negatif Negatif Negatif Negatif
A
VIC(katG) Negatif Negatif Negatif Negatif
B
VIC(inhA) Negatif Negatif Negatif Negatif
Negative control
FAM(gyrA) Negatif Negatif Negatif Negatif
C
VIC(embB) Negatif Negatif Negatif Negatif
D FAM(rpsL) Negatif Negatif Negatif Negatif
E FAM(rrs) Negatif Negatif Negatif Negatif
FAM(rpoB) Positif Positif Positif Positif
A VIC(katG) Positif Positif Positif Positif
B VIC(inhA) Positif Positif Positif Positif
Positive control FAM(gyrA) Positif Positif Positif Positif
C VIC(embB) Positif Positif Positif Positif
D FAM(rpsL) Positif Positif Positif Positif
E FAM(rrs) Positif Positif Positif Positif
A
VIC(katG) Positif Positif Positif Positif
B VIC(inhA) Negatif Negatif Negatif Negatif
P1 FAM(gyrA) Negatif Negatif Negatif Negatif
Positive C
Reference D FAM(rpsL) Positif Positif Positif Positif
A Positif Positif Positif Positif
P2 VIC(katG)
B FAM(rpoB) Positif Positif Positif Positif
-51-
C
A
B
P3
FAM(gyrA) Positif Positif Positif Positif
C
VIC(embB) Positif Positif Positif Positif
A
B
VIC(inhA) Positif Positif Positif Positif
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) Negatif Negatif Negatif Negatif
B
C FAM(gyrA) Negatif Negatif Negatif Negatif
-53-
A
B
N8
C
A
B
N9
C
A
B
N10
C
B
Repeatability VIC(inhA) Negatif Negatif Negatif Negatif
P3-1~P3-10 FAM(gyrA) Positif Positif Positif Positif
Reference
B
P3 FAM(gyrA) Positif Positif Positif Positif
A
LOD FAM(rpoB) Positif Positif Positif Positif
Reference P7 FAM(gyrA) Negatif Negatif Negatif Negatif
C
A
P9 B
C
-54-
Frekuensi Pencairan-Pembekuan Berulang
Sample Genotype kali ke-5 kali ke-6 kali ke-7 kali ke-8
A
B
Negative control
C
Positive control FAM(gyrA) Positif Positif Positif Positif
A
C
A
C
Positive FAM(rpoB) Negatif Negatif Negatif Negatif
Reference B
A VIC(katG) Negatif Negatif Negatif Negatif
B
C VIC(embB) Negatif Negatif Negatif Negatif
P5 FAM(rpoB) Negatif Negatif Negatif Negatif
B
C FAM(gyrA) Positif Positif Positif Positif
-55-
A
C
A
C
A VIC(katG) Negatif Negatif Negatif Negatif
B
C
A
B
P9
C
A
B
N1
C
A
Negative FAM(rpoB) Negatif Negatif Negatif Negatif
B
Reference VIC(inhA) Negatif Negatif Negatif Negatif
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) Negatif Negatif Negatif Negatif
C
-57-
B
Repeatability VIC(inhA) Negatif Negatif Negatif Negatif
P3-1~P3-10 FAM(gyrA) Positif Positif Positif Positif
Reference
B
A
LOD P7
Reference FAM(gyrA) Negatif Negatif Negatif Negatif
C
A
B
P9
C
4. Ringkasan Studi Stabilitas

Melalui stidi di atas, kami merangkum kesimpulan sebagai berikut:
Kit Deteksi Gen Ketahanan Obat untuk Mycobacterium Tuberculosis (PCR Real-Time) dapat
disimpan secara stabil selama 10 bulan dalam kondisi penyimpanan normal (-20℃) tanpa
perubahan kinerja yang signifikan. Masa berlaku kit ditentukan selama 8 bulan sesuai dengan
hasil studi stabilitas jangka panjang.
Ketika berulang kali menggunakan Kit Deteksi Gen Tahan Obat untuk Mycobacterium
Tuberculosis (PCR Real-Time) dalam periode yang valid, kinerja tes kit tidak akan
terpengaruh setelah berulang kali dibekukan dan dicairkan sebanyak 8 kali.
-58-

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

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Copyright:

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

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Copyright:

Available Formats

Drug-Resistant Gene Detection Kit for

Mycobacterium Tuberculosis (Real-

Applicant: CapitalBio Corporation

2. Long-term Stability Study

(1mL/copy) &A88G LOD and

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

Obat tuberculosis 105 cfu/mL

Obat tuberculosis 105 cfu/mL

Obat tuberculosis G1484T 105 cfu/mL

3.3 Standar Pengujian

Tabel 11 Hasil Uji Referensi Negatif

4. Ringkasan Studi Stabilitas

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