CN108753979A - A kind of kit and its application method for liver cancer early screening - Google Patents
A kind of kit and its application method for liver cancer early screening Download PDFInfo
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- CN108753979A CN108753979A CN201810746544.0A CN201810746544A CN108753979A CN 108753979 A CN108753979 A CN 108753979A CN 201810746544 A CN201810746544 A CN 201810746544A CN 108753979 A CN108753979 A CN 108753979A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The invention belongs to molecular biology fields, and in particular to a kind of kit and its application method for liver cancer early screening.Present invention firstly discovers that SCAND3 can be used as the marker that methylates for detecting liver cancer.The primer sequence that detection SCAND3 methylates is as follows：Forward primer sequence is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2；The fluorescence probe sequence is SEQ ID NO.3, and probe 5 ' holds flag F AM, 3 ' end label MGB.The present invention, for the marker that methylates for liver cancer early screening, is used for diagnosing cancer of liver, has the advantages that high sensitivity and high specificity with SCAND3, and especially in 400 μ g/L patients of AFP <, the SCAND3 incidences that methylate are up to 60%；It follows that the present invention is the marker that methylates with SCAND3, it is suitable for the diagnosis of liver cancer, is particularly suitable for the diagnosis of early liver cancer.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of kit and its use for liver cancer early screening
Method.
Background technology
Hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is the malignant tumour for threatening human life common
One of, and most Patients with Primary early symptom unobvious, Most patients have reached middle and advanced stage when making a definite diagnosis, therefore real
It now early diagnoses, obtain unique hope that radical excision is patient's long term survival.Due to lacking the tumor markers of specificity,
Belong to late period when most of patients is made a definite diagnosis, loses the chance of radical excision.
Currently, diagnosing cancer of liver method includes imageological examination, pathological examination and laboratory examination.But early liver cancer image
It learns performance not being true to type, and part checks there is radiativity and expensive.Needle biopsy of liver is diagnosis HCC
Goldstandard, but early liver cancer lesion is small, aspiration biopsy difficulty is big, and with a degree of traumatic, is not easy to be accepted by patients.
Tumor-marker analyte detection there is important supplement to be worth liver cancer iconography and pathological diagnosis, and by monitoring liver cancer mark
Object can find the Preventive of tumour or tumour in advance than other methods, the accuracy of prediction and diagnosis be improved, to improve
Curative effect improves prognosis.In primary hepatic carcinoma diagnosis, the widest tumor markers of clinical application are alpha-fetoprotein (AFP), but
Its diagnostic sensitivity and specificity are less than satisfactory, and the sensitivity of diagnosing liver cancer and specificity are respectively 41%~65% He
80%~94%.
In recent years the study found that there is free Tumour DNA in blood of cancer patients slurry, and with thin with tumour
The identical gene alterations of born of the same parents DNA, thus detect tumor patient peripheral circulation DNA be tumour early stage it is minimally invasive diagnose provide it is new
Means.
AFP is still the tumor markers of clinical application diagnosing liver cancer the most universal, using 400 μ g/L as clinical diagnosis liver
Cancer critical value；When the μ g/L of AFP >=400, diagnosis is higher；And for part early stage, patient can be cut off, i.e. 400 μ g/L of AFP < are easy
It is failed to pinpoint a disease in diagnosis.Thus, seek sensitivity and the higher tumor markers of specificity are of great significance to the early diagnosis of liver cancer, especially
It is to 400 μ g/L patients of AFP <.
Invention content
Present invention is primarily intended to provide a kind of kit and its application method for liver cancer early screening.The present invention
Find that the methylation of SCAND3 is related to liver cancer generation for the first time, and the journey that can methylate by SCAND3 in detection peripheral blood
Degree, the early detection for liver cancer and diagnosis.
To achieve the goals above, the present invention uses following technical scheme：
One of the object of the invention provides a kind of marker that methylates for liver cancer early screening, the mark that methylates
Object is SCAND3.
The two of the object of the invention provide the above-described marker that methylates in preparing liver cancer early screening diagnostic reagent
Application.
The three of the object of the invention provide one group of primer and probe for detecting the marker described above that methylates, described
Primer sequence is as follows：Forward primer sequence is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2；It is described
Fluorescence probe sequence is SEQ ID NO.3, and probe 5 ' holds flag F AM, 3 ' end label MGB.
The four of the object of the invention provide above-described primer and probe and are preparing the kit for liver cancer early screening
Application.
The five of the object of the invention provide a kind of kit, including above-described primer and probe, further include detection
The forward primer and probe of GAPDH, PCR Master Mix, lysate, Proteinase K, rinsing liquid, bisulfite solution and
ddH2O。
The six of the object of the invention provide the application method of kit described above, and steps are as follows：
(1) in sample genomic DNA extraction：Clinical samples are taken, lysate, Proteinase K, rinsing liquid and ddH are utilized2O
Extraction, obtains Genomic DNA solution；
(2) DNA methylation is modified：The Genomic DNA solution that step (1) extraction obtains is taken to be carried out with bisulfite solution
Modification obtains the DNA after modifying that methylates；
(3) DNA after modifying step (2) carries out PCR amplification.
Preferably, step (1) described sample is peripheral blood cells.
Preferably, PCR reaction solution is：20 μ L of PCR Master Mix, primer and each 1 μ L of probe, fluorescent quantitation reaction solution
3 μ L, 26 μ L of total amount/person-portion；PCR reaction conditions are：
The seven of the object of the invention provide application of the kit in preparing liver cancer early screening reagent.
The eight of the object of the invention provide the kit and are preparing the application in detecting SCAND3 methylating reagents
There is free Tumour DNA in peripheral blood, by detecting the methylation of gene in peripheral blood come diagnosing liver cancer,
The pain of liver cancer patient diagnosis can not only be mitigated, and can realize the early stage to liver cancer, quick diagnosis, early found conducive to patient
Early treatment.But in peripheral blood different genes methylation differ surely really reflection tissue true methylation status.This
For inventor in probing into the relationship that gene methylation occurs with liver cancer, screening obtains SCAND3 genes, is found for the first time by experiment
The gene methylation degree is related to the generation of liver cancer.In further research, find SCAND3 genes in 400 μ g/L of AFP <
Methylation is higher in liver cancer patient, is higher than known liver cancer marker gene SLIT2 and DAPK, it follows that SCAND3 exists
The early diagnosis of liver cancer has more potentiality.
Compared with prior art, the present invention has the advantage that：
(1) present invention is used for diagnosing cancer of liver, has the advantages that high sensitivity and high specificity using SCAND3 as target gene,
Especially in 400 μ g/L patients of AFP <, the SCAND3 incidences that methylate are up to 60%；It follows that the present invention is with SCAND3
For target gene, it is suitable for the diagnosis of liver cancer, is particularly suitable for the diagnosis of early liver cancer.
(2) present invention is minimally invasive diagnosis using peripheral blood in patients as sample, can greatly reduce patient pain's degree, reduces and suffers from
Person diagnoses expense, and quick diagnosis may be implemented.
Description of the drawings
The Figure of description for constituting the part of the present invention is used to provide further understanding of the present invention, and of the invention shows
Meaning property embodiment and its explanation are not constituted improper limitations of the present invention for explaining the present invention.
Fig. 1 is RT-PCR amplification curve diagrams：1-7 is 1-7 samples in the μ g/L liver cancer patient samples of AFP >=400, and 8-11 is
11-14 samples in 400 μ g/L liver cancer patients of AFP <.
Specific implementation mode
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific implementation mode, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or combination thereof.
In order to enable those skilled in the art can clearly understand technical scheme of the present invention, below with reference to tool
The embodiment of body and the comparative example technical solution that the present invention will be described in detail.
Embodiment 1SCAND3 and its primer and probe
Present invention firstly discovers that SCAND3, which methylates, can be used for early screening and the diagnosis of liver cancer.
It is as follows for detecting the primer sequence that SCAND3 methylates：
Forward primer sequence is：5 '-GTTATAAATTGAGCGGTAAGATATTTGC-3 ', such as SEQ ID NO：1 sequence institute
Show；
Reverse primer sequences are：5 '-CCTCGCCCAAACTACTCCG-3 ', such as SEQ ID NO：Shown in 2 sequences；,
Fluorescence probe sequence is：5 '-AATCGAGGTTATGACGGATA-3 ', such as SEQ ID NO：Shown in 3 sequences；Fluorescence
5 ' end flag F AM (fluorescent reporter group) of probe, 3 ' end label MGB (fluorescent quenching group).
Other primers and probe used are as follows in screening process of the present invention：
First group of primer and probe：
Forward primer sequence as shown in SEQ ID NO.4 (5 '-GCGAGATAGAAAATCGAGGTTATGAC-3 '),
Reverse primer sequences as shown in SEQ ID NO.5 (5 '-TATAAACCCCTCTCCCTCTCCG-3 '),
Fluorescence probe sequence is the fluorescence probe as shown in SEQ ID NO.6 (5 '-AAGGAAACGAAGATCGGAGTA-3 ')
5 ' end flag F AM (fluorescent reporter group), 3 ' end label MGB (fluorescent quenching group).
A kind of kit for liver cancer early screening of embodiment 2
The kit includes forward primer, and sequence is the reverse primer as shown in SEQ ID NO.1, and sequence is SEQ ID
NO.2 and fluorescence probe, sequence are SEQ ID NO.3；The probe 5 ' holds flag F AM, 3 ' end label MGB；Include additionally
Detect the forward primer and probe of GAPDH, PCR Master Mix, lysate, Proteinase K, rinsing liquid, bisulfite solution
And ddH2O；Primer and probe known in the art can be used in the forward primer and probe for detecting GAPDH.
The application method of kit described in embodiment 3
The application method of kit, specific as follows described in embodiment 2：
(1) extraction of peripheral blood cells genomic DNA：Take peripheral blood cells, using lysate, Proteinase K, rinsing liquid and
ddH2O is extracted, and obtains Genomic DNA solution；
The specific steps are：
200 μ L peripheral blood samples are drawn, are placed in 1.5mL centrifuge tubes, 1mL lysates 1 are added, then lasting vortex oscillation
Mixing 1min, mixes well, and 1.5mL centrifuge tubes are then placed in 70 DEG C of water-bath 5min.
1.5mL centrifuge tubes are taken out out of water-bath, 12,000rpm centrifugation 5min slowly draw 300 μ L supernatants to new
1.5mL centrifuge tubes in.
200 μ L lysates 2 and 20 μ L Proteinase Ks, vortex mixing are sequentially added into new 1.5mL centrifuge tubes.
New 1.5mL centrifuge tubes are placed in 70 DEG C of water-bath 10min.
Add 200 μ L isopropanols, vortex mixing.
Previous step acquired solution is all added in an adsorption column and (adsorption column is inserted in collecting pipe), 12,000rpm
30sec is centrifuged, waste liquid is abandoned.
600 μ L rinsing liquids are added into adsorption column, 12,000rpm centrifugation 30sec abandon waste liquid.
Aforesaid operations are repeated, 12,000rpm centrifugation 2min abandon waste liquid；Adsorption column is stored at room temperature 2min, thoroughly to volatilize
Remaining rinsing liquid in adsorption column.
Adsorption column is transferred in 1.5mL centrifuge tubes, 50 μ L eluents are vacantly added dropwise to adsorbed film centre position, are stored at room temperature
2min, 12,000rpm centrifugation 2min, solution is collected into centrifuge tube.
(2) DNA methylation is modified：The Genomic DNA solution that step (1) extraction obtains is taken to be carried out with bisulfite solution
Modification obtains the DNA after modifying that methylates；
The DNA eluents that step (1) is extracted are fully transferred in 0.2mL centrifuge tubes, 150 μ L bisulfites of addition are molten
Liquid；By flick centrifuge tube or pipettor softly blow and beat operation mixing after of short duration centrifugation.
Centrifuge tube is positioned in temperature cycles thermode and is arranged according to the following conditions：
①98℃10min
②64℃1.5h
③4℃(≦20h)
500 μ L combination liquid are added into adsorption column, then the mixed liquor of previous step is transferred in adsorption column, lid upper tube cap is simultaneously
Reverse mixing, 12,000rpm centrifugation 30sec, abandons waste liquid.
600 μ L rinsing liquids are added into adsorption column, 12,000rpm centrifugation 30sec abandon waste liquid.
It is primary to repeat aforesaid operations, 12,000rpm centrifugation 2min abandon waste liquid；Adsorption column is stored at room temperature 2min, with thorough
Remaining rinsing liquid in volatilization adsorption column.
Adsorption column is transferred in 1.5mL centrifuge tubes, 30 μ L eluents are vacantly added dropwise to adsorbed film centre position, are stored at room temperature
Solution is collected into centrifuge tube after 2min, 12,000rpm centrifugation 2min.
(3) DNA after modifying step (2) carries out PCR amplification.
PCR reaction solution is configured according to following table：
Table 1
After PCR reaction solution mixes well, it is sub-packed in eight unions of PCR by 26 μ L volumes of every pipe, and shifted after marking
To sample process area.
By BisDNA to be checked (DNA after the conversion that methylates) sample, the PCR eight dispensed is added to by the amount of 2 μ L of every hole
In union, compressing pipe lid, and of short duration centrifugation, tube wall liquid is centrifuged to tube bottom.
Eight unions of PCR are placed on the corresponding position of instrument sample slot, and records and puts order.Instrument sense channel selects
It selects：Reporter Dye1：FAM, Quencher Dye1：none；Reporter Dye2 (GAPDH) Cy5, Quencher
Dye2：none；Passive Reference：none.The setting of corresponding detection hole：Before amplified reaction starts, by sample to be checked
It is set as " Unknown " with quality-control product.
It is reacted by the following conditions setting PCR
Table 2
Test example 1
Strong Laboratory of medical test in May, 2017~2018 year Clinicopathologic Diagnosis in May is reached collected from Wuhu for liver cancer to suffer from
Person (20 people, wherein 400 μ g/L patients of AFP <, 10 people, the μ of AFP >=400 g/L), hepatitis (5 people) and normal person (20 people)
Peripheral blood.
It is detected by 3 method of embodiment, the CT values of the detailed testing result of each sample are shown in Table 3.
The CT values of 3 each sample fluorescence quantitative PCR detection result of table
(2) according to table 3, sensitivity and specificity are calculated according to following formula：
Sensitivity=testing result is liver cancer sample number/corresponding liver cancer total sample number of the positive；
Specificity=testing result is the non-liver cancer total sample number of negative non-liver cancer sample number/total；
The results are shown in Table 4.
The specificity of 4 present invention detection liver cancer of table and sensitivity
SCAND3 is in the μ g/L liver cancer patients of AFP >=400 it can be seen from table 3,4 testing result of table, detection sensitivity
It is 70%, in 400 μ g/L liver cancer patients of AFP <, detection sensitivity 60%.From the above results, SCAND3 is in liver cancer
There is preferable application potential in early diagnosis.
Test example 2
It is detected with the sample described in test example 1 of the present invention, liver is carried out using first group of primer in embodiment 1 and probe
The detection of cancer, testing result are as shown in table 5 below.
5 first groups of primer specificities of table and sensitivity technique result
By above-mentioned table 5 it is found that compared with first group of primer and probe, the primer and probe of the present invention have preferably spirit
Sensitivity and specificity.
Finally illustrate, the foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, to the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, for those skilled in the art, still can be with
It modifies to the technical solution recorded in previous embodiment, or equivalent replacement is carried out to which part.It is all the present invention
Within spirit and principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Although the above-mentioned specific implementation mode to the present invention is described, it is not intended to limit the protection scope of the present invention, affiliated neck
Field technique personnel should be understood that based on the technical solutions of the present invention those skilled in the art need not pay creativeness
The various modifications or changes that can be made work still within protection scope of the present invention.
SEQUENCE LISTING
<110>Anhui reaches strong medical science and technology Co., Ltd
<120>A kind of kit and its application method for liver cancer early screening
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213>Artificial sequence
<400> 1
gttataaatt gagcggtaag atatttgc 28
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
cctcgcccaa actactccg 19
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
aatcgaggtt atgacggata 20
<210> 4
<211> 26
<212> DNA
<213>Artificial sequence
<400> 4
gcgagataga aaatcgaggt tatgac 26
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
tataaacccc tctccctctc cg 22
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
aaggaaacga agatcggagt a 21
Claims (10)
1. a kind of marker that methylates for liver cancer early screening, which is characterized in that the marker that methylates is SCAND3.
2. the application of marker in preparing liver cancer early screening diagnostic reagent as described in claim 1 that methylates.
3. one group of primer and probe for the marker that methylates described in test right requirement 1, which is characterized in that the primer sequence
Row are as follows：Forward primer sequence is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2；The fluorescence is visited
Needle sequence is SEQ ID NO.3, and probe 5 ' holds flag F AM, 3 ' end label MGB.
4. a kind of primer as claimed in claim 3 and probe are in the application for preparing the kit for liver cancer early screening.
5. a kind of kit, which is characterized in that further include detecting GAPDH just including the primer and probe described in claim 3
To primer and probe, PCR Master Mix, lysate, Proteinase K, rinsing liquid, bisulfite solution and ddH2O。
6. the application method of kit according to claim 5, which is characterized in that steps are as follows：
(1) in sample genomic DNA extraction：Clinical samples are taken, lysate, Proteinase K, rinsing liquid and ddH are utilized2O is extracted,
Obtain Genomic DNA solution；
(2) DNA methylation is modified：The Genomic DNA solution that step (1) extraction obtains is taken to be repaiied with bisulfite solution
Decorations obtain the DNA after modifying that methylates；
(3) DNA after modifying step (2) carries out PCR amplification.
7. the application method of kit according to claim 6, which is characterized in that step (1) described sample is that peripheral blood is thin
Born of the same parents.
8. the application method of kit according to claim 6, which is characterized in that PCR reaction solution is：PCR Master Mix
20 μ L, primer and each 1 μ L of probe, 3 μ L of fluorescent quantitation reaction solution, 26 μ L of total amount/person-portion；PCR reaction conditions are：
9. application of the kit according to claim 5 in preparing liver cancer early screening reagent.
10. kit is preparing the application in detecting SCAND3 methylating reagents according to claim 5.
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| CN201810746544.0A CN108753979B (en) | 2018-07-09 | 2018-07-09 | Kit for early screening of liver cancer and use method thereof |
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| CN201810746544.0A CN108753979B (en) | 2018-07-09 | 2018-07-09 | Kit for early screening of liver cancer and use method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825583A (en) * | 2019-03-01 | 2019-05-31 | 清华大学 | Marker and its application of people's repeat element DNA methylation as hepatocarcinoma early diagnosis |
| CN110669831A (en) * | 2019-11-11 | 2020-01-10 | 益善生物技术股份有限公司 | Human SGIP1, SCAND3 and MYO1G gene methylation detection kit |
| CN111549134A (en) * | 2020-05-18 | 2020-08-18 | 浙江大学医学院附属第一医院 | Liver cancer early detection kit based on polygenic mutation |
| CN112063717A (en) * | 2020-09-17 | 2020-12-11 | 山东大学深圳研究院 | Application of MDM2 as marker in early diagnosis of hepatitis B virus-related hepatocellular carcinoma and detection kit |
| CN114634981A (en) * | 2020-12-16 | 2022-06-17 | 广州达健生物科技有限公司 | Primer probe combination for detecting liver cancer gene methylation as well as kit and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825583A (en) * | 2019-03-01 | 2019-05-31 | 清华大学 | Marker and its application of people's repeat element DNA methylation as hepatocarcinoma early diagnosis |
| CN109825583B (en) * | 2019-03-01 | 2021-08-17 | 清华大学 | Marker for early diagnosis of liver cancer by DNA methylation of human repeat element and application of marker |
| CN110669831A (en) * | 2019-11-11 | 2020-01-10 | 益善生物技术股份有限公司 | Human SGIP1, SCAND3 and MYO1G gene methylation detection kit |
| CN110669831B (en) * | 2019-11-11 | 2023-09-19 | 益善生物技术股份有限公司 | Human SGIP1, SCAND3 and MYO1G gene methylation detection kit |
| CN111549134A (en) * | 2020-05-18 | 2020-08-18 | 浙江大学医学院附属第一医院 | Liver cancer early detection kit based on polygenic mutation |
| CN111549134B (en) * | 2020-05-18 | 2023-03-28 | 浙江大学医学院附属第一医院 | Liver cancer early detection kit based on polygenic mutation |
| CN112063717A (en) * | 2020-09-17 | 2020-12-11 | 山东大学深圳研究院 | Application of MDM2 as marker in early diagnosis of hepatitis B virus-related hepatocellular carcinoma and detection kit |
| CN114634981A (en) * | 2020-12-16 | 2022-06-17 | 广州达健生物科技有限公司 | Primer probe combination for detecting liver cancer gene methylation as well as kit and application thereof |
| CN114634981B (en) * | 2020-12-16 | 2024-01-26 | 广州达健生物科技有限公司 | Liver cancer gene methylation detection primer probe combination, kit and application thereof |
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| CN108753979B (en) | 2021-10-26 |
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