CN116926215A - Kit for detecting tuberculosis typing based on three-gene scoring method and application - Google Patents

Kit for detecting tuberculosis typing based on three-gene scoring method and application Download PDF

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CN116926215A
CN116926215A CN202310929171.1A CN202310929171A CN116926215A CN 116926215 A CN116926215 A CN 116926215A CN 202310929171 A CN202310929171 A CN 202310929171A CN 116926215 A CN116926215 A CN 116926215A
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seq
tuberculosis
parp14
epha4
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夏江
高学娟
赵雁林
欧喜超
赵冰
邱倩
黄飞
余晧
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Pilot Gene Technologies Hangzhou Co ltd
Chinese Center for Disease Control and Prevention
Chongqing Public Health Medical Center
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Pilot Gene Technologies Hangzhou Co ltd
Chinese Center for Disease Control and Prevention
Chongqing Public Health Medical Center
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Abstract

The application provides a kit for detecting tuberculosis typing based on a three-gene scoring method and application thereof, wherein the transcription levels of three genes of STAT1 gene, PARP14 gene and EphA4 gene are detected simultaneously in different channels of the same amplification system by utilizing a digital PCR amplification technology, and normalization of housekeeping genes is not needed. In order to avoid the difference caused by different template total amounts during amplification, the scheme utilizes the detection values of the transcription levels of the STAT1 gene, the PARP14 gene and the EphA4 gene to score three genes of patients, and utilizes the score values to distinguish tuberculosis latency, active tuberculosis and normal people, thereby having the effect of high specificity.

Description

Kit for detecting tuberculosis typing based on three-gene scoring method and application
Technical Field
The application relates to the field of tuberculosis typing, in particular to a kit for detecting tuberculosis typing based on a three-gene scoring method and application thereof.
Background
Tuberculosis is an infectious disease caused by mycobacterium tuberculosis, and can be divided into two states: tuberculosis latency and active tuberculosis. Tuberculosis latency refers to the fact that a person infects mycobacterium tuberculosis, but disease symptoms do not appear yet, and in the tuberculosis latency state, the infected person has no symptoms of active tuberculosis, has infectivity and possibly suffers from active tuberculosis; active tuberculosis refers to the inability of the immune system of an infected person to control the growth of mycobacterium tuberculosis, resulting in the appearance of disease symptoms and infectivity. Since it is a patient with tuberculosis latency or active tuberculosis, the body contains mycobacterium tuberculosis, which results in that the prior art digital PCR technique cannot directly distinguish between normal, tuberculosis latency and active tuberculosis patients.
Specifically, although digital PCR can detect the nucleic acid of mycobacterium tuberculosis, by comparing the copy number or expression level of a target gene, the presence and amount of mycobacterium tuberculosis can be quantitatively analyzed, but only information about the presence and amount of mycobacterium tuberculosis is provided, and tuberculosis latency and active tuberculosis cannot be distinguished, because in different states of tuberculosis, there may be overlap or insignificant change in the nucleic acid level of mycobacterium tuberculosis, and DNA or RNA of mycobacterium tuberculosis may exist, whether active tuberculosis or tuberculosis latency, thereby leading to digital PCR technology capable of distinguishing only whether it is tuberculosis infection but not active tuberculosis, and further limiting the application of digital PCR in the field of tuberculosis detection.
Disclosure of Invention
The application provides a kit for detecting tuberculosis typing based on a three-gene scoring method and application thereof, wherein the detection of the transcription levels of STAT1 genes, PARP14 genes and EphA4 genes is utilized, the scoring value is obtained by utilizing the three-gene scoring method by taking the PARP14 transcription level detection value with moderate transcription level as a denominator, and normal people, tuberculosis latent patients and active tuberculosis patients are effectively distinguished based on the scoring value.
The scheme selects three genes of STAT1 gene, PARP14 gene and EphA4 as tuberculosis infection host marker genes, and the three genes all have certain changes when dealing with pathogen infection: STAT1 proteins play a critical role in innate immunity by directing transcription reactions to interferons and other cytokines, thereby protecting the host from pathogen infection; PARP14 is considered a partner of STAT6 (CoaSt 6) because it is able to promote IL-4 dependent transcription, its macroscopic domain can enhance IL-4 induced gene expression by cooperating with STAT6, helping to transduce IL-4 signals, promoting metabolic adaptation and survival of mature B cells; in addition, PARP14 also affects class distribution, affinity repertoire and recall capacity of antibody responses, and requires protein mono ADP ribosylation for T-helper differentiation; ephA4 may inhibit the anti-inflammatory polarization of monocytes/macrophages through mTOR, p-Akt and NF- κb pathway regulation, and EphA4 deletion or attenuation of expression of the LPS-stimulated pro-inflammatory gene in vitro following EphA4 peptide inhibition suggests that EphA4 may be responsible for increased inflammation and secondary injury following TBI. It was shown by the results of the study that inhibition of EphA4 may shift the inflammatory response to a pro-lytic state compared to IL-10, arginase 1, angpt2 and Tgfβ, as can be seen by the altered expression of IL-6, IL-8, IL-12 and TNF. EphA 4-deficient monocytes/macrophages are polarized to the M1 pro-inflammatory phenotype, which exhibit fewer CD86, IL-12p40, ccr2 and Mcp1.
The present inventors found that STAT1 gene plays an important role in the pathogenesis and immune response of tuberculosis. STAT1 deficiency or dysfunction may lead to impaired immune responses, increasing the risk of infection with mycobacterium tuberculosis (Mycobacterium tuberculosis); excessive activation of the PARP14 gene may lead to immune cell dysfunction, affecting the capacity of clearing mycobacterium tuberculosis, and some studies have found that variation of the PARP14 gene may be related to susceptibility and severity of tuberculosis; ephA4 plays a role in inflammation and immune response. In addition, although infection of different disease strains also causes the change of three genes, namely STAT1 gene, PARP14 gene and EphA4, the change amount caused by active tuberculosis and tuberculosis latency is different, so the scheme is characterized by scoring by the detection value of the transcription level of the three genes, distinguishing normal people, active tuberculosis and tuberculosis latency based on the score obtained by scoring, and having strong specificity.
In addition, the detection system developed by the scheme uses different channels of the same amplification system to detect three genes simultaneously, namely, three genes of one sample are amplified in the same amplification tube, so that the advantage is that the sample loading amount can be ensured to be completely consistent without normalization of a housekeeping gene. And because the content difference of mononuclear cells of different patients is large, the difference of the collection amount of mononuclear cells is also large, so that the difference of the total RNA amount of different samples exists, even if templates with the same volume are added during detection, the unavoidable detection values also have large differences, in order to avoid the difference caused by the difference of the total amount of the templates during amplification, the scheme selects the PARP14 detection value with moderate transcription level in three genes as a denominator, the detection values of other genes are used as molecules for carrying out detection value standardization treatment, the detection values of the transcription levels of the three genes are used for scoring, and whether the patient is infected by mycobacterium tuberculosis or not is judged based on the obtained score, and the type of infection belongs to the stage of tuberculosis latency or active tuberculosis.
In a first aspect, the present scheme provides an application of a kit for detecting tuberculosis typing based on a three-gene scoring method, wherein detected genes are STAT1 gene, PARP14 gene and EphA4 gene, and the kit comprises: the transcription levels of STAT1 gene, PARP14 gene and EphA4 gene are obtained, and three gene scores are calculated according to the following formula:
three gene score = STAT1/2PARP14-EphA4/parp14+1/2;
wherein STAT1 is the transcript level of STAT1, PARP14 is the transcript level of PARP14, ephA4 is the transcript level of EphA4, and if the three gene score is in the first interval, it is judged as normal, in the second interval it is latent, and the upper threshold value greater than the second interval is active tuberculosis.
In some embodiments, a three gene score between-1.5 and 0 is judged normal, and a three gene score between 0 and 1.0 is judged latent, and a score greater than 1.0 is judged active tuberculosis.
In some embodiments, the detection of three gene transcript levels is performed by a digital PCR method. The detection principle of the digital PCR is to detect fluorescent signals of all reaction units after partition amplification, directly calculate the concentration or copy number of target molecules by utilizing digital model correction, and realize absolute quantitative analysis without contrasting standard samples and standard curves and depending on CT values.
In some embodiments, three genes are detected in different channels of the same amplification system, and the detected value of the corresponding channel is obtained as the transcription level of the gene.
In some embodiments, mixing a STAT1 gene, a PARP14 gene, an EphA4 gene and an exogenous internal reference primer probe to obtain a primer probe mixed solution, mixing the primer probe mixed solution to prepare a ddPCR reaction system, mixing RNA extracted from a blood sample to be detected with the ddPCR reaction system, and performing amplification detection under a set amplification program to obtain the detection of transcription levels of the STAT1 gene, the PARP14 gene and the EphA4 gene.
In some embodiments, the primer sequences for specifically amplifying STAT1 gene are shown in SEQ ID NO. 1 and SEQ ID NO. 2, the probe sequences are shown in SEQ ID NO. 3, and the corresponding marker sequences are shown in SEQ ID NO. 0:14. In some embodiments, the modified fluorophores that specifically detect the probe sequence of the STAT1 gene are FAM and MGB.
In some embodiments, the primer sequences for specifically amplifying PARP14 gene are shown in SEQ ID NO. 4 and SEQ ID NO. 5, the probe sequences are shown in SEQ ID NO. 6, and the corresponding marker sequences are shown in SEQ ID NO. 0 and 15. In some embodiments, the modified fluorophores that specifically detect the probe sequence of the PARP14 gene are ROX and MGB.
In some embodiments, the primer sequences for specifically amplifying the EphA4 gene are shown in SEQ ID No. 7 and SEQ ID No. 8, the probe sequences are shown in SEQ ID No. 9, and the corresponding tag sequences are shown in SEQ ID No. 0:16. In some embodiments, the modified fluorophores that specifically detect the probe sequence of EphA4 gene are VIX and MGB.
The scheme also randomly generates random primers shown as SEQ ID NO. 10-11 and probes shown as SEQ ID NO. 12 as exogenous internal references, and the corresponding marker sequence is shown as SEQ ID NO. 0-13.
The specific sequences are shown in Table one:
list one sequence table
In a second aspect, the present scheme provides a kit for detecting tuberculosis typing based on a three-gene scoring method, comprising:
the primer sequences of the specificity amplification STAT1 gene are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the probe sequences are shown in SEQ ID NO. 3;
the primer sequences for specifically amplifying the PARP14 gene are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the probe sequences are shown as SEQ ID NO. 6;
the primer sequences for specifically amplifying the EphA4 gene are shown in SEQ ID NO. 7 and SEQ ID NO. 8, and the probe sequence is shown in SEQ ID NO. 9.
In some embodiments, the kit detects STAT1 gene, PARP14 gene and EphA4 gene in a digital PCR manner, detects in different channels of the same amplification tube, obtains detection values of the different channels as transcription levels of the corresponding genes, and calculates a three-gene score according to the following formula:
three gene score = STAT1/2PARP14-EphA4/parp14+1/2;
wherein STAT1 is the transcript level of STAT1, PARP14 is the transcript level of PARP14, ephA4 is the transcript level of EphA4, and if the three gene score is in the first interval, it is judged as normal, in the second interval it is latent, and the upper threshold value greater than the second interval is active tuberculosis.
In some embodiments, the kit further comprises random primers as set forth in SEQ ID NOS 10-11 and probes as set forth in SEQ ID NO 12 as exogenous internal references.
In some embodiments, the digital PCR detection kit further comprises: water, ddPCR Mix, a marker sequence shown as SEQ ID No. 0:13.
In some embodiments, the amplification procedure is: 55 ℃ for 10 min; 95 ℃ for 5min; [ 95 ℃ 15s,60 ℃ 30s ] 45.
In some embodiments, the test is performed on a blood sample of the patient to be tested.
In a third aspect, the present scheme provides an application of a kit for detecting tuberculosis typing based on a three-gene scoring method, for tuberculosis detection for non-diagnostic purposes, and for distinguishing latent tuberculosis or active tuberculosis.
Compared with the prior art, the technical scheme has the following characteristics and beneficial effects:
the scheme utilizes the digital PCR amplification technology to detect the transcription levels of STAT1 genes, PARP14 genes and EphA4 genes in different channels of the same amplification tube at the same time, and does not need normalization of housekeeping genes. In order to avoid the difference caused by different template total amounts during amplification, the scheme selects the detection value of the PARP14 gene with moderate transcription level in three genes as a denominator to carry out standardized treatment on gene detection values of other channels, calculates three-gene scoring by utilizing the change relation of the transcription level of the three genes, judges tuberculosis infection by the three-gene scoring, can efficiently identify tuberculosis latency and active tuberculosis, and has the effect of higher specificity and more accurate result. In addition, the scheme designs primer probe sequences for three genes, and can more accurately detect the corresponding gene transcription level.
Drawings
Fig. 1 is a scatter plot of test results for 100 patient clinical blood samples.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which are derived by a person skilled in the art based on the embodiments of the application, fall within the scope of protection of the application.
It will be appreciated by those skilled in the art that in the present disclosure, the terms "longitudinal," "transverse," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," etc. refer to an orientation or positional relationship based on that shown in the drawings, which is merely for convenience of description and to simplify the description, and do not indicate or imply that the apparatus or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore the above terms should not be construed as limiting the present application.
It will be understood that the terms "a" and "an" should be interpreted as referring to "at least one" or "one or more," i.e., in one embodiment, the number of elements may be one, while in another embodiment, the number of elements may be plural, and the term "a" should not be interpreted as limiting the number.
In order to verify the feasibility of the scheme, the inventor constructs a digital PCR detection system and performs the following experimental verification:
preparation:
designing a primer probe: according to the analysis results of STAT1, PARP14 and EphA4 gene sequences, adopting Primer Express software to respectively design a pair of primers and a probe; meanwhile, a group of exogenous internal reference primer probes are matched, and the obtained sequence table is shown in the table one.
Sample selection: bacterial RNA sample: and (5) outsourcing inactivated bacteria, and extracting genome RNA by a magnetic bead method. Human DNA sample: extracting RNA from blood by a column method. Exogenous internal reference template: the biological company synthesizes the materials, and the materials are dissolved and diluted for later use.
Construction of a digital PCR amplification system:
mixing to obtain a primer probe mixed solution, wherein the mixed system of the primer probe mixed solution is shown in the following table II:
mixed liquid system of surface two primer probe
Component (A) Stock solution (mu M) Volume (mu L/person)
STAT1-F 100 0.1
STAT1-R 100 0.1
STAT1-P 100 0.03
PARP14-F 100 0.1
PARP14-R 100 0.1
PARP14-P 100 0.03
EphA4-F-1 100 0.1
EphA4-R-1 100 0.1
EphA4-P-1 100 0.03
IC-F 100 0.15
IC-R 100 0.15
IC-P 100 0.045
Totalizing / 1.035
。
Construction of ddPCR reaction system: mixing to obtain a ddPCR reaction system, wherein the ddPCR reaction system is shown in the following Table III:
table three ddPCR reaction system
Reagent(s) Volume (mu L) Final concentration
Water and its preparation method 4.965
Primer probe mixed liquid 1.035
5XddPCR Mix 3 1X
IC-S 1
DNA 5
Totalizing 15
。
RNA is the RNA of the sample to be tested.
The amplification procedure was as follows:
55℃10min；95℃5min；【95℃15s，60℃30s】*45。
and (3) verification: 8 normal blood samples are selected for detection, RNA is extracted by a column method, and common infectious microbe RNA samples are selected: and (5) outsourcing inactivated bacteria, and extracting genome RNA by a magnetic bead method. The test results are shown in Table IV below:
table IV shows the results of verification
Therefore, the specificity of the system detection for various common bacteria and fungi is normal, and the blood samples of 8 normal people are scored between-0.33- (-0.17) by the kit detection, so that the repeatability is good.
Embodiment one: blank detection limit (LoB) value:
extracting 20 parts of deionized water every day as a template, respectively using the ddPCR reaction systems on different amplification instruments for three continuous days, analyzing whether the detection result is subjected to normal distribution by using spss23.0 software S-W except that all detection values are 0, and analyzing LoB values by using a parameter method if the detection result is subjected to normal distribution; if normal distribution is not obeyed, the LoB value is analyzed by a nonparametric method. The results are shown in Table five below:
five blank detection limit value
STAT1 PARP14 EphA4
First day 0.09 0.09 0.18
The next day 0 0.09 0.09
Third day 0.09 0 0
。
The detection target takes a maximum value of LoB, namely STAT1 and PARP14 of 0.09 copies/. Mu.l and EphA4 of 0.18 copies/. Mu.l, from 3 calculations.
Example 2 blank detection Limit (LoD) value
Minimum detection limit (LoD): if LoB =0, a probability unit scheme is adopted; if LoB is not equal to 0, the classical scheme is adopted. The maximum LOD in 3 batches of reagents was formulated as the final reported value.
Probability unit scheme: the DNA of known concentrations of different target genes was incorporated as positive samples using ultrapure water as a substrate. Then, ultrapure water was used for gradient dilution.
The same set of instrument, including a droplet generator, a PCR amplification instrument and a chip reader, is used for repeatedly detecting different concentration gradients of each target gene specimen 30 times respectively by using 3 batch reagents.
And respectively calculating the positive detection rate of each concentration gradient of each target gene specimen, and selecting the lowest concentration with the positive detection rate more than or equal to 95% as the LoD value of each target gene.
Classical scheme
The DNA of known concentrations of different target genes was incorporated as positive samples using ultrapure water as a substrate. Then, the samples were diluted to five concentrations of 1X LoB, 2X LoB, 3X LoB, 4X LoB and 5X LoB, respectively, using the corresponding ultrapure water, and used as low-value samples.
The same set of instrument, including a droplet generator, a PCR amplification instrument and a chip reader, is used for repeatedly detecting five low-value samples of each target gene at least 12 times respectively by using 3 batch reagents, and the total of at least 60 low-value sample measurement results are obtained.
The method for calculating the LoD value of each target gene comprises the following steps:
1) The data distribution of at least 60 low value specimen measurements per lot reagent is analyzed.
2) If the data are normally distributed, the analysis is carried out by adopting a parameter statistical method.
3) If the data is distributed in a biased state, adopting a non-parameter statistical method to analyze.
The results are shown in Table six below:
table six test results
STAT1 PARP14 EphA4
LoD 0.18copies/μl 0.27copies/μl 0.36copies/μl
。
The limit of detection LoD value STAT1 was 0.18 copies/. Mu.l, PARP14 was 0.27 copies/. Mu.l, and EphA4 was 0.36 copies/. Mu.l.
Example 3 clinical threshold line partitioning
150 cases of gamma interferon release test positive and tuberculosis patients, 150 cases of gamma interferon release test positive and non-tuberculosis patients without BCG vaccine, and 150 cases of gamma interferon release test negative volunteer test results. The four-gene scoring detection threshold line for the latent tuberculosis of normal people is marked as 0, and the detection threshold line for the latent tuberculosis and the active tuberculosis is marked as 1.0.
As shown in table seven below:
table seven score
Example 4 authentication of authentic samples
On the basis of the detection threshold line, 100 patient blood samples with unknown clinical information of the contact history of the tuberculosis patient are detected, the sample information of the patient is recorded in detail in the detection process, and the clinical information of the patient is checked according to the detection result. Based on the detection results, clinical information of the batch of patients is checked, and the detection results are made into a scatter diagram record as shown in fig. 1. The clinical information is checked to obtain: six of the three genes with scoring values greater than 1 are tuberculosis patients undergoing treatment; 56 positions between the three gene scoring values of 0-1 are gamma interferon release test to detect positive non-diseased patients without BCG; the other 38 cases were all patients with negative gamma interferon release assay.
The present application is not limited to the above-mentioned preferred embodiments, and any person who can obtain other various products under the teaching of the present application can make any changes in shape or structure, and all the technical solutions that are the same or similar to the present application fall within the scope of the present application.

Claims (10)

1. An application of a kit for detecting tuberculosis typing based on a three-gene scoring method, wherein detected genes are STAT1 gene, PARP14 gene and EphA4 gene, and the kit is characterized by comprising the following components: the transcription levels of STAT1 gene, PARP14 gene and EphA4 gene are obtained, and three gene scores are calculated according to the following formula:
three gene score = STAT1/2PARP14-EphA4/parp14+1/2;
wherein STAT1 is the transcript level of STAT1, PARP14 is the transcript level of PARP14, ephA4 is the transcript level of EphA4, and if the three gene score is in the first interval, it is judged as normal, in the second interval it is latent, and the upper threshold value greater than the second interval is active tuberculosis.
2. The use of a kit for detecting tuberculosis typing based on a three-gene scoring method according to claim 1, wherein if the three-gene score is between-1.5 and 0, the kit is judged to be normal, and if the three-gene score is between 0 and 1.0, the kit is judged to be latent to tuberculosis, and if the three-gene score is greater than 1.0, the kit is judged to be active tuberculosis.
3. The use of a kit for detecting tuberculosis typing based on a three-gene scoring method according to claim 1, wherein the detection of the transcription levels of STAT1 gene, PARP14 gene and EphA4 gene is performed based on a digital PCR method.
4. The application of the kit for detecting tuberculosis typing based on the three-gene scoring method as claimed in claim 1, wherein the primer sequence for specifically amplifying the STAT1 gene is shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the probe sequence is shown as SEQ ID NO. 3;
the primer sequences for specifically amplifying the PARP14 gene are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the probe sequences are shown as SEQ ID NO. 6;
the primer sequences for specifically amplifying the EphA4 gene are shown in SEQ ID NO. 7 and SEQ ID NO. 8, and the probe sequence is shown in SEQ ID NO. 9.
5. The use of a kit for detecting tuberculosis typing based on a three-gene scoring method as claimed in claim 3, wherein STAT1 gene, PARP14 gene and EphA4 gene are detected in different channels of the same amplification system, and the detected values of the corresponding channels are obtained as the transcription level of the tuberculosis infection host marker gene.
6. Kit for detecting tuberculosis typing based on three-gene scoring method, which is characterized by comprising:
the primer sequences of the specificity amplification STAT1 gene are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the probe sequences are shown in SEQ ID NO. 3;
the primer sequences for specifically amplifying the PARP14 gene are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the probe sequences are shown as SEQ ID NO. 6;
the primer sequences for specifically amplifying the EphA4 gene are shown in SEQ ID NO. 7 and SEQ ID NO. 8, and the probe sequence is shown in SEQ ID NO. 9.
7. The kit for detecting tuberculosis typing based on the three-gene scoring method as claimed in claim 6, wherein the kit comprises a random primer as shown in SEQ ID NO. 10-11 and a probe as shown in SEQ ID NO. 12 as exogenous internal reference.
8. The kit for detecting tuberculosis typing based on the three-gene scoring method as defined in claim 6, wherein detection is performed in different channels of the same amplification tube, detection values of the different channels are obtained as transcription levels of the corresponding genes, and the three-gene scoring is calculated according to the following formula:
three gene score = STAT1/2PARP14-EphA4/parp14+1/2;
wherein STAT1 is the transcript level of STAT1, PARP14 is the transcript level of PARP14, ephA4 is the transcript level of EphA4, and if the three gene score is in the first interval, it is judged as normal, in the second interval it is latent, and the upper threshold value greater than the second interval is active tuberculosis.
9. Use of a kit for detecting tuberculosis typing based on a three-gene scoring method according to any one of claims 5 to 8, characterized by tuberculosis latency and active tuberculosis detection for non-diagnostic purposes.
10. The application of the kit for detecting tuberculosis typing based on the three-gene scoring method according to claim 9, wherein a primer probe mixture is obtained by mixing primer probes for specifically amplifying the STAT1 gene, the PARP14 gene and the EphA4 gene and an exogenous internal reference, a ddPCR reaction system is prepared by mixing the primer probe mixture, RNA extracted from a blood sample to be detected and the ddPCR reaction system are mixed, and amplification detection is carried out under a set amplification program to obtain the detection of the transcription levels of the STAT1 gene, the PARP14 gene and the EphA4 gene.
CN202310929171.1A 2023-07-26 2023-07-26 Kit for detecting tuberculosis typing based on three-gene scoring method and application Pending CN116926215A (en)

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