CN113444823B - Primer group and method for identifying streptomyces albidoflavus W68 by using primer group - Google Patents
Primer group and method for identifying streptomyces albidoflavus W68 by using primer group Download PDFInfo
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Abstract
The invention belongs to the technical field of primer design, and discloses a primer group, which is P2939123F and P2939374R and/or P1751437F and P1751612R. The primer group provided by the invention has strong specificity, high sensitivity and high detection efficiency, and can be used for effectively identifying streptomyces albidoflavus W68.
Description
Technical Field
The invention belongs to the technical field of primer design.
Background
After releasing a target microorganism into a natural environment, the existence state or dynamic change of a target microorganism individual is monitored or traced from an environmental sample, and at present, a section of DNA gene fragment is implanted artificially into a target microorganism genome to be used as a marker, and then the detection is carried out by a genetic engineering means. The most commonly used gene labeling techniques include various fluorescent gene markers, antibiotic gene markers, auxotrophic gene markers. However, the strains marked by the means belong to genetic engineering strains, potential safety hazards exist after the strains are released into the natural environment, and controversy exists at home and abroad. Therefore, the method for searching the specific nucleic acid sequence from the genome sequence of the target microorganism as a molecular marker or a molecular label has the advantages of environmental friendliness, safety and reliability.
The DNA molecular marker is a direct reflection of the diversity of genetic materials on the DNA level, and can accurately reveal interspecies and intraspecies differences. The molecular marker is different from the traditional morphological, cytological and biochemical marking technologies, is independent of the expression of genes, is not influenced by environment and development stages, and is a direct reaction to genetic variation. The DNA molecular marker technology is often applied to diagnosis and prognosis of diseases, screening of pathogenic microorganisms in clinical medicine, identification of plant varieties or resources, and research on variety of plants in agriculture, but no research has been reported for tracing the source or destination of a species individual by the DNA molecular marker technology.
The specific segment obtained by PCR amplification belongs to the second generation molecular marker technology. A general PCR system only comprises a pair of primers, and a nucleic acid fragment is synthesized through PCR amplification. Multiplex PCR (multiplex PCR), also known as multiplex PCR, is a novel PCR amplification technology developed on the basis of conventional PCR, i.e., more than two pairs of primers are added to the same PCR reaction system, so that multiple nucleic acid fragments can be simultaneously amplified, which was proposed by Chamber hinan in 1988. The multiplex PCR can be applied to the simultaneous detection of various microorganisms, and has high efficiency, systematicness, economy and simplicity. At present, the multiplex PCR technology is most commonly used in the aspects of detection of pathogenic microorganisms, detection of food safety, research of plant genotyping, exclusive identification of microorganism species and the like in various clinics and agriculture. How to find out the primer for identifying the streptomyces albidoflauvs W68 is still a technical problem.
Disclosure of Invention
The invention discloses a primer group, which is a primer P2939123F and a primer P2939374R and/or a primer P1751437F and a primer P1751612R.
In a specific embodiment of the present invention, the primer P1751437F has the base sequence shown by SEQ ID NO.3 of the sequence Listing and the primer P1751612R has the base sequence shown by SEQ ID NO.4 of the sequence Listing.
In a specific embodiment of the present invention, the primer P2939123F has the base sequence shown by SEQ ID NO.5 of the sequence Listing and the primer P2939374R has the base sequence shown by SEQ ID NO.6 of the sequence Listing.
The invention also discloses a method for identifying the streptomyces albidoflauvs W68 by using the nuclear primer group, which comprises the following steps: designing a corresponding primer group according to the sequence information of the streptomyces albidoflauvs W68 nucleic acid fragment; adding the primer group into a PCR amplification system, and amplifying by using the streptomyces albidoflavus W68 genome DNA as a template according to a PCR amplification program to obtain an amplification product.
In an embodiment of the present invention, the sequence information of the nucleic acid fragment corresponds to the corresponding primer set by:
in an embodiment of the present invention, the PCR amplification system is: 2uL of 10 Xbuffer solution, 1.6uL of 2.5mM base triphosphate deoxynucleotide, 0.8uL of each of a forward primer and a reverse primer of 10uM, 0.2uL of DNA polymerase, 0.8uL of genomic DNA0.8978 uL of Streptomyces albidoflavus W68, and 20uL of water supplement.
In a specific embodiment of the present invention, the PCR amplification procedure is: firstly, denaturation at 94 ℃ for 4 min; secondly, denaturation at 94 ℃, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 40s for 30 cycles; and thirdly, extension is carried out for 10min at 72 ℃.
In a specific embodiment of the invention, the minimum amount of the primer P2939123F and the primer P2939374R is 0.3 uL.
In a specific embodiment of the invention, the minimum amount of the primer P1751437F and the primer P1751612R is 0.5 uL.
In a specific embodiment of the invention, the concentration of the Streptomyces albidoflauvs W68 is not less than 0.05 ng/uL.
The primer group provided by the invention has strong specificity, high sensitivity and high detection efficiency, and can be used for effectively identifying streptomyces albidoflavus W68.
Drawings
FIG. 1 is an electrophoretogram of the W68 genomic sequence amplified by primer sets P1751437F and P1751612R.
FIG. 2 is an electrophoretogram of the primers P1751437F and P1751612R verifying that W68 is derived from different strains of S.albidoflauvs from other 21 strains.
FIG. 3 is an electrophoretogram of the W68 genomic sequence amplified by primer sets P2939123F and P2939374R.
FIG. 4 is an electrophoretogram of Streptomyces albidoflavus from which the primer sets P2939123F and P2939374R verified that W68 was derived differently from the other 21 strains.
FIG. 5 is an electrophoresis diagram of two sets of primers for detecting genomic DNA of Streptomyces albidoflauvs W68 with different concentration gradients.
FIG. 6 is an electrophoretogram of two sets of primers identifying Streptomyces albidoflavus W68 and different sources of strain 21.
Detailed Description
The invention relates to a primer group, which is a primer P2939123F and a primer P2939374R and/or a primer P1751437F and a primer P1751612R.
According to 1751401-1751700 with specific sequences located in genome DNA of Streptomyces albidoflavus W68, a primer P1751437F and a primer P1751612R of a PCR primer group are designed, the primer group can amplify nucleic acid with the length of 176bp, the sequence of the nucleic acid is the base sequence described by SEQ ID NO.1 in the sequence table, and the annotation information is a hypothetic protein.
P1751437F：5’-TGCTCAACCAGTTCACCGTCAC-3’
P1751612R：5’-TCACCTCCGTCAGCTTGCCT-3’
According to 2939101-2939400 with specific sequences located in the genomic DNA of Streptomyces albidoflavus W68, a primer P2939123F and a primer P2939374R of a PCR primer group are designed, the primer group can amplify nucleic acid with the length of 252bp, the sequence of the nucleic acid is the base sequence shown in SEQ ID NO.2 in the sequence table, and the annotation information is hypothetic protein.
P2939123F：5’-CGAGAGCCCGAACACGAAGAAG-3’
P2939374R：5’-TCTGCGGCGAACAGCCAGTA-3’
The invention relates to a detection method of multiplex PCR, which adds two groups of primers, template DNA, Buffer and dNTPs into a PCR system at the same time, and can amplify simultaneously to obtain two target nucleic acid fragments with different sizes.
The primer group provided by the invention has strong specificity, high sensitivity and high detection efficiency, and can be used for effectively identifying streptomyces albidoflavus W68.
Example 1
Material method
Streptomyces albidoflavus (Streptomyces albicorflavus) W68 is separated from marine sludge, is a Streptomyces strain with remarkable biological control effect on plant fungal diseases, can be applied to microbial fertilizers or microbial pesticides, is preserved in the China general microbiological culture Collection center on 3-14 th month in 2016, has the preservation number of CGMCC No.12210, and has the preservation unit address of No.3 Homew No.1 of North Chen West Lu of the sunward area in Beijing, and the preservation unit is abbreviated as CGMCC.
The genomic sequence of Streptomyces albidoflavus W68, which has been filed on 19.11.2020 at NCBI database under GenBank accession number CP 064783.1. The genome size of the strain is 6796629 bp.
The PCR amplification system is as follows: 2uL of 10 Xbuffer (10 XTaq Buffer), 1.6uL of 2.5mM base triphosphate deoxynucleotide (dNTPs), 0.8uL of each of 10uM forward primer and reverse primer, 0.2uL of DNA polymerase (TaqDNA polymerase), 0.8uL of template DNA (genome DNA of a strain to be tested), and 20uL of water.
In all experiments this time, the initial concentration of primers was 10 uM.
The PCR amplification procedure was: the first step is as follows: denaturation at 94 deg.C for 4 min; the second step is that: denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 40s for a total of 30 cycles; the third step: extension for 10min at 72 ℃. The amplification products were detected by electrophoresis on a 2% agarose gel.
The DNA of 21 other strains of Streptomyces albidoides used in the invention is derived from the literature:
Yisong Li,Adrián A.Pinto-Tomás,Xiaoying Rong,Kun Cheng,Minghao Liu,Ying Huang*2019, position genetics instruments in optical analysis and scientific Differentiation in Streptomyces, AEM,85(7), e02555-18. the detailed information is shown in Table 1:
table 1: other 21 Streptomyces albidoflavus strains
Example 2
Screening of Streptomyces albidoflavus W68 genome sequence DNA molecular marker
Based on the complete genome sequence of the streptomyces albidoflauvs W68, the following steps are adopted to screen specific sequences:
cutting the genome sequence into 300bp window bins;
comparing the obtained bins sequence with a bacterial genome database;
filtering out bins with the alignment length of more than 100bp and the homology of more than 80 percent;
combining the filtered continuous bins.
Through the steps, 232 nucleic acid sequence fragments with different lengths are obtained;
further, the 232 nucleic acid sequences are artificially screened to obtain 10 target sequences for verification;
primer6 software is used to design primers for the 10 nucleic acid sequences, different templates are subjected to PCR amplification, and the following two specific nucleic acid fragments are finally screened out.
Example 3
Design and detection of sequence 1751401-
Primer set P1751437F and P1751612R were obtained by inputting nucleic acid sequence fragment 1751401 and 1751700 using primer6 software, and the information about the primers is shown in Table 2
Table 2: indices of primer groups P1751437F and P1751612R
After the primer sets P1751437F and P1751612R were added to the PCR amplification system in example 1 in proportion, the general PCR amplification reaction was performed according to the PCR amplification procedure in example 1.
As shown in FIG. 1, M represents Marker, and the fragment size is 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100b from top to bottomp; lane No.1 is ddH2O is a template, and no amplification product is detected; the lane numbered 2 is a target fragment of 176bp, which is determined that the base sequence of the amplification product is the same as expected by sequencing, and the amplification product is between 100bp and 200bp and is slightly close to 200bp after PCR amplification reaction by using the genome of streptomyces albidoflavus W68 as a template.
After the primer sets P1751437F and P1751612R were added to the PCR amplification system of example 1, the PCR amplification procedure of example 1 was followed to perform a general PCR amplification reaction using genomic DNAs of Streptomyces albidoflauvs of 21 strains of example 1 as templates. As shown in FIG. 2, M represents Marker, and the fragment size is 700bp, 600bp, 500bp, 400bp, 300bp, 200bp and 100bp sequentially from top to bottom; "+" represents the amplified product obtained by using the genome DNA of Streptomyces albidoflavus W68 as a template, and is located between 100bp and 200bp and is slightly close to 200bp, and the amplified product is the expected 176bp target fragment after sequencing verification; "-" represents ddH2And O is used as a template for amplification, namely a negative control.
As shown in FIG. 2, it can be seen that any amplified band was not detected; lanes numbered 1-21 represent amplification products using genomic DNAs of other 21 strains of Streptomyces albidoflauvs as templates, and the results show that no amplification band is detected between 100bp and 200bp, which is the same as the negative control; the bright band less than 100bp in the figure is primer dimer.
The above experimental results show that: the primer groups P1751437F and P1751612R are only effective on the Streptomyces albidoflavus W68, namely only can be combined with the genome of the Streptomyces albidoflavus W68, and a nucleic acid fragment with the size of 176bp is obtained through PCR amplification. The primer group obtains a nucleic acid sequence from the genomic DNA of other 21 strains of the streptomyces albidoflauvs without amplification. It follows that the primer sets P1751437F and P1751612R can specifically detect, identify or monitor whether the sample contains S.albidoflavus W68.
Example 4
Design and detection of sequence 2939101-2939400 specific primer
After inputting the nucleic acid sequence fragments 2939101-2939400 by using primer6 software, primer groups P2939123F and P2939374R were obtained, and the information about the primers is shown in Table 3
TABLE 3 indices of primer sets P2939123F and P2939374R
After the primer sets P2939123F and P2939374R were added to the PCR amplification system in example 1 in proportion, the general PCR amplification reaction was performed according to the PCR amplification procedure in example 1.
As shown in FIG. 3, M represents Marker, and the fragment size is 700bp, 600bp, 500bp, 400bp, 300bp, 200bp and 100bp sequentially from top to bottom; lane No.1 is ddH2O is a template, and no amplification product is detected; the lane numbered 2 is a target fragment with the length of 200bp-300bp detected by PCR amplification reaction with genome of Streptomyces albidoflavus W68 as template, and basically located at the middle position, and the base sequence of the amplification product is determined to be the same as expected by sequencing, and the amplification product is 252 bp. After the primer sets P2939123F and P2939374R were added to the PCR amplification system of example 1, the PCR procedure of example 1 was followed to perform a general PCR amplification reaction using genomic DNAs of Streptomyces albidoflauvs of 21 strains of example 1 as templates.
As shown in FIG. 4, M represents Marker, and the fragment size is 700bp, 600bp, 500bp, 400bp, 300bp, 200bp and 100bp sequentially from top to bottom; "+" represents the amplified product obtained by using the genomic DNA of Streptomyces albidoflavus W68 as a template, is located between 200bp and 300bp and is almost located at the middle position of 200bp to 300bp, and the product is the expected 252bp target fragment after sequencing verification; "-" represents ddH2And O is used as a template for amplification, namely a negative control. It can be seen from the figure that no amplified band was detected; lane generations Nos. 1 to 21The results of the amplification products using 21 strains of Streptomyces albidoflauvs genomic DNA as templates, respectively, show that no amplified band is detected between 200bp and 300bp, as in the negative control.
The above experimental results show that: the primer groups P2939123F and P2939374R are only effective on the Streptomyces albidoflavus W68, namely only can be combined with the genome of the Streptomyces albidoflavus W68, and a nucleic acid fragment with the size of 252bp is amplified through a PCR reaction. The primer group obtains a nucleic acid sequence from the genomic DNA of other 21 strains of the streptomyces albidoflauvs without amplification. It follows that the primer sets P2939123F and P2939374R can specifically detect, identify or monitor whether the sample contains S.albidoflavus W68.
Example 5
Optimization of multiplex PCR amplification system
In order to find the optimal ratio of the two groups of primer combinations, the multiple PCR reaction conditions are optimized on the basis of single PCR, mainly the optimization of primer concentration.
Two groups of primers are added into a PCR amplification reaction system at the same time, the volume of each primer is 0.8uL according to the PCR amplification system in the embodiment 1, and the result shows that the amplification product comprises two nucleic acid fragments, one 176bp fragment and one 252bp fragment.
The volumes of two groups of primers were optimized, including 0.8uL, 0.7uL, 0.6uL, 0.5uL, 0.4uL, 0.3uL, 0.2uL, 0.1 uL.
In the case where the volumes of the fixed primer groups P1751437F and P1751612R were 0.8uL, respectively, the amplification efficiencies of the primer groups P2939123F and P2939374R were tested from 0.8uL to 0.1uL, respectively. The results show that the volumes of the primer groups P1751437F and P1751612R are 0.8uL respectively, and the volumes of the primer groups P2939123F and P2939374R are 0.3uL respectively, so that obvious nucleic acid fragments can be obtained by amplification.
According to the above results, the amplification efficiencies of the primer sets P1751437F and P1751612R from 0.8uL to 0.1uL in the case where the optimal use volumes of the fixed primer sets P2939123F and P2939374R were 0.3uL, respectively. The results show that the volumes of the primer groups P2939123F and P2939374R are 0.3uL respectively, and the volumes of the primer groups P1751437F and P1751612R are 0.5uL respectively, so that obvious nucleic acid fragments can be obtained by amplification.
An optimized multiplex PCR amplification system was obtained by the above experiments, 2uL of 10 Xbuffer (10 XTaq Buffer), 1.6uL of 2.5mM deoxynucleotide triphosphate (dNTPs), 0.3uL of 10uM primer sets P2939123F and P2939374R, 0.5uL of 10uM primer sets P1751437F and P1751612R, 0.2uL of DNA polymerase (TaqDNA polymerase), 0.8uL of template DNA0.8uL, and water to 20 uL.
Example 6
Detection of minimum detected concentration of streptomyces albidoflavus W68 genome DNA by multiplex PCR system
The genomic DNA of the Streptomyces albidoflavus W68 was tested at different concentration gradients using the multiplex PCR amplification system of example 5, and the genomic DNA concentrations of the template Streptomyces albidoflavus W68 were 10ng/uL (lane No. 1), 5ng/uL (lane No. 2), 1ng/uL (lane No. 3), 0.5ng/uL (lane No. 4), 0.1ng/uL (lane No. 5), 0.05ng/uL (lane No. 6), and 0.01ng/uL (lane No. 7), respectively.
As shown in FIG. 5, M represents Marker, and the fragment sizes thereof are, in order from top to bottom, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, and 100 bp. The lane numbered 1 represents the concentration of the genomic DNA of Streptomyces albidoflavus W68 added to the multiplex PCR system at 10ng/uL, the lane numbered 2 represents 5ng/uL, the lane numbered 3 represents 1ng/uL, the lane numbered 4 represents 0.5ng/uL, the lane numbered 5 represents 0.1ng/uL, the lane numbered 6 represents 0.05ng/uL, and the lane numbered 7 represents 0.01 ng/uL.
The experimental result shows that the multiplex PCR system can detect at least the genomic DNA of the streptomyces albidoflavus W68 with the concentration of 0.05-0.1 ng/uL.
Example 7
Application of multiplex PCR amplification system
In order to test the specificity of the multiplex PCR amplification system, the genomic DNA of 21 other strains of Streptomyces albidoflauvs was selected, and the specific information was as described in example 1.
As shown in FIG. 6, the result is represented by "W68" which represents an amplification product using the genomic DNA of Streptomyces albidoflavus W68 as a template, "-" which represents a negative resultControl, i.e. using equal volume of ddH for template DNA2O, lanes 1-21 represent the amplification products using genomic DNA from 21 other strains of Streptomyces albidoides as templates. As can be seen from the figure, only the genomic DNA of the streptomyces albidoflavus W68 is taken as a template to amplify two nucleic acid fragments which are respectively 176bp and 252bp target fragments after sequencing; no amplified band was detected in any of the other amplification systems using the genomic DNA of 21 strains of Streptomyces albidoflauvs as a template, which was the same as the negative control. The result shows that the two groups of primers and the multiplex PCR amplification reaction system can be used in the related fields of detection, identification, monitoring and the like of the streptomyces albidoflavus W68. The primer group provided by the invention has strong specificity, high sensitivity and high detection efficiency, and can be used for effectively identifying streptomyces albidoflavus W68.
The two groups of primer nucleic acid fragments can distinguish the streptomyces albidoflavus W68 from other streptomyces albidoflavus strains, and the identification method has the advantages of strong specificity, high detection rate, sample saving and the like.
Sequence listing
<110> institute of microbiology of Chinese academy of sciences
<120> primer group and method for identifying streptomyces albidoflavus W68 by using same
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 176
<212> DNA
<213> Streptomyces albidoflavus (Streptomyces albiciflavus)
<400> 1
tgctcaacca gttcaccgtc acccccgacg acatgcccga cctcgacgcc ggatccggcg 60
aacagcccga catcccaccc cacgagccga cgcagaagaa gaaccgccga ggccgcaata 120
ccgtcttcga cgacgtcggc cccgccgcgg ccgacgaggc aagctgacgg aggtga 176
<210> 2
<211> 252
<212> DNA
<213> Streptomyces albidoflavus (Streptomyces albiciflavus)
<400> 2
cgagagcccg aacacgaaga agaacgcgaa agcctgggag cagcgcaccg tactcttcca 60
ggaagagcgc caccgttgcg agtggcacgg aaaaagactg tgggaccgcg atcgcgtcca 120
tttctctctc cctctgcagg cgcatgacgg ccgggtcctc attggcatct tcaccgacca 180
cctcgacacc tgaggcgatc gcgcgctccg tgcggctgtc tccaggtgcc cgtactggct 240
gttcgccgca ga 252
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tgctcaacca gttcaccgtc ac 22
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cgagagcccg aacacgaaga ag 22
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Claims (7)
1. The primer group is characterized by comprising a primer P2939123F and a primer P2939374R and/or
Primer P1751437F and primer P1751612R;
the nucleotide sequence of the primer P1751437F is shown as SEQ ID NO.3 in the sequence table;
the nucleotide sequence of the primer P1751612R is shown as SEQ ID NO.4 in the sequence table;
the nucleotide sequence of the primer P2939123F is shown as SEQ ID NO.5 in the sequence table;
the nucleotide sequence of the primer P2939374R is shown as SEQ ID NO.6 in the sequence table.
2. The method for identifying Streptomyces albidoflauvs W68 by using the primer set according to claim 1, comprising the following steps:
designing a corresponding primer group according to the sequence information of the streptomyces albidoflauvs W68 nucleic acid fragment;
adding the primer group into a PCR amplification system, and obtaining an amplification product after amplification according to a PCR amplification program by taking the streptomyces albidoflavus W68 genome DNA as a template;
the sequence information of the streptomyces albidoflavus W68 nucleic acid fragment and the corresponding primer group have the corresponding relation:
3. the method of claim 2, wherein the PCR amplification system is: 10 Xbuffer 2uL,2.5mM triphosphate base deoxynucleotide 1.6uL,10uM forward primer and reverse primer each 0.8uL, DNA polymerase 0.2uL, Streptomyces albidoflavus W68 genomic DNA0.8uL, and water to 20 uL.
4. The method of claim 3, wherein the PCR amplification procedure is:
the first step is as follows: denaturation at 94 deg.C for 4 min;
the second step is that: denaturation at 94 ℃, 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 40s for a total of 30 cycles;
the third step: extension at 72 ℃ for 10 min.
5. The method of claim 2, wherein the minimum amount of primer P2939123F and primer P2939374R is 0.3 uL.
6. The method of claim 2, wherein the minimum amount of primer P1751437F and primer P1751612R is 0.5 uL.
7. The method according to claim 2, wherein the concentration of Streptomyces albidoflauvs W68 is not less than 0.05 ng/uL.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103667461A (en) * | 2013-11-25 | 2014-03-26 | 盎亿泰地质微生物技术（北京）有限公司 | PCR primers for amplification of Streptomyces, and method and kit for detecting Streptomyces |
| CN105886428A (en) * | 2016-04-05 | 2016-08-24 | 中国科学院微生物研究所 | Streptomyces albidoflavus and applications thereof in microbial fertilizers |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667461A (en) * | 2013-11-25 | 2014-03-26 | 盎亿泰地质微生物技术（北京）有限公司 | PCR primers for amplification of Streptomyces, and method and kit for detecting Streptomyces |
| CN105886428A (en) * | 2016-04-05 | 2016-08-24 | 中国科学院微生物研究所 | Streptomyces albidoflavus and applications thereof in microbial fertilizers |
Non-Patent Citations (1)
| Title |
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| Candicidin Isomer Production Is Essential for Biocontrol of Cucumber Rhizoctonia Rot by Streptomyces albidoflavus W68;Xueyan Yao等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20210531;第87卷(第9期);1-14 * |
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