CN111621607A - Method and kit for detecting HBV genotype and/or X region mutation, CDS standard sequence of HBx, primer and application - Google Patents
Method and kit for detecting HBV genotype and/or X region mutation, CDS standard sequence of HBx, primer and application Download PDFInfo
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Abstract
The invention discloses a method and a kit for detecting HBV genotype and/or X region mutation, a CDS standard sequence of HBx, a primer and application. Selecting CDS of HBx in the middle of the sequencing sequence and CDS standard sequences of 10 HBx to perform homology analysis and comparison to obtain the genotype and/or X region mutation result of the HBV to be detected; further, single genotype and mixed genotype can be judged according to the peak nesting condition in the sequencing peak image, and if no peak nesting exists or the number is less than 10, the single genotype is judged; if the number of the set peaks is more than or equal to 10, the mixed genotype is judged. The invention provides a set of convenient and high-accuracy scheme for simultaneously detecting 10 HBV genotypes and X region mutation types, and can provide reference basis for HBV infected person disease progress risk assessment, antiviral population screening, antiviral mode selection and curative effect prediction.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method and a kit for detecting 10 HBV genotypes and X region mutations by combining PCR (polymerase chain reaction) directional sequencing with standard sequence comparison, a CDS (coding sequence) standard sequence, primers and application of HBx.
Background
China is the country with the highest incidence and death rate of liver cancer worldwide, and it is estimated that the number of deaths from liver cancer in China is about half of the number of deaths from liver cancer worldwide. Persistent infection with Hepatitis B Virus (HBV) is the leading cause of hepatoma in our country, with about 76% of hepatoma occurrences being associated with (de Martel C, Mahort-BoulchD, Plummer M, et al. world-wide relative distribution of hepatomas B and Cviruses in hepatocellular cancer. hepatology,2015,62(4): 1190-. It is currently believed that the levels of HBV DNA and HBsAg are key contributors to the prognosis of chronic hepatitis b, but HBV genotype and HBV genomic mutations are closely related to the progression and prognosis of chronic hepatitis b. Patients with low HBV DNA and HBsAg levels are at the lowest Risk of progressing to cirrhosis/liver cancer, while high HBV DNA and HBsAg levels are at the highest Risk of combining specific genotype HBV infection with HBV genomic mutations (Chen, C.J., et al, Risk of hepatogenic Carcinoma biological diagnosis of serum hepatitides B viral DNA levels, 2006.295(1): p.65-73.; Yang, H.I., et al, associates B viral genes and mutations and the Risk of hepatocellular Carcinoma. J Natl cancer Insts, 2008.100(16): p.1134-43.).
Based on the differences in HBV genome-wide sequences, 10 genotypes have been identified (A-J, sequence differences of more than 7.5%). HBV of different genotypes has significant differences in geographical distribution, transmission pathways, disease outcome, and response to existing treatments. The main popularity in European and American countries is A and D, the most popular in our country is B and C, and southern is dominated by B-type flow and northern is dominated by C-type flow. Forms a and D are mainly transmitted horizontally across the population; compared with infection type D, the treatment effect of infection type A by adopting IFN-alpha is better, but the rate of the progression to cirrhosis and liver cancer is higher. Type B and C are primarily transmitted vertically by mother and infant, patients infected with type B have better IFN- α treatment and have a lower rate of progression to cirrhosis and liver cancer than type C (Rajiiya N, Combet C, Zoulim F, et al. how much viral genetic variants and genetic infections B. time for an induced viral approach J Hepatol,2017,67(6): 1281-1297).
HBV X region gene mutation detection and significance: the HBV genome is about 3200bp in total length and consists of P, S, X and C regions which are completely or partially overlapped, wherein a typical X region is a region of 1374-1838 and is HBx CDS, and the HBx protein of 154 amino acids in total length is coded. And the X region contains the elements of an HBV Enhancer II (Enhancer II, 1636-1744) and a basic core promoter (basal core promoter, BCP, 1751-1769), and a great deal of research shows that the mutation of a specific site in the region is closely related to the disease progression and poor prognosis of hepatitis B. For example: G1613A, C1653T, T1753V, A1762T, G1764A and 3' terminal fragment deletion mutation in gene mutation in the X region can obviously increase the risk of hepatocellular carcinoma, wherein the simultaneous mutation of A1762T/G1764A double sites is an independent risk factor for HBV-related liver cancer; T1753V, A1762T, G1764A, C1766T and T1768A significantly increase The risk of The development of chronic plus acute liver failure (Lin CL, Kao JH. Naturalhistory of acid and chronic hepatitis B: The role of HBV genetics and pharmaceuticals. best practice Res Clin Gastroenterol,2017,31(3): 249-255).
The current principle of HBV genotyping is based on the sequence difference of HBV of different genotypes in a specific region, and the genotyping method mainly comprises restriction fragment length polymorphism analysis, type specificity probe method detection, gene chip method analysis, gene sequencing method identification and the like. Restriction fragment length polymorphisms are rarely used because of their low detection efficiency. At present, the most used parting methods are a type specific probe method and a gene chip method, the type specific probe method has high sensitivity and more complex operation, each genotype needs to be designed with a corresponding specific probe, and the detection cost is not low; the chip used in the gene chip method is complicated to manufacture and has high detection cost. And the typing specificity probe method and the gene chip method can not obtain the HBV sequence information, so that the mutation information can not be obtained. The gene sequencing method mainly includes whole gene sequencing, S gene sequencing and the like. The whole gene sequencing is the 'gold standard' of HBV genotyping, has the highest typing accuracy, can obtain complete mutation site information, and is time-consuming, labor-consuming and high in cost; the S gene sequencing and typing can obtain partial sequence information, the typing accuracy is high, but the sequence information of an X region containing more known pathogenic mutations cannot be obtained.
At present, no report related to a method and a reagent for typing by using X-region sequencing and simultaneously detecting the X-region mutation of HBV exists.
Disclosure of Invention
The primary object of the present invention is to provide a method for detecting all 10 known genotypes of HBV and simultaneously detecting the mutation in the X region, which is accurate, rapid, simple, convenient and economical. The method is mainly based on the full-length sequence of the virus X region to detect the genotype of HBV and the mutation of the X region.
A method for detecting mutations in the genotype and/or X region of HBV comprising the steps of:
(1) designing a universal primer capable of amplifying 10 genotype HBV virus genome X regions aiming at conserved regions at two ends of the X region of HBV to carry out PCR (polymerase chain reaction) directional amplification, and then sequencing;
(2) selecting CDS of HBx in the middle of the sequencing sequence and CDS standard sequences of 10 HBx to perform homology analysis and comparison to obtain the genotype and/or X region mutation result of the HBV to be detected;
the CDS standard sequence of HBx corresponding to HBV-A type standard whole genome sequence is shown in SEQ ID NO. 13;
the CDS standard sequence of HBx corresponding to HBV-B type standard whole genome sequence is shown in SEQ ID NO. 14;
the CDS standard sequence of HBx corresponding to HBV-C type standard whole genome sequence is shown in SEQ ID NO. 15;
the CDS standard sequence of HBx corresponding to HBV-D type standard whole genome sequence is shown in SEQ ID NO. 16;
the CDS standard sequence of HBx corresponding to HBV-E type standard whole genome sequence is shown in SEQ ID NO. 17;
the CDS standard sequence of HBx corresponding to HBV-F type standard whole genome sequence is shown in SEQ ID NO. 18;
the CDS standard sequence of HBx corresponding to HBV-G type standard whole genome sequence is shown in SEQ ID NO. 19;
the CDS standard sequence of HBx corresponding to HBV-H type standard whole genome sequence is shown in SEQ ID NO. 20;
the CDS standard sequence of HBx corresponding to HBV-I type standard whole genome sequence is shown in SEQ ID NO. 21;
the CDS standard sequence of HBx corresponding to HBV-J type standard whole genome sequence is shown in SEQ ID NO. 22.
The invention obtains the standard sequences of known 10 HBV genotypes (A-J) and the standard HBx CDS (see a sequence table) of the corresponding X region thereof respectively based on the analysis and statistics of HBV whole genome sequences of known genotypes in a GenBank database. Wherein HBV-A-S is obtained by counting 55 HBV whole genome sequences known as type A, HBV-B-S is obtained by counting 50 HBV whole genome sequences known as type B, HBV-C-S is obtained by counting 51 HBV whole genome sequences known as type C, HBV-D-S is obtained by counting 61 HBV whole genome sequences known as type D, HBV-E-S is obtained by counting 46 HBV whole genome sequences known as type E, HBV-F-S is obtained by counting 37 HBV whole genome sequences known as type F, HBV-G-S is obtained by counting 26 HBV whole genome sequences known as type G, HBV-H-S is obtained by counting 15 HBV whole genome sequences known as type H, and HBV-I-S is obtained by counting 27 HBV whole genome sequences known as type I. Since only one HBV-J sequence is reported in the literature, and there are only 1 HBV-J sequence in the GenBank database, HBV-J-S is the sequence recorded in the database.
TABLE 1
The whole genome standard sequence of the HBV-A-S, HBV-B-S, HBV-C-S, HBV-D-S, HBV-E-S, HBV-F-S, HBV-G-S, HBV-H-S, HBV-I-S, HBV-J-S is shown in SEQ ID NO.3-12 in the sequence table.
The above rules for determining the whole genome sequence of standard HBV: putting the whole genome sequence of the known genotype in each GenBank database in the table into sequence comparison software according to the genotype, arranging and comparing by using a Cluster W Method, and counting a sequence with the most conservative genotype according to the principle that each site is a base with the highest occurrence frequency, wherein the conservative sequence is a standard sequence of the genotype. The determination of the standard whole genome sequence is illustrated by taking HBV-A-S as an example, as shown in FIG. 1. Among 55 sequences known as type A, for site 1, 51 sequences were T, 3 sequences were C, and 1 sequence was G, so site 1 of HBV-A-S sequence was identified as "T"; for site 2, all 55 sequences were T, so site 2 of HBV-A-S sequence was identified as "T"; for site 10, 28 sequences are T, 26 sequences are C, 1 sequence is G, so site 10 of HBV-A-S sequence is determined as "T", and other positions are the same, and the final HBV-A-S sequence is shown as an arrow in FIG. 1, which represents the most conservative HBV whole genome sequence most representative of type A based on 55 known type A HBV whole genome sequences.
The 10 standard HBx CDS are obtained by extracting HBx CDS from corresponding 10 HBV standard sequences. See SEQ ID NO.13-22 of the sequence Listing.
In the standard sequence analysis of 10 HBV genotypes (A-J), the invention finds that the difference between HBV whole genome sequences is similar (homology is 84.7% -93.3%), the homology between HBx CDS sequences of HBV with different genotypes is 84.5% -96.8%, and the difference of about 15-72 nucleotide sequences exists, as shown in Table 2 below. Thus, differences in HBx CDS sequence can be exploited for HBV genotyping.
Table 210 HBV whole genome standard sequences and 10 HBx CDS standard sequence homology comparison results
Based on the obtained standard HBV sequence comparison and analysis, the invention designs a universal primer capable of amplifying 10 genotype HBV X regions aiming at conserved regions at two ends of the X region. The fragment amplified by the primer is about 618bp (corresponding to the region of 1262-1839 HBV genome) and comprises the complete HBx CDS (corresponding to the region of 1374-1838 HBV genome), the 3 'end of the upstream primer X1 is about 92 nucleotides away from the initiation codon (ATG) of the HBx CDS, and the 3' end of the downstream primer X2 is 22 nucleotides away from the termination codon (TAA) of the HBx CDS, so that the sequencing peak maps corresponding to the region (from ATG to TAA) of the HBx CDS in the sequencing peak map obtained by using X1 as the sequencing primer are stable peak maps.
FIG. 2 shows the alignment results of the whole genome sequence of 10 standard HBV, and the region targeted by the universal primers is the conserved region of 10 genotypes.
The detection method of the present invention can exclude the diagnosis purpose of genotyping HBV virus, mutational site analysis and judgment of single genotype or mixed genotype, and is only limited to scientific research as an intermediate result.
The detection method comprises the following steps: firstly extracting HBV DNA, and carrying out PCR directional amplification by using a designed and synthesized universal primer of an X region. And recovering the amplification product by using an agarose gel kit, and carrying out DNA sequencing reaction on the recovered PCR product by using an upstream primer of the universal primer in the X region used in the amplification stage. Respectively extracting CDS of HBx from all obtained sequencing results, performing homology analysis and comparison with CDS standard sequences of 10 HBx, and determining which genotype the sequence to be detected has the highest homology with which standard sequence to obtain the genotype of HBV to be detected and/or the mutation result of the X region; further, single genotype and mixed genotype can be judged according to the peak nesting condition in the sequencing peak image, and if no peak nesting exists or the number is less than 10, the single genotype is judged; if the number of the set peaks is more than or equal to 10, the mixed genotype is judged.
The standard for judging the peak is as follows: there are two peaks superimposed on each other at the same location, with the lower peak occupying at least 1/5 the height of the higher peak.
Description of the genotype judgment criteria: the invention adopts a threshold value of the homology of a sequence to be detected and which standard sequence has the highest homology instead of setting the homology as the standard for judging the genotype. Because it is not practical to determine the genotype by setting a threshold for the magnitude of homology, it makes typing more complicated and is not suitable for the sequence to be examined for deletion mutation. For example: if the homology with any HBx CDS standard sequence (465bp) is set to be more than 97 percent, then the sequence is found to be deletion mutant with only 456bp (such as HBx CDS detected in serum of sample No.2 in example 2), the sequence has 9 nt difference with all standard sequences in nature, the homology with any standard sequence is less than 97 percent (79.4 to 96.1 percent of homology with the standard sequence) in comparison result, and if the threshold value is specified, the genotype cannot be judged by the deletion mutant sequence; if only the highest homology is determined to which standard sequence, it can still be genotyped (sample No.2 in example 2 has a homology of 96.1% to HBx-C-S for HBx CDS). This is also one of the advantages of the present invention, as compared to type-specific probe methods, which are based on the design of probes for typing based on several nucleotide sequences characteristic between different genotypes, the method is ineffective if the very characteristic sequence is mutated or deleted. The invention carries out typing depending on dozens or dozens of different sites of different genotypes in the 465bp range, and if a certain characteristic small break mutation or deletion in the sequence to be detected still can carry out typing according to the different site information contained in other segments.
Description of the Mixed genotype judgment method: the threshold set for mixed genotype determination based on the set of peaks is the approximate threshold set for sequencing alignment combined with subjective determination. Since the number of differential nucleotides between the HBx CDS of the standard sequence is between 15 and 72, theoretically at least 15 sets of peaks should appear for a mixture of different genotypes. While the maximum number of sets of peaks in 14 samples was 4, the theoretical and practical trade-offs were defined at 10 sets of peaks. According to the invention, for sequencing peak graphs with more than 10 nested peaks, the sequence given by sequencing is still adopted for typing, and the obtained sequence is a genotype sequence with relatively high abundance. If we want to further find out which genotypes are mixed specifically, we can make the PCR amplified product undergo the process of blunt end cloning, then transform it into competent colibacillus plating, pick up a certain number of colonies (generally about 10 colonies) and continue the sequencing with X1 (the obtained sequencing peak diagram is theoretically single peak at each position), and then make the sequencing sequence by genotyping of the invention, and we can get the exact combination of which genotypes.
The second object of the present invention is to provide a kit for detecting HBV genotype and/or mutation in X region, comprising:
the universal primers which are designed aiming at conserved regions at two ends of an X region of HBV and can amplify X regions of 10 genotypes of HBV virus genomes and CDS standard sequences of 10 HBx are as follows:
the CDS standard sequence of HBx corresponding to HBV-A type standard whole genome sequence is shown in SEQ ID NO. 13;
the CDS standard sequence of HBx corresponding to HBV-B type standard whole genome sequence is shown in SEQ ID NO. 14;
the CDS standard sequence of HBx corresponding to HBV-C type standard whole genome sequence is shown in SEQ ID NO. 15;
the CDS standard sequence of HBx corresponding to HBV-D type standard whole genome sequence is shown in SEQ ID NO. 16;
the CDS standard sequence of HBx corresponding to HBV-E type standard whole genome sequence is shown in SEQ ID NO. 17;
the CDS standard sequence of HBx corresponding to HBV-F type standard whole genome sequence is shown in SEQ ID NO. 18;
the CDS standard sequence of HBx corresponding to HBV-G type standard whole genome sequence is shown in SEQ ID NO. 19;
the CDS standard sequence of HBx corresponding to HBV-H type standard whole genome sequence is shown in SEQ ID NO. 20;
the CDS standard sequence of HBx corresponding to HBV-I type standard whole genome sequence is shown in SEQ ID NO. 21;
the CDS standard sequence of HBx corresponding to HBV-J type standard whole genome sequence is shown in SEQ ID NO. 22.
Furthermore, the sequence of an upstream primer and sequencing primer X1 capable of amplifying the X region of the 10 genotypes of HBV virus genome is shown as SEQ ID NO. 1; the sequence of a downstream primer X2 capable of amplifying the X region of 10 genotype HBV genomes is shown as SEQ ID NO. 2.
The third purpose of the present invention is to provide the CDS standard sequence of HBx in the above 10 HBV viral genomes; and a universal primer capable of amplifying the X region of the 10 genotypes of the HBV virus genomes.
The fourth purpose of the present invention is to provide the application of the CDS standard sequence of HBx in the above 10 HBV virus genomes and the universal primer capable of amplifying the X region of 10 genotype HBV virus genomes in the preparation of reagents for detecting 10 HBV genotypes and/or X region mutations.
The invention mainly aims to obtain an HBV typing, a mutation target point and an intermediate result of scientific research on whether the HBV typing and the mutation target point are mixed typing or not, and the HBV typing and the mutation target point are not used as diagnosis results. These intermediate results can further assist in determining the risk of HBV infected persons developing severe liver diseases such as liver cirrhosis and liver cancer, and the like, and can provide reference for the selection of different types of antiviral methods and the prediction of curative effect, and the method can be used for evaluating the risk of HBV infected persons developing, screening antiviral people, selecting antiviral modes and predicting the curative effect.
The invention has the following advantages and effects:
(1) wide typing spectrum: the X region amplification primer can amplify the X region of all genotypes of HBV, and the typing spectrum covers all the genotypes of the known HBV.
(2) The function is various: besides genotyping HBV, the invention can also obtain important information of related mutation of X region and whether HBV with mixed typing exists.
(3) Simple and economical: only one pair of universal primers is used for the whole-course detection of the known 10 genotypes, different primers or probes are not required to be designed for different genotypes, only one conventional PCR reaction and one sequencing reaction are required, and related mutation results of the genotypes and the X region are analyzed in batch, so that the operation is simple and convenient, and the cost is greatly reduced.
Drawings
FIG. 1 is a diagram illustrating the determination of a standard whole genome sequence, taking HBV-A-S as an example;
FIG. 2 shows the alignment of 10 standard HBV genome-wide sequences;
FIG. 3 is an electrophoretogram of PCR products of 14 samples in example 2;
FIG. 4 shows CDS sequences and alignment of HBx for 14 samples in example 2;
FIG. 5 is a graph showing the results of homology analysis (evolutionary tree) of the CDS sequences of HBx in 14 samples in example 2;
FIG. 6 shows the results of genotyping using real-time fluorescence PCR as performed in Saint Valer medical examination of Hunan for samples Nos. 4 and 14 in example 2;
FIG. 7 is the CDS sequences and alignment of HBx mixed in the ratio of 1:1(C + B), 2:1(2C + B) and 5:1(5C + B) from sera of samples No. 4 (known as type C) and No.14 (known as type B) in example 3;
FIG. 8 is a graph showing the results of homology analysis of CDS sequences of HBx mixed in the ratio of 1:1(C + B), 2:1(2C + B) and 5:1(5C + B) in sera of samples No. 4 (known as type C) and No.14 (known as type B) in example 3;
FIG. 9 is a sequencing peak corresponding to CDS of sample HBx No. 4 in example 2;
FIG. 10 is a graph showing a sequencing peak corresponding to the CDS of sample HBx No.14 in example 2;
FIG. 11 is a graph showing sequencing peaks corresponding to CDS of HBx in which sera of samples No. 4 (known as type C) and No.14 (known as type B) in example 3 were mixed at a ratio of 1:1(C + B), respectively;
FIG. 12 is a graph showing sequencing peaks corresponding to CDS of HBx in which sera of samples No. 4 (known as type C) and No.14 (known as type B) in example 3 were mixed at a ratio of 2:1(2C + B), respectively;
FIG. 13 is a graph showing sequencing peaks corresponding to CDS of HBx in which sera of samples No. 4 (known as type C) and No.14 (known as type B) in example 3 were mixed at a ratio of 5:1(5C + B), respectively;
FIG. 14 is a graph showing the results of homology analysis of 10 complete genome sequences each selected from the GenBank database for defining HBV genotypes A-I with the CDS sequence of HBx determined in the present invention in example 4.
Detailed Description
The invention will now be further described with reference to examples, but the practice of the invention is not limited thereto.
Example 1 the composition of the reagent of the invention is as follows:
HBV DNA extraction reagent: nucleic acid extraction or purification reagents (magnetic bead method) from san Xiang Biotechnology Ltd.
Primer: the sequence of the upstream primer of the X region is as follows: 5'-CGATCCATACTGCGGAACTC-3' (shown in SEQ ID NO: 1); the sequence of the downstream primer of the X region is as follows: 5'-CAGCTTGGAGGCTTGAACA-3' (shown in SEQ ID NO: 2). The primer is synthesized by Beijing Optimalaceae New Biotechnology Co.
Negative and positive controls: deionized water was used as a Negative Control (NC) and HBV1.3 plasmid (known as HBV-D type) was used as a Positive Control (PC).
PCR reaction reagent: the high-performance high-fidelity PCR amplification reagent KOD FX (cat: KFX-101) from TOYOBO was used, and it contained 2 XPCR buffer for KOD FX, 2mM dNTPs and KOD FX (1.0U/. mu.L).
Setting a PCR amplification program: on a PCR instrument (Eppendorf), pre-denaturation: 94 ℃ for 2 min; and (3) PCR circulation: circularly amplifying for 45 times at 98 ℃ for 10s, 60 ℃ for 30s and 68 ℃ for 38 s; finally, the temperature is 68 ℃ for 7 min.
And (3) identifying and purifying PCR products: the gel is cut and recovered after 1 percent agarose gel electrophoresis, and a DNA fragment with the band size of 618bp is recovered by using a gel recovery kit.
Sequencing: sequencing by Biotechnology Ltd of Kyoto encyclopedia
Example 2 detection of HBV genotype and mutation in X region by the method of example 1
For example, the genotype and X region mutation of HBV in peripheral blood samples of 14 hepatitis B infected persons were examined. And (3) detection flow: firstly, obtaining a clinical hepatitis B infected person peripheral blood sample, and quickly extracting HBV DNA; preparing a PCR reaction system for X-region directional amplification, recovering PCR amplification products, carrying out sequencing reaction, and finally comparing the sequencing result with CDS standard sequences (shown as sequence table SEQID No.13-22) of 10 HBx to determine the HBV genotype and X-region mutation condition corresponding to the sequenced sequence.
The method comprises the following specific steps:
1) serum HBV DNA extraction: extracting HBV DNA from 200 mu L serum of HBV infected person according to the operating method of Shengxiang biological nucleic acid extraction or purification reagent specification, and finally using 20 mu L deionized water to elute magnetic beads to obtain enriched and purified HBV DNA extracting solution.
2) And (3) PCR amplification:
the PCR reaction system was 20. mu.L, and was specifically prepared as shown in Table 3 below.
TABLE 3
And (3) PCR reaction: the reaction was performed on a PCR instrument (Eppendorf) and the amplification procedure was as follows:
3) and (3) identifying and purifying PCR products: 1% agarose gel electrophoresis, the results are shown in FIG. 3, and then the PCR product is recovered by cutting the gel using a gel recovery kit (Promega corporation) (II)
SV Gel and PCRClean-Up System, Cat No.: a9281) Recovering the amplified fragment of the purified X region.
4) Sequencing: the sample is sent to Beijing Optimalaceae New Biotechnology Co., Ltd, and the upstream amplification primer (SEQID: NO 1) of the X region is adopted as a sequencing primer for sequencing.
5) Data processing and analysis: the CDS of HBx (from the start codon ATG to the stop codon TAA, about 465bp) in the middle of the sequencing sequence and CDS standard sequences of 10 HBx (see the sequence table SEQ ID NO.13-22) are selected for homology analysis and alignment, and are shown in Table 4 and FIG. 5.
TABLE 4
| Patient numbering | Length (bp) of HBx CDS obtained by sequencing | Number of "nested peaks" in sequencing |
| 1 | 465 | 0 |
| 2 | 456 | 1 |
| 3 | 465 | 0 |
| 4 | 465 | 0 |
| 5 | 465 | 3 |
| 6 | 465 | 0 |
| 7 | 465 | 2 |
| 8 | 465 | 2 |
| 9 | 465 | 4 |
| 10 | 465 | 1 |
| 11 | 465 | 1 |
| 12 | 465 | 0 |
| 13 | 465 | 0 |
| 14 | 465 | 1 |
6) As a result: the genotype and mutation site were determined according to the sequence homology analysis and the alignment results, as shown in Table 5.
TABLE 5
In the table, del.1756-1764 represents deletion mutation of 1756-1764 sites of X region of patient No.2, and X regions of viruses in blood serum of other patients are point mutation; the literature reports that the deletion mutation at the 3' end of the X region is closely related to the occurrence of liver cancer.
G1613A, C1653T, T1753V, A1762T, G1764A and 3' terminal fragment deletion mutation in gene mutation in the X region can obviously increase the risk of hepatocellular carcinoma, wherein the simultaneous mutation of A1762T/G1764A double sites is an independent risk factor for HBV-related liver cancer; T1753V, A1762T, G1764A, C1766T and T1768A can obviously increase the risk of the occurrence of chronic plus acute liver failure
7) And (3) verification: the template of PC is HBV1.3 plasmid with known genotype as D type, and the genotype identified by the method is also D type, which is consistent with the actual situation; further selecting serum samples No. 4 and No.14, sending the serum samples to a Hunan Saint Valer medical test to perform genotype identification by adopting real-time fluorescence PCR, and obtaining results which are shown in figure 6 and are consistent with the genotype identified by the method.
Example 3 detection of different HBV genotype mixes using the method of example 1
Sample sera No. 4 (known as type C) and 14 (known as type B) were mixed in a ratio of 1:1(C + B), 2:1(2C + B) and 5:1(5C + B), respectively, and the total volume was 200. mu.L. The rest of the procedure was the same as in example 2.
Data processing and analysis are shown in FIGS. 6-12 and tables 6-7.
And (4) judging a result: judging single genotype or mixed genotype according to whether the sequencing peak graph corresponding to HBx CDS has scattered 'nested peaks' (two peaks superposed with each other at the same position, and the lower peak at least occupies the height of the upper peak) and the quantity: if no nested peaks exist or the number is less than 10, judging the gene type to be a single gene type; if the number of the nested peaks is more than or equal to 10, the mixed genotypes are considered. And (4) carrying out homology analysis and comparison result according to the read main peak sequence to judge main genotypes and mutation sites.
TABLE 6
| Test sample | Length (bp) of HBx CDS obtained by sequencing | Number of "nested peaks" in sequencing Peak map |
| C+B | 465 | 26 |
| 2C+B | 456 | 29 |
| 5C+B | 465 | 25 |
TABLE 7
Example 4 further evaluation of the accuracy of HBV genotyping methods using sequences based on region X
10 complete genome sequences (not containing the sequences subjected to statistics) of definite HBV genotypes A-I are selected from a GenBank database, HBx CDS sequences are extracted, 10 standard HBx sequences (shown in a sequence table SEQ ID No.13-22) provided by the genotyping method are compared for genotyping, and the applicability and the accuracy of the method are further evaluated. Since only 1 case of HBV-J type is reported at present, and only 1 genome sequence exists in the database, the HBV-J type can be classified and judged only by the sequence. The information of the test sequences is shown in table 8 below.
TABLE 8
The results of the sequence homology comparison are shown in FIG. 12, and show that the methods provided by the present invention are consistent with the results based on the whole genome typing in the database. Further illustrating the reliability of the method of the present invention.
Sequence listing
<110> Xiangya II Hospital of Zhongnan university
<120> method and kit for detecting HBV genotype and/or X region mutation, CDS standard sequence of HBx, primer and application
<160>22
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
cgatccatac tgcggaactc 20
<210>2
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
cagcttggag gcttgaaca 19
<210>3
<211>3221
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
ttccacagct ttccaccaag ctctgcaaga tcccagagtc aggggtctgt attttcctgc 60
tggtggctcc agttcaggaa cagtaaaccc tgttccgaat attgcctctc acatctcgtc 120
aatctcctcg aggactgggg accctgcgac gaacatggag aacatcacat caggattcct 180
aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240
gcagagtcta gactcgtggt ggacttctct caattttcta gggggatcac ccgtgtgtct 300
tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcctgtc ctccaatttg 360
tcctggttat cgctggatgt gtctgcggcg ttttatcata ttcctcttca tcctgctgct 420
atgcctcatc ttcttattgg ttcttctgga ttatcaaggt atgttgcccg tttgtcctct 480
aattccagga tcaacaacaa ccagtacggg accctgcaaa acctgcacga ctcctgctca 540
aggcaactct atgtttccct catgttgctg tacaaaacct acggatggaa attgcacctg 600
tattcccatc ccatcatctt gggctttcgc aaaataccta tgggagtggg cctcagtccg 660
tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720
tgtttggctt tcagctatat ggatgatgtg gtattggggg ccaagtctgt acaacatctt 780
gagtcccttt ataccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840
aacaaaacaa aaagatgggg ttattcccta aacttcatgg gttacgtaat tggaagttgg 900
ggaacattgc cacaggatca tattgtacaa aaaatcaaac actgttttag aaaacttcct 960
gttaacaggc ctattgattg gaaagtatgt caaagaattg tgggtctttt gggctttgct 1020
gctccattta cacaatgtgg atatcctgcc ttaatgcctt tgtatgcatg tatacaagct 1080
aaacaggctt ttactttctc gccaacttac aaggcctttc taagtaaaca gtatatgaac 1140
ctttaccccg ttgcccggca acggcctggt ctgtgccaag tgtttgctga cgcaaccccc 1200
actggctggg gcttggccat aggccatcag cgcatgcgtg gaacctttgt ggctcctctg 1260
ccgatccata ctgcggaact cctagccgct tgttttgctc gcagccggtc tggagcaaaa 1320
ctcatcggga ctgataattc tgtcgtcctt tctcggaaat atacatcgtt tccatggctg 1380
ctaggctgta ctgccaactg gattcttcgc gggacgtcct ttgtttacgt cccgtcggcg 1440
ctgaatcccg cggacgaccc ctctcggggc cgcttgggac tctatcgtcc ccttctccgt 1500
ctgccgtacc ggccgaccac ggggcgcacc tctctttacg cggtctcccc gtctgtgcct 1560
tctcatctgc cggtccgtgt gcacttcgct tcacctctgc acgttgcatg gagaccaccg 1620
tgaacgccca tcagatcctg cccaaggtct tacataagag gactcttgga ctcccagcaa 1680
tgtcaacgac cgaccttgag gcctacttca aagactgtgt gtttaaagac tgggaggagt 1740
tgggggagga gattaggtta aaggtctttg tattaggagg ctgtaggcat aaattggtct 1800
gcgcaccagc accatgcaac tttttcacct ctgcctaatc atctcttgta catgtcccac 1860
tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccttataa 1920
agaatttgga gctactgtgg agttactctc gtttttgcct tctgacttct ttccttccgt 1980
ccgagatcta ctagacaccg cctcagctct gtatcgggaa gccttagagt ctcctgagca 2040
ttgctcacct caccatacag cactcaggca agccattctc tgctgggggg aattaatgac 2100
tctagctacc tgggtgggta ataatttgga agatccagca tccagggatc tagtagtcaa 2160
ttatgttaat actaacatgg gcctaaagat caggcaacta ttgtggtttc atatttcttg 2220
ccttactttt ggaagagaaa ctgtacttga gtatttggtc tctttcggag tgtggattcg 2280
cactcctcca gcctatagac caccaaatgc ccctatctta tcaacacttc cggaaactac 2340
tgttgttaga cgacgggacc gaggcaggtc ccctagaaga agaactccct cgcctcgcag 2400
acgaagatct caatcgccgc gtcgcagaag atctcaatct cgggaatctc aatgttagta 2460
ttccttggac tcataaggtg ggaaatttta ctgggcttta ttcctctact gtacctatct 2520
ttaatcctga atggcaaact ccttcctttc ctaagattca tttacatgag gacattatta 2580
ataggtgtca acaatttgtg ggccctctca ctgtaaatga aaagagaaga ttgaaattaa 2640
ttatgcctgc tagattttat cctaacagca ctaaatattt gcccttagac aaaggaatta 2700
aaccttatta tccagatcag gtagttaatc attacttcca aacccgacat tatttacata 2760
ctctttggaa ggctggtatt ctatataaga gggaaactac acgtagcgcc tcattttgcg 2820
ggtcaccata ttcttgggaa caagagctac atcatgggag gttggtcatc aaaacctcgc 2880
aaaggcatgg ggacgaatct ttctgttccc aaccctctgg gattctttcc cgatcatcag 2940
ttggaccctg cattcggagc caactcaaac aatccagatt gggacttcaa ccccatcaag 3000
gaccactggc cacaagccaa ccaggtagga gtgggagcat tcgggccagg gttcacccct 3060
ccacacggag gtgttttggg gtggagccct caggctcagg gcatattgac cacagtgcca 3120
gcagttcctc ctcctgcctc caccaatcgg cagtcaggaa ggcagcctac tcccatctct 3180
ccacctctaa gagacagtca tcctcaggcc atgcagtgga a 3221
<210>4
<211>3215
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
ctccaccact ttccaccaaa ctcttcaaga tcccagagtc agggccctgt actttcctgc 60
tggtggctcc agttcaggaa cagtgagccc tgctcagaat actgtctctg ccatatcgtc 120
aatcttatcg aagactgggg accctgtacc gaacatggag aacatcgcat caggactcct 180
aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaaaaatcc tcacaatacc 240
acagagtcta gactcgtggt ggacttctct caattttcta gggggaacac ccgtgtgtct 300
tggccaaaat tcgcagtccc aaatctccag tcactcacca acctgttgtc ctccaatttg 360
tcctggttat cgctggatgt gtctgcggcg ttttatcatc ttcctctgca tcctgctgct 420
atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480
aattccagga tcatcaacaa ccagcaccgg accatgcaaa acctgcacga ctcctgctca 540
aggaacctct atgtttccct catgttgctg tacaaaacct acggacggaa actgcacctg 600
tattcccatc ccatcatctt gggctttcgc aaaataccta tgggagtggg cctcagtccg 660
tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720
tgtctggctt tcagttatat ggatgatgtg gttttggggg ccaagtctgt acaacatctt 780
gagtcccttt atgccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840
cacaaaacaa aaagatgggg atattccctt aacttcatgg gatatgtaat tgggagttgg 900
ggcacattgc cacaggaaca tattgtacaa aaaatcaaaa tgtgttttag gaaacttcct 960
gtaaacaggc ctattgattg gaaagtatgt caacgaattg tgggtctttt ggggtttgcc 1020
gcccctttca cgcaatgtgg atatcctgct ttaatgcctt tatatgcatg tatacaagca 1080
aaacaggctt ttactttctc gccaacttac aaggcctttc taagtaaaca gtatctgaac 1140
ctttaccccg ttgctcggca acggcctggt ctgtgccaag tgtttgctga cgcaaccccc 1200
actggttggg gcttggccat aggccatcag cgcatgcgtg gaacctttgt gtctcctctg 1260
ccgatccata ctgcggaact cctagccgct tgttttgctc gcagcaggtc tggggcaaaa 1320
ctcatcggga ctgacaattc tgtcgtgctc tcccgcaagt atacatcgtt tccatggctg 1380
ctaggctgtg ctgccaactg gatcctgcgc gggacgtcct ttgtttacgt cccgtcggcg 1440
ctgaatcccg cggacgaccc ctcccggggc cgcttggggc tctaccgccc gcttctccgc 1500
ctgttgtacc gaccgaccac ggggcgcacc tctctttacg cggactcccc gtctgtgcct 1560
tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccaccg 1620
tgaacgccca ccggaacctg cccaaggtct tgcataagag gactcttgga ctttcagcaa 1680
tgtcaacgac cgaccttgag gcatacttca aagactgtgt gtttactgag tgggaggagt 1740
tgggggagga gattaggtta aaggtctttg tactaggagg ctgtaggcat aaattggtgt 1800
gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac 1860
tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccgtataa 1920
agaatttgga gcttctgtgg agttactctc ttttttgcct tctgacttct ttccttctat 1980
tcgagatctc ctcgacaccg cctctgctct gtatcgggag gccttagagt ctccggaaca 2040
ttgttcacct caccatacgg cactcaggca agctattctg tgttggggtg agttgatgaa 2100
tctagccacc tgggtgggaa gtaatttgga agatccagca tccagggaat tagtagtcag 2160
ctatgtcaac gttaatatgg gcctaaaaat cagacaacta ttgtggtttc acatttcctg 2220
tcttactttt gggagagaaa ctgttcttga atatttggtg tcttttggag tgtggattcg 2280
cactcctcct gcatatagac caccaaatgc ccctatctta tcaacacttc cggaaactac 2340
tgttgttaga cgaagaggca ggtcccctag aagaagaact ccctcgcctc gcagacgaag 2400
gtctcaatcg ccgcgtcgca gaagatctca atctcgggaa tctcaatgtt agtattcctt 2460
ggacacataa ggtgggaaac tttacggggc tttattcttc tacggtacct tgctttaatc 2520
ctaaatggca aactccttct tttcctgaca ttcatttgca ggaggacatt gttgatagat 2580
gtaagcaatt tgtggggccc cttacagtaa atgaaaacag gagactaaaa ttaattatgc 2640
ctgctaggtt ttatcccaat gttactaaat atttgccctt agataaaggg atcaaaccgt 2700
attatccaga gcatgtagtt aatcattact tccagacgcg acattattta cacactcttt 2760
ggaaggcggg gatcttatat aaaagagagt ccacacgtag cgcctcattt tgcgggtcac 2820
catattcttg ggaacaagat ctacagcatg ggaggttggt cttccaaacc tcgaaaaggc 2880
atggggacaa atctttctgt ccccaatccc ctgggattct tccccgatca tcagttggac 2940
cctgcattca aagccaactc agaaaatcca gattgggacc tcaacccgca caaggacaac 3000
tggccggacg ccaacaaggt gggagtggga gcattcgggc cagggttcac ccctccccat 3060
gggggactgt tggggtggag ccctcaggct cagggcatac tcacaactgt gccagcagct 3120
cctcctcctg cctccaccaa tcggcagtca ggaaggcagc ctactccctt atctccacct 3180
ctaagggaca ctcatcctca ggccatgcag tggaa 3215
<210>5
<211>3215
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
ctccacaaca ttccaccaag ctctgctaga tcccagagtg aggggcctat actttcctgc 60
tggtggctcc agttccggaa cagtaaaccc tgttccgact actgcctctc ccatatcgtc 120
aatcttctcg aggactgggg accctgcacc gaacatggag agcacaacat caggattcct 180
aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240
acagagtcta gactcgtggt ggacttctct caattttcta gggggagcac ccacgtgtcc 300
tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcttgtc ctccaatttg 360
tcctggctat cgctggatgt gtctgcggcg ttttatcata ttcctcttca tcctgctgct 420
atgcctcatc ttcttgttgg ttcttctgga ctaccaaggt atgttgcccg tttgtcctct 480
acttccagga acatcaacta ccagcacggg accatgcaag acctgcacga ttcctgctca 540
aggaacctct atgtttccct cttgttgctg tacaaaacct tcggacggaa actgcacttg 600
tattcccatc ccatcatcct gggctttcgc aagattccta tgggagtggg cctcagtccg 660
tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720
tgtttggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acaacatctt 780
gagtcccttt ttacctctat taccaatttt cttttgtctt tgggtataca tttgaaccct 840
aataaaacca aacgttgggg ctactccctt aacttcatgg gatatgtaat tggaagttgg 900
ggtactttac cacaggaaca tattgtacta aaactcaagc aatgttttcg aaaactgcct 960
gtaaatagac ctattgattg gaaagtatgt caaagaattg tgggtctttt gggctttgct 1020
gcccctttta cacaatgtgg ctatcctgcc ttaatgcctt tatatgcatg tatacaatct 1080
aagcaggctt tcactttctc gccaacttac aaggcctttc tgtgtaaaca atatctgaac 1140
ctttaccccg ttgcccggca acggtcaggt ctctgccaag tgtttgctga cgcaaccccc 1200
actggatggg gcttggccat aggccatcag cgcatgcgtg gaacctttgt ggctcctctg 1260
ccgatccata ctgcggaact cctagcagct tgttttgctc gcagccggtc tggagcaaaa 1320
cttatcggca ccgacaactc tgttgtcctc tctcggaaat acacctcctt tccatggctg 1380
ctagggtgtg ctgccaactg gatcctgcgc gggacgtcct ttgtctacgt cccgtcggcg 1440
ctgaatcccg cggacgaccc gtctcggggc cgtttgggac tctaccgtcc ccttcttcgt 1500
ctgccgttcc ggccgaccac ggggcgcacc tctctttacg cggtctcccc gtctgtgcct 1560
tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccaccg 1620
tgaacgccca ccaggtcttg cccaaggtct tacataagag gactcttgga ctctcagcaa 1680
tgtcaacgac cgaccttgag gcatacttca aagactgtgt gtttaaagac tgggaggagt 1740
tgggggagga gattaggtta aaggtctttg tactaggagg ctgtaggcat aaattggtct 1800
gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac 1860
tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccgtataa 1920
agaatttgga gcttctgtgg agttactctc ttttttgcct tctgacttct ttccttctat 1980
tcgagatctc ctcgacaccg cctctgctct gtatcgggag gccttagagt ctccggaaca 2040
ttgttcacct caccatacag cactcaggca agctattctg tgttggggtg agttgatgaa 2100
tctggccacc tgggtgggaa gtaatttgga agacccagca tccagggaat tagtagtcag 2160
ctatgtcaat gttaatatgg gcctaaaaat cagacaacta ttgtggtttc acatttcctg 2220
tcttactttt ggaagagaaa ctgttcttga gtatttggtg tcttttggag tgtggattcg 2280
cactcctccc gcttacagac caccaaatgc ccctatctta tcaacacttc cggaaactac 2340
tgttgttaga cgacgaggca ggtcccctag aagaagaact ccctcgcctc gcagacgaag 2400
gtctcaatcg ccgcgtcgca gaagatctca atctcgggaa tctcaatgtt agtatccctt 2460
ggactcataa ggtgggaaac tttactgggc tttattcttc tactgtacct gtctttaatc 2520
ctgagtggca aactccctcc tttcctcaca ttcatttaca ggaggacatt attaatagat 2580
gtcaacaata tgtgggccct cttacagtta atgaaaaaag gagattaaaa ttaattatgc 2640
ctgctaggtt ctatcctaac cttaccaaat atttgccctt agataaaggc attaaacctt 2700
attatcctga acatgcagtt aatcattact tcaaaactag gcattattta catactctgt 2760
ggaaggctgg cattctatat aagagagaaa ctacacgcag cgcctcattt tgtgggtcac 2820
catattcttg ggaacaagag ctacagcatg ggaggttggt cttccaaacc tcgacaaggc 2880
atggggacga atctttctgt tcccaatcct ctgggattct ttcccgatca ccagttggac 2940
cctgcgttcg gagccaactc aaacaatcca gattgggact tcaaccccaa caaggatcac 3000
tggccagagg caaatcaggt aggagcggga gcattcgggc cagggttcac cccaccacac 3060
ggcggtcttt tggggtggag ccctcaggct cagggcatat tgacaacagt gccagcagca 3120
cctcctcctg cctccaccaa tcggcagtca ggaagacagc ctactcccat ctctccacct 3180
ctaagagaca gtcatcctcaggccatgcag tggaa 3215
<210>6
<211>3182
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
ctccacaacc ttccaccaaa ctctgcaaga tcccagagtg agaggcctgt atttccctgc 60
tggtggctcc agttcaggaa cagtaaaccc tgttccgact actgcctctc ccatatcgtc 120
aatcttctcg aggattgggg accctgcgct gaacatggag aacatcacat caggattcct 180
aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240
gcagagtcta gactcgtggt ggacttctct caattttcta gggggaacta ccgtgtgtct 300
tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcctgtc ctccaacttg 360
tcctggttat cgctggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420
atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480
aattccagga tcttcaacca ccagcacggg accatgcaga acctgcacga ctcctgctca 540
aggaacctct atgtatccct cctgttgctg taccaaacct tcggacggaa attgcacctg 600
tattcccatc ccatcatcct gggctttcgg aaaattccta tgggagtggg cctcagcccg 660
tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720
tgtttggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acagcatctt 780
gagtcccttt ttaccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840
aacaaaacaa aaagatgggg ttactcttta catttcatgg gctatgtcat tggatgttat 900
gggtcattgc cacaagatca catcatacag aaaatcaaag aatgttttag aaaacttcct 960
gttaacaggc ctattgattg gaaagtctgt caacgtattg tgggtctttt gggttttgct 1020
gcccctttta cacaatgtgg ttatcctgct ttaatgccct tgtatgcatg tattcaatct 1080
aagcaggctt tcactttctc gccaacttac aaggcctttc tgtgtaaaca atacctgaac 1140
ctttaccccg ttgcccggca acggccaggt ctgtgccaag tgtttgctga cgcaaccccc 1200
actggctggg gcttggtcat gggccatcag cgcatgcgtg gaacctttct ggctcctctg 1260
ccgatccata ctgcggaact cctagccgct tgttttgctc gcagcaggtc tggagcaaac 1320
attctcggga cggataactc tgttgttctc tcccgcaaat atacatcgtt tccatggctg 1380
ctaggctgtg ctgccaactg gatcctgcgc gggacgtcct ttgtttacgt cccgtcggcg 1440
ctgaatcccg cggacgaccc ttctcggggc cgcttgggac tctctcgtcc ccttctccgt 1500
ctgccgtttc gaccgaccac ggggcgcacc tctctttacg cggactcccc gtctgtgcct 1560
tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccaccg 1620
tgaacgccca ccaattcttg cccaaggtct tacataagag gactcttgga ctctctgtaa 1680
tgtcaacgac cgaccttgag gcatacttca aagactgttt gtttaaagac tgggaggagt 1740
tgggggagga gattagatta aaggtctttg tactaggagg ctgtaggcat aaattggtct 1800
gcgcaccagc accatgcaac tttttcacct ctgcctaatc atctcttgtt catgtcctac 1860
tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccttataa 1920
agaatttgga gctactgtgg agttactctc gtttttgcct tctgacttct ttccttcagt 1980
acgagatctt ctagataccg cctcagctct gtatcgggaa gccttagagt ctcctgagca 2040
ttgttcacct caccatactg cactcaggca agcaattctt tgctgggggg aactaatgac 2100
tctagctacc tgggtgggtg ttaatttgga agatccagca tctagggacc tagtagtcag 2160
ttatgtcaac actaatatgg gcctaaagtt caggcaacta ttgtggtttc acatttcttg 2220
tctcactttt ggaagagaaa cggtcataga gtatttggtg tctttcggag tgtggattcg 2280
cactcctcca gcttatagac caccaaatgc ccctatctta tcaacacttc cggagactac 2340
tgttgttaga cgacgaggca ggtcccctag aagaagaact ccctcgcctc gcagacgaag 2400
gtctcaatcg ccgcgtcgca gaagatctca atctcgggaa tctcaatgtt agtattcctt 2460
ggactcataa ggtgggaaac tttacggggc tttattcttc tactgtacct gtctttaacc 2520
ctcattggaa aacaccctct tttcctaata tacatttaca ccaagacatt atcaaaaaat 2580
gtgaacaatt tgtaggccca ctcacagtca atgagaaaag aagactgcaa ttgattatgc 2640
ctgctaggtt ttatccaaat gttaccaaat atttgccatt ggataagggt attaaacctt 2700
attatccaga acatctagtt aatcattact tccaaaccag acattattta cacactctat 2760
ggaaggcggg tatattatat aagagagaaa caacacatag cgcctcattt tgtgggtcac 2820
catattcttg ggaacaagag ctacagcatg gggcagaatc tttccaccag caatcctctg 2880
ggattctttc ccgaccacca gttggatcca gccttcagag caaacaccgc aaatccagat 2940
tgggacttca atcccaacaa ggacacctgg ccagacgccaacaaggtagg agctggagca 3000
ttcgggctgg gattcacccc accgcacgga ggccttttgg ggtggagccc tcaggctcag 3060
ggcatactac aaaccttgcc agcaaatccg cctcctgcct ctaccaatcg ccagtcagga 3120
aggcagccta ccccgctgtc tccacctttg agaaacactc atcctcaggc catgcagtgg 3180
aa 3182
<210>7
<211>3212
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
ttccacaaca ttccaccaag ctctgcagga tcccagagta agaggcctgt attttcctgc 60
tggtggctcc agttccggaa cagtgaaccc tgttccgact actgcctcac tcatctcgtc 120
aatcttctcg aggattgggg accctgcacc gaacatggaa agcatcacat caggattcct 180
aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaaaaatcc tcacaatacc 240
gcagagtcta gactcgtggt ggacttctct caattttcta gggggagctc ccgtgtgtct 300
tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcttgtc ctccaatttg 360
tcctggctat cgctggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420
atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480
aattccagga tcatcaacca ccagtacggg accctgccga acctgcacga ctcttgctca 540
aggaacctct atgtttccct catgttgctg ttcaaaacct tcggacggaa attgcacttg 600
tattcccatc ccatcatcat gggctttcgg aaaattccta tgggagtggg cctcagcccg 660
tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgccgggc tttcccccac 720
tgtctggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acaacatctt 780
gagtcccttt atacctctgt taccaatttt cttttgtctt tgggtataca tttaaatccc 840
aacaaaacaa aaagatgggg atattcccta aatttcatgg gttatgtaat tggaagttgg 900
gggtcattac cacaggaaca catcagaatg aaaatcaaag actgttttag aaaactccct 960
gttaaccggc ctattgattg gaaagtatgt caaagaattg tgggtctttt gggctttgct 1020
gcccctttta cacaatgtgg atatcctgct ttaatgcctc tgtatgcgtg tattcaatct 1080
aagcaggctt tcactttctc gccaacttac aaggcctttc tgtgtaaaca atacctgaac 1140
ctttaccccg ttgcccggca acggccaggt ctgtgccaag tgtttgctga tgcaaccccc 1200
actggctggg gcttggccat aggccatcag cgcatgcgtg gaacctttgt ggctcctctg 1260
ccgatccata ctgcggaact cctagccgct tgttttgctc gcagcaggtc tggagcgaaa 1320
cttatcggga cagataattc tgtcgttctc tcccggaaat atacatcatt tccatggctg 1380
ctaggctgtg ctgccaactg gatcctgcga gggacgtcct ttgtctacgt cccgtcagcg 1440
ctgaatcctg cggacgaccc gtctcggggt cgcttgggga tctatcgtcc ccttctccgt 1500
ctgccgttcc agccgaccac ggggcgcacc tctctttacg cggtctcccc gtctgtgcct 1560
tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccaccg 1620
tgaacgccca ccaaatcttg cccaaggtct tacataagag gactcttgga ctctctgcaa 1680
tgtcaacgac cgaccttgag gcatacttca aagactgttt gtttaaagac tgggaggagt 1740
tgggggagga gattagatta aaggtctttg tactaggagg ctgtaggcat aaattggtct 1800
gcgcaccagc accatgcaac tttttcacct ctgcctaatc atctcttgtt catgtcctac 1860
tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccttataa 1920
agaatttgga gctactgtgg agttactctc gtttttgcct tctgacttct ttccttcagt 1980
aagagatctt ctagataccg cctcagctct gtatcgggat gccttagaat ctcctgagca 2040
ttgttcacct caccacactg cactcaggca agccattctt tgctgggggg aactaatgac 2100
tctagctacc tgggtgggtg taaatttgga agatccagca tccagggacc tagtagtcag 2160
ttatgtcaat actaatatgg gcctaaagtt caggcaatta ttgtggtttc acatttcttg 2220
tctcactttt ggaagagaaa ccgtcataga gtatttggtg tcttttggag tgtggattcg 2280
cactcctcca gcttatagac caccaaatgc ccctatctta tcaacacttc cggagaatac 2340
tgttgttaga cgaagaggca ggtcccctag aagaagaact ccctcgcctc gcagacgaag 2400
atctcaatcg ccgcgtcgca gaagatctca atctccagct tcccaatgtt agtattcctt 2460
ggactcacaa ggtgggaaat tttacggggc tttactcttc tactatacct gtctttaatc 2520
ctaactggaa aactccatct tttcctgata ttcatttgca ccaggacatt attaacaaat 2580
gtgaacaatt tgtaggtcct ctaacagtaa atgaaaaacg aagattaaac ttagtcatgc 2640
ctgctagatt ttttcccatc tctacgaaat atttgcccct agagaaaggt ataaaacctt 2700
attatccaga taatgtagtt aatcattact tccaaaccag acactattta cataccctat2760
ggaaggcggg catcttatat aaaagagaaa ctacacgtag cgcctcattt tgtgggtcac 2820
cttattcttg ggaacaagag ctacatcatg gggctttctt ggacggtccc tctcgaatgg 2880
gggaagaatc attccaccac caatcctctg ggattttttc ccgaccacca gttggatcca 2940
gcattcagag caaacaccag aaatccagat tgggaccaca atcccaacaa agaccactgg 3000
acagaagcca acaaggtagg agtgggagca ttcgggccgg ggttcactcc cccacacgga 3060
ggccttttgg ggtggagccc tcaggctcaa ggcatgctaa aaacattgcc agcagatccg 3120
cctcctgcct ccaccaatcg gcagtcagga aggcagccta ccccaatcac tccacctttg 3180
agagacactc atcctcaggc catgcagtgg aa 3212
<210>8
<211>3215
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
ctccactcag ttccaccagg ctctgttaga tccgagggta agggctctgt attttcctgc 60
tggtggctcc agttcagaga cacagaaccc tgctccgact attgcctctc tcacatcatc 120
aatcttcttg aagactgggg gccctgctat gaacatggac aacatcacat caggactcct 180
aggacccctg ctcgtgttac aggcggtgtg tttcttgttg acaaaaatcc tcacaatacc 240
acagagtcta gactcgtggt ggacttctct caattttcta gggggactac ccgggtgtcc 300
tggccaaaat tcgcagtccc caacctccaa tcacttacca acctcctgtc ctccaacttg 360
tcctggctat cgttggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420
atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480
acttccagga tccacgacca ccagcacggg accatgcaaa acctgcacaa ctcttgctca 540
aggaacctct atgtttccct cttgttgctg ttccaaaccc tcggacggaa actgcacttg 600
tattcccatc ccatcatctt gggctttagg aaaataccta tgggagtggg cctcagcccg 660
tttctcctgg ctcagtttac tagtgcaatt tgttcagtgg tgcgtagggc tttcccccac 720
tgtctggctt ttagttatat ggatgatctg gtattggggg ccaaatctgt gcagcatctt 780
gagtcccttt ataccgctgt taccaatttt ctgttatctg tgggtatcca tttaaatacc 840
tctaaaacaa aaagatgggg ttatacccta catttcatgg gttatgttat tggcagttgg 900
ggatcattac cccaagatca cattgtacaa aaaatcaaag attgttttcg taaacttcct 960
gtaaatcgtc caattgattg gaaagtttgt caacgcattg tgggtctttt gggctttgcc 1020
gcccctttta ctcaatgtgg ttatcctgct ctcatgcctc tgtatgcctg tataactgct 1080
aaacaggctt ttgtcttttc gccaacttac aaggcctttc tatgtcaaca atacatgaac 1140
ctttaccccg ttgctcggca acggccaggc ctgtgccaag tgtttgctga cgcaaccccc 1200
actggttggg gcttggccat tggccatcag cgcatgcgtg gaacctttgt ggctcctctg 1260
ccgatccata ctgcggaact ccttgcagct tgtttcgctc gcagccggtc tggagcgaaa 1320
ctcatcggca cagacaactc tgttgtcctc tctaggaagt acacctcctt cccatggctg 1380
ctcggttgtg ctgccaactg gatcctgcgc gggacgtcct ttgtttacgt cccgtcggcg 1440
ctgaatcccgcggacgaccc ctctcggggt cgcttggggc tgtatcgccc ccttctccgt 1500
ctgccgttcc agccgacgac gggtcgcacc tctctttacg cggcctcccc gtctgttcct 1560
tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccaccg 1620
tgaacgcccc tcggagcttg ccaacagtct tacataagag gactcttgga ctttcaggac 1680
ggtcaatgac ctggatcgaa gaatacatca aagactgtgt atttaaggac tgggaggagc 1740
tgggggagga gattaggtta aaggtctttg tattaggagg ctgtaggcat aaattggtct 1800
gttcaccagc accatgcaac tttttcacct ctgcctaatc atcttttgtt catgtcctac 1860
tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccttataa 1920
agaatttgga gcttctgtgg aattactctc ttttttgcct tctgatttct tcccgtcagt 1980
tcgggaccta ctcgacaccg cttcagccct ctaccgggat gctttagagt caccggaaca 2040
ttgcacaccc aaccataccg ctctcaggca agctattttg tgctggggtg agttaatgac 2100
tttggcttcc tgggtgggca ataatttgga agaccctgca gctagggatc tagtagttaa 2160
ctatgtcaac actaacatgg gcctaaaaat tagacaattg ttgtggtttc acatttcctg 2220
ccttactttt ggaagagaaa cagttcttga gtatttggtg tcctttggag tgtggattcg 2280
cactcctcct gcttatagac caccaaatgc ccctatccta tccacacttc cggaaactac 2340
tgttgttaga cgacgaggca ggtcccctag aagaagaact ccctcgcctc gcagacgaag 2400
gtctcaatcg ccgcgtcgca gaagatctca atctccagct tcccaatgtt agtattcctt 2460
ggactcataa ggtgggaaat tttacgggac tctattcttc tactgttcct actttcaatc 2520
ctgactggtt aactccttct tttcctgata ttcatttaca tcaagatttg atatctaaat 2580
gtgaacaatt tgtaggccct ctcacaaaaa atgaattgag aagattaaaa ttggttatgc 2640
cagccagatt ttttcctaag gttaccaaat attttcctat ggaaaaagga attaaaccct 2700
attatcctga gcatgcagtt aatcattatt ttaagaccag acattatttg catactttat 2760
ggaaggcggg aattctatat aagagagaat ccacacgtag cgcctcattt tgtgggtcac 2820
catattcctg ggaacaagag ctacagcatg ggagcacctc tctcaacgac tcgaaggggc 2880
atgggacaga atctctctgt acccaatcct ctgggattct ttccagacca tcagctggat 2940
cctctattca gagcaaattc cagcagtccc gactgggact tcaacaaaaa caaggacaat 3000
tggccaatgg caaacaaggt aggagtggga ggatacggtc cagggttcac acccccacac 3060
ggtggcctgt tggggtggag ccctcaggca cagggtgtgc taacaacctt gccagcagat 3120
ccgcctcctg cttccaccaa tcggcggtcc gggagaaagc caaccccagt ctctccacct 3180
ctaagagaca ctcatccaca ggccatgcag tggaa 3215
<210>9
<211>3248
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
ctctacagca ttccaccaag ctctacaaaa tcccaaagtc aggggcctgt attttcctgc 60
tggtggctcc agttcaggga tagtgaaccc tgttccgact attgcctctc acatctcgtc 120
aatcttctcc aggattgggg accctgcacc gaacatggag aacatcacat caggattcct 180
aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240
gcagagtcta gactcgtggt ggacttctct caattttcta gggggagtgc ccgtgtgtcc 300
tggcctaaat tcgcagtccc caacctccaa tcactcacca atctcctgtc ctccaacttg 360
tcctggctat cgctggatgt gtctgcggcg ttttatcata ttcctcttca tcctgctgct 420
atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 480
gattccagga tcctcgacca ccagtacggg accctgcaaa acctgcacga ctcctgctca 540
aggcaactct atgtatccct catgttgctg tacaaaacct tcggacggaa attgcacctg 600
tattcccatc ccatcatctt gggctttcgc aaaataccta tgggagtggg cctcagtccg 660
tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720
tgtctggctt tcagctatat ggatgatgtg gtattggggg ccaaatctgt acaacatctt 780
gagtcccttt ataccgctgt taccaatttt cttttgtctt tgggtataca tctaaaccct 840
aacaaaacaa aaagatgggg ttattcctta aattttatgg gatatgtaat tggaagttgg 900
ggtactttgc cacaagaaca catcacacag aaaattaagc aatgttttcg gaaactccct 960
gttaacaggc caattgattg gaaagtctgt caacgaataa ctggtctgtt gggtttcgct 1020
gctcctttta cccaatgtgg ttaccctgcc ttaatgcctt tatatgcatg tatacaagct 1080
aagcaggctt ttactttctc gccaacttat aaggcctttc tctgtaaaca atacatgaac 1140
ctttaccccg ttgctaggca acggcccggt ctgtgccaag tgtttgctga cgcaaccccc 1200
actggttggg gcttggccat cggccatcagcgcatgcgtg gaacctttgt ggctcctctg 1260
ccgatccata ctgcggaact cctagctgct tgttttgctc gcagccggtc tggagcaaaa 1320
ctcattggga ctgacaattc tgtcgtcctt tctcggaaat atacatcctt tccatggctg 1380
ctaggctgtg ctgccaactg gatccttcgc gggacgtcct ttgtttacgt cccgtcagcg 1440
ctgaatccag cggacgaccc ctcccggggc cgtttggggc tctgtcgccc ccttctccgt 1500
ctgccgttcc tgccgaccac ggggcgcacc tctctttacg cggtctcccc gtctgttcct 1560
tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgttacatg gaaaccgcca 1620
tgaacacctc tcatcatctg ccaaggcagt tatataagag gactcttgga ctgtttgtta 1680
tgtcaacaac cggggtggag aaatacttca aggactgtgt ttttgctgag tgggaagaat 1740
taggcaatga gtccaggtta atgacctttg tattaggagg ctgtaggcat aaattggtct 1800
gcgcaccagc accatgtaac tttttcacct ctgcctaatc atctcttgtt catgtcctac 1860
tgttcaagcc tccaagctgt gccttgggtg gctttagggc atggatagaa caactttgcc 1920
atatggcctt tttggcttag acattgaccc ttataaagaa tttggagcta ctgtggagtt 1980
gctctcgttt ttgccttctg actttttccc gtctgttcgt gatcttctcg acaccgcttc 2040
agctttgtac cgggaatcct tagagtcctc tgatcattgt tcgcctcacc atacagcact 2100
caggcaagca atcctgtgct ggggtgagtt gatgactcta gctacctggg tgggtaataa 2160
tttggaagat ccagcatcca gagatttggt ggtcaattat gttaatacta atatgggttt 2220
aaaaatcagg caactattgt ggtttcacat ttcctgtctt acttttggga gagaaaccgt 2280
tcttgagtat ttggtgtctt ttggagtgtg gattcgcact cctcctgctt atagaccacc 2340
aaatgcccct atcctatcaa cacttccgga gactactgtt gttagacgaa gaggcaggtc 2400
ccctcgaaga agaactccct cgcctcgcag acgaagatct caatcgccgc gtcgcagaag 2460
atctgcatct ccagcttccc aatgttagta ttccttggac tcacaaggtg ggaaacttta 2520
cggggctgta ttcttctact atacctgtct ttaatcctga ttggcaaact ccttcttttc 2580
caaatatcca tttgcatcaa gacattataa ctaaatgtga acaatttgtg ggccctctca 2640
cagtaaatga gaaacgaaga ttaaaactag ttatgcctgc cagatttttc ccaaactcta 2700
ctaaatattt accattagac aaaggtatca aaccgtatta tccagaaaat gtagttaatc 2760
attacttcca gaccagacat tatttacata ccctttggaa ggcgggtatt ctatataaga 2820
gagaaacatc ccgtagcgct tcattttgtg ggtcaccata tacttgggaa caagatctac 2880
agcatggggc tttcttggac ggtccctctc gagtggggaa agaacctttc caccagcaat 2940
cctctaggat tccttcccga tcaccagttg gacccagcat tcagagcaaa taccaacaat 3000
ccagattggg acttcaatcc caaaaaggac ccttggccag aggccaacaa ggtaggagtt 3060
ggagcctatg gacccgggtt cacccctcca cacggaggcc ttttggggtg gagccctcag 3120
tctcagggca cactaacaac tttgccagca gatccgcctc ctgcctccac caatcgtcag 3180
tcagggaggc agcctactcc catctctcca ccactaagag acagtcatcc tcaggccatg 3240
cagtggaa 3248
<210>10
<211>3215
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
ctcaacacag ttccaccaag cactgttgga tccgagagta aggggtctgt attttcctgc 60
tggtggctcc agttcagaaa cacagaaccc tgctccgact attgcctctc tcacatcatc 120
aatcttctcg aagactgggg accctgctat gaacatggag aacatcacat caggactcct 180
aggacccctt ctcgtgttac aggcggtgtg tttcttgttg acaaaaatcc tcacaatacc 240
aaagagtcta gactcgtggt ggacttctct caattttcta ggggtaccac ccgggtgtcc 300
tggccaaaat tcgcagtccc caatctccaa tcacttacca acctcctgtc ctccaacttg 360
tcctggctat cgttggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420
atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tgtgtcctct 480
acttccagga tctacaacca ccagcacggg accctgcaaa acctgcacca ctcttgctca 540
aggaacctct atgtttccct cctgctgctg taccaaacct tcggacggaa attgcacctg 600
tattcccatc ccatcatctt gggctttcgg aaaataccta tgggagtggg cctcagcccg 660
tttctcttgg ctcagtttac tagtgcaatt tgttcagtgg tgcgtagggc tttcccccac 720
tgtctggctt ttagttatat ggatgatttg gtattggggg ccaaatctgt gcagcatctt 780
gagtcccttt ataccgctgt taccaatttt ttgttatctg tgggcatcca tttgaacaca 840
gctaaaacaa aatggtgggg ttattcctta cactttatgg gttatataat tgggagttgg 900
gggaccttgc ctcaggaaca tattgtgcat aaaatcaaag attgctttcg caaacttccc 960
gtgaatagac ccattgattg gaaggtttgt caacgcattg tgggtctttt gggctttgca 1020
gcccctttta ctcaatgtgg ttatcctgct ctcatgccct tgtatgcctg tattaccgct 1080
aagcaggctt ttgttttctc gccaacttac aaggcctttc tctgtaaaca atacatgaac 1140
ctttaccccg ttgctcggca acggccaggc ctttgccaag tgtttgctga cgcaaccccc 1200
actggctggg gcttggcgat tggccatcag cgcatgcgcg gaacctttgt ggctcctctg 1260
ccgatccata ctgcggaact cctagcagct tgtttcgctc gcagcaggtc tggagcggac 1320
attatcggca ctgacaactc cgttgtcctt tctcggaagt acacctcctt cccatggctg 1380
ctaggctgtg ctgccaactg gatcctgcgc gggacgtcct ttgtctacgt cccgtcggcg 1440
ctgaatcctg cggacgaccc ctctcgtggt cgcttggggc tctgccgccc tcttctccgc 1500
ctgccgttcc ggccgacgac gggtcgcacc tctctttacg cggactcccc gcctgtgcct 1560
tctcatctgc cggcccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccaccg 1620
tgaacgcccc ttggaacttg ccaacaacct tacataagag gactcttgga ctttcgcccc 1680
ggtcaacgac ctggattgag gaatacatca aagactgtgt atttaaggac tgggaggagt 1740
cgggggagga gttgaggtta aaggtctttg tattaggagg ctgtaggcat aaattggtct 1800
gttcaccagc accatgcaac tttttcacct ctgcctaatc atcttttgtt catgtcccac 1860
tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccttataa 1920
agaatttgga gcttctgtgg agttactctc atttttgcct tctgacttct tcccgtctgt 1980
ccgggaccta ctcgacaccg cttcagccct ctaccgagat gccttagaat cacccgaaca 2040
ttgcaccccc aaccacactg ctctcaggca agctattttg tgctggggtg agttgatgac 2100
cttggcttcc tgggtgggca ataatttaga ggatcctgca gcaagagatc tagtagttaa 2160
ttatgtcaat actaacatgg gcctaaaaat tagacaatta ttatggtttc acatttcctg 2220
ccttacattt ggaagagaaa ctgtgcttga gtatttggtg tcttttggag tgtggattcg 2280
cactccacct gcttatagac caccaaatgc ccctatccta tcaacacttc cggagactac 2340
tgttgttaga caacgaggca gggcccctag aagaagaact ccctcgcctc gcagacgaag 2400
atctcaatca ccgcgtcgca gaagatctca atctccagct tcccaatgtt agtattcctt 2460
ggactcataa ggtgggaaac tttaccggtc tttactcctc tactatacct gttttcaatc 2520
ctgactggtt aactccttct tttcctgaca ttcacttgca tcaagatctg atacaaaaat 2580
gtgaacaatt tgtaggccca ctcactacaa atgaaaggag acgattgaaa ctaattatgc 2640
cagctaggtt ttatcccaaa gttactaaat acttcccttt ggataaaggt attaagcctt 2700
actatccaga gaatgtggtt aatcattact ttaaaactag acattattta catactttgt 2760
ggaaggcagg aattctatat aagagagaat ccacacatag cgcctcattt tgtgggtcac 2820
catattcctg ggaacaagag ctacagcatg ggagcacctc tctcaacggc gagaaggggc 2880
atgggacaga atctttctgt gcccaatcct ctgggattct ttccagacca ccagttggat 2940
ccactattca gagcaaattc cagcagtccc gattgggact tcaacacaaa caaggacaat 3000
tggccaatgg caaacaaggt aggagtggga ggcttcggtc cagggttcac acccccacac 3060
ggtggccttc tggggtggag ccctcaggca cagggcattc tgacaacctc gccaccagat 3120
ccacctcctg cttccaccaa tcggaggtca ggaagaaagc caaccccagt ctctccacct 3180
ctaagggaca cacatccaca ggccatgcag tggaa 3215
<210>11
<211>3214
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
ctccacaacc ttccaccaag ctctacaaga tcccagaatc aggggcctgt attttcctgc 60
tggtggctcc agttcaggaa cagtaaaccc tgctccgaat attgcctctc acatctcatc 120
aatcttcacg aggattgggg accctgcaac gaacatggag aacatcacat caggattcct 180
cggacccctg ctcgtgttac aggcggggtt tttcttgttg acaaaaatcc tcacaatacc 240
gcagagtcta gactcgtggt ggacttctct caattttcta gggggagcac ccgtgtgtct 300
tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcctgtc ctccaatttg 360
tcctggctat cgctggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420
atgcctcatc ttcttgttgg ttcttctgga ttatcaaggt atgttgcccg tttgtcctct 480
aattccagga tcctcgacca ccagtacggg accatgcaaa acctgcacga ctcctgctca 540
aggcaactct atgtatccct catgttgctg taccaaacct tcggacggaa attgcacctg 600
tattcccatc ccatcatctt gggctttcgc aaaataccta tgggagtggg cctcagcccg 660
tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720
tgtctggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt acaacatctt 780
gagtcccttt ataccgctgt taccaatttt cttttgtctt tgggtataca tttaaattct 840
aacaaaacta agagatgggg ttattcctta aacttcatgg gatatgtaat tggaagttgg 900
ggtaccttgc cacaagatca tattatacag aaaatcaaac aatgttttag aaaactccct 960
gttaacaggc ccattgattg gaaagtatgt caaagaattt caggactctt gggctttgct 1020
gctccattta cacaatgtgg ttaccctgcg ttaatgcctt tgtatgcatg tatacaagct 1080
aaacaggctt tcactttctc gccaacttac aaggcctttc tgtgtaaaca atatatgaac 1140
ctttaccccg ttgcccggca acggcccggt ctgtgccaag tgtttgctga cgcaaccccc 1200
actggctggg gcttggcctt aggccatcag cgcatgcgtg gaacctttgt ggctcctctg 1260
ccgatccata ctgcggaact gctagctgcc tgttttgctc gcagcaggtc tggagcaaaa 1320
cttatcggga ctgataattc tgtcgtcctt tcgcggaaat atacatcatt tccatggctg 1380
ctaggctgtg ctgccaactg gatcctgcgc gggacgtcct ttgtttacgt cccgtcggcg 1440
ctgaatcctg cggacgaccc ctctcggggc cgcttgggga tctaccgtcc tcttcttcat 1500
ctgccgtacc gaccgtccac ggggcgcacc tctctttacg cggtctcccc gtttgtgcct 1560
tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccacca 1620
tgaacgccca cctgatcttg cccaaggtat tgcataagcg gactcttgga ctctcagcaa 1680
tgtcaacgac cgaccttgag gcatacttca aagactgtgt gtttaaagac tgggaggagt 1740
tgggggagga gattaggtta aagatctttgtactaggagg ctgtaggcat aaattggtct 1800
gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac 1860
tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccttataa 1920
agaatttgga gcttctgtgg agttactctc ttttttgcct tctgatttct ttccgtctat 1980
tcgggacctt ctcgacaccg catcagctct gtatcgggag gcattagagt ctccggaaca 2040
ttgttcacct caccatacag cactcaggca agcagttttg tgttggggtg agttgatgac 2100
tctagctacc tgggtgggaa gtaatttgga agaccctgcc tccagggatt tggtagtcag 2160
ctatgtcaat gttaatatgg gcctaaaaat tagacaacta tttggtttca catttcctgt 2220
cttacttttg gaagagaaac tgttcttgag tatttggtgt ctttcggagt gtggattcgc 2280
actcctcccg catacagacc accaaatgcc cctatcttat caacacttcc ggaaactact 2340
gttgttagac gacgaggcag gtcccctaga agaagaactc cctcgcctcg cagacgaaga 2400
tctcaatcgc cgcgtcgcag aagatctcaa tctcgggaat cccaatgtta gtattccttg 2460
gactcataag gtgggaaact ttaccgggct ttattcttct actgtacctg tctttaatcc 2520
tgagtggcaa actccctctt ttcctaacat tcatttgcat gaggacatta tccataggtg 2580
tcaacaattt gtgggccctc ttacagttaa tgaaaaaaga agattaaact taatcatgcc 2640
tgctaggttc tatcctaacc ttactaagta tttgccctta gataaaggca ttaaacctta 2700
ttatcctgag caggcagtta atcattactt caaaactagg cattatttac atactctgtg 2760
gaaagctggc attctatata agagagaaac aacacgcagc gcctcatttt gtgggtcacc 2820
atattcttgg gaacaagagc tacagcatgg gaggttggtc ttccaaacct cggaaaggca 2880
tggggacgaa tctttctgtt cccaatcctc tgggatttct tcccgatcat cagttggacc 2940
ctgcgttcgg agccaactca aacaatccag attgggactt caaccccaac aaggaccatt 3000
ggccacaagc ccatcaggta ggagtgggag cattcgggtc agggttcacc cctcctcacg 3060
gaggtctttt ggggtggagc cctcaggctc agggcatatt aacaaacgtg ccagcagttc 3120
ctcctcctgc ctccaccaat cggcagtcag gaaggcagcc aactcccatc tctccacctc 3180
taagagacag tcatcctcag gccatgcagt ggaa 3214
<210>12
<211>3182
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
ctccacggca ttccaccaag ctctacaaga tcccagagtg aagggcctgt atcatcctgc 60
tggtggctcc agttcaggga cagtgagccc tgttccgacc actgtctctc ccacctcgtc 120
aagcttcacc aagactgggg accttgttcc gaatatggag aacatcacat caggattcct 180
aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaaaaatcc tcacaattcc 240
tcagagtcta gactcgtggt ggacttctct caattttcta gggggagcac tcgtgtgtcc 300
tggccaaaat tcgcagtccc taacctccaa tcactcacca acctcttgtc ctccaacctg 360
tcctggctat cgctggatgt gtctgcggcg ttttatcatc ttcctcttca tcctgctgct 420
atgcctcatc ttcttgttgg ttcttctgga ttatcaaggt atgttgcccg tttgtcctct 480
gcttccagga tcaaccacca ccagcacggg accatgcaga acctgcacga tcactgctca 540
aggaacctct atgtttccct cttgttgctg tacaaaacct tcggacggaa attgcacctg 600
tattcccatc ccatcatcat gggctttcgc aaaattccta tgggagtggg cctcagtccg 660
tttctcctgg ctcagtttac tagcgccatt tgttcagtgg ttcgcagggc tttcccccac 720
tgtttggctt tcagttatat ggatgatctg gtattggggg ccaagtctgt acaacatctt 780
gaatcccttt ataccgctgt taccaatttt cttttgtctt tgggtataca tttaaaccct 840
cacaaaacaa aaagatgggg gtactccctc cacttcatgg gatatgtaat tggcagttgg 900
ggaactttgc cacaggacca tattatccag aaaatcactc aatgttttag gaaactacct 960
gtcaataggc ctattgattg gaaagtgtgt caacgaattg taggtctttt aggtttcgct 1020
gcccctttta cacaatgtgg atatccagcc ttaatgccct tatatgcatg tattcaagct 1080
aaacaggctt ttactttctc gccaacatac aaggcctttc tacgtacaca gtacatgaac 1140
ctttaccccg ttgctcggca acgaccgggc ttgtgccaag tgtttgctga cgcaaccccc 1200
actggttggg gcttggccat cggtcaccag cgcatgcgtg gaacctttgt ggctcctctg 1260
ccgatccata ctgcggaact cctagcagct tgttttgctc gcagcaggtc tggggcaaac 1320
ctactcggga cagataattc tgtggtttta tcacggaagt atacgtcctt cccatggctg 1380
ctaggctgtg ctgccaactg gatcctgcgc gggacgtcct ttgtctacgt cccgtcggcg 1440
ctgaatcccg cggacgaccc gtctcggggc aagttgggcc tctaccgtcc tcttctccgt 1500
ctgccgttcc gaccgaccac ggggcgcacc tctctttacg cggtctcccc gtctgtacct 1560
tctcatctgc cggcccgtgt gcacttcgct tcacctctgc acgttgcatg gagaccaccg 1620
tgaacgcccc ctggaatttg ccaagagtgt tacataagcg gactcttgga ctttcggaca 1680
tgtcaacgtc cgcaattgag acatacttca aggactgtgt atttaaagac tgggaggagt 1740
caggggagga gattaggtta atgatctttg tattaggagg ctgtaggcat aaattggtct 1800
gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcttgtt catgtcctac 1860
tgttcaagcc tccaagctgt gccttgggtg gctttagggc atggacattg acccttataa 1920
agaatttgga gcttctgtgg agttactctc ttttttgcct tctgatttct ttccgtcaat 1980
cagagacctc ctcgacaccg cctcagctct ataccgagaa gccttagagt ctccagaaca 2040
ttgctcacct caccatacag cacttaggca agctgtgcta tgttggggtg agttgatgaa 2100
tctggctacc tgggtgggaa gtaatttgga agacccagca tccagggaac ttgtagtcag 2160
ctatgttaac attaatatgg gcctaaaaat tagacaatta ttgtggtttc acatctcctg 2220
tcttactttt ggaagagaaa ctgttcttga atatttggtg tcttttggag tgtggattcg 2280
cactcctcct gcttacagac caccaaatgc ccctatcttg tcaacacttc cggaaactac 2340
tgttgttaga cgaagaggca ggtcccctag aagaagaact ccctcgcctc gcagacgaag 2400
gtctcaatcg ccgcgtcgca gaagatctca atctccatct tcccaatgtt agtattcctt 2460
ggacgcataa ggtgggagat tttactgggc tttattcttc tactgtacct gtttttaatc 2520
ctaaatggca aacaccctct tttccagata ttcatttaca tcaggatatt attgataagt 2580
gtcaacaatt tgtaggccct ctaacagtca atgaaaaacg aaggttaaaa ctaattatgc 2640
ctgctaggtt ctttccgaat gctaccaaat attttccccc agataaaggt ataaaacctt 2700
actatccaga gaatgtggtt aatcattatt tccaagccag acattatttg catactctat 2760
ggaaggcggg catcttatat aagagagaaa ccacacatag cgcctcattc tgtgggtcac 2820
cctattcttg ggaacaagag ctacatcatg ggggcaaacc tctctgtccc caatccgctg 2880
ggattcttcc cagagcatca gttggacccc gcattccgag ccaacacaag caatccagat 2940
tgggacttca accccaacaa agacaactgg ccggattcca caaaggtggg tgcgggagct 3000
ttcgggccag ggttcacccc accacatgga ggcattctgg ggtggagccc tcaggctcag 3060
ggactaacaa cattcctgcc agcagctcct cctcttgcct ccaccaatcg gcaagcaaga 3120
aggcaaccta cacccatatc gccaccactg agagacactc atcctcaggc catgcagtgg 3180
aa 3182
<210>13
<211>465
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
atggctgcta ggctgtactg ccaactggat tcttcgcggg acgtcctttg tttacgtccc 60
gtcggcgctg aatcccgcgg acgacccctc tcggggccgc ttgggactct atcgtcccct 120
tctccgtctg ccgtaccggc cgaccacggg gcgcacctct ctttacgcgg tctccccgtc 180
tgtgccttct catctgccgg tccgtgtgca cttcgcttca cctctgcacg ttgcatggag 240
accaccgtga acgcccatca gatcctgccc aaggtcttac ataagaggac tcttggactc 300
ccagcaatgt caacgaccga ccttgaggcc tacttcaaag actgtgtgtt taaagactgg 360
gaggagttgg gggaggagat taggttaaag gtctttgtat taggaggctg taggcataaa 420
ttggtctgcg caccagcacc atgcaacttt ttcacctctg cctaa 465
<210>14
<211>465
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
atggctgcta ggctgtgctg ccaactggat cctgcgcggg acgtcctttg tttacgtccc 60
gtcggcgctg aatcccgcgg acgacccctc ccggggccgc ttggggctct accgcccgct 120
tctccgcctg ttgtaccgac cgaccacggg gcgcacctct ctttacgcgg actccccgtc 180
tgtgccttct catctgccgg accgtgtgca cttcgcttca cctctgcacg tcgcatggag 240
accaccgtga acgcccaccg gaacctgccc aaggtcttgc ataagaggac tcttggactt 300
tcagcaatgt caacgaccga ccttgaggca tacttcaaag actgtgtgtt tactgagtgg 360
gaggagttgg gggaggagat taggttaaag gtctttgtac taggaggctg taggcataaa 420
ttggtgtgtt caccagcacc atgcaacttt ttcacctctg cctaa 465
<210>15
<211>465
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
atggctgcta gggtgtgctg ccaactggat cctgcgcggg acgtcctttg tctacgtccc 60
gtcggcgctg aatcccgcgg acgacccgtc tcggggccgt ttgggactct accgtcccct 120
tcttcgtctg ccgttccggc cgaccacggg gcgcacctct ctttacgcgg tctccccgtc 180
tgtgccttct catctgccgg accgtgtgca cttcgcttca cctctgcacg tcgcatggag 240
accaccgtga acgcccacca ggtcttgccc aaggtcttac ataagaggac tcttggactc 300
tcagcaatgt caacgaccga ccttgaggca tacttcaaag actgtgtgtt taaagactgg 360
gaggagttgg gggaggagat taggttaaag gtctttgtac taggaggctg taggcataaa 420
ttggtctgtt caccagcacc atgcaacttt ttcacctctg cctaa 465
<210>16
<211>465
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
atggctgcta ggctgtgctg ccaactggat cctgcgcggg acgtcctttg tttacgtccc 60
gtcggcgctg aatcccgcgg acgacccttc tcggggccgc ttgggactct ctcgtcccct 120
tctccgtctg ccgtttcgac cgaccacggg gcgcacctct ctttacgcgg actccccgtc 180
tgtgccttct catctgccgg accgtgtgca cttcgcttca cctctgcacg tcgcatggag 240
accaccgtga acgcccacca attcttgccc aaggtcttac ataagaggac tcttggactc 300
tctgtaatgt caacgaccga ccttgaggca tacttcaaag actgtttgtt taaagactgg 360
gaggagttgg gggaggagat tagattaaag gtctttgtac taggaggctg taggcataaa 420
ttggtctgcg caccagcacc atgcaacttt ttcacctctg cctaa 465
<210>17
<211>465
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
atggctgcta ggctgtgctg ccaactggat cctgcgaggg acgtcctttg tctacgtccc 60
gtcagcgctg aatcctgcgg acgacccgtc tcggggtcgc ttggggatct atcgtcccct 120
tctccgtctg ccgttccagc cgaccacggg gcgcacctct ctttacgcgg tctccccgtc 180
tgtgccttct catctgccgg accgtgtgca cttcgcttca cctctgcacg tcgcatggag 240
accaccgtga acgcccacca aatcttgccc aaggtcttac ataagaggac tcttggactc 300
tctgcaatgt caacgaccga ccttgaggca tacttcaaag actgtttgtt taaagactgg 360
gaggagttgg gggaggagat tagattaaag gtctttgtac taggaggctg taggcataaa 420
ttggtctgcg caccagcacc atgcaacttt ttcacctctg cctaa 465
<210>18
<211>465
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
atggctgctc ggttgtgctg ccaactggat cctgcgcggg acgtcctttg tttacgtccc 60
gtcggcgctg aatcccgcgg acgacccctc tcggggtcgc ttggggctgt atcgccccct 120
tctccgtctg ccgttccagc cgacgacggg tcgcacctct ctttacgcgg cctccccgtc 180
tgttccttct catctgccgg accgtgtgca cttcgcttca cctctgcacg tcgcatggag 240
accaccgtga acgcccctcg gagcttgcca acagtcttac ataagaggac tcttggactt 300
tcaggacggt caatgacctg gatcgaagaa tacatcaaag actgtgtatt taaggactgg 360
gaggagctgg gggaggagat taggttaaag gtctttgtat taggaggctg taggcataaa 420
ttggtctgtt caccagcacc atgcaacttt ttcacctctg cctaa 465
<210>19
<211>465
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
atggctgcta ggctgtgctg ccaactggat ccttcgcggg acgtcctttg tttacgtccc 60
gtcagcgctg aatccagcgg acgacccctc ccggggccgt ttggggctct gtcgccccct 120
tctccgtctg ccgttcctgc cgaccacggg gcgcacctct ctttacgcgg tctccccgtc 180
tgttccttct catctgccgg accgtgtgca cttcgcttca cctctgcacg ttacatggaa 240
accgccatga acacctctca tcatctgcca aggcagttat ataagaggac tcttggactg 300
tttgttatgt caacaaccgg ggtggagaaa tacttcaagg actgtgtttt tgctgagtgg 360
gaagaattag gcaatgagtc caggttaatg acctttgtat taggaggctg taggcataaa 420
ttggtctgcg caccagcacc atgtaacttt ttcacctctg cctaa 465
<210>20
<211>465
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
atggctgcta ggctgtgctg ccaactggat cctgcgcggg acgtcctttg tctacgtccc 60
gtcggcgctg aatcctgcgg acgacccctc tcgtggtcgc ttggggctct gccgccctct 120
tctccgcctg ccgttccggc cgacgacggg tcgcacctct ctttacgcgg actccccgcc 180
tgtgccttct catctgccgg cccgtgtgca cttcgcttca cctctgcacg tcgcatggag 240
accaccgtga acgccccttg gaacttgcca acaaccttac ataagaggac tcttggactt 300
tcgccccggt caacgacctg gattgaggaa tacatcaaag actgtgtatt taaggactgg 360
gaggagtcgg gggaggagtt gaggttaaag gtctttgtat taggaggctg taggcataaa 420
ttggtctgtt caccagcacc atgcaacttt ttcacctctg cctaa 465
<210>21
<211>465
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
atggctgcta ggctgtgctg ccaactggat cctgcgcggg acgtcctttg tttacgtccc 60
gtcggcgctg aatcctgcgg acgacccctc tcggggccgc ttggggatct accgtcctct 120
tcttcatctg ccgtaccgac cgtccacggg gcgcacctct ctttacgcgg tctccccgtt 180
tgtgccttct catctgccgg accgtgtgca cttcgcttca cctctgcacg tcgcatggag 240
accaccatga acgcccacct gatcttgccc aaggtattgc ataagcggac tcttggactc 300
tcagcaatgt caacgaccga ccttgaggca tacttcaaag actgtgtgtt taaagactgg 360
gaggagttgg gggaggagat taggttaaag atctttgtac taggaggctg taggcataaa 420
ttggtctgtt caccagcacc atgcaacttt ttcacctctg cctaa 465
<210>22
<211>465
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
atggctgcta ggctgtgctg ccaactggat cctgcgcggg acgtcctttg tctacgtccc 60
gtcggcgctg aatcccgcgg acgacccgtc tcggggcaag ttgggcctct accgtcctct 120
tctccgtctg ccgttccgac cgaccacggg gcgcacctct ctttacgcgg tctccccgtc 180
tgtaccttct catctgccgg cccgtgtgca cttcgcttca cctctgcacg ttgcatggag 240
accaccgtga acgccccctg gaatttgcca agagtgttac ataagcggac tcttggactt 300
tcggacatgt caacgtccgc aattgagaca tacttcaagg actgtgtatt taaagactgg 360
gaggagtcag gggaggagat taggttaatg atctttgtat taggaggctg taggcataaa 420
ttggtctgtt caccagcacc atgcaacttt ttcacctctg cctaa 465
Claims (10)
1. A method for detecting mutations in the genotype and/or X region of HBV comprising the steps of:
(1) designing a universal primer capable of amplifying 10 genotype HBV virus genome X regions aiming at conserved regions at two ends of the X region of HBV to carry out PCR (polymerase chain reaction) directional amplification, and then sequencing;
(2) selecting CDS of HBx in the middle of the sequencing sequence and CDS standard sequences of 10 HBx to perform homology analysis and comparison to obtain the genotype and/or X region mutation result of the HBV to be detected;
the CDS standard sequence of HBx corresponding to HBV-A type standard whole genome sequence is shown in SEQ ID NO. 13;
the CDS standard sequence of HBx corresponding to HBV-B type standard whole genome sequence is shown in SEQ ID NO. 14;
the CDS standard sequence of HBx corresponding to HBV-C type standard whole genome sequence is shown in SEQ ID NO. 15;
the CDS standard sequence of HBx corresponding to HBV-D type standard whole genome sequence is shown in SEQ ID NO. 16;
the CDS standard sequence of HBx corresponding to HBV-E type standard whole genome sequence is shown in SEQ ID NO. 17;
the CDS standard sequence of HBx corresponding to HBV-F type standard whole genome sequence is shown in SEQ ID NO. 18;
the CDS standard sequence of HBx corresponding to HBV-G type standard whole genome sequence is shown in SEQ ID NO. 19;
the CDS standard sequence of HBx corresponding to HBV-H type standard whole genome sequence is shown in SEQ ID NO. 20;
the CDS standard sequence of HBx corresponding to HBV-I type standard whole genome sequence is shown in SEQ ID NO. 21;
the CDS standard sequence of HBx corresponding to HBV-J type standard whole genome sequence is shown in SEQ ID NO. 22.
2. The method as claimed in claim 1, wherein the upstream primer and sequencing primer X1 capable of amplifying the X region of 10 genotypes HBV virus genome is shown in SEQ ID NO. 1; the sequence of a downstream primer X2 capable of amplifying the X region of 10 genotype HBV genomes is shown as SEQ ID NO. 2.
3. The method according to claim 1 or 2, wherein the primer amplified fragment is 618bp in length and comprises the entire HBx CDS.
4. The method according to claim 1 or 2, wherein the CDS of HBx in the middle of the sequencing sequence and the CDS standard sequences of 10 HBx are selected for homology analysis and comparison, and the sequencing sequence and which CDS standard sequence of HBx have the highest homology can be determined as which genotype, so as to obtain the result of HBV genotype to be detected and/or mutation of X region; further, single genotype and mixed genotype can be judged according to the peak nesting condition in the sequencing peak image, and if no peak nesting exists or the number is less than 10, the single genotype is judged; if the number of the set peaks is more than or equal to 10, the mixed genotype is judged.
5. A kit for detecting HBV genotype and/or X region mutation, comprising:
the universal primers which are designed aiming at conserved regions at two ends of an X region of HBV and can amplify X regions of 10 genotypes of HBV virus genomes and CDS standard sequences of 10 HBx are as follows:
the CDS standard sequence of HBx corresponding to HBV-A type standard whole genome sequence is shown in SEQ ID NO. 13;
the CDS standard sequence of HBx corresponding to HBV-B type standard whole genome sequence is shown in SEQ ID NO. 14;
the CDS standard sequence of HBx corresponding to HBV-C type standard whole genome sequence is shown in SEQ ID NO. 15;
the CDS standard sequence of HBx corresponding to HBV-D type standard whole genome sequence is shown in SEQ ID NO. 16;
the CDS standard sequence of HBx corresponding to HBV-E type standard whole genome sequence is shown in SEQ ID NO. 17;
the CDS standard sequence of HBx corresponding to HBV-F type standard whole genome sequence is shown in SEQ ID NO. 18;
the CDS standard sequence of HBx corresponding to HBV-G type standard whole genome sequence is shown in SEQ ID NO. 19;
the CDS standard sequence of HBx corresponding to HBV-H type standard whole genome sequence is shown in SEQ ID NO. 20;
the CDS standard sequence of HBx corresponding to HBV-I type standard whole genome sequence is shown in SEQ ID NO. 21;
the CDS standard sequence of HBx corresponding to HBV-J type standard whole genome sequence is shown in SEQ ID NO. 22.
6. The kit according to claim 5, wherein the sequence of the upstream primer and sequencing primer X1 capable of amplifying the X region of the 10 genotypes HBV virus genome is shown as SEQ ID NO. 1; the sequence of a downstream primer X2 capable of amplifying the X region of 10 genotype HBV genomes is shown as SEQ ID NO. 2.
The CDS standard sequence of HBx in the genome of 7.10 genotype HBV viruses is characterized by comprising the following specific sequences:
the CDS standard sequence of HBx corresponding to HBV-A type standard whole genome sequence is shown in SEQ ID NO. 13;
the CDS standard sequence of HBx corresponding to HBV-B type standard whole genome sequence is shown in SEQ ID NO. 14;
the CDS standard sequence of HBx corresponding to HBV-C type standard whole genome sequence is shown in SEQ ID NO. 15;
the CDS standard sequence of HBx corresponding to HBV-D type standard whole genome sequence is shown in SEQ ID NO. 16;
the CDS standard sequence of HBx corresponding to HBV-E type standard whole genome sequence is shown in SEQ ID NO. 17;
the CDS standard sequence of HBx corresponding to HBV-F type standard whole genome sequence is shown in SEQ ID NO. 18;
the CDS standard sequence of HBx corresponding to HBV-G type standard whole genome sequence is shown in SEQ ID NO. 19;
the CDS standard sequence of HBx corresponding to HBV-H type standard whole genome sequence is shown in SEQ ID NO. 20;
the CDS standard sequence of HBx corresponding to HBV-I type standard whole genome sequence is shown in SEQ ID NO. 21;
the CDS standard sequence of HBx corresponding to HBV-J type standard whole genome sequence is shown in SEQ ID NO. 22.
8. The universal primer capable of amplifying the X region of 10 genotypes HBV virus genomes is characterized in that the sequence of an upstream primer and a sequencing primer X1 is shown as SEQ ID NO. 1; the sequence of the downstream primer X2 is shown as SEQ ID NO. 2.
9. Use of the CDS standard sequence of HBx in 10 HBV viral genomes as defined in claim 7 and the universal primer capable of amplifying X region of 10 genotype HBV viral genomes as defined in claim 8 in the preparation of reagents for detecting mutations in 10 HBV genotypes and/or X region.
10. The use according to claim 9, wherein the reagent for detecting 10 HBV genotypes and/or X region mutations is subjected to PCR-directed amplification by using universal primers designed for conserved regions at both ends of the X region of HBV and capable of amplifying the X region of 10 genotype HBV genomes, and then sequencing; and selecting CDS of HBx in the middle of the sequencing sequence and CDS standard sequences of 10 HBx to perform homology analysis and comparison to obtain the result of HBV genotype and/or X region mutation to be detected.
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| CN116479182A (en) * | 2023-05-05 | 2023-07-25 | 赛立安生物技术(广州)有限公司 | Primer group for detecting mutation of HBV pre-C region and core promoter region, detection method and kit thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112725535A (en) * | 2021-02-03 | 2021-04-30 | 无锡中德美联生物技术有限公司 | Fluorescent quantitative PCR kit for simultaneously detecting full-length and truncated HBV pgRNA and application thereof |
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| CN116479182A (en) * | 2023-05-05 | 2023-07-25 | 赛立安生物技术(广州)有限公司 | Primer group for detecting mutation of HBV pre-C region and core promoter region, detection method and kit thereof |
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