CN114672591B - Primer group and kit for identifying novel coronavirus and application of primer group and kit - Google Patents

Primer group and kit for identifying novel coronavirus and application of primer group and kit Download PDF

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CN114672591B
CN114672591B CN202210026905.0A CN202210026905A CN114672591B CN 114672591 B CN114672591 B CN 114672591B CN 202210026905 A CN202210026905 A CN 202210026905A CN 114672591 B CN114672591 B CN 114672591B
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江永忠
方斌
高利芬
彭海
方治伟
李论
刘琳琳
叶国军
蔡昆
霍细香
雷亚克
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Wuhan Mingming Biotechnology Co ltd
Hubei Provincial Center For Disease Control And Prevention (hubei Academy Of Preventive Medicine)
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Abstract

The invention discloses a primer group and a kit for identifying a novel coronavirus and application thereof. The primer group comprises at least one pair of primer pair from No. 1 to No. 168, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the primer pair No. 1, the forward primer of the primer pair No. 1 to No. 168 and the reverse primer of the primer pair No. 168 are sequentially shown as SEQ ID NO:1 to SEQ ID NO: 336. The primer group provided by the invention has higher amplification specificity and stability for 168 MNP marker loci on a novel coronavirus genome; the kit using the primer group has good sensitivity, reproducibility and accuracy for identifying the novel coronavirus, and can be used for qualitative identification, quantitative identification, genetic variation identification, fingerprint database construction and fine typing identification and tracing of the novel coronavirus for non-diagnosis purposes.

Description

Primer group and kit for identifying novel coronavirus and application of primer group and kit
Technical Field
The invention relates to the technical field of biology, in particular to a primer group and a kit for identifying a novel coronavirus and application thereof.
Background
Coronaviruses belong to the genus coronaviruses (Coronavirus) in the phylogenetic classification, and RNA viruses whose genomes are linear single-stranded positive strands are a large group of viruses that widely exist in nature. The pathogenic bacteria of the new coronavirus outbreak of 2019, which is still prevalent worldwide, is the 7 th coronavirus (2019-nCoV or SARS-CoV-2) which is known to infect human.
After outbreak of new coronary pneumonia, various domestic organizations including thermo scientific and Huada Gene, etc. have successively introduced a method or a kit for detecting novel coronavirus based on a sequencing technology. The existing detection based on the second generation sequencing technology is to identify and analyze the new coronavirus through detecting Single Nucleotide Polymorphism (SNP) variation on the new coronavirus genome. The SNP marker is a binary marker, and has the defects of low polymorphism and high typing error rate when identifying microorganisms existing in a population form including viruses, and the prior art cannot be used for determining the absolute copy number of the novel coronavirus in a sample, which is not beneficial to the detection of the novel coronavirus.
Disclosure of Invention
In order to solve the problems in the prior art, the embodiment of the invention provides a primer group for identifying a novel coronavirus, a kit and application thereof. The technical scheme is as follows:
in one aspect, the present invention provides a primer set for identifying a novel coronavirus, wherein the primer set comprises at least one pair of a primer pair 1 to a primer pair 168, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the primer pair 1, the reverse primer of the primer pair 1 to the forward primer of the primer pair 168, and the reverse primer of the primer pair 168 are sequentially represented as SEQ ID NOs: 1 to SEQ ID NO:336, respectively.
In another aspect, the embodiments of the present invention provide a kit for identifying a novel coronavirus, wherein the kit comprises the primer set as described above.
Specifically, the kit also comprises a nucleotide sequence shown as SEQ ID NO:337 to SEQ ID NO:346, 9 internal standard DNAs.
Specifically, the kit further comprises a multiplex PCR premix.
In another aspect, an embodiment of the present invention provides an application of a primer set, where the application includes: and detecting at least one of 168 MNP marker loci on the novel coronavirus genome by using the primer group, and performing qualitative identification, quantitative identification, genetic variation identification, MNP fingerprint database construction and fine typing of the novel coronavirus with non-diagnosis purposes according to the sequence of the obtained MNP marker loci.
The technical scheme provided by the embodiment of the invention has the following beneficial effects: the primer group provided by the embodiment of the invention detects 168 MNP marker sites on the genome of the new coronavirus, the detection result has higher amplification specificity and stability, and the kit using the primer group has good sensitivity, reproducibility and accuracy for detecting the new coronavirus. The primer group is used for detecting the MNP marker locus provided by the embodiment of the invention, and can be used for qualitative identification, quantitative identification, genetic variation identification, construction of MNP fingerprint database and fine typing of novel non-diagnostic coronaviruses.
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In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is apparent that the drawings in the description below are only some embodiments of the present disclosure, and it is obvious for those skilled in the art that other drawings may be obtained according to the drawings without creative efforts.
FIG. 1 is a standard graph for quantification of novel coronaviruses provided by embodiments of the present disclosure.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.
The source of the novel coronavirus provided by the embodiment of the invention is donated by the Hubei disease prevention and control center.
Example one
The invention provides a primer group for identifying a novel coronavirus, which comprises at least one pair from a 1 st primer pair to a 168 th primer pair, wherein each primer pair comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair to the 168 th primer pair and the reverse primer of the 168 th primer pair are sequentially shown as SEQ ID NO:1 to SEQ ID NO:336, respectively.
In the implementation, the base of the primer set may be modified.
The embodiment of the invention provides a kit for identifying a novel coronavirus, which comprises the primer group.
Specifically, the kit can also comprise a nucleotide sequence shown as SEQ ID NO:337 to SEQ ID NO:346, 9 internal standard DNAs shown.
Specifically, the kit may further comprise a multiplex PCR master mix.
The embodiment of the invention provides application of a primer group, wherein the primer group is used for detecting at least one of 168 MNP marker loci on a novel coronavirus genome, and is used for qualitative identification, quantitative identification, genetic variation identification, MNP fingerprint database construction and fine typing of the novel coronavirus for non-diagnosis purposes. Meanwhile, the primer set provided by the embodiment of the invention and the corresponding MNP marker site are shown in Table 1.
Table 1 shows the sequences of the primer sets and the corresponding MNP marker sites
Figure GDA0003620850150000021
Figure GDA0003620850150000031
Figure GDA0003620850150000041
Figure GDA0003620850150000051
Figure GDA0003620850150000061
Figure GDA0003620850150000071
Figure GDA0003620850150000081
The performance of the primer set was evaluated as follows:
the ribonucleic acid national standard (GBW (E) 091099) of SARS-CoV-2 with known copy number is added into 2ng of human genome DNA, the source of which is human Huh-7 cell line stored in the laboratory, and 1 copy/test, 10 copy/test and 100 copy/test of the novel coronavirus simulant samples are respectively prepared, meanwhile, 1 uL of internal standard DNA is added into each simulant sample, and two negative controls consisting of sterile water and 1 uL of internal standard DNA are set. Results a total of 5 samples of 2 negative controls, 1 copy/test, 10 copies/test and 100 copies/test as shown in table 2 were tested. The kit provided by the embodiment of the invention is used for amplifying 5 samples every day, 3 times of amplification are carried out each time to obtain an amplification product, and the amplification product is connected with a purchased commercial sample label to obtain a library suitable for high-throughput sequencing. Construction was continued for 4 days, i.e., 12 libraries were obtained per sample, and 60 libraries were obtained in total from 5 samples. The 60 libraries were mixed in equimolar amounts and high throughput sequencing was performed after mixing.
The preparation method of the internal standard DNA comprises the following steps:
selecting 1 or more MNP marker sites from 168 MNP marker sites provided by the embodiment of the invention;
at least 3 DNA sequences which are consistent with the selected MNP marker locus amplification primers and have the same length as the amplification region but different DNA sequences with all MNP marker loci are respectively designed for each selected MNP marker locus, and 3 DNA sequences are set in the embodiment;
connecting each DNA sequence to a plasmid vector to generate 3 DNA plasmid templates;
the copy numbers of the 3 DNA plasmid templates per unit volume are quantified and mixed in proportion to obtain internal standard DNA containing 3 DNA plasmid templates with 10 copies, 100 copies and 1000 copies per unit volume (mu L).
In order to ensure the accuracy of the quantification, 3 MNP marker loci are selected in this example, 3 sets of internal standard DNAs are obtained in total, and the sequences shown in SEQ ID NOs: 337 to SEQ ID NO:346, and 9 internal standard DNA sequences. 3 sets of internal standard DNAs were mixed to prepare 3 pieces of internal standard DNAs used in this example, each of which had 10 copies, 100 copies and 1000 copies of DNA plasmid templates per final unit volume (. Mu.L). In the quantitative identification of the novel coronavirus, the average value of the sequencing fragment numbers corresponding to 3 internal standard DNAs with the same copy number in the same detection is adopted.
TABLE 2 sensitivity and stability assays for the detection of novel coronaviruses
Figure GDA0003620850150000082
Figure GDA0003620850150000091
The high throughput sequencing results are shown in table 2, in 12 repeated experiments (3 times per day, for 4 days in total) of each sample, the detection effect of the MNP marker site of the novel coronavirus is stable between the repetition of the two negative control samples and the repetition of the novel coronavirus nucleic acid standard, and it can be seen that the stability of the kit provided by the embodiment of the invention for detecting the novel coronavirus is good. Due to the extreme sensitivity of the MNP marker detection method, the sequences of the novel coronavirus are also detected in a partial negative control, and the result that false positive results easily appear due to data pollution in the detection process can be seen. Therefore, the following quality control scheme is formulated in the embodiment of the invention to avoid the occurrence of false positives.
The quality control scheme is as follows:
1) The sequencing data amount is more than 250 megabases, and the internal standard DNA is required to be detected completely. The measuring and calculating basis is that according to the multiple tests of the sample with the known copy number of the novel coronavirus, when the sequencing data volume is not more than 250 megabases, the sample containing 10 copies/tests and more of the novel coronavirus can have less than 6 MNP marker sites, and the condition that the MNP marker sites are detected to reach 6 cannot be met by increasing the sequencing volume; and when the sequencing data amount is more than 250 Mbp, the detected MNP marker sites of all samples containing 10 copies/tests and more of the novel coronavirus are not less than 6.
2) Calculating the signal-to-noise ratio of the novel coronavirus in the sample, wherein the signal-to-noise ratio refers to the signal index S of the novel coronavirus in the test sampleTAnd the signal index Sc in the same batch of negative controls, including: a sterile water negative control and an internal standard DNA negative control, wherein:
the signal index of the novel coronavirus is calculated according to formula (1):
a
In formula (1):
s is the signal index (%) of the novel coronavirus;
n is the number of sequencing fragments of the novel coronavirus in the sample;
n is the number of sequencing fragments of the internal standard DNA in the sample;
STthe signal index of the novel coronavirus in a sample to be detected;
Sc(ii) a signal index for the novel coronavirus in the negative control sample;
in this example, the signal-to-noise ratio of the novel coronavirus in each sample is specifically shown in table 3.
TABLE 3 SNR of the novel coronavirus in the sample
Figure GDA0003620850150000092
Figure GDA0003620850150000101
As can be seen from Table 3, the maximum value of the signal index in the internal standard DNA negative control is 0.04%, while the average value of the signal index in 1 copy/test is 0.08%, the average value of the signal index in 10 copies/test is 0.73%, and the maximum value of the signal index of the novel coronavirus in the sterile water negative control is 0.72%, it can be seen that the signal index of the sterile water negative control is equivalent to the average value of the signal index of the sample of 10 copies/test, which makes it difficult to accurately identify the sample of 10 copies/test and the concentration below, and when the internal standard DNA is used as the negative control, the sensitivity of the detection can be improved to 10 copies/test or even 1 copy/test. Therefore, the embodiment of the invention adopts the internal standard DNA as the negative control, and can obviously improve the detection sensitivity.
The maximum signal-to-noise ratio for the novel coronavirus at 1 copy/sample under test was 9 (0.18%/0.02%) with internal standard DNA as a negative control. Therefore, the determination standard of the present invention is one of the conditions for determining that the nucleic acid of the novel coronavirus is detected in the sample by using the internal standard DNA as a negative control and by setting the signal to noise ratio of the novel coronavirus in the sample to be greater than 10. A maximum of 6 MNP marker sites were detected in the negative control samples (see table 2). In order to avoid high signal-to-noise ratio caused by preferential amplification of a few sites, the determination criteria for positivity of the novel coronavirus nucleic acid in the embodiment of the invention are as follows:
1) When the number of the detected marker sites of the new coronavirus in the sample to be detected is not less than 6 and the signal to noise ratio is not less than 10, judging that the new coronavirus nucleic acid is detected in the sample to be detected;
2) When the number of the detected marker sites of the novel coronavirus in the sample to be detected is less than 6 or the signal to noise ratio is less than 10, judging that the novel coronavirus nucleic acid is not detected in the sample to be detected.
For sensitivity
In order to demonstrate the effect of internal standard DNA as a negative control on improving detection sensitivity, this example also tested a parallel experiment with sterile water as a negative control. The sequencing results are shown in Table 3, and it can be seen from Table 3 that the sensitivity of the test sample can be improved to 10 copies/test or even 1 copy/test when the internal standard DNA is used as a negative control.
Evaluation for reproducibility and accuracy
Based on 12 replicates per sample, whether the major genotype with the co-detected MNP marker site was reproducible in any two replicates was used to assess the reproducibility and accuracy of the detection of the novel coronavirus. Specifically, 12 sets of data from 100 copies/test samples in Table 2 were compared separately, and the genotype of the MNP marker sites was compared 1721 in total. The alignment results are shown in Table 4, the number of MNP marker loci with differences in major genotypes is 0; the evaluation is carried out according to the principle that the reproducible genotypes are considered to be accurate among 2 repeated experiments, the accuracy rate a =1- (1-r)/2 =0.5+0.5r, wherein r represents the reproduction rate, and the reproduction rate is the ratio of the number of the reproducible loci of the main genotype to the number of the shared loci. The results are shown in Table 4.
Table 4 shows the evaluation of the reproducibility and accuracy
Figure GDA0003620850150000102
As can be seen from table 4, the log difference between different libraries and different lots of the MNP-labeled master genotype in the reproducibility test was 0, i.e., the reproducibility r =100% and the accuracy a =100%.
Assessment for specificity
The nucleic acids of the novel coronavirus, the human coronavirus HCoV-229E, HCoV-HKU1, HCoV-NL63, HCoV-OC43, the human parainfluenza virus, the metapneumovirus, the human rhinovirus, the mumps virus, the measles virus, the respiratory syncytial virus, the influenza A virus, the influenza B virus, the influenza C virus, the avian influenza virus and the Zika virus are mixed according to equal copy number to prepare a mixed template, and the internal standard DNA is used as a negative control to detect the novel coronavirus in the mixed template by adopting the primer set provided by the embodiment of the invention. After 3 repeated experiments are carried out and analyzed according to the quality control scheme and the judgment threshold value, sequencing fragments obtained in the 3 repeated experiments can only be specifically compared with MNP (MNP) marker sites of the novel coronavirus, in the 3 repeated experiments, the number of the MNP marker sites detected by the novel coronavirus is 153, 155 and 155 respectively, and the signal-to-noise ratio is 893, 690.4 and 785.4 in sequence. According to the quality control scheme and the judgment standard provided by the invention, the detection rate of the novel coronavirus reaches 100% in 3 times of repetition, and the primer group provided by the embodiment of the invention has higher specificity in detecting the novel coronavirus in a complex template.
Identification of novel coronaviruses in clinical samples
The kit provided by the embodiment of the invention is used for identifying the novel coronavirus in 38 collected pharyngeal swab samples, wherein the 38 pharyngeal swab samples are all from Hubei province disease control and prevention control center, the number of sequencing fragments which can be compared with MNP (MNP) labeled sites of the novel coronavirus and are obtained from each sample is at least 1,510,000, the sequencing fragments cover all 168 MNP labeled sites at most, and the average coverage multiple of each labeled site reaches nearly 9000 times. 9 internal standard DNAs in each sample are detected, so that the quality control conclusion of 38 samples is qualified.
Table 5 shows the detection of the novel coronavirus in 38 clinical samples by the kit
Figure GDA0003620850150000111
And (3) displaying a detection result: the kit provided by the embodiment of the present invention is contrary to the results of the conventional nucleic acid detection method based on fluorescence quantitative PCR in 4 cases (S22, S26, S27, S28), and the detection results provided by the embodiment of the present invention are confirmed to be correct in further review.
Example two
The embodiment of the invention provides an application of a primer group, which comprises the following steps: 168 MNP marker loci on the novel coronavirus genome are detected by utilizing a primer group, and the novel coronavirus with non-diagnostic purpose is quantitatively identified according to the sequence of the obtained MNP marker loci.
The method for preparing the MNP (Polynucleotide polymorphism) marker site is briefly described as follows:
based on 547 genomes of HCoV-229E published on the web, 518 genomes of HCoV-HKU1, 913 genomes of HCoV-NL63, 1426 genomes of HCoV-OC43, 1438 genomes of MERS-CoV, 40855 genomes of SARS-CoV-2 and 44326 genomes of SARS-CoV as reference sequences, a genome region conserved at both ends, which is specific for a novel coronavirus and has a plurality of nucleotide polymorphisms within a species, is screened by sequence alignment; designing multiple PCR primers for the regions, and testing by taking the national standard of ribonucleic acid (GBW (E) 091099) of the novel coronavirus as a template to finally obtain 168 MNP marker sites for detecting the novel coronavirus;
the MNP marker sites as shown in table 1 were obtained and the starting positions of the MNP marker sites on the reference sequence are shown in table 1.
For species without genomic data on the net, the genomic sequence information of the representative microspecies of the microbial species to be detected can also be obtained by high-throughput sequencing, wherein the high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure polymorphism of the marker screened, the genomic sequence of at least 10 isolates is generally used as a reference.
In this example, the internal standard DNA provided in the kit was added to each sample; counting the number of sequencing sequences detected by each internal standard DNA in the sequencing data of the sample; and (3) drawing a graph of the copy number of the internal standard DNA and the corresponding sequencing sequence number, and generating a calculation formula of the DNA copy number and the sequencing sequence number by using an excel self-contained analysis tool. In the present embodiment, the calculation formula is y =0.9091x-0.0662 (R)2= 0.9667). And (3) obtaining the absolute copy number of the novel coronavirus in the sample by using a generated calculation formula according to the detection sequencing sequence number of the MNP marker site which has the same primer with the internal standard DNA in the sample, thereby realizing the absolute quantitative detection of the novel coronavirus.
In this embodiment, in order to avoid the internal standard DNA detection failure and the bias caused by a single marker site, the internal standard DNA includes 3 amplification primers of MNP marker sites, and the internal standard DNA of each pair of primer sets has 3 concentration gradients, which are 10 copies, 100 copies, and 1000 copies, respectively. In plotting the standard curve, the average of the corresponding sequencing sequences at the same copy number was used.
In this example, the 1 copy/test, 10 copy/test and 100 copy/test new coronavirus ribonucleic acid standard shown in table 2 were first detected, a standard curve was plotted and quantified, and the results are shown in fig. 1, and the standard curve was plotted according to the copy number of the internal standard DNA and the corresponding sequencing sequence number in 36 sets of sequencing data of the new coronavirus ribonucleic acid standard generated in table 2. Counting the number of effective sequences of the novel coronavirus detected from the MNP marker site which is the same as the internal standard DNA detection primer, wherein the effective sequences refer to the number of sequences which are compared with the target MNP marker site. The copy number of the novel coronavirus in each sample was then calculated according to the formula y =0.9091 x-0.0662. The result shows that the positive and negative deviation of the quantitative result and the expected result of 1 copy/test sample is about 20 percent, and the positive and negative deviation of the quantitative result and the known result of 10 copies/test and 100 copies/test sample is less than 5 percent, thereby proving that the primer group provided by the embodiment of the invention is used for detecting 168 MNP marker loci on the novel coronavirus genome so as to realize accurate quantification of the novel coronavirus.
The quantitative method provided by the embodiment of the invention is adopted to carry out quantitative determination on the copy number of the new coronavirus in 38 samples detected in the table 5, the copy number is 4 copies at the lowest, and the highest copy number can reach 6362 copies.
Therefore, the 168 MNP marker loci have high polymorphism, low detection error rate and high genome coverage, so that the primer group and the kit provided by the embodiment of the invention can be used for qualitatively and quantitatively identifying the novel coronavirus in the sample to be detected with high sensitivity and high accuracy.
EXAMPLE III
The novel coronavirus is qualitatively and quantitatively identified by adopting 36 pairs of primer pairs, namely the 1 st primer pair to the 36 th primer pair, in the primer set provided by the embodiment of the invention. Based on the detection result of the standard substance of 1 copy/test in Table 2, 36 primer pairs of 36 MNP marker sites detected in 1 copy/test of S-1 to S-12 samples are selected, wherein the primer pairs of 3 MNP marker sites of the internal standard DNA for amplification are contained, 38 samples in Table 5 are qualitatively and quantitatively detected, the qualitative and quantitative detection effect of the 36 primer pairs is equivalent to the qualitative and quantitative detection effect of 168 primer pairs, namely, the 36 primer pairs can be used independently to accurately detect the positive samples of the novel coronavirus. Therefore, the accurate and sensitive qualitative and quantitative identification of the novel coronavirus can be realized by using the partial primer pairs provided by the invention under the condition of containing the internal standard DNA primer pairs. And 168 pairs of primers can be used for better performing the application of identifying virus variation and constructing a database, identifying more comprehensive virus variation and constructing a more refined fingerprint database. In this embodiment, 36 primer pairs of the 168 primer pairs are used for qualitative identification and quantitative identification, and in other embodiments, any one or any combination of the 168 primer pairs can be used for preparing internal standard DNA, and qualitative identification and quantitative identification can be performed.
Example four
The embodiment of the invention provides an application of a primer group, which comprises the following steps: 168 MNP marker loci on the novel coronavirus genome are detected by utilizing a primer group, and genetic variation identification of the novel coronavirus with non-diagnosis purpose is carried out according to the sequence of the obtained MNP marker loci.
Since the outbreak of new coronaviruses, the new coronaviruses have been mutated at many positions in the genome, resulting in different variant strains, such as Delta (Delta) and Omicron (Omicron) variant strains. The MNP marker locus covers most of the genome region of the novel coronavirus, and the sequence variation of the MNP marker locus is analyzed through sequencing, so that the genetic variation of strains contained in samples can be accurately revealed. In this example, the results of sequence alignment of virus MNP genotypes detected in 7 samples (S1-S7) with 150 or more MNP marker sites among 38 clinical samples shown in table 5 are shown in table 6.
Table 6 is a comparative analysis of genotypes of partially infected strains
Figure GDA0003620850150000131
As can be seen from Table 6, the major genotypes of the 2-5 MNP marker loci of the new coronavirus in some samples were different, indicating that the infected strains had variation. The primer group is used for identifying the genetic variation among strains, and can also be used for ensuring the genetic consistency of the same-named novel coronavirus strains in different laboratories, thereby ensuring the comparability of research results, and the primer group has important significance for the scientific research of novel coronaviruses. In this example, the 168 MNP marker loci are used together to identify genetic variants of a novel coronavirus, thereby identifying a relatively comprehensive virus variant. In other embodiments, any one or combination of the 168 MNP marker loci can be used for genetic variation identification of the novel coronavirus.
EXAMPLE five
The embodiment of the invention provides an application of a primer group, which comprises the following steps: and (3) detecting 168 MNP marker loci on the novel coronavirus genome by using a primer group, and constructing a novel coronavirus MNP fingerprint database according to the sequence of the obtained MNP marker loci.
After sequence comparison is carried out on the sequencing data of 38 samples detected in the first embodiment, the main genotype of each strain at the MNP marker site provided by the embodiment of the invention is obtained, so that the MNP fingerprint of each strain is formed and recorded into a database file to construct a novel coronavirus MNP fingerprint database. The constructed MNP fingerprint database is based on the gene sequences of the detected strains and is therefore compatible with all high throughput sequencing data. The MNP fingerprint spectrum of the strains obtained by each detection is compared with the established MNP fingerprint database, and the MNP fingerprint database established by the MNP fingerprint spectrum of the strains with different main genotypes realizes co-establishment sharing and real-time updating of the database. In this embodiment, the 168 MNP marker loci are used together to construct the MNP fingerprint database, so as to construct a refined fingerprint database. In other embodiments, any one or any combination of 168 MNP marker sites can be adopted for construction of the MNP fingerprint database.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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<210> 6
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tactgaatgc cttcgagttc tgcta 25
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ttcagtacgg tcgtagtggt gag 23
<210> 8
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
accagctcct ttattaccgt tctta 25
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
catagttacg gcgccgatct 20
<210> 10
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tgctatgttt agtgttccag ttttct 26
<210> 11
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ctatgtcgat aacaacttct gtggc 25
<210> 12
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
caataaagtc cagttgttcg gacaa 25
<210> 13
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ttgacactaa gaggggtgta tactg 25
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aaaaggtgtc tgcaattcat agctc 25
<210> 15
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
accttttgaa attaaattgg caaaga 26
<210> 16
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
caagcttttt cttttcaacc cttgg 25
<210> 17
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tggctttatg ggtagaattc gatct 25
<210> 18
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
caaaattcgc aagtggcttt aacaa 25
<210> 19
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
attttgtggc actgagaatt tgact 25
<210> 20
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ttatggtatt cggcaagact atgct 25
<210> 21
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gtgttctctt atgttggttg ccata 25
<210> 22
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gaaccttctc caacaacacc tgtat 25
<210> 23
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gcctggaata ttggtgaaca gaaat 25
<210> 24
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gcacagaatt ttgagcagtt tcaag 25
<210> 25
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gatgttcaca tctgatttgg ctact 25
<210> 26
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
caaggacggg tttgagtttt tcata 25
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gtccttgatt ggcttgaaga gaag 24
<210> 28
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
caatttcaca agcacaggtt gagat 25
<210> 29
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
taaatttttg gctttgtgtg ctgac 25
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
acacactttc tgtacaatcc ctttg 25
<210> 31
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ttgtattaac gggcttatgt tgctc 25
<210> 32
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
acttctatca cagtgtcatc accaa 25
<210> 33
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
tagaagtgca aggttacaag agtgt 25
<210> 34
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
caggcgaact catttacttc tgtac 25
<210> 35
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tggcagatgc tgtcataaaa acttt 25
<210> 36
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tgaagccaat ttaaactcac cagac 25
<210> 37
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tggtactgaa gatgattacc aaggt 25
<210> 38
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
tcatcatcta accaatcttc ttcttgc 27
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
aagacggcag tgaggacaat c 21
<210> 40
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cttcaatagt ctgaacaact ggtgt 25
<210> 41
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
tgaagtgaat agttttagtg gttatttaaa a 31
<210> 42
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
aggtaaacat tggctgcatt aacaa 25
<210> 43
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
aataaggcta ctaacaatgc catgc 25
<210> 44
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gacaacatga agacagtgtt tagca 25
<210> 45
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ttatcagctg gtatttttgg tgctg 25
<210> 46
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
tccaaaaagc ttgaaacaag tttgt 25
<210> 47
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
tttggaaatg aagagtgaaa agcaa 25
<210> 48
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
tgttttctct gttcaactga aggtt 25
<210> 49
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
tcaaagcttg tgttgaagaa gttac 25
<210> 50
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cagaatctgg atgaagattg ccatt 25
<210> 51
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
actcttgtta gtgacattga catcac 26
<210> 52
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
aggtataacc acagcagtta aaacac 26
<210> 53
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tatacctact aaaaaggctg gtggc 25
<210> 54
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gcctcctcta cagtgtaacc attta 25
<210> 55
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gaggcaaaga cagtgcttaa aaagt 25
<210> 56
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
ggcattaatt tgcgtgtttc ttctg 25
<210> 57
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
agcgtcactt atcaacacac ttaac 25
<210> 58
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
gtgaagaaac agaaactgta gctgg 25
<210> 59
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tcttcacctg atgctgttac ag 22
<210> 60
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
ttataggaac cagcaagtga gatgg 25
<210> 61
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ttctggacaa tctacacaac taggt 25
<210> 62
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
agtgtcttaa gattgtcaaa ggtga 25
<210> 63
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
ggacaacagt ttggtccaac ttatt 25
<210> 64
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
tagtactcaa aagcctcaac acgta 25
<210> 65
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
tggaaatacc cacaagttaa tggtt 25
<210> 66
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
cccttgctct gtaataagca tcttg 25
<210> 67
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
aagctgctaa cttttgtgca cttat 25
<210> 68
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
acgttcaaga ctcttttgca agaat 25
<210> 69
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
ggtgtgtaaa acttgtggac aacag 25
<210> 70
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
tttgtagctt gtttaccaca cgtac 25
<210> 71
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
acatttactt gtgctagtga gtacac 26
<210> 72
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
tttgtagaaa acatccgtaa taggac 26
<210> 73
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
ttatttcaca gagcaaccaa ttgat 25
<210> 74
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
agctctcttg aagcaggttt cttat 25
<210> 75
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
ttaaatggtg atgtggtggc tattg 25
<210> 76
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
caggtatttg gtttatacgt ggctt 25
<210> 77
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
tacctggtgt atacgttgtc tttgg 25
<210> 78
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
tttccactac ttcttcagag actgg 25
<210> 79
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
ccatacagaa agacgttctt gagtg 25
<210> 80
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
cataagcagc cattagatct gtgtg 25
<210> 81
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
gcttatgtag acaattctag tcttactat 29
<210> 82
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
agggacacta ttaacagcag ctaaa 25
<210> 83
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
tgtcccttgg gatactatag ctaat 25
<210> 84
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
ttagtacaaa cacggtttaa acacc 25
<210> 85
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
tgaagtcacc taatttttct aaactga 27
<210> 86
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
tgtaaccagt acagtaagaa ggcat 25
<210> 87
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
ggttacagag aaggctattt gaact 25
<210> 88
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
agtttctaaa gaaggatagg tgtct 25
<210> 89
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
gggatttaac tgcttttggc ttagt 25
<210> 90
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
tgcaaaatag ctgaaaaaca attgca 26
<210> 91
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
cccgatttca gctatggtta gaatg 25
<210> 92
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
tgtacattcg actcttgttg ctcta 25
<210> 93
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
ttgtgataca ttctgtgctg gtagt 25
<210> 94
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
atggatggaa ccattcttca ctgta 25
<210> 95
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
tgttaactta gacaacctga gagct 25
<210> 96
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
ataggttgac acataagctg actgt 25
<210> 97
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
actgttacta gatcaggcat tagtgt 26
<210> 98
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
tccattggta cgttaaaagt tgatga 26
<210> 99
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
aatctgacat agaagttact ggcga 25
<210> 100
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
actttttgct acctgcgcat taata 25
<210> 101
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
ttgtctgaac aactacgaaa acaaa 25
<210> 102
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
ccacccttaa gtgctatctt tgttg 25
<210> 103
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
tacacttgtg ttcctttttg ttgct 25
<210> 104
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
ccaccatcaa tagccttgta tccta 25
<210> 105
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
cactcgtgac atagcatcta cagat 25
<210> 106
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
aatcaatggg caagctttgt catta 25
<210> 107
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
catctgttac acaccatcaa aactt 25
<210> 108
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
gcaacagaac cttctagtac attgg 25
<210> 109
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
tcagaagctg gtgtttgtgt atcta 25
<210> 110
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
ccaataggtt gaattagtgg tgtaa 25
<210> 111
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
acctggtgtt tattctgtta tttacttgt 29
<210> 112
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
tccagaaagg tactaaaggt gtgaa 25
<210> 113
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
cgctgtgcac ctttttgtta aata 24
<210> 114
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
cttctctgta gctagttgta tccat 25
<210> 115
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
aaggctctca atgacttcag taact 25
<210> 116
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
tgggaatgcc atttttctaa aacca 25
<210> 117
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
ccatctggta aagttgaggg ttgta 25
<210> 118
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
cacatgtctt ggacagtaaa ctacg 25
<210> 119
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
cctctgaaga catgcttaac cctaa 25
<210> 120
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
agaatgtcca ataaccctga gttga 25
<210> 121
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
cagccaatcc taagacacct aagta 25
<210> 122
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
gaacccttaa tagtgaaatt gggcc 25
<210> 123
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
tccttaatgg ttcatgtggt agtgt 25
<210> 124
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
aaagttacct tctaagtctg tgcca 25
<210> 125
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
gtacgctgct gttataaatg gagac 25
<210> 126
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
gtttgagcag aaagaggtcc tagta 25
<210> 127
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
ctgcaaaatg gtatgaatgg acgta 25
<210> 128
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
tacccttgat tgttcttttc actgc 25
<210> 129
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
tccagagtac tcaatggtct ttgtt 25
<210> 130
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
ttgacaaaca tcattgcaaa agcag 25
<210> 131
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
gtttttgtta ccttctcttg ccact 25
<210> 132
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
gctgatgcat acataacaca gtctt 25
<210> 133
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
agaactgtgt atgatgatgg tgcta 25
<210> 134
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
cagagattat aagagcccac atgga 25
<210> 135
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
atgtttttgg ccagaggtat tgttt 25
<210> 136
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
aacaaaagag gccaaagtaa caagt 25
<210> 137
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
tactcaaccg ctactttaga ctgac 25
<210> 138
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
caccaacacc caacaattta atgtt 25
<210> 139
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
catctaaatt gtgggctcaa tgtgt 25
<210> 140
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
tgcatggaaa gcaaaacaga aagta 25
<210> 141
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
ccttacaagc tatagcctca gagtt 25
<210> 142
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
gccacattca aagacttctt caact 25
<210> 143
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
gacaagaggg caaaagttac tagtg 25
<210> 144
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
tggctgctgt tgtaagaggt attat 25
<210> 145
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
atccaacagg ttgtagatgc agata 25
<210> 146
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
aaagctgtta caataagagg ccatg 25
<210> 147
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
taatgagctt agtcctgttg cacta 25
<210> 148
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
aagtacaaac ctacctccct ttgtt 25
<210> 149
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
cgatttacag gatttgaaat gggct 25
<210> 150
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
gtgtgtctgt aacaaaccta caagg 25
<210> 151
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
cctaaaggtc ctaaagtgaa gtatttat 28
<210> 152
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
gataatacag ttgaattggc aggca 25
<210> 153
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
tactggtcag gcaataacag ttaca 25
<210> 154
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
ggatttggat gatctatgtg gcaac 25
<210> 155
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
tacctacaac ttgtgctaat gaccc 25
<210> 156
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
ttaaaaacga ttgtgcatca gctga 25
<210> 157
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
ctagtactga tgtcgtatac agggc 25
<210> 158
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
gtcatcttcg tccttttctt ggaag 25
<210> 159
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
tgctttaagg cattttgatg aaggt 25
<210> 160
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
cgtatacgcg taatatatct gggtt 25
<210> 161
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
aacagtacaa ttctgtgatg ccatg 25
<210> 162
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
aatctacaac aggaactcca ctacc 25
<210> 163
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
gtagattctt attattcatt gttaatgcct 30
<210> 164
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
tttaacctct cttccgtgaa gtcat 25
<210> 165
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
aactgtttgg atgacagatg cattc 25
<210> 166
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
tctgaagtgg tatccagttg aaact 25
<210> 167
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
aaggaattac ttgtgtatgc tgctg 25
<210> 168
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
aattaccggg tttgacagtt tgaaa 25
<210> 169
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
aaacacttct tctttgctca ggatg 25
<210> 170
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
ccaccatcgt aacaatcaaa gtact 25
<210> 171
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
caacctagac aaatcagctg gtttt 25
<210> 172
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
ggatgacatt acgttttgta tatgcg 26
<210> 173
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
agctggtgtc tctatctgta gtact 25
<210> 174
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
cagtttttaa catgttgtgc caacc 25
<210> 175
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
tgggttggga ttatcctaaa tgtga 25
<210> 176
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
tatagaaacg gtgtgacaag ctaca 25
<210> 177
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
tgtggcggtt cactatatgt taaac 25
<210> 178
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
cggcaatttt gttaccatca gtaga 25
<210> 179
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
acttatgcat ctcaaggtct agtgg 25
<210> 180
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
tgttgagagc aaaattcatg aggtc 25
<210> 181
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
tgaacggttc gtgtctttag ctata 25
<210> 182
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
tgtcctgtta actcatcatg tagct 25
<210> 183
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
tgcttactaa tgataacact tcaaggt 27
<210> 184
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
gtatgcaagc accacatctt aatga 25
<210> 185
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
tgttaatccg tatgtttgca atgct 25
<210> 186
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
ttgtccatta gcacacaatg gaaaa 25
<210> 187
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 187
tgtgttggta gcgataatgt tactg 25
<210> 188
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 188
gagtctttca gtacaggtgt tagct 25
<210> 189
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 189
tcatgggaag ttggtaaacc tagac 25
<210> 190
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 190
ggtaaacaac agcatcacca tagtc 25
<210> 191
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 191
gccacaagag cactatgtta gaatt 25
<210> 192
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 192
ggtccctgga gtgtagaata ctttt 25
<210> 193
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 193
tgcactatgt gagaaggcat taaaa 25
<210> 194
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 194
tcaggcaatg catttacagt acaaa 25
<210> 195
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 195
agttgtcttt gatgaaattt caatggc 27
<210> 196
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 196
ttctagtgtg cccttagtta gcaat 25
<210> 197
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 197
tgaaaactat aggtccagac atgtt 25
<210> 198
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 198
tgagctgatt tgtctttatg tgctt 25
<210> 199
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 199
caaataggcg tggtaagaga attcc 25
<210> 200
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 200
tagtcccaaa atctttgagg ctaca 25
<210> 201
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 201
actcaaactg ttgattcatc acagg 25
<210> 202
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 202
agtatgccta cttttgctct ggtaa 25
<210> 203
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 203
gtggcaactt tacaagctga aaatg 25
<210> 204
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 204
atgccaggta tgtcaacaca taaac 25
<210> 205
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 205
gcgaagaagc tataagacat gtacg 25
<210> 206
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 206
taggtacagc aactaggtta acacc 25
<210> 207
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 207
gcctggagat caatttaaac acctc 25
<210> 208
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 208
cccataagac aaatacgact ctgtc 25
<210> 209
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 209
gggttttaca ggtaacctac aaagc 25
<210> 210
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 210
tattcaatag tccagtcaac acgct 25
<210> 211
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 211
acgacattgg taaccctaaa gctat 25
<210> 212
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 212
tgaatttgtc agaatgtgtg gcata 25
<210> 213
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 213
tcctgctaat tccattgttt gtaga 25
<210> 214
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 214
ttttatcaaa agctggtgtg tggaa 25
<210> 215
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 215
ccatttttct attactctga cagtcca 27
<210> 216
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 216
caccacctaa attgcaacgt gttat 25
<210> 217
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 217
tataacatga tgatctcagc tggct 25
<210> 218
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 218
tgttgtccat caaagtgtcc cttat 25
<210> 219
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 219
accagtacca gaggtgaaaa tactc 25
<210> 220
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 220
tggtttcttg gctatgtcag tcata 25
<210> 221
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 221
ttgatggtag agttgatggt caagt 25
<210> 222
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 222
tccattaaga ctagcttgtt tggga 25
<210> 223
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 223
cccaggagtc aaatggaaat tgatt 25
<210> 224
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 224
tccaatcagt agatgtaaac cacct 25
<210> 225
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 225
tggactagct aaacgtttta aggaa 25
<210> 226
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 226
gaacacacac acttagatga acctg 25
<210> 227
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 227
ggccatgtag aaacatttta cccaa 25
<210> 228
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 228
gcctttaggt aatgttgcac tatca 25
<210> 229
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 229
aggcataatg atgaatgtcg caaaa 25
<210> 230
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 230
ccagcaccaa aatgtataac tctca 25
<210> 231
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 231
caggtacagc tgttttaaga cagtg 25
<210> 232
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 232
gtatgtacag ttgcacaatc accaa 25
<210> 233
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 233
agtgatatgt acgaccctaa gacta 25
<210> 234
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 234
tgttatcttt atagccacgg aacct 25
<210> 235
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 235
aatgtgaatg cgtcatcatc tgaag 25
<210> 236
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 236
aggaagacaa ctgaattgga tttgt 25
<210> 237
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 237
aattaagggg tactgctgtt atgtc 25
<210> 238
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 238
tgttcgttta gttgttaaca agaaca 26
<210> 239
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 239
cttacaacca gaactcaatt acccc 25
<210> 240
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 240
aggtaagaac aagtcctgag ttgaa 25
<210> 241
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 241
ggaccaatgg tactaagagg tttga 25
<210> 242
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 242
agtagtacca aaaatccagc ctctt 25
<210> 243
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 243
attgttaata acgctactaa tgttgttatt 30
<210> 244
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 244
aaaagtgcaa ttattcgcac tagaa 25
<210> 245
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 245
cagccttttc ttatggacct tgaag 25
<210> 246
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 246
tgagggagat cacgcactaa attaa 25
<210> 247
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 247
tttagaacca ttggtagatt tgcca 25
<210> 248
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 248
tgaagaagaa tcaccaggag tcaaa 25
<210> 249
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 249
gctgcagctt attatgtggg ttatc 25
<210> 250
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 250
tacactttgt ttctgagaga gggtc 25
<210> 251
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 251
tattacaaac ttgtgccctt ttggt 25
<210> 252
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 252
acttaaaagt ggaaaatgat gcgga 25
<210> 253
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 253
tctctgcttt actaatgtct atgcaga 27
<210> 254
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 254
ataacgcagc ctgtaaaatc atctg 25
<210> 255
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 255
caatcttgat tctaaggttg gtggt 25
<210> 256
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 256
aaaccttcaa caccattaca aggtg 25
<210> 257
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 257
caccttgtaa tggtgttgaa ggttt 25
<210> 258
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 258
ctggtgcatg tagaagttca aaaga 25
<210> 259
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 259
accaaccata cagagtagta gtact 25
<210> 260
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 260
tgcctgttaa accattgaag ttgaa 25
<210> 261
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 261
gcacaggtgt tcttactgag tctaa 25
<210> 262
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 262
aagaatctca agtgtctgtg gatca 25
<210> 263
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 263
atacttctaa ccaggttgct gttct 25
<210> 264
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 264
tgtagaataa acacgccaag tagga 25
<210> 265
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 265
aaacacgtgc aggctgttta atag 24
<210> 266
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 266
tgatggattg actagctaca ctacg 25
<210> 267
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 267
catttgtggt gattcaactg aatgc 25
<210> 268
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 268
tcttgggtgt ttttgtcttg ttcaa 25
<210> 269
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 269
ccaagaagtt tttgcacaag tcaaa 25
<210> 270
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 270
aaatgacctc ttgcttggtt ttgat 25
<210> 271
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 271
ctggcttcat caaacaatat ggtga 25
<210> 272
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 272
agcaatcatt tcatctgtga gcaaa 25
<210> 273
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 273
ctcaatacac ttctgcactg ttagc 25
<210> 274
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 274
aacattctgt gtaactccaa tacca 25
<210> 275
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 275
accaaaatgc acaagcttta aacac 25
<210> 276
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 276
ctatcaattt gcacttcagc ctcaa 25
<210> 277
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 277
gtttgcagac atatgtgact caaca 25
<210> 278
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 278
tagccctttc cacaaaaatc aactc 25
<210> 279
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 279
gggctatcat cttatgtcct tccc 24
<210> 280
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 280
agtgtgcttt tccatcatga caaat 25
<210> 281
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 281
acacaaagga atttttatga accaca 26
<210> 282
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 282
atctaactcc tccttgaatg agtct 25
<210> 283
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 283
gataaatatt ttaagaatca tacatcacca ga 32
<210> 284
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 284
gaggcggtca atttcttttt gaatg 25
<210> 285
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 285
cctcaatgag gttgccaaga attta 25
<210> 286
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 286
gcaatcaagc cagctataaa accta 25
<210> 287
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 287
ttatgctttg ctgtatgacc agttg 25
<210> 288
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 288
tgtaatttga ctcctttgag cactg 25
<210> 289
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 289
attgttggcg ttgcacttct tg 22
<210> 290
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 290
gagcaaaagg tgtgagtaaa ctgt 24
<210> 291
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 291
agcccctttt ctctatcttt atgct 25
<210> 292
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 292
ccagcaaaga aaatagttgg catca 25
<210> 293
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 293
aattgtcatt acttcaggtg atggc 25
<210> 294
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 294
acagctggta atagtctgaa gtgaa 25
<210> 295
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 295
acttcttttt cttgctttcg tggta 25
<210> 296
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 296
agactcacgt taacaatatt gcagc 25
<210> 297
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 297
tctagagttc ctgatcttct ggtct 25
<210> 298
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 298
taatagtacc gttggaatct gccat 25
<210> 299
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 299
gctccttgaa caatggaacc tagta 25
<210> 300
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 300
caagctaaag ttactggcca taaca 25
<210> 301
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 301
tagcttgttt tgtgcttgct gc 22
<210> 302
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 302
tgaaagaagc aatgaagtag ctgag 25
<210> 303
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 303
catgtggtca ttcaatccag aaact 25
<210> 304
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 304
tccgattacg agttcacttt ctaga 25
<210> 305
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 305
aagaaatcac tgttgctaca tcacg 25
<210> 306
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 306
tttatagttg ccaatcctgt agcga 25
<210> 307
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 307
acaacagatg tttcatctcg ttgac 25
<210> 308
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 308
atgatgtaat caagattcca aatgga 26
<210> 309
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 309
agctttatca ctaccaagag tgtgt 25
<210> 310
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 310
ctagaggatg aaatggtgaa ttgcc 25
<210> 311
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 311
agctgataac aaatttgcac tgact 25
<210> 312
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 312
agttcttgaa cttcctcttg tctga 25
<210> 313
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 313
tcttattgtt gcggcaatag tgttt 25
<210> 314
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 314
tagcagaaag gctaaaaagc acaaa 25
<210> 315
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 315
acgcctaaac gaacatgaaa tttct 25
<210> 316
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 316
agaatagaag tgaataggac acggg 25
<210> 317
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 317
aggagctaga aaatcagcac cttta 25
<210> 318
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 318
cccaatttag gttcctggca attaa 25
<210> 319
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 319
tagagtatca tgacgttcgt gttgt 25
<210> 320
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 320
tggttactgc cagttgaatc tgag 24
<210> 321
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 321
taaccagaat ggagaacgca gtg 23
<210> 322
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 322
gttaattgga acgccttgtc ctc 23
<210> 323
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 323
ttaacaccaa tagcagtcca gatga 25
<210> 324
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 324
ggcccagttc ctaggtagta gaaat 25
<210> 325
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 325
gacttcccta tggtgctaac aaaga 25
<210> 326
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 326
cttgaggaag ttgtagcacg attg 24
<210> 327
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 327
ctcttctcgt tcctcatcac gtag 24
<210> 328
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 328
ctcaagctgg ttcaatctgt caag 24
<210> 329
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 329
ttgagagcaa aatgtctggt aaagg 25
<210> 330
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 330
gtatgcttta gtggcagtac gtttt 25
<210> 331
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 331
aaggaactga ttacaaacat tggcc 25
<210> 332
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 332
atccaatttg atggcacctg tgtag 25
<210> 333
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 333
catacaaaac attcccacca acaga 25
<210> 334
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 334
tgcaattgtt tggagaaatc atcca 25
<210> 335
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 335
aattgcaaca atccatgagc agtg 24
<210> 336
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 336
aattcattct gcacaagagt agact 25
<210> 337
<211> 179
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 337
aataaggcta ctaacaatgc catgcataaa cctaagtgca ttgcatttga cacccgggtg 60
taaattggac gttctatgta attactggtt acaatgcgtg ttgtgatcaa cacaacaggg 120
ttaatgagtg atattaactc tcgagctaac actttgctaa acactgtctt catgttgtc 179
<210> 338
<211> 179
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 338
aataaggcta ctaacaatgc catgcaaata aggtaaataa ggaaatcgag ttacatgctt 60
gccttaagga gaggtttcag ggtgctcccg agcaattagt aggttgcctg ccagatattc 120
ttttcctaat tacctagcat tctgcttaat aaattgctaa acactgtctt catgttgtc 179
<210> 339
<211> 179
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 339
aataaggcta ctaacaatgc catgcaaaca gagactttac aagatgcgta tatcaccggt 60
gctacagggt tgccgtggct tcaattaaag atttccaatt gtgagcatag agaaaactac 120
tcttttattt tcaccgcttt gattggaata atggtgctaa acactgtctt catgttgtc 179
<210> 340
<211> 196
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 340
tataacatga tgatctcagc tggctaagac tttccgataa ttaaaagcaa gccaggctga 60
tgttaactat gttagacttg aataaaatgg ttcagagtta ttcgttcgtt agtatccgat 120
aatccactga gcgtgttgta atggcaaaaa gtatttttat aaagctcttc tataagggac 180
actttgatgg acaaca 196
<210> 341
<211> 196
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 341
tataacatga tgatctcagc tggctaaaag actacagaaa atgattttac taggtgaagc 60
agatcaacgt ccttcttagt cattgttagt ctagagtaga tttttaacca atcggaattg 120
tagatcgtgc ttgattggga cctgtaactt cacttattaa tggcaaaatt tataagggac 180
actttgatgg acaaca 196
<210> 342
<211> 196
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 342
tataacatga tgatctcagc tggctcaaag cctcattatt aggatttaat actcatgaca 60
ggtgatttcg atacagtcac gtacttccct tgtaataaag tggtattttt taacctgtaa 120
aggtggaaca aaagttctat ttttaagaga ctttgacagg tgtatgaaga cataagggac 180
actttgatgg acaaca 196
<210> 343
<211> 197
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 343
tagagtatca tgacgttcgt gttgtcagac ccataagcac ccgctgtgcg accggtcggg 60
ctccttaatc aacaacaaag ccttacaccc tatagtactg ttcctgagaa acttgccagg 120
acagacatgt gttcgttcaa aaggctgagg attgaggtac aaaaagccca acgctcagat 180
tcaactggca gtaacca 197
<210> 344
<211> 197
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 344
tagagtatca tgacgttcgt gttgtggatc cttggccaca tagaccatga gtcactcaca 60
gccctcgtac ttgccaagcg gtcttaaacc aacgaaccac taaaaacctg gtatacgaag 120
gtatcttgct ggcccagtat gatacggaga ccccgaaaac gagtacagtg tagctcagat 180
tcaactggca gtaacca 197
<210> 345
<211> 197
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 345
tagagtatca tgacgttcgt gttgttgtta acgctgtgca gaaccaaata gcctacctca 60
ataagagcgc tggatcaaac agggccatcg cgtttataag cagcaatagc gcgcatagtc 120
tgtactcgag cctggcacct acgttcggtt aaacaacacg cctacgagaa accctcagat 180
tcaactggca gtaacca 197

Claims (5)

1. A primer group for identifying a novel coronavirus is characterized by comprising a 1 st primer pair to a 36 th primer pair, wherein each primer pair comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the forward primer of the 1 st primer pair to the 36 th primer pair and the reverse primer of the 36 th primer pair are sequentially shown as SEQ ID NO:1 to SEQ ID NO:72 is shown;
or, the primer group consists of a 1 st primer pair to a 168 th primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the forward primer of the 1 st primer pair to the 168 th primer pair and the reverse primer of the 168 th primer pair are sequentially shown as SEQ ID NO:1 to SEQ ID NO: 336.
2. A kit for identifying a novel coronavirus, wherein the kit comprises the primer set of claim 1.
3. The kit of claim 2, further comprising the amino acid sequence set forth in SEQ ID NO:337 to SEQ ID NO:346, 9 internal standard DNAs.
4. The kit of claim 2, further comprising a multiplex PCR premix.
5. Use of the primer set according to claim 1, wherein the use comprises: the primer group is utilized to detect 36 or 168 MNP marker loci on the novel coronavirus genome, and qualitative identification, quantitative identification, genetic variation identification, MNP fingerprint database construction and fine typing of the novel coronavirus with non-diagnostic purpose are carried out according to the sequence of the obtained MNP marker loci, wherein the 168 MNP marker loci are shown as the following table:
Figure FDA0003835191350000011
Figure FDA0003835191350000021
Figure FDA0003835191350000031
Figure FDA0003835191350000041
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