1. The LAMP primer for detecting serotypes 2a and XV of the Shigella flexneri is characterized by comprising an LAMP primer for detecting wzx gene of the Shigella flexneri, an LAMP primer for detecting gtrII gene of the Shigella flexneri, an LAMP primer for detecting gtrX gene of the Shigella flexneri and an LAMP primer for detecting opt gene of the Shigella flexneri;

the LAMP primer for detecting the shigella flexneri wzx gene comprises the following components:

outer forward primer F3: 5'-GTGCAGAATAAAATGCAACTT-3', respectively;

outer reverse primer B3: 5'-CTGGTTTAATATTTGCAAGAGTT-3', respectively;

inner forward primer FIP: 5'-GGCGGTTGGATGACAGTAAGTAATATGCCCCCATAATATTTGACA-3', respectively;

inner reverse primer BIP: 5'-ACAAAAATGAACCCCCGAAATTTTTTCGATTTTAGTCTCTGCCTAT-3', respectively;

the LAMP primer for detecting the gene gtrII of the Shigella flexneri comprises the following components:

outer forward primer F3: 5'-CGAAATGAGAAAAATATTCGTGA-3', respectively;

outer reverse primer B3: 5'-AGGAATCAGTAACCACTTGT-3', respectively;

inner forward primer FIP: 5'-TGATAGCTTTGGTTAATAGCAGGTAAGTTCATGTTTATCTCTATCCCTCA-3', respectively;

inner reverse primer BIP: 5'-TGCAAGCGATATTAAATGGCTTGTTTTAAACCAGTGGGAGGATT-3', respectively;

the LAMP primer for detecting the gene gtrX of the Shigella flexneri comprises the following components:

outer forward primer F3: 5'-ATTGGCAAATGAAAGTGTTTG-3', respectively;

outer reverse primer B3: 5'-TGTCAGGAAGAGGGGTTA-3', respectively;

inner forward primer FIP: 5'-GTTGGCAGTTGAGGAACAGAATCTTATTGTCGCATTTTCTATGG-3', respectively;

inner reverse primer BIP: 5'-ATGCGTTTGTTTGTTTCAATGCATACATTGCAATAAGGATGACTGAA-3', respectively;

the LAMP primer for detecting the opt gene of the Shigella flexneri comprises the following components:

outer forward primer F3: 5'-ATAGTCCTCCCTTCCGTAT-3', respectively;

outer reverse primer B3: 5'-AGAGGTTTAAGAGGACAGTT-3', respectively;

inner forward primer FIP: 5'-ACTTGAGGAAAATGGTTACCATACATGATAGCATTAAACTCAATGGGTC-3', respectively;

inner reverse primer BIP: 5'-TCGATCTCTACTTTAGATGCTTGGCCCTCATCATATCAAATGACCG-3' are provided.

2. The LAMP primer according to claim 1, characterized in that the LAMP primer for detecting the Shigella flexneri wzx gene further comprises: loop primer LF: 5'-GGTGTATTTTGATCGATTTATAG-3', respectively; and/or

The LAMP primer for detecting the shigella flexneri gtrII gene also comprises: loop primer LF: 5'-TGAGATGTACTCGTTCGA-3' and loop primer LB: 5'-CAGCCATAGATAATATATATAACTTC-3', respectively; and/or

The LAMP primer for detecting the shigella flexneri gtrX gene also comprises: loop primer LF: 5'-CTGCCAGTTGAGGTTTTG-3', respectively; and/or

The LAMP primer for detecting the opt gene of the Shigella flexneri also comprises the following components: the loop primer LB: 5'-CAAGGCCAGAATTATCTT-3' are provided.

3. A detection reagent or kit comprising the LAMP primer according to claim 1 or 2.

4. Use of the LAMP primer of claim 1 or 2 or the detection reagent or kit of claim 3 for detection of Shigella flexneri serotype 2a and XV for non-disease diagnostic and therapeutic purposes.

5. The shigella flexneri serotype 2a and Xv detection method is characterized by comprising the following steps:

1) extracting DNA in a sample to be detected;

2) performing LAMP amplification reaction by using the DNA extracted in the step 1) as a template and using the LAMP primer according to claim 2;

3) judging an amplification result;

the methods are for non-disease diagnostic and therapeutic purposes.

6. The method of claim 5, wherein the 25 μ L reaction system used in step 2) is: 12.5 mu L of premix, 1.4 mu L of primer mixture, 1 mu L of DNA template and 10.1 mu L of sterile water;

wherein, in the primer mixture, 40pmol of each of the primers FIP and BIP, 10pmol of each of the primers F3 and B3, and 20pmol of each of the primers LF and/or LB are contained.

7. The method of claim 6, wherein the reaction conditions used in step 2) are: at 61 ℃ for 20 minutes.

8. The method according to any one of claims 5 to 7, wherein the decision criterion of step 3) is: if the amplification result shows that the gtrII and wzx genes are positive and the gtrX and opt genes are negative, judging the serotype 2 a; and if the amplification result shows that the gtrX, opt and wzx genes are positive and the gtrII gene is negative, judging the serotype as the XV.

9. The method according to claim 8, wherein the amplification result is determined in step 3) by any one of the following (i) to (iii):

a fluorescent staining method: after the amplification reaction is finished, judging the amplification condition of the target gene according to the color change of the reaction system before and after the reaction;

② agarose gel electrophoresis method: if the amplification product presents a characteristic ladder-shaped band on the agarose gel, the target gene is contained in the sample to be detected;

③ turbidity detection of magnesium pyrophosphate: whether LAMP amplification reaction occurs or not is judged by observing the turbid condition after reaction by naked eyes, or the absorbance of the LAMP amplification reaction at 400nm is detected by a turbidimeter, so that the detection of the target gene is realized.

Background

According to statistics, the serotypes of Shigella flexneri which are mainly popular in China are 2a serotypes and XV serotypes. According to the statistical data of 1991-2000, the main epidemic Shigella flexneri in China is 86 percent. The dominant serotype is 2a, and the proportion reaches 80%. The XV serotype appeared in Henan in the first 2000 years, and then the 2a serotype was replaced in 2002-2006 to become the dominant serotype. In 2007, shigella isolated in gansu and anhui provinces, the Xv serotype was also the dominant serotype, accounting for 67% and 54% of isolation, respectively. Monitoring data from 2003 to 2013, based on national sentry hospitals, showed that of isolated 2912 shigella flexneri was 1610 strain, of which 2a (n ═ 500, 31.1%) and Xv (n ═ 433, 26.9%) serotypes were the dominant serotypes of shigella flexneri. Data show that in the monitoring data of 10 provinces and 20 national monitoring points of an infectious disease epidemic monitoring network in China, 148 strains of Shigella flexneri are separated in 2015, wherein the 2a serotype 68 strain and the XV serotype 19 strain are separated; shigella flexneri 103 strain was isolated in 2016, with serotype 2a 21 strain; shigella flexneri 88 strain was isolated in 2017, of which serotype 2a 59 strain; shigella flexneri 66 strains were isolated in 2018, of which serotype 2a 52 strains were isolated.

The conventional identification of the serotype of Shigella flexneri mainly adopts commercial diagnostic antiserum to carry out a slide agglutination reaction with a strain, and judgment is carried out according to an immunoagglutination result. The Xv serotype requires group-specific antisera 7; 8 and the monoclonal antibody MASF IV-1. However, the commercial monoclonal antibody MASF IV-1 is expensive, and is not easily obtained by basic disease control departments, so that the application of the monoclonal antibody is limited; moreover, the result of the serum agglutination reaction is influenced by the culture condition and the growth state of the bacteria, and the result judgment subjective factor has large influence.

The diversity of Shigella flexneri serotypes is determined by type-specific epitopes (e.g., I-V and IC) and group-specific epitopes (e.g., 3, 4; 6; and 7, 8). According to the Shigella flexneri Xv serotype O anti-modification and the generation mechanism thereof, specific primers are designed aiming at type-specific antigenic determinants and group-specific antigenic determinants respectively, and a multiple PCR system is designed to be used for detecting serotypes. However, the multiplex PCR method has high requirements for reaction reagents, the PCR reaction requires at least about 2 hours, gel electrophoresis is required for result interpretation, and the result interpretation is complex.

In addition to the multiplex PCR method, a fluorescent quantitative PCR method was designed based on Shigella flexneri serotype-specific epitopes and population-specific epitope genes. The current fluorescent quantitative PCR system needs a plurality of reactions to be carried out simultaneously to detect the serotype, and needs a fluorescent quantitative PCR instrument, which is not beneficial to popularization and use of the basic layer.

Disclosure of Invention

The invention aims to provide LAMP primers and a detection method for detecting serotypes 2a and XV of Shigella flexneri.

In order to realize the purpose of the invention, in the first aspect, the invention provides LAMP primers for detecting serotypes 2a and XV of Shigella flexneri, which comprise an LAMP primer for detecting wzx gene of Shigella flexneri, an LAMP primer for detecting gtrII gene of Shigella flexneri, an LAMP primer for detecting gtrX gene of Shigella flexneri and an LAMP primer for detecting opt gene of Shigella flexneri.

The LAMP primer for detecting the shigella flexneri wzx gene comprises (SEQ ID NO: 1-4):

outer forward primer F3: 5'-GTGCAGAATAAAATGCAACTT-3', respectively;

outer reverse primer B3: 5'-CTGGTTTAATATTTGCAAGAGTT-3', respectively;

inner forward primer FIP: 5'-GGCGGTTGGATGACAGTAAGTAATATGCCCCCATAATATTTGACA-3', respectively;

inner reverse primer BIP: 5'-ACAAAAATGAACCCCCGAAATTTTTTCGATTTTAGTCTCTGCCTAT-3', respectively;

the LAMP primer for detecting the gtrII gene of the Shigella flexneri comprises (SEQ ID NO: 6-9):

outer forward primer F3: 5'-CGAAATGAGAAAAATATTCGTGA-3', respectively;

outer reverse primer B3: 5'-AGGAATCAGTAACCACTTGT-3', respectively;

inner forward primer FIP: 5'-TGATAGCTTTGGTTAATAGCAGGTAAGTTCATGTTTATCTCTATCCCTCA-3', respectively;

inner reverse primer BIP: 5'-TGCAAGCGATATTAAATGGCTTGTTTTAAACCAGTGGGAGGATT-3', respectively;

the LAMP primer for detecting the gene of the Shigella flexneri gtrX comprises (SEQ ID NO: 12-15):

outer forward primer F3: 5'-ATTGGCAAATGAAAGTGTTTG-3', respectively;

outer reverse primer B3: 5'-TGTCAGGAAGAGGGGTTA-3', respectively;

inner forward primer FIP: 5'-GTTGGCAGTTGAGGAACAGAATCTTATTGTCGCATTTTCTATGG-3', respectively;

inner reverse primer BIP: 5'-ATGCGTTTGTTTGTTTCAATGCATACATTGCAATAAGGATGACTGAA-3', respectively;

the LAMP primer for detecting the opt gene of the Shigella flexneri comprises (SEQ ID NO: 17-20):

outer forward primer F3: 5'-ATAGTCCTCCCTTCCGTAT-3', respectively;

outer reverse primer B3: 5'-AGAGGTTTAAGAGGACAGTT-3', respectively;

inner forward primer FIP: 5'-ACTTGAGGAAAATGGTTACCATACATGATAGCATTAAACTCAATGGGTC-3', respectively;

inner reverse primer BIP: 5'-TCGATCTCTACTTTAGATGCTTGGCCCTCATCATATCAAATGACCG-3', respectively;

preferably, the LAMP primer for detecting the shigella flexneri wzx gene further comprises: loop primer LF: 5'-GGTGTATTTTGATCGATTTATAG-3' (SEQ ID NO: 5).

Preferably, the LAMP primer for detecting the shigella flexneri gtrII gene further comprises: loop primer LF: 5'-TGAGATGTACTCGTTCGA-3' and loop primer LB: 5'-CAGCCATAGATAATATATATAACTTC-3' (SEQ ID NOS: 11-12).

Preferably, the LAMP primer for detecting the shigella flexneri gtrX gene further comprises: loop primer LF: 5'-CTGCCAGTTGAGGTTTTG-3' (SEQ ID NO: 16).

Preferably, the LAMP primer for detecting the Shigella flexneri opt gene further comprises: the loop primer LB: 5'-CAAGGCCAGAATTATCTT-3' (SEQ ID NO: 21).

In a second aspect, the invention provides a detection reagent or kit containing the LAMP primer.

In a third aspect, the invention provides the application of the LAMP primer or the detection reagent or kit containing the LAMP primer in the detection of Shigella flexneri serotype 2a and XV (the application includes non-disease diagnosis and treatment purposes).

In a fourth aspect, the present invention provides a method for detecting shigella flexneri serotypes 2a and Xv (said method including non-disease diagnostic and therapeutic purposes), comprising the steps of:

1) extracting DNA in a sample to be detected;

2) performing LAMP amplification reaction by using the LAMP primer by using the DNA extracted in the step 1) as a template (the DNA template can be prepared by using a water boiling method);

3) and (5) judging an amplification result.

The 25 μ L reaction system used in step 2) is: 12.5. mu.L of premix, 1.4. mu.L of primer mixture, 1. mu.L of DNA template, and 10.1. mu.L of sterile water.

Wherein, in the primer mixture, 40pmol of each of the primers FIP and BIP, 10pmol of each of the primers F3 and B3, and 20pmol of each of the primers LF and/or LB are contained.

The premix may be obtained from New England Biolabs, Inc. (USA) using the WarmStart Colorimetric LAMP 2 × Master premix.

Preferably, the reaction conditions are: at 61 ℃ for 20 minutes.

The judgment standard of the step 3) is as follows: if the amplification result shows that the gtrII and wzx genes are positive and the gtrX and opt genes are negative, judging the serotype 2 a; and if the amplification result shows that the gtrX, opt and wzx genes are positive and the gtrII gene is negative, judging the serotype as the XV.

Further, the amplification result can be determined by any one of the following methods (i) to (iii):

a fluorescent staining method: after the amplification reaction is finished, judging the amplification condition of the target gene according to the color change of the reaction system before and after the reaction;

② agarose gel electrophoresis method: if the amplification product presents a characteristic ladder-shaped band on the agarose gel, the target gene is contained in the sample to be detected;

③ turbidity detection of magnesium pyrophosphate: whether LAMP amplification reaction occurs or not is judged by observing the turbid condition after reaction by naked eyes, or the absorbance of the LAMP amplification reaction at 400nm is detected by a turbidimeter, so that the detection of the target gene is realized.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the operation is simple, fast and efficient. After a reaction system is prepared, a water boiling template is added, and the result can be observed by naked eyes after incubation for 20 minutes at 61 ℃.

(II) high sensitivity and high specificity. The sensitivity of the four genes to the shigella flexneri poaching template is 1 pg/mu L (wzx gene), 100 fg/mu L (gtrII gene), 1 pg/mu L (gtrX gene) and 100 fg/mu L (opt gene) respectively. The verification in 19 different serotypes of Shigella flexneri and other species of Shigella flexneri proves that the method has good specificity.

And (III) no expensive equipment is needed. The reaction can be realized only by a water bath tank. The reaction conditions of the method are 61 ℃ and 20 minutes. The reaction condition is mild and controllable.

Drawings

FIG. 1 shows the positions of LAMP primers of the present invention on genes, and primers are designed based on wzx (A), gtrII (B), gtrX (C) and opt (D).

FIG. 2 is a diagram showing LAMP reaction of Shigella flexneri serotype 2a and XV in a preferred embodiment of the present invention. A: the results of LAMP reactions of serotypes 2a and XV were visually observed. B: results of LAMP reactions of serotypes 2a and Xv observed using a nephelometer. C: and (5) agarose gel electrophoresis results. Wherein, every four tubes/columns/lanes are in a group, which represents a group of LAMP reaction systems of four genes. Tubes 1-4/columns 1-4 (B1)/lanes 1-4: shigella flexneri 2a positive reaction; tubes 9-12/columns 1-4 (B2)/lanes 9-12: shigella flexneri serotype Xv positive reaction. Tubes 5-8/column 5-8 (B1)/lanes 5-8 and tubes 13-16/column 5-8 (B2)/lanes 13-16, negative control (template water).

Detailed Description

The LAMP primers are designed according to type-specific antigenic determinants and group-specific antigenic determinant genes of main serotypes 2a and XV of Shigella flexneri, wherein the serotype 2a is (gtrII and wzx gene are positive, gtrX and opt gene are negative), and the serotype XV is (gtrX, opt and wzx gene are positive, gtrII gene is negative). The method does not need expensive equipment and instruments, and can quickly, accurately and specifically detect the serotype of Shigella flexneri 2a and XV.

The invention adopts the following technical scheme:

the invention designs LAMP primers respectively based on four O antigen determinant genes (gtrII, wzx, gtrX and opt genes), and can rapidly detect shigella flexneri serotype 2a and Xv. The primer information is shown in Table 1.

TABLE 1 LAMP primer sequences

The LAMP amplification reaction conditions are as follows: 61 ℃ for 20 minutes. The sensitivity of the four genes to the shigella flexneri poaching template is 1 pg/mu L (wzx gene), 100 fg/mu L (gtrII gene), 1 pg/mu L (gtrX gene) and 100 fg/mu L (opt gene) respectively. The detection results of the four genes in different serotypes of 19 shigella flexneri are shown in table 2.

TABLE 2 detection results of four genes in 19 different serotypes of Shigella flexneri

The detection is negative in 50 other Shigella species, and the 50 Shigella species comprise 20 Shigella sonnei, 12 Shigella dysenteriae and 18 Shigella boydii. The detection results are negative in 46 other intestinal common bacteria. The method comprises the following steps: EAEC2 strain, EHEC 5 strain, EIEC 5 strain, EPEC 10 strain, ETEC 10 strain, UPEC 1 strain, nonpathogenic Escherichia coli 2 strain, Listeria monocytogenes 5 strain, Vibrio parahaemolyticus 1 strain, Salmonella 4 strain and Yersinia enterocolitica 1 strain.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Example 1 DNA template preparation

DNA templates were prepared using a water-boiling method. The single clone on the overnight cultured Shigella plate was removed and placed in an eppendorf tube containing 100. mu.L of sterile water and mixed well with shaking. The mixture was boiled in water at 100 ℃ for 10 minutes, placed on ice for 5 minutes, centrifuged at 13,000 rpm for 10 minutes, and the supernatant was used as a template.

Example 2 LAMP primer design

Primer design of wzx, gtrII, gtrX and opt genes was performed using Primer Explorer V5 software (https:// Primer Explorer. jp/V5_ manual/index. html) software. The LAMP primers include a pair of inner primers (FIP and BIP) and a pair of outer primers (F3 and B3). A pair or a single loop primer (LF and/or LB) may be added to the reaction system to increase the specificity of the LAMP reaction and speed up the reaction. The specificity of the primers was determined by the BLAST method in the NCBI database.

Table 1 shows the information of the designed LAMP primers, and the positions of the primers on the genes are shown in FIG. 1.

Example 3 LAMP reaction protocol

LAMP reactions were performed using WarmStart Colorimetric LAMP 2X Master premix (New England Biolabs Inc., USA). The LAMP reaction system consisted of 25. mu.L of a primer mixture (40 pmol each of FIP and BIP primers, 10pmol each of F3 and B3 primers, and 20pmol each of LF and/or LB primers), 12.5. mu.L of a premix and 1. mu.L of a DNA template. The mixed LAMP reaction tubes were incubated at 61 ℃ for 20 minutes. The color change is observed by naked eyes, and the LAMP premix for the positive reaction changes from red to yellow. The color of the negative reaction LAMP premixed solution is kept unchanged.

Example 4 LAMP specificity evaluation

Specific evaluation is carried out in 19 serotypes of Shigella flexneri, and the result proves that the method can specifically detect the serotypes of Shigella flexneri 2a and XV (Table 2).

The detection is negative in 50 other Shigella species, and the 50 Shigella species comprise 20 Shigella sonnei, 12 Shigella dysenteriae and 18 Shigella boydii. The detection results are negative in 46 other intestinal common bacteria. The method comprises the following steps: EAEC2 strain, EHEC 5 strain, EIEC 5 strain, EPEC 10 strain, ETEC 10 strain, UPEC 1 strain, nonpathogenic Escherichia coli 2 strain, Listeria monocytogenes 5 strain, Vibrio parahaemolyticus 1 strain, Salmonella 4 strain and Yersinia enterocolitica 1 strain.

The LAMP reaction scheme of Shigella flexneri serotype 2a and XV is shown in FIG. 2.

Example 5 LAMP sensitivity evaluation

Sensitive assays were performed using shigella flexneri serotype 2a serotype 301 and Xv serotype 2002017. Nucleic acids of Shigella flexneri 301 and 2002017 were obtained by water boiling, and nucleic acid quantification was performed by a Nanodrop ND-1000 instrument, and template concentrations were adjusted to 1 ng/. mu.L, 100 pg/. mu.L, 10 pg/. mu.L, 1 pg/. mu.L, 100 fg/. mu.L, 10 fg/. mu.L, and 1 fg/. mu.L using AE buffer (Qiagen), for a total of 7 concentrations. The LAMP reaction proves that the sensitivities of the four genes to the shigella flexneri water boiling template are respectively 1 pg/muL (wzx gene), 100 fg/muL (gtrII gene), 1 pg/muL (gtrX gene) and 100 fg/muL (opt gene).

The experimental result is coded, and the LAMP system of the method is sensitive and specific. Expensive equipment is not needed, and the operation is simple, rapid and efficient.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> infectious disease prevention and control institute of China center for disease prevention and control

<120> LAMP primer for detecting serotype 2a and XV of Shigella flexneri and detection method

<130> KHP211115206.9

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gtgcagaata aaatgcaact t 21

<210> 2

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctggtttaat atttgcaaga gtt 23

<210> 3

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggcggttgga tgacagtaag taata 60

tgcccccata atatttgaca 46

<210> 4

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acaaaaatga acccccgaaa tttt 60

ttcgatttta gtctctgcct at 47

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggtgtatttt gatcgattta tag 23

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgaaatgaga aaaatattcg tga 23

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aggaatcagt aaccacttgt 20

<210> 8

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tgatagcttt ggttaatagc aggtaa 60

gttcatgttt atctctatcc ctca 51

<210> 9

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tgcaagcgat attaaatggc ttgt 60

tttaaaccag tgggaggatt 45

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgagatgtac tcgttcga 18

<210> 11

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cagccataga taatatatat aacttc 26

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

attggcaaat gaaagtgttt g 21

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgtcaggaag aggggtta 18

<210> 14

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gttggcagtt gaggaacaga at 60

cttattgtcg cattttctat gg 45

<210> 15

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgcgtttgt ttgtttcaat gcata 60

cattgcaata aggatgactg aa 48

<210> 16

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ctgccagttg aggttttg 18

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atagtcctcc cttccgtat 19

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

agaggtttaa gaggacagtt 20

<210> 19

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

acttgaggaa aatggttacc ataca 60

tgatagcatt aaactcaatg ggtc 50

<210> 20

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tcgatctcta ctttagatgc ttggc 60

cctcatcata tcaaatgacc g 47

<210> 21

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

caaggccaga attatctt 18