CN112725410A

CN112725410A - Primer group for detecting pathogenic microorganisms

Info

Publication number: CN112725410A
Application number: CN202011633514.2A
Authority: CN
Inventors: 余成鹏; 邹树勇; 朱鹏远; 吴春求; 陈丹; 张东东; 陈嘉昌; 柳俊; 胡朝晖
Original assignee: Guangzhou Jinqirui Biotechnology Co ltd
Current assignee: Guangzhou Jinqirui Biotechnology Co ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-04-30
Anticipated expiration: 2040-12-31
Also published as: CN112725410B

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a primer group for detecting pathogenic microorganisms. The multi-system provided by the invention can detect 98 pathogenic microorganisms at most simultaneously, has high detection accuracy and good specificity, does not generate cross contamination among samples outside the range, hardly generates primer dimer among primers, and is suitable for batch detection of samples containing unknown microorganisms.

Description

Primer group for detecting pathogenic microorganisms

Technical Field

The invention relates to the technical field of nucleic acid detection, in particular to a primer group for detecting pathogenic microorganisms.

Background

Infectious diseases caused by microorganisms are still the major diseases that currently threaten global human health. Infectious diseases are mainly controlled from the aspects of discovery, diagnosis, treatment and prevention. Among them, diagnosis is very important for early detection and early control of infectious diseases. At present, the diagnosis of the microorganism in China mainly depends on serological diagnosis and microorganism culture. These diagnostic methods often take several tens of hours to several days from sampling, culturing to identification, and in some remote areas, it takes a longer time, and it is more difficult to complete the relevant differential diagnosis in a short time. On the other hand, with the addition of WTO in China, foreign trade in China develops rapidly, and the import and export of various foods and cosmetics increases rapidly. Import and export quarantine faces tremendous pressure. Therefore, there is an urgent need for new technologies and devices to meet the high throughput and increasing low cost detection of various genetic information.

Polymerase Chain Reaction (PCR) has been widely used in medicine, genetics, microbiology, and even throughout life sciences. Multiplex PCR is a novel amplification technique developed on the basis of conventional PCR, i.e., two or more pairs of primers can be added into a reaction system to simultaneously amplify a plurality of nucleic acid fragments. The multiplex PCR has important application in the disciplines of microorganism, genetic disease, tumor, pharmacogenomics and the like.

A multiple PCR detection system and a method based on pathogenic microorganisms are urgently needed by departments such as clinical laboratories, disease control centers, import and export quarantine inspection and the like.

Disclosure of Invention

The first aspect of the present invention relates to a primer set comprising at least 50 of 175 primer pairs;

175 pairs of primer pairs have nucleotide sequences shown as SEQ ID NO: 1-175, and the nucleotide sequence corresponding to the upstream primer in sequence is shown as SEQ ID NO: 175-350 as shown in the specification.

Optionally, a primer set as described above, said primer set comprising at least 100 of the 175 primer pairs.

Optionally, a primer set as described above, said primer set comprising at least 140 of the 175 primer pairs.

A second aspect of the present invention relates to a kit comprising a primer set as described above.

Optionally, a kit as described above, comprising at least one of the following reagents:

DNA polymerase, dNTPs, lysis and/or washing buffers, a solid support for enriching nucleic acids, nucleic acid elution reagents, dilution buffers, water, molecular weight marker, proteinase K and neutralization reagents.

Alternatively, a kit as described above, comprising a positive control for the nucleic acid to be detected of known sequence and concentration.

Optionally, the kit as described above, comprising at least one of a terminal repair enzyme, a ligation buffer, a DNA ligase, and an adaptor fragment.

A third aspect of the invention relates to a method for sequencing and pooling a plurality of target nucleic acids, the method comprising the steps of:

the genomic DNA of the sample to be detected is used as a template, and the primer set or the kit is used for performing multiplex PCR amplification.

A fourth aspect of the invention relates to a method for non-diagnostic purposes of detecting a plurality of pathogenic microorganisms, characterized in that it comprises:

the method described above was used for pooling, sequencing to determine the presence of pathogenic microorganisms.

Alternatively, the method of sequencing is high throughput sequencing, as described above.

The invention has the beneficial effects that:

the multi-system provided by the invention can detect 98 pathogenic microorganisms at most simultaneously, has high detection accuracy and good specificity, does not generate cross contamination among samples outside the range, hardly generates primer dimer among primers, and is suitable for batch detection of samples containing unknown microorganisms.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the amplification effect of 6 samples of MT1-MT6 based on a primer system designed based on a penalty mechanism according to an embodiment of the present invention;

FIG. 2 shows the amplification effect of 6 samples of MT1-MT6 without a primer system designed based on a penalty mechanism in one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In some embodiments, the primer set comprises at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100, or at least 110, or at least 120, or at least 130, or at least 140, or at least 150, or at least 160, or at least 170 of the 175 primer pairs.

In some embodiments, the primer set comprises a primer for detecting any one of the microbial species defined in table 1.

In some embodiments, the primer set is capable of detecting at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90 of the 98 pathogenic microorganism species defined in table 1.

The term "kit" refers to any article of manufacture (e.g., a package or container) comprising at least one device, the kit may further comprise instructions for use, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.

In some embodiments, the kit comprises at least one of the following reagents:

The term "buffer" as used herein refers to an aqueous solution or composition that resists changes in pH when an acid or base is added to the solution or composition. This resistance to pH changes is due to the buffer properties of such solutions. Thus, a solution or composition that exhibits buffering activity is referred to as a buffer or buffer solution. Buffers generally do not have the unlimited ability to maintain the pH of a solution or composition. Rather, they are generally capable of being maintained at a pH within a specified range, for example, pH 7-pH 9. Generally, Buffers are capable of maintaining a pH at their pKa and within the next logarithm (see, e.g., Mohan, Buffers, A guide for the preparation and use of Buffers in biological systems, CALBIOCHEM, 1999). Buffers and buffer solutions are generally prepared from buffered salts or preferably non-ionic buffer components such as TRIS and HEPES. The buffer which can be used in the method of the invention is preferably selected from the group consisting of phosphate buffer, phosphate buffered saline buffer (PBS), 2-amino-2 hydroxymethyl-1, 3-propanediol (TRIS) buffer, TRIS buffered saline solution (TBS) and TRIS/edta (te).

In some embodiments, the water is nuclease-free water, such as double distilled or deionized water.

In some embodiments, the DNA polymerase is selected from any of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, Klenow fragment.

As used herein, a "solid support," preferably an "enrichment particle," can be made from any number of known materials. Examples of such materials include: minerals, natural polymers and synthetic polymers. Specific examples of these materials include: cellulose, cellulose derivatives, acrylics, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene, polyacrylamide, latex gel, dextran, rubber, silica gel, plastic, nitrocellulose, natural sponge, silica gel, control pore glass (control pore glass), metal, cross-linked dextran (e.g., Sephadex TM), Sepharose (TM), and other solid supports known to those skilled in the art.

As used herein, "particle" refers to a discrete small object, such as a sphere (e.g., bead), capsule, polyhedron, etc., that can be of various shapes. The particles may be macroscopic or microscopic, such as microparticles or nanoparticles. The particles may be non-magnetic or magnetic. The magnetic particles may contain a ferromagnetic substance, and the ferromagnetic substance may be Fe, Ni, Co, iron oxide, or the like.

The kit may further contain a nucleic acid extraction reagent which can be used for extracting nucleic acid by performing phenol chloroform method, NaOH method, resin extraction method, salting-out method, hexadecyl trimethyl ammonium bromide method, silica gel membrane adsorption method, FTA card method, silica bead method or magnetic bead extraction method.

In some embodiments, the kit includes a positive control for the nucleic acid to be detected of known sequence and concentration.

In some embodiments, the kit comprises at least one of a terminal repair enzyme, a ligation buffer, a DNA ligase, and an adaptor fragment.

A fourth aspect of the present invention relates to a method for detecting a plurality of pathogenic microorganisms, comprising:

The term "detecting" and similar terms are used in this application to generally refer to a process or the discovery or determination of the presence or absence, as well as the degree, quantity or level, or probability of occurrence of something. For example, the term "detecting" when used in reference to a target nucleic acid sequence can refer to finding or determining the presence, absence, level, or amount, and the probability or likelihood of the presence or absence, of the sequence. It is to be understood that the expressions "detecting the presence or absence", "detecting the presence or absence" and related expressions include both qualitative and quantitative detections. For example, quantitative detection includes determining the level, amount, or quantity of a nucleic acid sequence associated with parainfluenza virus type 3 in a sample.

The term "test agent" refers to any composition that contains or is suspected of containing nucleic acid from a pathogenic microorganism. The term includes whole blood, plasma, serum, cells, throat swab, saliva, urine, feces, cerebrospinal fluid, pleural effusion, amniotic fluid, vaginal secretions.

In some embodiments of the invention, the sequencing method is high throughput sequencing, also known as next generation sequencing ("NGS"). Second generation sequencing produces thousands to millions of sequences simultaneously in a parallel sequencing process. NGS is distinguished from "Sanger sequencing" (one generation sequencing), which is based on electrophoretic separation of chain termination products in a single sequencing reaction. Sequencing platforms for NGS useful in the present invention may be commercially available, including but not limited to Illumina MiniSeq, NextSeq 550, and the like.

In some embodiments, the method is a method of non-diagnostic interest.

Embodiments of the present invention will be described in detail with reference to examples.

Example 1

The invention provides a method for detecting pathogens by high-throughput sequencing, which is used for a pathogenic microorganism ultra-multiplex PCR primer design device system output primer combination mode. The invention designs a specific primer combination scheme according to a specific sequence of a sample to be detected, the specific primer combination scheme is used for ultra-multiplex PCR amplification after verification, an amplification product is prepared and sequenced through a library, and the types of microorganisms and pathogenic/drug-resistant genes in the sample are detected. Take 98 kinds of pathogenic microorganism detection as an example.

98 genome sequences of 98 common pathogenic microorganisms are selected through literature search, and the sequences of the pathogenic microorganisms are downloaded on Genebank. Aiming at the functional regions, conserved regions and sequences of tandem repeat fragments supported by the existing literature of the pathogenic microorganisms, mega software is used for carrying out multi-sequence comparison analysis, and the obtained intergeneric sequence, interspecies specific sequence and subspecies conserved sequence of the subspecies under the classification are compared, and the sequences of pathogenic genes and drug-resistant genes related to the pathogenic microorganisms account for 175 target amplification regions.

Aiming at the sequences, designing primers, confirming no non-specific amplification risk through primer specificity comparison, and storing the primers in a primer pool, wherein the primer design rule is up to the following standard:

(1) the species specificity interval and the family conservation interval, the drug resistance gene and the virulence gene are selected, and 5 pairs of primers are designed for each pathogen to ensure the sensitivity and the specificity.

(2) The length of the primer fragment is between 18 and 24bp, and the length of the amplification product is between 110 and 120 bp;

(3) the annealing temperature of the primer is between 55 and 65 ℃;

(4) the GC content of the primer is between 40 and 60 percent;

(5) the 3' end of the primer is C or G; 6. each primer does not have 4 or more continuous A or T or C or G;

aiming at the obtained primer pool, screening and pairing are carried out according to the following conditions, and an optimal primer combination scheme is calculated:

(1) the head and the tail of each primer have the condition of complementary pairing of less than 2 continuous bases to obtain 2 points, the head and the tail of each primer have the condition of complementary pairing of 2-3 continuous bases to obtain 1 point, and the head and the tail of each primer have the condition of complementary pairing of more than 3 continuous bases to obtain 0 point;

(2) the deviation degree of the primer Tm value and the Tm average value is within 2 ℃, each primer is divided into +1, the deviation degree of the primer Tm value and the Tm average value is above 2 ℃, and each primer is divided into + 0;

(3) the GC content of the primers is within 5 percent of the average GC content, each primer is divided into +1 part, the GC content of the primers is more than 5 percent of the average GC content, and each primer is divided into +0 part;

(4) taking a primer pair with the minimum deviation Tm average value as a standard, constructing a binary matrix by using the standard primer pair, and respectively carrying out complementary analysis on the binary matrix and other primers, wherein the initial score of each 2 primers is 5 minutes, and the complementary pairing condition of more than 2 continuous bases exists between each primer and each primer, and the score is reduced by 2 when each base exceeds 1 base; and returning the primers with the scores of less than 3 points or the occurrence frequency of the primers with the scores of less than 3 points and other primers with the complementary analysis score of more than 50 percent to the primer pool, replacing the primers with the other primers and recalculating.

After the screening of the steps, a primer combination scheme with the highest score is output, and the obtained primers are shown in table 1.

SEQ ID NO: the upstream primers shown in 1 to 175 correspond to 1 to 175 in the sequence numbers in Table 1.

TABLE 1 primer information

The pathogenic microorganism ultra-multiplex PCR specific primers completely cover 175 regions of a target, the primer combination is used for ultra-multiplex PCR, and the amplification product is used for library construction and high-throughput sequencing.

The total of 846 known positive samples of 98 microorganisms within the primer design coverage and 48 known samples of 3 microorganisms outside the primer design coverage are selected, and 894 samples are subjected to DNA extraction, PCR amplification, library preparation and machine sequencing.

All samples covered by the primer design can be correctly detected, the corresponding pathogenic microorganisms have a positive coincidence rate of 100 percent, and the positive coincidence rate is shown in table 2. Pathogenic microorganisms are not detected in all samples outside the design coverage range of the primers, and the negative coincidence rate is 100 percent as shown in table 3. The cross contamination phenomenon does not occur between samples within the designed coverage range or outside the designed coverage range of the primers, and the specificity is better.

TABLE 2 results of sample detection within primer design coverage

Note: part of positive samples are difficult to collect, and the test adopts synthetic false viruses or commercial quality control products are purchased.

TABLE 3 detection results of samples outside the primer design coverage

Serial number	Pathogenic microorganism	Total number of	Negative count	Negative match rate (%)
					1	Aspergillus terreus	5	5	100
2	Burkholderia cepacia	28	28	100
					3	Pneumocystis yeri	15	15	100

Meanwhile, 8 positive samples of respiratory syncytial virus type B were selected, and the Sanger sequencing method and the tNGS method based on the combination of the ultra-multiple PCR primers described in this example were used respectively, and the Sanger sequencing was used as a standard to verify the consistency of the different methodologies. The results show that Sanger sequencing and tNGS can correctly detect respiratory syncytial virus B types in 8 samples, and the consistency is good.

The results are combined, and the primer combination designed by the ultra-multiplex PCR primer design device can accurately detect positive samples or negative samples in a detection range.

Example 2

The primer combination for the ultra-multiplex PCR described in example 1 was selected, and the Coxsackie virus A6, which is a closely related species of enterovirus not designed in the above primer design, was selected and detected, and it was revealed that 2 cases of Coxsackie virus A6 were not within the range of primer design, but the sequences of the universal primers suggested that the enterovirus was of another type. The results of the 2 cases are consistent with known positive results, which prove that the primer combination has the capability of detecting or predicting unknown samples, and the results are shown in Table 4.

TABLE 4 kindred species detection

Serial number	Pathogenic microorganism	Total number of	Positive counts	Positive match rate (%)
					1	Coxsackie virus type A6	2	2	100

Example 3

The target sequence described in example 1 was selected for primer design without penalty mechanism to obtain primer combinations, and 6 samples were amplified with the primer combinations described in example 1, and the amplification effect was compared with the concentration of amplified product and distribution of amplified fragments.

The concentration of 6 samples after PCR amplification is analyzed by amplifying reaction systems of different primer systems with the same template amount and analyzing the fragment length of an amplification product by using Qsep100, and the result shows that the concentration of the primer combinations designed based on the penalty mechanism after amplification is higher than that of the primer combinations not designed based on the penalty mechanism, as shown in Table 5; the primer combination based on the penalty mechanism has no peak in the range of 40-60bp, and the primer combination not based on the penalty mechanism has a peak in the range of 40-60bp, i.e., the primer combination giving the penalty mechanism has significantly less primer dimer after amplification than the primer combination not designed based on the penalty mechanism, as shown in FIG. 1 and FIG. 2.

TABLE 5 comparison of concentrations of different primer combinations after amplification

In conclusion, the penalty mechanism described in this patent, especially the self-constructed binary matrix, can effectively eliminate primer dimers possibly generated between primer pairs, thereby effectively improving the primer amplification specificity and amplification efficiency of the super-multiplex PCR.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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<212> DNA

<213> artificial sequence

<400> 71

accaggaaaa ttctgccaca ga 22

<210> 72

<211> 24

<212> DNA

<213> artificial sequence

<400> 72

aacttgtgga agagtggcct tttc 24

<210> 73

<211> 21

<212> DNA

<213> artificial sequence

<400> 73

atgtaaactc catgcattta a 21

<210> 74

<211> 23

<212> DNA

<213> artificial sequence

<400> 74

ttacaccaaa tgaaccaggg aaa 23

<210> 75

<211> 23

<212> DNA

<213> artificial sequence

<400> 75

cagacaaaaa ggactcatca tca 23

<210> 76

<211> 22

<212> DNA

<213> artificial sequence

<400> 76

tgcaaaataa gggaataagg tg 22

<210> 77

<211> 21

<212> DNA

<213> artificial sequence

<400> 77

taccatccag cttgtgactg c 21

<210> 78

<211> 21

<212> DNA

<213> artificial sequence

<400> 78

tggccgacaa ggagacatca t 21

<210> 79

<211> 23

<212> DNA

<213> artificial sequence

<400> 79

aggtccatca cgaccagggg gta 23

<210> 80

<211> 22

<212> DNA

<213> artificial sequence

<400> 80

agaactgaag ataaggccca ag 22

<210> 81

<211> 21

<212> DNA

<213> artificial sequence

<400> 81

cggccggcgg cacccccggg t 21

<210> 82

<211> 22

<212> DNA

<213> artificial sequence

<400> 82

cggtcagcgg gatgtcgtac ag 22

<210> 83

<211> 21

<212> DNA

<213> artificial sequence

<400> 83

ggtttccacc cgggtaattg c 21

<210> 84

<211> 24

<212> DNA

<213> artificial sequence

<400> 84

ggccgcaaaa tttctgacgc caca 24

<210> 85

<211> 21

<212> DNA

<213> artificial sequence

<400> 85

aatgtgtcat tctttggcat t 21

<210> 86

<211> 24

<212> DNA

<213> artificial sequence

<400> 86

tgctcaaatt tgatagcagt ctga 24

<210> 87

<211> 24

<212> DNA

<213> artificial sequence

<400> 87

agatcaacaa atcaatgcaa agct 24

<210> 88

<211> 24

<212> DNA

<213> artificial sequence

<400> 88

ctaatgttgg agaaaatcac aaac 24

<210> 89

<211> 23

<212> DNA

<213> artificial sequence

<400> 89

atccggaaag ttggctgatg acg 23

<210> 90

<211> 22

<212> DNA

<213> artificial sequence

<400> 90

tttccagaag tcctgttgcg tc 22

<210> 91

<211> 21

<212> DNA

<213> artificial sequence

<400> 91

ccgataatat gtccacctca c 21

<210> 92

<211> 22

<212> DNA

<213> artificial sequence

<400> 92

ctcgtgaaac actgaggcaa ta 22

<210> 93

<211> 24

<212> DNA

<213> artificial sequence

<400> 93

tgggatactg gctacgctaa ggtt 24

<210> 94

<211> 24

<212> DNA

<213> artificial sequence

<400> 94

ttacgacatg ccttgcgatg tttt 24

<210> 95

<211> 23

<212> DNA

<213> artificial sequence

<400> 95

gatgaacgcg aacaaacact taa 23

<210> 96

<211> 24

<212> DNA

<213> artificial sequence

<400> 96

taaaattccg acggcagctg tatt 24

<210> 97

<211> 22

<212> DNA

<213> artificial sequence

<400> 97

ggtatgattt cttgacccac aa 22

<210> 98

<211> 23

<212> DNA

<213> artificial sequence

<400> 98

cgcttctcgc caatcttttc ggg 23

<210> 99

<211> 22

<212> DNA

<213> artificial sequence

<400> 99

gaagctgctc gatcagttta ac 22

<210> 100

<211> 22

<212> DNA

<213> artificial sequence

<400> 100

tcagcggaat gctgtttgtg gt 22

<210> 101

<211> 22

<212> DNA

<213> artificial sequence

<400> 101

aaatacaaaa tgttaacgac gc 22

<210> 102

<211> 24

<212> DNA

<213> artificial sequence

<400> 102

ctccggttat cataaaacgg gaca 24

<210> 103

<211> 24

<212> DNA

<213> artificial sequence

<400> 103

agtttcttcg gatactatga tatt 24

<210> 104

<211> 22

<212> DNA

<213> artificial sequence

<400> 104

tgttgtggcg tttatcaatt ag 22

<210> 105

<211> 22

<212> DNA

<213> artificial sequence

<400> 105

tttgagacgc agcgtttatt gt 22

<210> 106

<211> 21

<212> DNA

<213> artificial sequence

<400> 106

tactttaaaa tcggctcgac t 21

<210> 107

<211> 23

<212> DNA

<213> artificial sequence

<400> 107

tatctcttat ggtttgcgtg gtg 23

<210> 108

<211> 22

<212> DNA

<213> artificial sequence

<400> 108

taagtgaata ccagcgaata tt 22

<210> 109

<211> 0

<212> DNA

<213> artificial sequence

<400> 109

<210> 110

<211> 23

<212> DNA

<213> artificial sequence

<400> 110

cccccgggtt cgacctcaac gtc 23

<210> 111

<211> 21

<212> DNA

<213> artificial sequence

<400> 111

gcagatgact cacctgttgg a 21

<210> 112

<211> 21

<212> DNA

<213> artificial sequence

<400> 112

cgactgcacc tggagcagct a 21

<210> 113

<211> 22

<212> DNA

<213> artificial sequence

<400> 113

ttttaaaaag gccaagcaaa ag 22

<210> 114

<211> 24

<212> DNA

<213> artificial sequence

<400> 114

tctttatcga tgaaattgat gctg 24

<210> 115

<211> 23

<212> DNA

<213> artificial sequence

<400> 115

tccttcgctt ttatggcaca agc 23

<210> 116

<211> 21

<212> DNA

<213> artificial sequence

<400> 116

gaacaggatg tcaaagatct a 21

<210> 117

<211> 21

<212> DNA

<213> artificial sequence

<400> 117

gtgtcaaggt gtctttcgga t 21

<210> 118

<211> 24

<212> DNA

<213> artificial sequence

<400> 118

gtgtagatag agagactgag gatg 24

<210> 119

<211> 22

<212> DNA

<213> artificial sequence

<400> 119

tgttagtgga gatccttcta ta 22

<210> 120

<211> 23

<212> DNA

<213> artificial sequence

<400> 120

gaggaacacc cggtgaatac cta 23

<210> 121

<211> 23

<212> DNA

<213> artificial sequence

<400> 121

tgggccgtga acaagactgg gct 23

<210> 122

<211> 21

<212> DNA

<213> artificial sequence

<400> 122

tttatatggt tgggtagtgg a 21

<210> 123

<211> 23

<212> DNA

<213> artificial sequence

<400> 123

aatcgtgcaa gtaaaccaaa taa 23

<210> 124

<211> 24

<212> DNA

<213> artificial sequence

<400> 124

caatgaatca agcgatgcag ctag 24

<210> 125

<211> 24

<212> DNA

<213> artificial sequence

<400> 125

attctcacag gcttgcttgc tgga 24

<210> 126

<211> 24

<212> DNA

<213> artificial sequence

<400> 126

agcgtaggcg tcggtgacaa aggc 24

<210> 127

<211> 21

<212> DNA

<213> artificial sequence

<400> 127

ccctcacggt tcagggttag c 21

<210> 128

<211> 24

<212> DNA

<213> artificial sequence

<400> 128

gatgtcaaga acttcgacgg ccgt 24

<210> 129

<211> 24

<212> DNA

<213> artificial sequence

<400> 129

gttcctttct tgggcagagc acgc 24

<210> 130

<211> 24

<212> DNA

<213> artificial sequence

<400> 130

ttgcttattt ggtttccacc cggg 24

<210> 131

<211> 21

<212> DNA

<213> artificial sequence

<400> 131

atcttcatct ggtccaggcc c 21

<210> 132

<211> 22

<212> DNA

<213> artificial sequence

<400> 132

attggtgaag agtttggacc ct 22

<210> 133

<211> 23

<212> DNA

<213> artificial sequence

<400> 133

tcgtgggctg cctgttgttt tac 23

<210> 134

<211> 22

<212> DNA

<213> artificial sequence

<400> 134

ttgtcgttgc agtgattacg at 22

<210> 135

<211> 21

<212> DNA

<213> artificial sequence

<400> 135

tggaaatgag ctggttcgtt t 21

<210> 136

<211> 24

<212> DNA

<213> artificial sequence

<400> 136

gcatgaagac ggattcaaac gtgc 24

<210> 137

<211> 22

<212> DNA

<213> artificial sequence

<400> 137

gaagtcttgg aggtatgcaa gc 22

<210> 138

<211> 24

<212> DNA

<213> artificial sequence

<400> 138

gggaccagcg gatatgaacg aaca 24

<210> 139

<211> 21

<212> DNA

<213> artificial sequence

<400> 139

ttcagctact tgcaagattg g 21

<210> 140

<211> 22

<212> DNA

<213> artificial sequence

<400> 140

ttcgggcaga caagctgctt tt 22

<210> 141

<211> 22

<212> DNA

<213> artificial sequence

<400> 141

aggcaaaatc gtgtttacgg ga 22

<210> 142

<211> 24

<212> DNA

<213> artificial sequence

<400> 142

gcctggtcaa aatgggcgcg aaaa 24

<210> 143

<211> 23

<212> DNA

<213> artificial sequence

<400> 143

aggctgcctg gaaggtgtcg ggt 23

<210> 144

<211> 22

<212> DNA

<213> artificial sequence

<400> 144

cctcggtgcg accccaactc ga 22

<210> 145

<211> 23

<212> DNA

<213> artificial sequence

<400> 145

gatatgccgt cgaaagtcct gag 23

<210> 146

<211> 24

<212> DNA

<213> artificial sequence

<400> 146

cgaaccggcg gattataaaa gtgt 24

<210> 147

<211> 22

<212> DNA

<213> artificial sequence

<400> 147

tgttgttcag gttttcataa gt 22

<210> 148

<211> 21

<212> DNA

<213> artificial sequence

<400> 148

tcgttttcgg cgcggcattc c 21

<210> 149

<211> 24

<212> DNA

<213> artificial sequence

<400> 149

tacaagaaag tcgtcctatt ttaa 24

<210> 150

<211> 23

<212> DNA

<213> artificial sequence

<400> 150

gaggcggtaa cgatgaacga gaa 23

<210> 151

<211> 23

<212> DNA

<213> artificial sequence

<400> 151

gatcattcgt tggcactgac atc 23

<210> 152

<211> 22

<212> DNA

<213> artificial sequence

<400> 152

cactgtatac cgatgaaact tt 22

<210> 153

<211> 23

<212> DNA

<213> artificial sequence

<400> 153

tatcaagggt cgatttttcc agc 23

<210> 154

<211> 22

<212> DNA

<213> artificial sequence

<400> 154

tgaatgtcac tagtaatgat gc 22

<210> 155

<211> 21

<212> DNA

<213> artificial sequence

<400> 155

cgctgctgat gctgctggac t 21

<210> 156

<211> 21

<212> DNA

<213> artificial sequence

<400> 156

aaaggtatcc atgccgcggc g 21

<210> 157

<211> 23

<212> DNA

<213> artificial sequence

<400> 157

ccatcatcca cagcgagaac tgg 23

<210> 158

<211> 22

<212> DNA

<213> artificial sequence

<400> 158

tgcggctgct gatagaacgc cg 22

<210> 159

<211> 24

<212> DNA

<213> artificial sequence

<400> 159

tcgtttggaa ggcttgactc ttgt 24

<210> 160

<211> 23

<212> DNA

<213> artificial sequence

<400> 160

tttaacattt cttgtgcaaa ctc 23

<210> 161

<211> 23

<212> DNA

<213> artificial sequence

<400> 161

gttatccctt tctcttctca agc 23

<210> 162

<211> 23

<212> DNA

<213> artificial sequence

<400> 162

agcgttttgt atttgcaatg cca 23

<210> 163

<211> 22

<212> DNA

<213> artificial sequence

<400> 163

gactcaggtc cctataggct tt 22

<210> 164

<211> 23

<212> DNA

<213> artificial sequence

<400> 164

aaccataatc cgaacgaagt cca 23

<210> 165

<211> 24

<212> DNA

<213> artificial sequence

<400> 165

tgataatgca ctgggggcat atct 24

<210> 166

<211> 23

<212> DNA

<213> artificial sequence

<400> 166

atctgctgct caatcgctcg ggt 23

<210> 167

<211> 24

<212> DNA

<213> artificial sequence

<400> 167

gacgcttaac atcttatcgt catg 24

<210> 168

<211> 21

<212> DNA

<213> artificial sequence

<400> 168

ttacaatgtc gagcctctag g 21

<210> 169

<211> 24

<212> DNA

<213> artificial sequence

<400> 169

tggttacggc taaaaatgtc agca 24

<210> 170

<211> 23

<212> DNA

<213> artificial sequence

<400> 170

attatcgtcc tactgatttg gcg 23

<210> 171

<211> 24

<212> DNA

<213> artificial sequence

<400> 171

ttgggccttt ttgagaatct gcac 24

<210> 172

<211> 22

<212> DNA

<213> artificial sequence

<400> 172

gcaactaaag attccagctc gg 22

<210> 173

<211> 22

<212> DNA

<213> artificial sequence

<400> 173

gaaagccgtc caatcttaac ag 22

<210> 174

<211> 24

<212> DNA

<213> artificial sequence

<400> 174

aggtgggaca tgtaaagttt tccc 24

<210> 175

<211> 22

<212> DNA

<213> artificial sequence

<400> 175

gttttagtaa tgttttagta gg 22

<210> 176

<211> 24

<212> DNA

<213> artificial sequence

<400> 176

aggatggtca aaagttatat cttc 24

<210> 177

<211> 24

<212> DNA

<213> artificial sequence

<400> 177

gcgtcgtgtt cgggagttgg tgga 24

<210> 178

<211> 23

<212> DNA

<213> artificial sequence

<400> 178

aagtcccttt aagagctcaa tga 23

<210> 179

<211> 21

<212> DNA

<213> artificial sequence

<400> 179

agcatgagag cttttaattt c 21

<210> 180

<211> 24

<212> DNA

<213> artificial sequence

<400> 180

atatcttcat atctgatttt atca 24

<210> 181

<211> 23

<212> DNA

<213> artificial sequence

<400> 181

aattccatat ctgattgtat tga 23

<210> 182

<211> 22

<212> DNA

<213> artificial sequence

<400> 182

ttttgggaca tcttccacat gt 22

<210> 183

<211> 22

<212> DNA

<213> artificial sequence

<400> 183

tcaagatcat ctgttatcaa tg 22

<210> 184

<211> 23

<212> DNA

<213> artificial sequence

<400> 184

cgattgcaga tccaacacct aac 23

<210> 185

<211> 23

<212> DNA

<213> artificial sequence

<400> 185

atctataaga tgagggtgtt ttt 23

<210> 186

<211> 23

<212> DNA

<213> artificial sequence

<400> 186

ttcaaaatgg taatcacctt ttg 23

<210> 187

<211> 23

<212> DNA

<213> artificial sequence

<400> 187

ccatttaggg cgttttggac aaa 23

<210> 188

<211> 23

<212> DNA

<213> artificial sequence

<400> 188

gggtgtctga caagttgtat tgc 23

<210> 189

<211> 21

<212> DNA

<213> artificial sequence

<400> 189

ggtaacagtt gctgtaggct t 21

<210> 190

<211> 23

<212> DNA

<213> artificial sequence

<400> 190

accgatttgc ttctctacct cat 23

<210> 191

<211> 21

<212> DNA

<213> artificial sequence

<400> 191

tattaccttg ctcctgccac t 21

<210> 192

<211> 24

<212> DNA

<213> artificial sequence

<400> 192

atcctagtgt cttgagaaaa tacc 24

<210> 193

<211> 24

<212> DNA

<213> artificial sequence

<400> 193

tccctaatcc caaagaggct aatg 24

<210> 194

<211> 22

<212> DNA

<213> artificial sequence

<400> 194

aagtatacac attcaccgtt at 22

<210> 195

<211> 23

<212> DNA

<213> artificial sequence

<400> 195

ttggagcttg ttgaatgggt tga 23

<210> 196

<211> 24

<212> DNA

<213> artificial sequence

<400> 196

aaacagtatt accataagta gtaa 24

<210> 197

<211> 23

<212> DNA

<213> artificial sequence

<400> 197

aaataataaa gaaattacaa aaa 23

<210> 198

<211> 22

<212> DNA

<213> artificial sequence

<400> 198

tgcattagtt tcaggattaa aa 22

<210> 199

<211> 23

<212> DNA

<213> artificial sequence

<400> 199

aaaacgattt tcataaaatg att 23

<210> 200

<211> 22

<212> DNA

<213> artificial sequence

<400> 200

taactatttc aagtctagcc gg 22

<210> 201

<211> 23

<212> DNA

<213> artificial sequence

<400> 201

aacagctaag aaaaccagtt tgt 23

<210> 202

<211> 23

<212> DNA

<213> artificial sequence

<400> 202

cgacatcagt actagtgcct gtg 23

<210> 203

<211> 21

<212> DNA

<213> artificial sequence

<400> 203

tttcttagtg acagtttggc c 21

<210> 204

<211> 23

<212> DNA

<213> artificial sequence

<400> 204

tcctaggtaa gctctaactt ctt 23

<210> 205

<211> 22

<212> DNA

<213> artificial sequence

<400> 205

aaatctatgt atgttagcac ag 22

<210> 206

<211> 21

<212> DNA

<213> artificial sequence

<400> 206

tgtgacactg ttatatggta t 21

<210> 207

<211> 23

<212> DNA

<213> artificial sequence

<400> 207

ataggtatgt cgagtaccgt cag 23

<210> 208

<211> 22

<212> DNA

<213> artificial sequence

<400> 208

ccatcccgca attactcrtt ac 22

<210> 209

<211> 21

<212> DNA

<213> artificial sequence

<400> 209

gcacatttca gtaaactttt t 21

<210> 210

<211> 23

<212> DNA

<213> artificial sequence

<400> 210

gcattgcatg catcattgaa ttt 23

<210> 211

<211> 23

<212> DNA

<213> artificial sequence

<400> 211

tcagtgaact tcttgagcca act 23

<210> 212

<211> 24

<212> DNA

<213> artificial sequence

<400> 212

ctgtgaactt cttaagccat gatt 24

<210> 213

<211> 21

<212> DNA

<213> artificial sequence

<400> 213

ttcaagatgt tgcaggcgag c 21

<210> 214

<211> 24

<212> DNA

<213> artificial sequence

<400> 214

tatggactcc tgattattaa taaa 24

<210> 215

<211> 21

<212> DNA

<213> artificial sequence

<400> 215

ttgggcaggc ttgaagcatc a 21

<210> 216

<211> 21

<212> DNA

<213> artificial sequence

<400> 216

tatgtttgga catgagttgc t 21

<210> 217

<211> 24

<212> DNA

<213> artificial sequence

<400> 217

ggcattgctc acacactcgg ggac 24

<210> 218

<211> 21

<212> DNA

<213> artificial sequence

<400> 218

gtctcgtacg tcgtgaccta c 21

<210> 219

<211> 22

<212> DNA

<213> artificial sequence

<400> 219

gtctcgtaca tcgtgaccta cc 22

<210> 220

<211> 24

<212> DNA

<213> artificial sequence

<400> 220

tggcggccgt gcacgtcgcc ttaa 24

<210> 221

<211> 24

<212> DNA

<213> artificial sequence

<400> 221

cgtctgtaca gctcgacgat atcg 24

<210> 222

<211> 24

<212> DNA

<213> artificial sequence

<400> 222

gttttccatc agaaaactat atac 24

<210> 223

<211> 22

<212> DNA

<213> artificial sequence

<400> 223

gggggtcggg ctgggccgcc ag 22

<210> 224

<211> 24

<212> DNA

<213> artificial sequence

<400> 224

ggggagaggg aaggcgactc gccc 24

<210> 225

<211> 21

<212> DNA

<213> artificial sequence

<400> 225

cgtgtgcgcg aaagcctgtt c 21

<210> 226

<211> 22

<212> DNA

<213> artificial sequence

<400> 226

ggtataggta atgctactgt ga 22

<210> 227

<211> 24

<212> DNA

<213> artificial sequence

<400> 227

aaaagcaaac agtattttgt ttgc 24

<210> 228

<211> 23

<212> DNA

<213> artificial sequence

<400> 228

atcactgttt tccagcatga aaa 23

<210> 229

<211> 22

<212> DNA

<213> artificial sequence

<400> 229

aaagggtatt tgtagtgcta ta 22

<210> 230

<211> 21

<212> DNA

<213> artificial sequence

<400> 230

acttgttcta ttaagcgtgc c 21

<210> 231

<211> 23

<212> DNA

<213> artificial sequence

<400> 231

tttcaagcat gaagaatggt atc 23

<210> 232

<211> 24

<212> DNA

<213> artificial sequence

<400> 232

ccaagttttt ctccccatgc cgtc 24

<210> 233

<211> 21

<212> DNA

<213> artificial sequence

<400> 233

tctatttgta gttcttgaaa t 21

<210> 234

<211> 21

<212> DNA

<213> artificial sequence

<400> 234

ataaaaggaa acacggmcac c 21

<210> 235

<211> 21

<212> DNA

<213> artificial sequence

<400> 235

aacttcttta gccatgaggc a 21

<210> 236

<211> 21

<212> DNA

<213> artificial sequence

<400> 236

gatgcgctct gtttctgagc a 21

<210> 237

<211> 24

<212> DNA

<213> artificial sequence

<400> 237

ttgaattttt ttagccatga tgcg 24

<210> 238

<211> 23

<212> DNA

<213> artificial sequence

<400> 238

acaatctgaa tacctttggt aga 23

<210> 239

<211> 21

<212> DNA

<213> artificial sequence

<400> 239

tttcattttt gtctggtcct t 21

<210> 240

<211> 22

<212> DNA

<213> artificial sequence

<400> 240

taatatcaga agcatagtct ag 22

<210> 241

<211> 24

<212> DNA

<213> artificial sequence

<400> 241

gaagtaggtg tctgtggcgc gsgc 24

<210> 242

<211> 21

<212> DNA

<213> artificial sequence

<400> 242

cgccgcggat gtcaaagtag g 21

<210> 243

<211> 22

<212> DNA

<213> artificial sequence

<400> 243

caatgccaaa tgtgtttgtg gt 22

<210> 244

<211> 20

<212> DNA

<213> artificial sequence

<400> 244

gcgttttgct cttcttcttc 20

<210> 245

<211> 21

<212> DNA

<213> artificial sequence

<400> 245

ggtggttgaa gggatttacg t 21

<210> 246

<211> 24

<212> DNA

<213> artificial sequence

<400> 246

tctcaaagct tctgccattt ctgc 24

<210> 247

<211> 22

<212> DNA

<213> artificial sequence

<400> 247

cctcgaatac cttcaacata tc 22

<210> 248

<211> 24

<212> DNA

<213> artificial sequence

<400> 248

acaataacca ccaggcatgt catt 24

<210> 249

<211> 21

<212> DNA

<213> artificial sequence

<400> 249

ttcttcaaag gttaagattg a 21

<210> 250

<211> 24

<212> DNA

<213> artificial sequence

<400> 250

ttgcagagtt tgtatacctt tgaa 24

<210> 251

<211> 23

<212> DNA

<213> artificial sequence

<400> 251

aagatcatcg tggttttcat ttt 23

<210> 252

<211> 23

<212> DNA

<213> artificial sequence

<400> 252

aagatctaga gatgtacaaa caa 23

<210> 253

<211> 23

<212> DNA

<213> artificial sequence

<400> 253

atggccgccc ttgaccaggt tgc 23

<210> 254

<211> 24

<212> DNA

<213> artificial sequence

<400> 254

tggcccccct gctggtggcc gcca 24

<210> 255

<211> 21

<212> DNA

<213> artificial sequence

<400> 255

ctttcaggtg agacgctttt g 21

<210> 256

<211> 24

<212> DNA

<213> artificial sequence

<400> 256

gatgtcctcg acctcgtcct tcag 24

<210> 257

<211> 21

<212> DNA

<213> artificial sequence

<400> 257

agtcgctgtt cgcggccgcg c 21

<210> 258

<211> 24

<212> DNA

<213> artificial sequence

<400> 258

agacgcccgt cggttgcccg taca 24

<210> 259

<211> 21

<212> DNA

<213> artificial sequence

<400> 259

agagtagttc cgtgttgccg a 21

<210> 260

<211> 24

<212> DNA

<213> artificial sequence

<400> 260

aatgccatct tgttctaatt cccg 24

<210> 261

<211> 24

<212> DNA

<213> artificial sequence

<400> 261

ttctttttta ttggataatc tcct 24

<210> 262

<211> 22

<212> DNA

<213> artificial sequence

<400> 262

aatttcattt tgcaacttct tt 22

<210> 263

<211> 23

<212> DNA

<213> artificial sequence

<400> 263

gtttgggtat tgggttgaca tat 23

<210> 264

<211> 23

<212> DNA

<213> artificial sequence

<400> 264

caacacactc tcctgcaaca cgc 23

<210> 265

<211> 22

<212> DNA

<213> artificial sequence

<400> 265

tgaaatgccg gctgtgtgat ca 22

<210> 266

<211> 23

<212> DNA

<213> artificial sequence

<400> 266

gacccatacc tcaattgaga cgc 23

<210> 267

<211> 24

<212> DNA

<213> artificial sequence

<400> 267

tcaactcgct tatcaactgt aaaa 24

<210> 268

<211> 21

<212> DNA

<213> artificial sequence

<400> 268

acccgctggg aacaaccaga t 21

<210> 269

<211> 21

<212> DNA

<213> artificial sequence

<400> 269

aattcttcgt caacagagat a 21

<210> 270

<211> 22

<212> DNA

<213> artificial sequence

<400> 270

gacctggacg aagaagggct gg 22

<210> 271

<211> 21

<212> DNA

<213> artificial sequence

<400> 271

ctttcctcaa gaatctgtct a 21

<210> 272

<211> 22

<212> DNA

<213> artificial sequence

<400> 272

atgaaatatt aactaataaa ca 22

<210> 273

<211> 22

<212> DNA

<213> artificial sequence

<400> 273

accggccgaa cgcggcggct tc 22

<210> 274

<211> 24

<212> DNA

<213> artificial sequence

<400> 274

ctcctgctcg cgcagaaacc ggga 24

<210> 275

<211> 24

<212> DNA

<213> artificial sequence

<400> 275

agaccgttat caccacaaac ggca 24

<210> 276

<211> 23

<212> DNA

<213> artificial sequence

<400> 276

cgatgtatca acctgcttag tat 23

<210> 277

<211> 23

<212> DNA

<213> artificial sequence

<400> 277

gatgaatgta tgtttacaat gtt 23

<210> 278

<211> 22

<212> DNA

<213> artificial sequence

<400> 278

gggttaaagc cataaagaaa at 22

<210> 279

<211> 23

<212> DNA

<213> artificial sequence

<400> 279

aattttaccc ctcaatttta aaa 23

<210> 280

<211> 23

<212> DNA

<213> artificial sequence

<400> 280

ccgaccatgg cgatttacag tta 23

<210> 281

<211> 23

<212> DNA

<213> artificial sequence

<400> 281

ttccctgtat agaacgggtg cga 23

<210> 282

<211> 21

<212> DNA

<213> artificial sequence

<400> 282

taatccgcgg cgaaaaaaga c 21

<210> 283

<211> 22

<212> DNA

<213> artificial sequence

<400> 283

tcgtcaaatt aatggcgaca tc 22

<210> 284

<211> 22

<212> DNA

<213> artificial sequence

<400> 284

gaaacgactg cgactacaag gg 22

<210> 285

<211> 23

<212> DNA

<213> artificial sequence

<400> 285

gacgtcctcg atctcgtcct tca 23

<210> 286

<211> 21

<212> DNA

<213> artificial sequence

<400> 286

tctcctcgcg cggcccgagg g 21

<210> 287

<211> 21

<212> DNA

<213> artificial sequence

<400> 287

aacgtaattt tatcggccac c 21

<210> 288

<211> 21

<212> DNA

<213> artificial sequence

<400> 288

atcttgatca tcatcatcag c 21

<210> 289

<211> 24

<212> DNA

<213> artificial sequence

<400> 289

gtcgcagcga tgacgataat ccct 24

<210> 290

<211> 22

<212> DNA

<213> artificial sequence

<400> 290

cgggatccca gcggtaggta gc 22

<210> 291

<211> 21

<212> DNA

<213> artificial sequence

<400> 291

ttcaatatgc catttagccc t 21

<210> 292

<211> 23

<212> DNA

<213> artificial sequence

<400> 292

ccttcaaaat ggctctcccc act 23

<210> 293

<211> 21

<212> DNA

<213> artificial sequence

<400> 293

acttctcctc gcaatccagg a 21

<210> 294

<211> 24

<212> DNA

<213> artificial sequence

<400> 294

ctgaatcgca atttttttcc ctct 24

<210> 295

<211> 22

<212> DNA

<213> artificial sequence

<400> 295

ccttactgtc cacttggaac gg 22

<210> 296

<211> 23

<212> DNA

<213> artificial sequence

<400> 296

tgcaacactt cataaaagta gtt 23

<210> 297

<211> 21

<212> DNA

<213> artificial sequence

<400> 297

gccaaccatt ctgatagagc t 21

<210> 298

<211> 23

<212> DNA

<213> artificial sequence

<400> 298

ttagttaaat agttccattc tac 23

<210> 299

<211> 24

<212> DNA

<213> artificial sequence

<400> 299

aaggcgcatc caccagatga taac 24

<210> 300

<211> 23

<212> DNA

<213> artificial sequence

<400> 300

tcctgaggag gtgatcccgc caa 23

<210> 301

<211> 21

<212> DNA

<213> artificial sequence

<400> 301

ggccacagcc cgtcccgccg a 21

<210> 302

<211> 21

<212> DNA

<213> artificial sequence

<400> 302

ccagccgccg cgagctgcgc g 21

<210> 303

<211> 21

<212> DNA

<213> artificial sequence

<400> 303

ccaacgtctc tatacaattg g 21

<210> 304

<211> 22

<212> DNA

<213> artificial sequence

<400> 304

tccggtgcac cagcgtctcg ac 22

<210> 305

<211> 21

<212> DNA

<213> artificial sequence

<400> 305

ggtcgtagac gcccgtcggt t 21

<210> 306

<211> 22

<212> DNA

<213> artificial sequence

<400> 306

acggtggagt actgcaacca gc 22

<210> 307

<211> 24

<212> DNA

<213> artificial sequence

<400> 307

gtattcaaag ggttttgagg tgaa 24

<210> 308

<211> 22

<212> DNA

<213> artificial sequence

<400> 308

gcctcgtgct tttaatgtct ca 22

<210> 309

<211> 23

<212> DNA

<213> artificial sequence

<400> 309

aggttggttt acgtaatttt att 23

<210> 310

<211> 21

<212> DNA

<213> artificial sequence

<400> 310

aacaagcttt ttattatcta a 21

<210> 311

<211> 21

<212> DNA

<213> artificial sequence

<400> 311

ttgatagata taattgctgt c 21

<210> 312

<211> 24

<212> DNA

<213> artificial sequence

<400> 312

ccttgcaatt tcattgaaag ctct 24

<210> 313

<211> 24

<212> DNA

<213> artificial sequence

<400> 313

cagaggcaga acgttgtccg aagc 24

<210> 314

<211> 24

<212> DNA

<213> artificial sequence

<400> 314

gcaattattg tcgggatctt agtt 24

<210> 315

<211> 23

<212> DNA

<213> artificial sequence

<400> 315

ggggtctatc aaatgatcat cta 23

<210> 316

<211> 24

<212> DNA

<213> artificial sequence

<400> 316

cgaagtgttt tcaaacagga tgtt 24

<210> 317

<211> 23

<212> DNA

<213> artificial sequence

<400> 317

ccaccgcaca caggaacgtc ccc 23

<210> 318

<211> 24

<212> DNA

<213> artificial sequence

<400> 318

gtgaggacgg cgctgcgtgc ggcg 24

<210> 319

<211> 23

<212> DNA

<213> artificial sequence

<400> 319

gacgaattgc gcgccgcgct ggc 23

<210> 320

<211> 23

<212> DNA

<213> artificial sequence

<400> 320

ggccgatgtg tgacttttcg gcc 23

<210> 321

<211> 21

<212> DNA

<213> artificial sequence

<400> 321

acccgggaag acggagaaac g 21

<210> 322

<211> 24

<212> DNA

<213> artificial sequence

<400> 322

cgtgtttacg ggaggccgga ccaa 24

<210> 323

<211> 21

<212> DNA

<213> artificial sequence

<400> 323

gtacgtccac aatgattgtg g 21

<210> 324

<211> 24

<212> DNA

<213> artificial sequence

<400> 324

tgaagtattg tccaaagtga ttaa 24

<210> 325

<211> 22

<212> DNA

<213> artificial sequence

<400> 325

aaagctggat ctaaaacatc ac 22

<210> 326

<211> 0

<212> DNA

<213> artificial sequence

<400> 326

<210> 327

<211> 22

<212> DNA

<213> artificial sequence

<400> 327

gatgttggat cattcgttct gc 22

<210> 328

<211> 21

<212> DNA

<213> artificial sequence

<400> 328

cctaaagtaa tgacaatcgt t 21

<210> 329

<211> 23

<212> DNA

<213> artificial sequence

<400> 329

cggcgctctc tagcggaacc aaa 23

<210> 330

<211> 21

<212> DNA

<213> artificial sequence

<400> 330

cagcaggccg atgtgcacca g 21

<210> 331

<211> 23

<212> DNA

<213> artificial sequence

<400> 331

cggcagcttg tcgtccggta cgt 23

<210> 332

<211> 22

<212> DNA

<213> artificial sequence

<400> 332

agccgcagcg ggcacggcaa tc 22

<210> 333

<211> 23

<212> DNA

<213> artificial sequence

<400> 333

ggcagccggg ctggaagtgg gag 23

<210> 334

<211> 23

<212> DNA

<213> artificial sequence

<400> 334

tctgccaatt tcacgatatc ttc 23

<210> 335

<211> 21

<212> DNA

<213> artificial sequence

<400> 335

tttcgtgaca agatgatcca g 21

<210> 336

<211> 23

<212> DNA

<213> artificial sequence

<400> 336

cctatccctt taaaagagcc tgt 23

<210> 337

<211> 23

<212> DNA

<213> artificial sequence

<400> 337

taaccctgcc ttcctggcat cat 23

<210> 338

<211> 22

<212> DNA

<213> artificial sequence

<400> 338

aaaataaata ttgggattta ac 22

<210> 339

<211> 24

<212> DNA

<213> artificial sequence

<400> 339

acacagacaa aaacacgcca gaaa 24

<210> 340

<211> 21

<212> DNA

<213> artificial sequence

<400> 340

gatccacacc cggcaaattc c 21

<210> 341

<211> 23

<212> DNA

<213> artificial sequence

<400> 341

tgaggagccg ccattgaaga tct 23

<210> 342

<211> 24

<212> DNA

<213> artificial sequence

<400> 342

acgtgcctca atataatgcc aata 24

<210> 343

<211> 24

<212> DNA

<213> artificial sequence

<400> 343

tattaattca attttataag cata 24

<210> 344

<211> 21

<212> DNA

<213> artificial sequence

<400> 344

gctcgtttga cgacttcacg a 21

<210> 345

<211> 21

<212> DNA

<213> artificial sequence

<400> 345

ttttagttca gctaccgtgg c 21

<210> 346

<211> 24

<212> DNA

<213> artificial sequence

<400> 346

aggaccggat gtattccctt gcgg 24

<210> 347

<211> 23

<212> DNA

<213> artificial sequence

<400> 347

gcggggctgt tggccacgac gga 23

<210> 348

<211> 24

<212> DNA

<213> artificial sequence

<400> 348

agggatttac ttggaacaac caaa 24

<210> 349

<211> 23

<212> DNA

<213> artificial sequence

<400> 349

taaccggtaa cgcaactgtg gca 23

<210> 350

<211> 22

<212> DNA

<213> artificial sequence

<400> 350

aactatagta gttagaacaa gt 22

Claims

1. A primer set, wherein the primer set comprises at least 50 of 175 primer pairs;

2. The primer set of claim 1, wherein said primer set comprises at least 100 of 175 primer pairs.

3. The primer set of claim 2, wherein said primer set comprises at least 140 of 175 primer pairs.

4. A kit comprising the primer set according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that it comprises at least one of the following reagents:

6. The kit of claim 4 or 5, wherein the kit comprises a positive control for the nucleic acid to be detected of known sequence and concentration.

7. The kit of claim 4 or 5, wherein the kit comprises at least one of a terminal repair enzyme, a ligation buffer, a DNA ligase, and an adaptor fragment.

8. A method for sequencing a library of a plurality of target nucleic acids, said method comprising the steps of:

performing multiplex PCR amplification using the genomic DNA of an object to be detected as a template, using the primer set according to any one of claims 1 to 3, or the kit according to any one of claims 4 to 7.

9. A non-diagnostic method of interest for detecting a plurality of pathogenic microorganisms, comprising:

using the method of claim 8 for pooling, sequencing to determine the presence of pathogenic microorganisms.

10. The method of claim 9, wherein the sequencing method is high throughput sequencing.