KR100745763B1 - Method for designing probe set probe set designed by the method microarray comprising the probe set and method for identifying target sequence using the probe set - Google Patents

Abstract

The present invention relates to a method of designing a probe set used for identification of a target sequence by hybridization. The present invention also provides a primer and probe set designed by the method, a kit comprising the primer and probe set, a computer readable medium having recorded thereon a program for allowing a computer to perform the method, and the primer and probe set. It relates to a method for identifying a target sequence. According to the present invention, it is possible to easily design a probe set capable of quickly identifying a large number of target sequences and accurately identifying two or more target sequences simultaneously.

Probe, design, target sequence identification, microarray

Description

Method for designing probe set, probe set designed by the method, microarray including the probe set designed by the method, a microarray including the probe set, and a method for identifying a target sequence using the probe set probe set, and method for identifying target sequence using the probe set}

1 is a flowchart illustrating a method of designing a probe set according to the present invention.

2 is a schematic diagram showing an example of a method for designing a probe set of the present invention using two conserved sequences.

3 is a schematic diagram showing another example of a probe set design method of the present invention using two conserved sequences.

4 is a schematic diagram showing an example of a method for designing a probe set of the present invention using three conserved sequences.

5 is a schematic diagram showing another example of the method for designing a probe set of the present invention using three conserved sequences.

FIG. 6 illustrates an example of a spotting arrangement of a microarray including a probe set designed by FIG. 2 and a method of identifying target sequences using the same.

FIG. 7 illustrates an example of a spotting arrangement of a microarray including a probe set designed by FIG. 3 and a target sequence identification method using the same.

FIG. 8 illustrates an example of a spotting arrangement of a microarray including a probe set designed by FIG. 4 and a method of identifying target sequences using the same.

FIG. 9 illustrates an example of a spotting arrangement of a microarray including a probe set designed by FIG. 5 and a method of identifying target sequences using the same.

The present invention relates to a method of designing a probe set that can be used for identification of a target sequence, a probe set designed thereby, a microarray including the probe set, and a computer readable medium having recorded thereon a program for allowing a computer to perform the method. And a method for identifying a target sequence using the probe set.

A microarray is a group of polynucleotides immobilized at a high density on a substrate, and the polynucleotide groups are microarrays each immobilized in a predetermined region. Such microarrays are well known in the art. Microarrays are disclosed, for example, in US Pat. Nos. 5,445,934 and 5,744,305. In addition, a method using photolithography is generally known as a method for producing the microarray. If photolithography is used, the micronucleotides of the polynucleotide may be repeated by exposing certain regions on the substrate surface to which the monomers protected with the removable groups are applied to an energy source to remove the protecting groups and coupling the monomers protected with the removable groups. Arrays can be made. In this case, the polynucleotides immobilized on the microarray are synthesized by extending the monomers one by one. In addition, in the case of the spotting method, the microarray is immobilized by immobilizing the already synthesized polynucleotide in a fixed position. Methods of making such microarrays are disclosed, for example, in US Pat. Nos. 5,744,305, 5,143,854, and 5,424,186. The above documents on microarrays and methods for their preparation are hereby incorporated by reference in their entirety.

Polynucleotides (also referred to as "probes" or "probe nucleic acids" or "probe polynucleotides") immobilized on a microarray can be specifically hybridized with a target nucleic acid and used to detect or identify the target nucleic acid. Conventional probe DNA sets criteria for selecting probe DNA for each target sequence and selects DNA sequences that meet the criteria. The above criteria and other requirements are assayed for the selected DNA sequence and then the most preferred probe sequence is selected. The criteria may include the length of the probe, the Tm value of the probe (the temperature at which 50% of the double stranded DNA molecules dissociate into two single strands), and the limit of sequence homology with other DNA. When a candidate probe DNA that meets the above criteria is selected, whether the candidate probe DNA is unique to the target sequence or not is a sequence that is easy to induce cross hybridization, and the like is examined through Tm and sequence homology. Among the candidate probe DNAs satisfying these standards, the most preferred is selected as the sequence that specifically binds to the target DNA sequence. However, since the probe set design method selects only the specific sequence as the probe that hybridizes to each target sequence but does not cross hybridize to the other sequence, the sequence sequence between the target sequences is high or the number of target sequences to be identified is large. There was a problem in designing specific probes when losing.

For example, the consensus sequence of the plurality of bacteria, particularly the 16S rRNA site, has been conventionally used to identify which species are present in the sample among the plurality of bacteria constituting a particular bacterial group. That is, sequences common to the species of the 16S rRNA site were used as primers, and sequences unique to each species were used as probes. The method can be used to identify several bacteria, but the 16S rRNA site is highly conserved, making it difficult to use for the identification of more than 10 bacteria. For example, for a total of 71 species of 37 bacteria related to sepsis and 34 bacteria related to contamination or bacteremia in blood culture, 14 sequence homology was 100% in 16S rRNA sequence 1,002 bp 97% sequence homology of the species is at least 70%, and the average of the 71 species sequence homology is highly conserved at 83%. Therefore, when designing probes for identifying the 71 species by using the conventional method, only 12 species can be identified when designed to have a homology of 80% or less between each probe.

23S rRNA, which is a gene for identifying another species, has a different sequence, so that more species can be identified when using 16S rRNA. However, the 23S rRNA sequence is often not disclosed, there is a problem that additional costs are required to identify the sequence. For example, in the case of the 71 kinds of bacteria, only about 43 or more of about 2,600 bp 23S rRNA sequences are disclosed. Although 30 species of bacteria were identified using the 23S rRNA sequence ("Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array, JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2000, pp. 781-788), the 23S rRNA sequence is not disclosed in the paper, there is still a problem that can not identify more than 30 species.

In short, designing probes that can identify multiple species using existing probe set design methods has the following limitations. Firstly, it is difficult to obtain a published sequence of identical positions for multiple species except for 16S rRNA, and secondly, 16S rRNA is well preserved in order to distinguish it from other species in the same genus. In order to secure sequences for all species in the case of genes or sequences of specific positions on the genome, which are relatively different from one species to another, it is necessary to perform separate experiments to obtain sequences. There is a delay in the development period.

The inventors of the present invention, while continuing to research to solve the above-mentioned conventional problems, by comparing one conserved sequence of a plurality of target sequences to group the plurality of target sequences, consisting of one target sequence of the group Select a target sequence specific probe if one is selected, and if the group probe consists of two or more target sequences, repeat the procedure for other conserved sequences of target sequences of the group consisting of the two or more target sequences. The present invention was completed by confirming that repeating could design a probe set capable of identifying more than 30 target sequences.

It is an object of the present invention to provide a method of designing a set of probes used for identification of a target sequence.

Another object of the present invention is to provide a probe set designed according to the method.

Still another object of the present invention is to provide a microarray including the probe set.

It is a further object of the present invention to provide a computer readable medium having recorded thereon a program which enables a computer to carry out the method.

Still another object of the present invention is to provide a method for identifying a target sequence using the probe set.

In order to achieve the object of the present invention, the present invention is a) comparing a single conserved sequence of a plurality of target sequences, each target sequence comprising a polynucleotide having a predetermined criteria contained in the one conserved sequence Grouping; b) in the case of a group consisting of a target sequence of one of the groups of step a), selecting an oligonucleotide that specifically binds to the polynucleotide having the predetermined criteria as a target sequence specific probe step; c) in the case of a group consisting of two or more target sequences of the groups of step a), selecting oligonucleotides that specifically bind to the polynucleotide having the predetermined criteria as a group probe of each group; And d) in the case of a group consisting of two or more target sequences of the groups of step a), there is a group consisting of two or more target sequences relative to other conserved sequences of a plurality of target sequences of each group consisting of the two or more target sequences. It provides a method of designing a probe set used for identification of the target sequence comprising repeating steps a) to c) until not.

In the probe set design method according to the present invention, the predetermined criteria are sequence homology, base length, hybridization melting point (Tm), hybridization melting point difference (ΔTm), GC content, self-alignment, mutation position, repetition It may be at least one selected from the group consisting of sequence degree and 3 'terminal base configuration.

In the probe set design method according to the present invention, the predetermined criteria are preferably 100% homology with respect to polynucleotides of the same group and 90% homology with respect to other groups of polynucleotides.

In the probe set design method according to the present invention, the conserved sequence may be selected from the group consisting of 16S rRNA, 23S rRNA, sodA, gyrA, groEL and rpoB.

In order to achieve another object of the present invention, the present invention provides a probe set designed according to the probe set design method.

In order to achieve another object of the present invention, the present invention provides a microarray for identifying a target sequence, characterized in that the probe set is immobilized on a substrate.

The substrate may be coated with an active group selected from the group consisting of amino-silane, poly-L-lysine and aldehyde.

In addition, the substrate may be selected from the group consisting of silicon wafer, glass, quartz, metal and plastic.

In order to achieve another object of the present invention, the present invention provides a computer readable medium having recorded thereon a program for allowing a computer to perform the probe set design method.

In order to achieve another object of the present invention, the present invention provides a method for identifying a target sequence using the probe set.

A method of identifying a target sequence according to the present invention comprises the steps of: a) applying a reaction sample comprising a target sequence to a microarray according to the present invention; b) hybridizing the probe and the target sequence; c) washing to remove nonspecific reactions; And d) detecting the fluorescence signal by hybrid formation.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

One aspect of the invention relates to a method of designing a probe set for identifying a target sequence.

1 is a flowchart illustrating a probe set design method according to the present invention.

The probe set design method according to the present invention comprises: a) comparing one preservation sequence of a plurality of target sequences, and grouping each of the target sequences including polynucleotides having a predetermined criterion included in the one conserved sequence. Steps.

As used herein, "target sequence" refers to a polynucleotide selected for identification by binding to a probe. The target sequence includes genomic DNA, DNA fragments cut by restriction enzymes, PCR products, and the like. Generally, genomic DNA fragments obtained by amplifying a specific region of genomic DNA by PCR are frequently used. The method of the present invention is a method of designing a probe set applicable to a case where there are a plurality of target sequences.

As used herein, the term "consensus sequence" refers to a polynucleotide having the same or similar base sequence located at the same site in the target sequences. The conserved sequence can be any gene of the corresponding target sequences. For example, the conserved sequence compared in step a) may be any one selected from the group consisting of 16S rRNA site, 23S rRNA site, sodA site, gyrA site, groEL site and rpoB.

 In the probe set design method according to the present invention, the "criteria" may be a conventional conventional criterion for selecting a probe. That is, the criteria are based on sequence homology, base length, hybridization melting point (Tm), hybridization melting point difference (ΔTm), GC content, self-alignment, mutation position, degree of repetition, and base configuration of 3 'end. It may be one or more selected from the group consisting of. In addition, the "schedule" means 100% homology to polynucleotides of the same group and 90% or less homology to polynucleotides of other groups, 18-25 bp base length, hybridization melting point of 72-76 ℃, 30 ~ Preference is given to a GC content of 70% and a 3 'terminal base configuration of G or C.

In addition, according to the present invention, a method for designing a probe set includes b) a target sequence specificity of an oligonucleotide that specifically binds to a polynucleotide having the predetermined criteria in the case of a group consisting of a target sequence of one of the groups of step a). Selecting as a target sequence specific probe.

In the present invention, the selection of the probe can be determined using a conventionally known method according to the above criteria. That is, criteria for selecting probe DNA are set, DNA sequences meeting the criteria are selected, the criteria and other requirements are assayed for the selected DNA sequence, and the most preferred sequence is selected as the probe DNA. When a candidate probe DNA that meets the above criteria is selected, the most preferred candidate DNA satisfying the above criteria is selected as a probe sequence that specifically binds to the target DNA sequence. Two or more probe DNAs may be selected for one target DNA as long as the probe DNA is capable of specifically binding to the target DNA.

In addition, the probe set design method according to the present invention c) in the case of a group consisting of two or more target sequences of the group of step a), the oligonucleotide that specifically binds to the polynucleotide having the predetermined criterion Selecting as a group probe.

In addition, the probe set design method according to the present invention is d) in the case of a group consisting of two or more target sequences of the group of step a), a different conserved sequence of a plurality of target sequences of each group consisting of the two or more target sequences Repeating steps a) to c) until no group consisting of two or more target sequences is present.

The other conserved sequence may be any gene of a plurality of target sequences of each group consisting of the two or more target sequences. The other conserved sequences compared in step d) may be any one selected from conserved sequences except for one conserved sequence compared in step a). For example, when 16S rRNA sites are compared in step a), the other conserved sequence of step d) may be any one selected from the group consisting of 23S rRNA site, sodA site, gyrA site, groEL site and rpoB. In addition, the conserved sequences compared in each group do not necessarily need to be identical. For example, when 16S rRNA sites are compared in step a), one group may be compared to 23S rRNA sites and the other group may be compared to sodA sites in step d).

This step is repeated until there are no groups of two or more target sequences, ie all groups are composed of one target sequence and a target sequence specific probe is selected for each one target sequence of each group.

A plurality of group probes and target sequence specific probes obtained by the above process are selected as probe sets for use in identifying the plurality of target sequences.

2 is a schematic diagram showing an example of a method for designing a probe set of the present invention using two conserved sequences.

Although six target sequences are used in this example for the sake of understanding, the skilled person will readily understand that the present invention is more powerful as its number is increased.

Referring to FIG. 2, by conserving conserved sequence A of six target sequences, grouping target sequences comprising target sequence 1, target sequence 2 and target sequence 3 comprising polynucleotide a into group I, and comprising polynucleotide b Target sequences 4, target sequence 5 and target sequence 6 are grouped into group II. Since both Group I and Group II consist of two or more target sequences, the polynucleotide a and polynucleotide b are selected as group probes of Group I and Group II, respectively.

The other conserved sequences of the plurality of target sequences of each of Group I and Group II are then compared. Since this step is performed independently for each group, different conserved sequences may be used for each group. For example, conserved sequence B can be compared for group I and conserved sequence C can be compared for group II. Referring back to FIG. 2, target sequence 1, target sequence 2, and conserved sequence B of target sequence 3 of group I are compared to compare target sequence 1 comprising polynucleotide a 'to group I-1, polynucleotide b'. Target sequence 2 comprising comprises grouping target sequence 3 comprising group I-2 and polynucleotide c 'into group I-3. Further, by comparing the target sequence 4 of the group II, the target sequence 5 of the target sequence 5 and the conserved sequence B of the target sequence 6, the target sequence 4 comprising the polynucleotide d 'and the target sequence 5 comprising the group II-1, polynucleotide e' Target group 6 comprising group II-2 and polynucleotide c 'to group II-3. Since each of the above groups consists of one target sequence, the polynucleotide a ', polynucleotide b', polynucleotide c ', polynucleotide d' and polynucleotide e 'are selected as target sequence specific probes.

If the group probes of the two target sequences are different, such as target sequence 3 and target sequence 6 of the above example, their target sequence specific probes may be the same.

3 is a schematic diagram showing another example of a probe set design method of the present invention using two conserved sequences.

Referring to FIG. 3, by comparing conserved sequence A of six target sequences, target sequence 1 and target sequence 2 comprising polynucleotide a are grouped into group I, target sequence 3 comprising polynucleotide b, target sequence 4 and target sequence 5 are grouped into group II and target sequence 6 comprising polynucleotide c is grouped into group III. Since group III consists of one target sequence, the polynucleotide c is selected as the target sequence specific probe of target sequence 6. On the other hand, since the group I and group II are composed of two or more target sequences, the polynucleotide a and the polynucleotide b are selected as the group probes of the group I and the group II, respectively. Next, target sequence 1 of group I and conserved sequence B of target sequence 2 are compared, and target sequence 1 comprising polynucleotide a 'is selected from group I-1 and target sequence 2 comprising polynucleotide b' is selected from Group I. Group by -2. Since each group consists of one target sequence, the polynucleotides a 'and b' are selected as target sequence specific probes of target sequences 1 and 2. Similarly, comparing target sequence 3 of target group II, target sequence 4 of target group II, and conserved sequence B of target sequence 5, target sequence 3 comprising polynucleotide c 'and target sequence 4 comprising group II-1, polynucleotide d' Groups target sequence 5 comprising group II-2 and e 'into group II-3. Since each group consists of one target sequence, the polynucleotides c ', d' and e 'are selected as target sequence specific probes of target sequences 3, 4 and 5.

4 is a schematic diagram showing an example of a probe set design method of the present invention using three conserved sequences, and FIG. 5 is a schematic diagram showing another example of a probe set design method of the present invention using three conserved sequences.

4 and 5, when one or more groups are composed of two or more target sequences even after comparing two conserved sequences as in the above example, again different from the group consisting of the two or more target sequences. Conservation sequences can be compared. The specific method is the same as described above. Although four conserved sequences are used in FIGS. 4 and 5, when there are a large range of target sequences to be identified, four or more conserved sequences may be used.

Another aspect of the present invention relates to a probe set designed according to the probe set design method of the present invention.

Another aspect of the invention relates to a microarray characterized in that the probe set is immobilized on a substrate.

Microarrays according to the invention may be prepared by conventional methods known to those skilled in the art using the probe set according to the invention.

That is, the substrate may be coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde. In addition, the substrate may be selected from the group consisting of silicon wafer, glass, quartz, metal and plastic. As a method of immobilizing the probe set according to the present invention, a micropipetting method using a piezoelectric method, a method using a pin-shaped spotter, etc. may be used. .

Another aspect of the invention relates to a method for identifying a target sequence using the probe set. The identification method is preferably performed using the microarray. That is, the target sequence identification method according to the present invention comprises the steps of: a) applying a reaction sample comprising the target sequence to the microarray according to the present invention; b) hybridizing the probe and the target sequence; c) washing to remove nonspecific reactions; And d) detecting the fluorescence signal by hybrid formation.

6 to 9 illustrate an example of a spotting arrangement of a microarray including a probe set designed by FIGS. 2 to 5 and a target sequence identification method using the same.

In the microarray according to the present invention, the probe set may be spotted by conserved sequences from which it is derived, as shown in FIGS. 6 to 9, but is not particularly limited to a specific spotting arrangement.

Referring to FIG. 6A, polynucleotides a and polynucleotide b, which are group probes derived from conserved sequence A, are arranged in one column, and each target sequence specific probes derived from conserved sequence B are polynucleotides a 'and b'. , c ', d' and e 'were arranged in different columns to prepare a microarray. Referring to FIG. 6B, hybridization reactions between the probe a and the probe c ′ were detected by performing the target sequence identification method of the present invention using the microarray prepared above. Referring again to FIG. 2, probe a means group I and probe c ′ means target sequence 3. Therefore, what is contained in the sample from the said result can be identified as target sequence 3.

Similarly, referring to FIG. 7A, polynucleotides a and polynucleotide b, which are group probes derived from conserved sequence A, and polynucleotide c, which are target sequence specific probes, are arranged in one column, and each derived from conserved sequence B Polynucleotides a ', b', c ', d' and e ', which are target sequence specific probes, were arranged in different columns to prepare microarrays. Referring to FIG. 7B, a hybridization reaction was detected between probe b and probe d ′ as a result of performing the target sequence identification method of the present invention using the prepared microarray. Referring back to FIG. 3, it can be identified from the results that the target sequence 4 is included in the sample.

8A, polynucleotides a and b, which are group probes derived from conserved sequence A, are arranged in one column, and polynucleotides a ′, which are group probes or target sequence specific probes derived from conserved sequence B, b 'and c' were arranged in different columns, and target sequences specific probes derived from the conserved sequence C, polynucleotides a ", b" and c ", were arranged in different columns. Microarrays were prepared. When the target sequence identification method of the present invention was performed using the microarray prepared above, hybridization reaction was detected on probe b, probe c ', and probe a ". Referring back to FIG. 4, it can be identified from the results that the target sequence 6 is included in the sample.

9A, polynucleotides a, b and c, which are group probes or target sequence specific probes derived from conserved sequence A, are arranged in one column, and group probes or target sequence specificities derived from conserved sequence B are arranged. Polynucleotides a ', b', and c ', the red probes, were arranged in different columns, and target sequence specific probes polynucleotides a "and b", derived from the conserved sequence C, were arranged in different columns to prepare microarrays. . Referring to FIG. 9B, hybridization reactions to probe a, probe a 'and probe b "were detected by performing the target sequence identification method of the present invention using the prepared microarray. Referring to FIG. From the above results, it can be identified that the target sequence 2 is included in the sample.

Another aspect of the present invention relates to a computer readable medium having recorded thereon a program for allowing a computer to perform the method for designing a probe set according to the present invention.

The present invention can be embodied as code that can be read by a computer (including all devices having an information processing function) in a computer-readable recording medium. The computer-readable recording medium includes all kinds of recording devices in which data that can be read by a computer system is stored. Examples of computer-readable recording devices include ROM, RAM, CD-ROM, magnetic tape, floppy disks, optical data storage devices, and the like.

In an embodiment of the present invention, a probe set capable of identifying the bacteria may be selected from a total of 71 bacteria including 37 bacteria associated with sepsis and 34 bacteria associated with contamination or bacteremia. Design was made. As a result, a probe set consisting of a total of 80 of 24 group probes and 56 target sequence specific probes was designed that can identify a total of 64 species.

Conventional probe design method using only 16S rRNA site was able to identify 35 out of 71 species when designed to have 90% or less homology between each probe, and 12 out of 71 when designed to have 80% or less homology. I could identify the species. However, the present invention was able to identify 64 species even when designed to have a homology of 80% or less between each probe.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1

Design of a Probe Set That Can Identify 71 Bacteria

In this example, a total of 71 species including 37 bacteria (Nos. 1 to 37) associated with sepsis induction and 34 bacteria (Nos. 38 to 71) associated with contamination or bacteremia induction in blood culture are shown in Table 1. We wanted to design a set of probes that could identify the bacteria in bacteria.

First, 16S rRNA site sequences, one conserved sequence of the 71 target species, were compared to have a homology of 100% for the same group of polynucleotides and 80% or less for the other group of polynucleotides. Groups containing 25 bp of polynucleotides were grouped and each of these polynucleotides was selected as a probe of each group. The 16S rRNA sequences of each species can vary depending on the strain and are readily available from known sequence databases such as GenBank. An example of the sequence is shown in Table 1 by GenBank accession number.

Next, independent comparisons of 23S rRNA, sodA, gyrA, groEL or rpoB site sequences, which are different conserved sequences, for a plurality of species in each of the groups above were used to compare 100% homology to other species of polynucleotides of the same species Groups containing 18-25 bp polynucleotides with up to 80% homology to the polynucleotides were grouped together. The grouping was repeated until all groups consisted of one species, with each polynucleotide selected as species specific probe of each species. The conserved sequence of each species can vary depending on the strain and can be readily obtained from known sequence databases such as GenBank.

Some contents of the group probes and species specific probes designed by the above procedure are shown in Table 2. In this example, a total of 80 probe sets of 24 group probes and 56 target sequence specific probes were designed, and using these probe sets, two low-frequency occurrences associated with sepsis induction among 71 bacteria were detected ( Bacteroides). frequency of appearance is less related to fragilis, Proteus penneri) and bacteremia (bacteremia) cause could be identified on the 64 kinds other than the five kinds (Enterococcus gallinarum, Lactobacillus fermentum, Propionibacterium acnes, Corynebacterium jeikeium, Aeromonas hydrophila).

TABLE 1

number Species GenBank Accession No. 16S rRNA site sequence One Bacteroides fragilis NC_006347 2 Clostridium perfringens AB075767 3 Chlamydophila pneumoniae NC_005043 4 Enterobacter aerogenes AY186054 5 Enterococcus avium AY442814 6 Enterococcus casseliflavus AJ420804 7 Enterobacter cloacae AY736548 8 Escherichia coli NC_004431 9 Enterococcus durans AY683836 10 Enterococcus faecium AY723748 11 Enterococcus faecalis NC_004668 12 Enterococcus raffinosus AJ301838 13 Enterobacter sakazakii AY702097 14 Haemophilus influenzae NC_000907 15 Klebsiella oxytoca AJ630270 16 Klebsiella pneumoniae AY736552 17 Listeria monocytogenes NC_002973 18 Mycobacterium avium X74495 19 Mycobacterium tuberculosis AJ536031

Table 1 (continued)

number Species GenBank Accession No. 16S rRNA site sequence 20 Neisseria gonorrhoeae AF398329 21 Neisseria meningitidis AY573194 22 Pseudomonas aeruginosa AY631058 23 Proteus mirabilis AJ605736 24 Proteus penneri AJ634474 25 Proteus vulgaris (Proteus vulgaris) AY186048 26 Rickettsia rickettsii AY573599 27 Streptococcus agalactiae NC_004116 28 Staphylococcus aureus NC_002952 29 Streptococcus bovis AY327523 30 Salmonella enteritidis AY186056 31 Staphylococcus epidermidis AY728198 32 Serratia Massasesense marcescens ) AY730005 33 Streptococcus mitis AY005045 34 Streptococcus pneumoniae NC_003098 35 Streptococcus pyogenes NC_004070 36 Salmonella typhi Z47544 37 Yersinia enterocolitica AJ639645

Table 1 (continued)

number Species GenBank Accession No. 16S rRNA site sequence 38 Acinetobacter baumannii Z93435 39 Acinetobacter calcoaceticus AY800383 40 Aeromonas hydrophila AB182089 41 Acinetobacter lwoffii Z93441 42 Corynebacterium diphtheriae BX248357 43 Citrobacter freundii AB182200 44 Cardiobacterium hominis AY360343 45 Corynebacterium jeikeium X84250 46 Campylobacter jejuni AY830883 47 Enterococcus gallinarum AY346316 48 Fusobacterium nucleatum AJ810282 49 Haemophilus aphrophilus AY362906 50 Haemophilus parainfluenzae AY362908 51 Lactobacillus fermentum AJ617543 52 Micorococcus luteus AB182215 53 Morganella morganii AB182240 54 Propionibacterium acnes ) AF076032 55 Pseudomonas fluorescens NC_005043 56 Pseudomanas putida AY789573 57 Staphylococcus capitis AY688039 58 Staphylococcus cohnii AJ717378

Table 1 (continued)

number Species GenBank Accession No. 16S rRNA site sequence 59 Staphylococcus haemolyticus AY688062 60 Staphylococcus hominis AJ717375 61 Streptococcus intermedius Z69040 62 Stenotrophomonas maltophilia AY826621 63 Streptococcus oralis AY281080 64 Streptococcus salivarius AY669233 65 Streptococcus sanguinis AY691542 66 Staphylococcus saprophyticus AY688090 67 Staphylococcus simulans AY688101 68 Salmonella typhimurium (Salmonella typhimurium) NC_003197 69 Streptococcus vestibularis AY581143 70 Staphylococcus warneri AY688106 71 Staphylococcus xylosus AY688109

TABLE 2

group Group probe sequence The corresponding species in the group Species specific probe sequence I SEQ ID NO: 1 Cardiobacterium hominis SEQ ID NO: 14 II SEQ ID NO: 2 Enterobacter aerogenes Escherichia coli Enterobacter sakazakii Salmonella typhimurium Salmonella typhi Morganella morganii Pseudomonas aeruginosa Proteus mirabilis Proteus vulgaris Streptococcus intermedius Salmonella enteritidis Yersinia enterocolitica SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26 III SEQ ID NO: 3 Pseudomonas fluorescens Pseudomonas putida SEQ ID NO: 27 SEQ ID NO: 28 IV SEQ ID NO: 4 Acinetobacter baumannii cinetobacter calcoaceticus Acinetobacter lwoffii SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 V SEQ ID NO: 5 Haemophilus aphrophilus Haemophilus influenzae SEQ ID NO: 32 SEQ ID NO: 33 VI SEQ ID NO: 6 Enterobacter cloacae Klebsiella oxytoca SEQ ID NO: 34 SEQ ID NO: 35

Table 2 (continued)

group Group probe sequence The corresponding species in the group Species specific probe sequence VII SEQ ID NO: 7 Aeromonas hydrophila SEQ ID NO: 36 VIII SEQ ID NO: 8 Mycobacterium avium ycobacterium  tuberculosis SEQ ID NO: 37 SEQ ID NO: 38 IX SEQ ID NO: 9 Neisseria gonorrhoeae Neisseria  meningitides Stenotrophomonas maltophilia SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 41 X SEQ ID NO: 10 Streptococcus bovis Streptococcus mitis Streptococcus pyogenes SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 44 XI SEQ ID NO: 11 Staphylococcus aureus Staphylococcus capitis Staphylococcus cohnii Staphylococcus epidermidis Staphylococcus saprophyticus Staphylococcus haemolyticus Staphylococcus hominis Staphylococcus simulans Staphylococcus warneri Staphylococcus xylosus SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50 SEQ ID NO: 51 SEQ ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 54

Table 2 (continued)

group Group probe sequence The corresponding species in the group Species specific probe sequence XII SEQ ID NO: 12 Enterococcus avium Enterococcus durans Enterococcus faecalis Enterococcus raffinosus SEQ ID NO 55 SEQ ID NO 56 SEQ ID NO 57 SEQ ID NO 58 XIII SEQ ID NO: 13 Streptococcus mitis Streptococcus pyogenes Streptococcus agalactiae Streptococcus oralis Streptococcus pneumoniae Streptococcus salivarius Streptococcus sanguinis Septococcus vestibularis SEQ ID NO: 59 SEQ ID NO: 60 SEQ ID NO: 61 SEQ ID NO: 62 SEQ ID NO: 63 SEQ ID NO: 64 SEQ ID NO: 65 SEQ ID NO: 66

According to the present invention, it is possible to easily design a probe set capable of quickly identifying a large number of target sequences and accurately identifying two or more target sequences simultaneously.

Attach sequence list electronic file

Claims (11)

a) comparing one consensus sequence of a plurality of target sequences and grouping each of the target sequences comprising a polynucleotide having a predetermined criterion included in the one conserved sequence; b) in the case of a group consisting of a target sequence of one of the groups of step a), selecting an oligonucleotide that specifically binds to the polynucleotide having the predetermined criteria as a target sequence specific probe step; c) in the case of a group consisting of two or more target sequences of the groups of step a), selecting oligonucleotides that specifically bind to the polynucleotide having the predetermined criteria as a group probe of each group; And d) for a group consisting of two or more target sequences of the groups of step a), there is no group consisting of two or more target sequences for other conserved sequences of a plurality of target sequences of each group consisting of the two or more target sequences Repeating steps a) to c) until none is used. The method of claim 1, The schedule consists of sequence homology, base length, hybridization melting point (Tm), hybridization melting point difference (ΔTm), GC content, self-alignment, mutation position, degree of repetition and base configuration of 3 'end. At least one selected from the group. The method of claim 1, Wherein said predetermined criteria is 100% homology with respect to polynucleotides of the same group and 90% homology with respect to another group of polynucleotides. The method of claim 1, wherein the conserved sequence is 16S rRNA, 23S rRNA, superoxide dismutase subunit A (sodA), DNA gyrase subunit A (gyrA), groEL protein coding region (groEL) and DNA-directed RNA polymerase beta chain protein (rpoB) ) Is selected from the group consisting of. A probe set designed according to the method of claim 1. The probe array of claim 5, wherein the probe set is immobilized on a substrate. The method of claim 6, The substrate is a microarray characterized in that the active group selected from the group consisting of amino-silane (amino-silane), poly-L- lysine (poly-L--sine) and aldehyde (aldehyde) is coated. The method of claim 6, The substrate is a microarray, characterized in that selected from the group consisting of silicon wafer, glass, quartz, metal and plastic. A computer-readable medium selected from the group consisting of a ROM, a RAM, a CD-ROM, a magnetic tape, a floppy disk, and an optical data storage device having recorded thereon a program which enables a computer to carry out the method according to claim 1. A method of identifying a target sequence using the probe set of claim 5. The method of claim 10, The method is a) applying a reaction sample comprising a target sequence to a microarray according to claim 6; b) hybridizing the probe and the target sequence; c) washing to remove nonspecific reactions; And d) detecting a fluorescence signal by hybrid formation.

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