JP6386234B2 - Method and kit for simultaneously detecting two or more detection target organisms - Google Patents

Description

  The present invention relates to a method and kit for simultaneously detecting two or more organisms to be detected.

  Phytoplasma is a pathogenic bacterium that parasitizes the phloem tissue of plants. Phytoplasma has not been successfully cultured, and there are many unclear points about its ecology. Phytoplasma has been reported to infect a variety of crops including fruit trees and flowers, which causes leaf yellowing, atrophy and crowding, fruit atrophy, quality loss, and tree death .

  In Europe fruit trees and African and Caribbean coconuts, etc., the economic damage caused by phytoplasma infection is becoming increasingly serious. For example, apple phytoplasma disease that erupted in Germany and Italy in 2001 caused damages of up to € 25 million and € 100 million, respectively (Strauss, 2009).

  In Japan, phytoplasma infection is known for trees and hydrangeas of flowers. On the other hand, the phytoplasma that infects fruit trees such as apples, pears, grapes and peaches, which have caused great damage in Europe and the United States, has not yet invaded Japan, so the target plants are strictly inspected by import quarantine. Yes.

  Conventional detection methods for phytoplasmas are mainly PCR methods, and PCR methods using primer sets for a wide range of phytoplasmas have been reported (Heinrich et al., 2001; Deng and Hiruki, 1991). In order to identify whether the amplified fragment obtained by this PCR method is truly derived from phytoplasma, and to which species or pathogenic group, a method for decoding the base sequence of the amplified fragment, and an RFLP method ( A method of confirming a phytoplasma-specific band pattern or a specific band pattern for each species or pathogenic group by a technique such as restriction enzyme fragment length polymorphism (Sawayanagi et al., 1999; Martini and Lee , 2013).

  However, it takes time to decode the base sequence, and it is difficult to use it at a quarantine site. The RFLP method has disadvantages such as the cost required for the necessary enzyme and the need for sufficient examination of the processing time. Furthermore, phytoplasma of multiple species or pathogenic groups may be mixed and in this case, it is technically possible to detect all of the mixed phytoplasma by both base sequencing and RFLP methods. It is impossible and there is a risk of detection leaking.

  Patent Document 1 discloses a method for detecting a wide range of phytoplasma using a specific antibody against a phytoplasma membrane protein. However, since this method takes time and cost, it is difficult to use it in a quarantine place.

JP 2001-281254 A

  Plant import and export quarantine requires the rapid and accurate detection of infected phytoplasma species or pathogenic groups so that new phytoplasmas do not enter the country or flow out to export destinations. It has been. Also in Japan, it is necessary to suppress the spread of infection damage by quickly diagnosing the presence or absence of plant phytoplasma infection.

  Some species or pathogenic groups of phytoplasmas infect and infest a wide range of host plants and vector insects. Therefore, in order to prevent secondary infection to plant species other than those to be examined, it is necessary to individually identify phytoplasma species or pathogenic groups infected or parasitic on plants and vector insects.

  However, as described above, the conventional method has a problem that it takes time and cost to specify the species or pathogenic group. Moreover, when phytoplasma of a plurality of species or pathogenic groups is mixed and infected, it is impossible to detect each of these phytoplasmas quickly and accurately by the conventional method. In particular, the PCR method using a primer set for a wide range of phytoplasms has many false positives, and multiple phytoplasma species or pathogenic groups cannot be analyzed at once.

  The present invention has been made in view of the above-described problems, and an object of the present invention is to provide a detection method and a kit capable of detecting two or more types of detection target organisms simultaneously and quickly.

  In order to solve the above-mentioned problems, the present inventors have found differences in single nucleotide polymorphism (SNP) in the phytoplasma 16S rDNA sequence between species and pathogenic groups. And, by using the ligase chain reaction (LCR; hereinafter referred to as “LCR”) method using this SNP difference, a method that can distinguish highly closely related species or pathogenic groups with high sensitivity. developed. In addition, by using the LCR method using a combination of various oligomers designed based on the difference in SNPs, multiple phytoplasmas can be simultaneously and quickly identified even when they are mixed. I found it possible. And the present inventors completed this invention based on these knowledge.

  The present invention uses a ligase chain reaction step in which ligase chain reaction is performed using two or more types of oligomer sets corresponding to each of two or more types of detection target organisms as a template, and each of the ligase chain reaction. 4 for the ligase chain reaction designed by sandwiching an arbitrary single nucleotide polymorphism (SNP) of the corresponding organism to be detected. Provided is a method for simultaneously detecting two or more detection target organisms composed of two oligomers.

  The present invention also includes two or more kinds of oligomer sets corresponding to each of two or more kinds of detection target organisms, and the oligomer set is designed to sandwich any single nucleotide polymorphism (SNP) of the corresponding detection target organisms. Provided is a kit for simultaneously detecting two or more detection target organisms composed of four oligomers for ligase chain reaction.

The present invention also provides the method and kit, wherein the four oligomers are
A first oligomer comprising a sequence complementary to the 5 ′ region of the SNP site in the (+) strand;
A second oligomer comprising a sequence complementary to the 3 ′ region of the SNP site in the (+) strand;
A third oligomer comprising a sequence complementary to the 3 ′ region of the SNP site in the (−) strand; and a third sequence comprising a sequence complementary to the 5 ′ region of the SNP site in the (−) strand. Any one of the first oligomer and the second oligomer includes a base of the SNP site, and any one of the third oligomer and the fourth oligomer includes a base of the SNP site. , Methods and kits are provided.

  The present invention also provides the method and kit, wherein the detection target organism is phytoplasma and the detection target sample is a plant-derived DNA sample.

  Further, the present invention provides the method and kit, wherein the organism to be detected is Phytoplasma mali, Phytoplasma prunorum, Phytoplasma pyri, Phytoplasma pyri. pruni, Phytoplasma aurantifolia, Phytoplasma vitis, Phytoplasma castaneae and Phytoplasma phoenicium, and Phytoplasma phoenicium, Phytoplasma phoenicium At least two members selected from the group consisting of 16SrX group composed of Pyri and Phytoplasma purnoram, wherein the SNP is the 308th base, the 402nd base of 16S rDNA of Phytoplasma mari (SEQ ID NO: 1) Group, 560th base, 617th base or 1398 base, 178th base, 415th base, 1090th base or 1400th base of Phytoplasma purnorum 16S rDNA (SEQ ID NO: 2), 16S rDNA of Phytoplasma pyri (SEQ ID NO: 3) 203rd base, 258th base or 678th base, Phytoplasma piri PD-TW strain 16S rDNA (SEQ ID NO: 4) 179th base, 396th base or 1224th base, Phytoplasma 167 base, 182 base, 195 base, 202 base, 230 base, 448 base, 461 base, 462 base, 823 base, 823 base, 828 base of 16S rDNA (SEQ ID NO: 5) 975th base, 1017th base, 1218th base, 1221th base, 1267th base, 1270th base, 1418th base, 1437th base or 1462th base, 16S rDNA of Phytoplasma orantifolia (SEQ ID NO: 6) 429th base, 441th base, 442nd base, 443th base, 450th base, 453th salt Group, 454 base, 458 base, 459 base, 464 base, 473 base, 572 base, 572 base, 575 base, 582 base, 583 base, 588 base, 593 base, 594 base, 613 base, 619 base, 627 base, 629 base, 630 base, 650 base, 718 base, 945 base, 945 base, 972 base, 985 base, 986 base, 990 base, 996 base Base, 1014 base, 1080 base, 1174 base, 1226 base, 1240 base, 1240 base, 1248 base, 1256 base or 1258 base, 186th base of 16S rDNA of Phytoplasma vitis (SEQ ID NO: 7) , 206th base, 554th base, 557th base, 622nd base, 761st base, 982nd base, 1019th base, 1028th base, 1080th base, 1084th base, 1104th base, 1159th base, 1st base 1205 base, 1207 base, 1217 base, 1250 base, 1337 base, 1337 base, 1359 base, 1430 base or 1443 base, 16S rDNA of Phytoplasma castaneae (SEQ ID NO: 8) 63rd base, 124th base, 125th base, 151st base, 152nd base, 230th base, 246th base, 260th base, 276th base, 461st base, 462nd base, 485th base , 577 bases, 585 bases, 595 bases, 596 bases, 620 bases, 629 bases, 882 bases, 905 bases, 907 bases, 987 bases, 996 bases, 1122 bases, 1142 bases, 1155 bases, 1220 bases, 1328 bases, 1399 bases, 1400 bases, 1401 bases, 1424 bases, 1429 bases, 1431 bases or 1452 bases, and 16S of Phytoplasma forenium 336 base, 377 base, 414 base, 445 base, 573 base, 590 base, 803 base, 804 base, 964 base, 983 base, 996 base of rDNA (SEQ ID NO: 9) 1083 bases, 1098 bases, 1216 bases, 1217 bases or 1380 bases, and the 126th base of SEQ ID NO: 3 to identify the 16SrX group, the 136th base, 137th base, 189th base, 190th base, 192nd base, 193rd base, 205th base, 209th base, 219th base, 289th base, 308th base, 438th base, 439th base, 449th Base, base 453, base 500, base 513, base 534, base 584, base 603, base 604, base 696, base 701, base 807, base 818, base 821, 1049 base, 1093 base, 1097 base, 1100 base, 1100 base, 1114 base, 1241 base, 1298 base, 1331 base, 1398 base, 1413 base or 1416 base Methods and kits are provided.

  In addition, the present invention provides the presence or absence of the amplified DNA fragment by detecting the size of the DNA fragment amplified by the ligase chain reaction in the detection step. Methods and kits for detecting are provided.

  Further, the present invention provides the method described above, wherein at least one of the four oligomers constituting the oligomer set is labeled with a labeled molecule, and the labeled molecule of the DNA fragment amplified by the ligase chain reaction is detected in the detection step. Methods and kits for detecting the presence or absence of amplified DNA fragments are provided.

  The present invention also provides a kit further comprising a thermostable DNA ligase.

  According to the present invention, two or more kinds of organisms to be detected can be detected simultaneously and quickly and accurately. By using the present invention, it is possible to rapidly detect the presence or absence of pathogen infection and the species of the pathogen infected. The method of the present invention is capable of identifying pathogens at the species level. Further, according to the present invention, even when a plurality of pathogens are infected, the species of each pathogen can be rapidly detected. Therefore, for example, it is possible to quickly diagnose whether the target plant is infected with phytoplasma and the species of phytoplasma infected.

The figure which shows the result of having electrophoresed the PCR product in one Example of this invention. The figure which shows the result of having electrophoresed the product after LCR in one Example of this invention. The figure which shows the result of having electrophoresed the product after LCR in one Example of this invention. The figure which shows the result of having electrophoresed the product after LCR in one Example of this invention. The figure which shows an example of the location of SNP discovered within the 16SrDNA arrangement | sequence of phytoplasma.

  The method and kit according to the present invention are a method and kit for simultaneously detecting two or more kinds of organisms to be detected.

  The organism to be detected may be any organism and includes, for example, animals, plants, insects, bacteria, fungi, viruses and phytoplasmas. The present invention is particularly useful for the rapid detection of pathogens such as bacteria, fungi, viruses and phytoplasmas that infect or parasite other organisms.

  In the present specification, “two or more species” means that there are a plurality of groups (groups) including “species” as a unit of biological classification or two or more closely related species. That is, the method and kit of the present invention are methods and kits for simultaneously detecting a plurality of species or groups of detection target organisms.

[Method of detecting two or more species to be detected simultaneously]
The method for simultaneously detecting two or more detection target organisms according to the present invention includes a ligase chain reaction step (hereinafter also referred to as “LCR step”) and a detection step.

[Ligase chain reaction process]
The ligase chain reaction step is a step for performing a ligase chain reaction using two or more types of oligomer sets corresponding to two or more types of detection target organisms simultaneously and using the detection target sample as a template.

(Oligomer set)
The oligomer set is composed of four oligomers for ligase chain reaction designed by sandwiching any single nucleotide polymorphism (SNP) of the corresponding detection target organism. In the present invention, “oligomer” means a single-stranded DNA composed of several to 100 bases.

  Ligase chain reaction is a method of amplifying and detecting a specific gene sequence by performing a temperature cycling reaction (repetition of heating and cooling) using thermostable DNA ligase (Barany, 1991; Wiedmann et al. , 1994). First, two adjacent oligomers and template DNA that anneal to the (+) strand and (-) strand of the target DNA, respectively, are heated to separate the DNA into single strands. Subsequently, two adjacent oligomers anneal to the (+) strand and (-) strand of the template DNA by cooling. Next, adjacent oligomers are ligated by heat-resistant DNA ligase. By repeating this series of reactions, the ligation product is amplified and an amplified DNA fragment is generated.

  Examples of thermostable DNA ligases include Taq DNA Ligase (New England Biolabs, MA, USA), Ampligase (Epicentre, WI, USA), and Ape DNA Ligase (Thermophilic Enzyme Laboratory, Hyogo Prefecture, Japan) ) Etc. can be used.

  The four oligomers that make up the oligomer set were designed by sandwiching any single nucleotide polymorphism (SNP) of the corresponding organism to be detected, and (2) adjacent oligomers that anneal to the (+) strand, and (-) It consists of two adjacent oligomers that anneal to the chain. In this specification, “design with SNP in between” means that the SNP site is designed with 5 ′ side or 3 ′ side in between. That is, two types of adjacent oligomers sandwiching the SNP are designed so that either one contains the base of the SNP site. The four oligomers generate amplified DNA fragments in the LCR using the corresponding organism's DNA as a template, but do not amplify in different species of DNA, i.e. template DNA with different SNP site bases. Designed.

  As used herein, “single nucleotide polymorphism (SNP)” refers to a single nucleotide variation that is conserved in only one species or group in the genomic sequence of a given population of organisms. The SNP utilized in the present invention can be any SNP in the genomic sequence, and may be in the gene coding region or non-coding region. For example, when individually identifying a phytoplasma species or pathogenic group, it can be a SNP in a 16S rDNA sequence. The SNP of the 16S rDNA sequence can be, for example, the location shown in FIG.

  In the present specification, “(+) strand” and “(−) strand” mean one strand of double-stranded DNA and the other strand having a sequence complementary thereto, such as sense strand and anti-strand. It may be a sense strand.

The four oligomers constituting the oligomer set (first oligomer, second oligomer, third oligomer and fourth oligomer, hereinafter also referred to as oligomers A to D) may be the following four oligomers: Good:
First oligomer (oligomer A): an oligomer consisting of a sequence complementary to the 5 'region of the SNP site in the (+) strand,
Second oligomer (oligomer B): an oligomer consisting of a sequence complementary to the 3 'region of the SNP site in the (+) strand,
Third oligomer (oligomer C): oligomer consisting of a sequence complementary to the region 3 ′ of the SNP site in the (−) chain, and fourth oligomer (oligomer D): 5 of the SNP site in the (−) chain 'A fourth oligomer consisting of a sequence complementary to the side region.
Either the first oligomer or the second oligomer contains a base at the SNP site, and either one of the third oligomer or the fourth oligomer contains a base at the SNP site.

For example, the four oligomers that make up the oligomer set may be the following four oligomers:
First oligomer (oligomer A): an oligomer composed of a sequence complementary to the 5 ′ side region from the base adjacent to the 5 ′ side of the base of the SNP site in the (+) chain,
Second oligomer (oligomer B): an oligomer comprising a base at the SNP site in the (+) chain and a sequence complementary to the region 3 ′ from the base adjacent to the 3 ′ side of the base,
Third oligomer (oligomer C): an oligomer comprising a base at the SNP site in the (−) chain and a sequence complementary to a region 3 ′ from the base adjacent to the 3 ′ side of the base, and the fourth Oligomer (oligomer D): an oligomer having a sequence complementary to the region 5 ′ from the base adjacent to the 5 ′ side of the base of the SNP site in the (−) chain.

Further, the four oligomers constituting the oligomer set may be the following four oligomers:
First oligomer (oligomer A): an oligomer comprising a base at the SNP site in the (+) chain and a sequence complementary to the region 5 ′ from the base adjacent to the 5 ′ side of the base,
Second oligomer (oligomer B): an oligomer consisting of a sequence complementary to the region 3 ′ to the base adjacent to the 3 ′ side of the base of the SNP site in the (+) chain,
Third oligomer (oligomer C): an oligomer composed of a sequence complementary to the region 3 ′ from the base adjacent to the 3 ′ side of the base of the SNP site in the (−) chain, and the fourth oligomer (oligomer D) ): An oligomer comprising a base at the SNP site in the (−) chain and a sequence complementary to a region 5 ′ from the base adjacent to the 5 ′ side of the base.

Further, the four oligomers constituting the oligomer set may be the following four oligomers:
First oligomer (oligomer A): an oligomer comprising a base at the SNP site in the (+) chain and a sequence complementary to the region 5 ′ from the base adjacent to the 5 ′ side of the base,
Second oligomer (oligomer B): an oligomer consisting of a sequence complementary to the region 3 ′ to the base adjacent to the 3 ′ side of the base of the SNP site in the (+) chain,
Third oligomer (oligomer C): an oligomer comprising a base at the SNP site in the (−) chain and a sequence complementary to a region 3 ′ from the base adjacent to the 3 ′ side of the base, and the fourth Oligomer (oligomer D): an oligomer having a sequence complementary to the region 5 ′ from the base adjacent to the 5 ′ side of the base of the SNP site in the (−) chain.

Further, the four oligomers constituting the oligomer set may be the following four oligomers:
First oligomer (oligomer A): an oligomer composed of a sequence complementary to the 5 ′ side region from the base adjacent to the 5 ′ side of the base of the SNP site in the (+) chain,
Second oligomer (oligomer B): an oligomer comprising a base at the SNP site in the (+) chain and a sequence complementary to the region 3 ′ from the base adjacent to the 3 ′ side of the base,
Third oligomer (oligomer C): an oligomer composed of a sequence complementary to the region 3 ′ from the base adjacent to the 3 ′ side of the base of the SNP site in the (−) chain, and the fourth oligomer (oligomer D) ): An oligomer comprising a base at the SNP site in the (−) chain and a sequence complementary to a region 5 ′ from the base adjacent to the 5 ′ side of the base.

  Since the oligomers A to D are designed with the SNP sandwiched in this way, if the DNA of the organism to be detected is contained in the LCR template, the oligomer AB and the fragment AB to which the oligomer A and B are linked in the LCR process As a result, an amplified DNA fragment hybridized with the fragment CD to which the oligomer C and the oligomer D are linked is produced. On the other hand, with respect to DNA other than the detection target organism, that is, template DNA having a different base at the SNP site, oligomers cannot be ligated in the LCR step, and no amplified DNA fragment is generated.

  The 5 'end of oligomer A and the 5' end of oligomer D can be phosphorylated for ligation with oligomer B or C, respectively, in the LCR.

  The length of each oligomer is not particularly limited as long as LCR and subsequent detection are possible, and may be, for example, 20 to 100 bp.

  Fragment AB and fragment CD can be fragments of the same length having sequences complementary to each other. However, the present invention is not limited thereto, and the fragment AB and the fragment CD may partially have non-complementary sequences, and may have the same length or different lengths.

  The amplified DNA fragment can be designed to have an arbitrary length. Without limitation, the oligomer set can be designed to produce amplified DNA fragments, eg, 50-100 base pairs long. The length of the amplified DNA fragment obtained by hybridizing the fragment AB and the fragment CD may be the same depending on the oligomer set or may be different from each other. When the lengths are different from each other, the presence or absence of an amplified DNA fragment derived from each oligomer set can be detected by the difference in size.

  At least one of the oligomers constituting the oligomer set may be labeled. For example, any one or more of the 3 'end of oligomer A, the 5' end of oligomer B, the 5 'end of oligomer C and the 3' end of oligomer D may be labeled. Oligomers can be labeled by any method, for example, directly detectable label molecules such as fluorescent dyes and radioisotopes, labeled molecules indirectly detectable using immunological techniques, and others It can be labeled with a detectable labeling molecule.

  The fluorescent dye may generally be a fluorescent dye that can be detected by labeling a nucleic acid, such as fluorescein or a derivative thereof (for example, fluorescein-isothiocyanate (FITC)), rhodamine, Texas red ( Texas Red), Alexa 488, Alexa 532, cy3, cy5, 6-joe and EDANS.

  Labeled molecules that can be detected indirectly using immunological techniques include, for example, biotin, digoxigenin, antigens and coenzymes.

  By labeling the oligomer, an amplified DNA fragment in which the fragment AB and the fragment CD are hybridized can be easily detected. As a detection method, fluorescence emission measurement, radiation dose measurement, ELISA (Enzyme-Linked ImmunoSorbent Assay), immunostrip assay, and the like can be used.

  For example, the 3 'end of oligomer A or the 5' end of oligomer C and the 5 'end of oligomer B or the 3' end of oligomer D may be labeled with different labeling molecules. For example, an amplified DNA fragment obtained by hybridizing the fragment AB and the fragment CD may be labeled so as to be detectable using an immunological technique such as ELISA.

(Sample to be detected)
The detection target sample is a sample that may contain the DNA of the detection target organism for detecting the presence or absence of the detection target organism. The detection target sample may be, for example, a substance suspected of being mixed with the detection target organism, an organism suspected of being infected or carrying the detection target organism, and a DNA sample derived from this organism. The DNA sample may be DNA extracted from an organism and amplification products obtained by PCR or the like performed on the extracted DNA. Known methods can be used as a method for extracting DNA from an organism and an amplification method using PCR or the like.

(Ligase chain reaction (LCR))
In the present invention, the ligase chain reaction is performed by using each oligomer set corresponding to each of two or more kinds of organisms to be detected and using the sample to be detected as a template.

  As an example of the ligase chain reaction, first, a reaction solution containing each oligomer set, a sample to be detected and a heat-resistant DNA ligase is denatured by heating to separate the DNA into single strands. The temperature and time for heating can be set as appropriate.

  Subsequently, the reaction solution is cooled. The temperature and time for cooling can be set as appropriate. Here, when the detection target sample contains DNA derived from the detection target organism, two types of neighboring DNAs corresponding to the (+) strand and the (−) strand of the template DNA separated into single strands, respectively. The existing oligomers anneal. Next, adjacent oligomers are linked by a heat-resistant DNA ligase.

  By repeating this series of heating and cooling reactions, the ligation product (oligomer ligation product) is exponentially amplified. The number of times this series of reactions is repeated can be set as appropriate.

  On the other hand, if the sample to be detected contains DNA derived from a different species of organism from the organism to be detected, this DNA has an SNP site base different from that of the organism to be detected. Cannot be completely annealed and no ligation product is produced. That is, for DNA derived from organisms other than the detection target organism, there is a mismatch with the template at the boundary site between adjacent oligomers, ligation does not occur, and DNA is not amplified.

  Therefore, by performing the LCR process and the detection process described later, the detection target organism can be identified at the species level by the difference in the base of the SNP site.

The ligase chain reaction can be performed, for example, by the following steps:
(1) denature at 94 ° C. for 2 minutes;
(2) denature for 90 seconds at 94 ° C;
(3) Annealing at 55 ° C for 6 minutes;
(4) Repeat the above 2 and 3 cycles twice;
(5) denature at 91 ° C. for 30 seconds;
(6) Annealing at 55 ° C for 6 minutes;
(7) Repeat the above cycles (5) and (6) 20 times.
Alternatively, it can be carried out by the following steps:
(1) denature at 94 ° C. for 2 minutes;
(2) denature at 94 ° C for 1 minute;
(3) Annealing at 55 ° C for 6 minutes;
(4) Repeat the above cycles (2) and (3) 30 times.

[Detection process]
The detection step is a step of detecting the presence or absence of amplified DNA fragments (also referred to as “amplified fragments” in this specification) derived from each oligomer set by ligase chain reaction. In other words, in the detection step, it is detected from which oligomer set the DNA fragment amplified by the ligase chain reaction is derived.

  The amplified fragment can be detected using any method for detecting a nucleic acid, and can be detected by, for example, electrophoresis, detection using an immunological technique, base sequencing, and the like.

  When the length of the amplified fragment differs depending on each oligomer set, the presence or absence of the amplified fragment derived from each oligomer set can be easily detected by detecting the size of the amplified fragment by electrophoresis or the like. A high separation gel or the like can be used for electrophoresis.

  When the oligomer is labeled with a labeled molecule, the amplified fragment can be detected by an immunological technique. For detection by immunological techniques, for example, one end of the amplified fragment is labeled with an immobilizable label molecule such as biotin, and the other end can be detected with a fluorescent dye or antibody such as FITC, rhodamine or Texas Red. It may be labeled with a labeling molecule such as DIG. In this case, the amplified fragment is detected by immobilizing one end of the amplified fragment on an avidin plate or avidin-labeled particles (such as colloidal gold and latex particles) and detecting luminescence from the other end. Can do. Alternatively, after the label molecule at the other end is bound to a fluorescent dye or an antibody bound to an enzyme, the product of fluorescence or enzyme reaction may be detected.

  The amplified fragment may be labeled in different ways depending on the oligomer set. For example, it may be labeled with different types of fluorescent molecules depending on the oligomer set. Thereby, in the detection step, it is possible to easily detect which oligomer set-derived amplified fragment is present by the detected fluorescence.

[PCR process]
In the method of the present invention, a PCR step may be provided before the LCR step. The PCR step is a step of performing PCR for amplifying the DNA of an organism to be detected and a group of organisms including the species close thereto, using the sample to be detected as a template as a primary screening.

  For PCR (polymerase chain reaction), any commonly used method and conditions can be used.

  As a primer used for PCR, a primer that produces an amplified fragment by PCR can be used only when the DNA of an organism to be detected and a group of organisms including related species are used as templates. The fragment amplified by PCR may contain a region where the oligomer for LCR anneals.

  By detecting the presence or absence of a PCR-amplified fragment by the PCR step, it is possible to identify the presence or absence of a detection target organism and a group of organisms including species closely related thereto. The presence or absence of a PCR-amplified fragment can be detected using any method for detecting a nucleic acid, and can be detected by, for example, electrophoresis, detection using an immunological technique, and base sequence decoding.

  In the method of the present invention, the LCR step and the detection step may be performed only when amplification by the PCR step is observed. Thereby, cost can be reduced.

  Further, by performing the PCR process on a plurality of organism groups, one or a plurality of organism groups that are likely to contain the detection target organism may be specified from the plurality of organism groups. By identifying a group of organisms that are likely to contain a detection target organism by the PCR process, depending on the detection target sample, species or groups of the detection target organism can be narrowed down. For example, when the detection target sample is a plant body or a vector insect, and the phytoplasma is specified as a group of organisms likely to contain the detection target organism, the phytoplasma to be detected by the type of the plant body or vector insect Species or pathogenic group candidates can be narrowed down in advance. Therefore, the number of times of performing the LCR process and the detection process can be reduced, the cost can be reduced, and rapid detection can be performed.

  In addition, when a sequence including a region where the LCR oligomer is annealed is amplified in the PCR process, a part of the PCR amplified fragment may be used as a template for the LCR process. In this case, since there is a sufficient amount of template DNA in the LCR process, generation of non-specific amplification bands can be prevented and accuracy can be improved.

〔kit〕
The kit of the present invention is a kit for simultaneously detecting two or more detection target organisms, and includes two or more kinds of oligomer sets as described above corresponding to each of the two or more detection target organisms. As described above, the oligomer set is composed of four oligomers for ligase chain reaction designed by sandwiching any SNP of the corresponding organism to be detected. The four oligomers that make up the oligomer set consist of two adjacent oligomers that anneal to the (+) strand across any single nucleotide polymorphism (SNP) of the corresponding organism to be detected, and the SNP ( -) Consists of two adjacent oligomers that anneal to the chain. Two types of adjacent oligomers sandwiching the SNP are designed so that either one contains the base of the SNP site. The four oligomers generate amplified DNA fragments in the LCR using the corresponding organism's DNA as a template, but do not amplify in different species of DNA, i.e. template DNA with different SNP site bases. Designed. For example, the four oligomers that make up the oligomer set may be the following four oligomers:
First oligomer (oligomer A): an oligomer consisting of a sequence complementary to the 5 'region of the SNP site in the (+) strand,
Second oligomer (oligomer B): an oligomer consisting of a sequence complementary to the 3 'region of the SNP site in the (+) strand,
Third oligomer (oligomer C): oligomer consisting of a sequence complementary to the region 3 ′ of the SNP site in the (−) chain, and fourth oligomer (oligomer D): 5 of the SNP site in the (−) chain 'A fourth oligomer consisting of a sequence complementary to the side region.
Either the first oligomer or the second oligomer contains a base at the SNP site, and either one of the third oligomer or the fourth oligomer contains a base at the SNP site.

  The 5 'end of oligomer A and the 5' end of oligomer D can be phosphorylated for ligation with oligomer B or C, respectively, in the LCR.

  The length of each oligomer is not particularly limited as long as LCR and subsequent detection are possible, and may be, for example, 20 to 100 bp.

  Each oligomer set can be designed to yield amplified DNA fragments, eg, 50-100 base pairs long.

  The kit of this invention can be utilized suitably for the method mentioned above. By using the kit of the present invention, the presence / absence of two or more detection target organisms can be simultaneously and easily detected from the detection target sample.

  In the kit of the present invention, the four oligomers constituting each oligomer set may be included separately from each other or may be mixed. Moreover, when the four oligomers of each oligomer set are mixed, each of the oligomer sets may be included separately from each other or may be mixed.

  The kit of the present invention may further contain a thermostable DNA ligase that can be used for LCR. The kit of the present invention may further contain a reagent for detecting the LCR amplification product, a reagent for labeling the oligomer, a primer for primary screening, and the like.

  In the present invention, the organism to be detected may be, for example, phytoplasma. In that case, the sample to be detected may be a DNA sample derived from a plant or a vector that is suspected of being infected or carrying bacteria.

  The organisms to be detected include, for example, Phytoplasma mali, Phytoplasma prunorum, Phytoplasma pyri, Phytoplasma pruni, Phytoplasma prunirum (Phytoplasma pruni), Phytoplasma prunirum (Phytoplasma pruni) There may be at least two species selected from Phytoplasma aurantifolia, Phytoplasma vitis, Phytoplasma castaneae, and Phytoplasma phoenicium. Further, at least one of the organisms to be detected may be a group including a plurality of species phylogeny based on the sequence of ribosomal RNA, for example, Phytoplasma Mali, Phytoplasma pylori, and Phytoplasma purnoram. (16SrX group) etc. comprised by these.

  When the organism to be detected is phytoplasma as described above, the SNP can be any SNP, and the SNP of the 16S rDNA sequence can be, for example, the location shown in FIG. Further, for example, it may be SNP in 16S rDNA as found in Examples described later.

  According to the present invention, for example, even when a plant body is mixed with phytoplasma, the species or pathogenic group of each phytoplasma can be simultaneously and rapidly detected. Therefore, the present invention can be used for plant import and export quarantine and disease diagnosis. The present invention is useful not only in Japan but also in countries that are severely damaged by phytoplasma. For example, plant quarantine can prevent new phytoplasma from entering the country. In export quarantine, it is possible to quickly inspect whether the plant to be exported is infected with phytoplasma and to notify the partner country of the result. On the other hand, in Japan, the spread of infection damage can be suppressed by quickly diagnosing the presence or absence of infection by existing phytoplasma.

  The present invention can be used for identifying various organisms to be detected by designing and preparing a large number of oligomer sets for identifying the organism to be detected, and appropriately combining them.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims.

  Hereinafter, examples will be shown and the embodiment of the present invention will be described in more detail. However, the present invention is not limited to the following examples.

[Example 1]
[Selection of identification SNPs]
Various phytoplasma 16S rDNA sequences were extracted from public DNA sequence databases such as NCBI GenBank. Arbitrarily selected 82 sequences were aligned using sequence analysis software MEGA5.2. By alignment analysis, we found SNPs (single nucleotide polymorphisms) conserved in only one species or pathogenic group in the 16S rDNA sequence.

The following were identified as SNPs for identification of each phytoplasma:
Phytoplasma mali (hereinafter also referred to as “P. mali”): 308th base, 402th base, 560th base, 617th base and 1398th base of 16S rDNA (SEQ ID NO: 1; from GenBank number X68375) base;
Phytoplasma prunorum (hereinafter also referred to as “P. prunorum”): 178th base, 415th base, 1090th base and 1400th base of 16S rDNA (SEQ ID NO: 2; from GenBank number AY029540);
Phytoplasma pyri (hereinafter also referred to as “P. pyri”): 16S rDNA (SEQ ID NO: 3; from GenBank No. AJ542543), base 203, base 258 and base 678 Phytoplasma pyri ) PD-TW strain: 179th base, 396th base and 1224th base of 16S rDNA (SEQ ID NO: 4; from GenBank number DQ011588);
Phytoplasma pruni (hereinafter also referred to as “P. pruni”): 167th, 182nd, 195th, 202th, 230th, 16S rDNA (SEQ ID NO: 5; from GenBank number JQ044396) Base, 448 base, 461 base, 462 base, 823 base, 828 base, 975 base, 975 base, 1017 base, 1218 base, 1221 base, 1267 base, 1270 base, 1418 base, Bases 1437 and 1462;
Phytoplasma aurantifolia (Phytoplasma aurantifolia): 429th, 441th, 442th, 443th, 450th, 453th, 454th, 454th of 16S rDNA (SEQ ID NO: 6; from GenBank number AB295056) Base, 458 base, 459 base, 464 base, 473 base, 572 base, 575 base, 582 base, 583 base, 588 base, 593 base, 594 base, 613 base, 619 base, 627 base, 629 base, 630 base, 650 base, 718 base, 945 base, 945 base, 972 base, 985 base, 986 base, 990 base, 996 base, 1014 Base, base 1080, base 1174, base 1226, base 1240, base 1248, base 1256, base 1258;
Phytoplasma vitis: 186th base, 206th base, 554th base, 557th base, 557th base, 622th base, 761th base, 982th base of 16S rDNA (SEQ ID NO: 7; from GenBank number AF122912), 1019 base, 1028 base, 1080 base, 1084 base, 1104 base, 1104 base, 1159 base, 1205 base, 1207 base, 1217 base, 1250 base, 1337 base, 1359 base, 1430 Base and 1443 base;
Phytoplasma castaneae: 63S base, 124th base, 125th base, 151st base, 152nd base, 230th base, 246th base, 16S rDNA (SEQ ID NO: 8; from GenBank number AB054986), 260th base, 276th base, 461st base, 462nd base, 485th base, 577th base, 585th base, 595th base, 596th base, 620th base, 629th base, 882th base, 905th base Base, 907 base, 987 base, 996 base, 1122 base, 1142 base, 1155 base, 1155 base, 1220 base, 1328 base, 1399 base, 1400 base, 1401 base, 1424 base, 1429 base, 1431 base and 1452 base;
Phytoplasma phoenicium: 16S rDNA (SEQ ID NO: 9; from GenBank number AF515637), 336th base, 377th base, 414th base, 445th base, 573th base, 590th base, 803th base, 804 base, 964 base, 983 base, 996 base, 1083 base, 1098 base, 1098 base, 1216 base, 1217 base and 1380 base.

Moreover, the following was discovered as SNPs for identification of the group (16SrX group) containing P. mali, P. prunorum, and P. pyri:
126th base, 136th base, 137th base, 189th base, 190th base, 192nd base, 193rd base, 205th base, 16th rDNA (SEQ ID NO: 3) of Phytoplasma pyri 209 bases, 219 bases, 289 bases, 308 bases, 438 bases, 439 bases, 449 bases, 453 bases, 500 bases, 513 bases, 534 bases, 584 bases, 603 bases , 604 base, 696 base, 701 base, 807 base, 807 base, 818 base, 821 base, 1049 base, 1093 base, 1097 base, 1100 base, 1114 base, 1241 base, 1st base 1298 bases, 1331 bases, 1398 bases, 1413 bases and 1416 bases.

[Oligomer design]
Four oligomers were designed for use in the LCR method for P. pyri, P. mali, P. prunorum and P. pruni. The oligomers were designed based on the SNPs for identification of each phytoplasma and its peripheral sequence (target sequence). Specifically, oligomer A consisting of a sequence complementary to the 5 'region of the SNP site in the (+) strand of the target sequence and oligomer B consisting of a sequence complementary to the 3' region of the SNP site, In addition, oligomer C consisting of a sequence complementary to the 3 ′ region of the SNP site in the (−) strand and oligomer D consisting of a sequence complementary to the 5 ′ region of the SNP site were designed. One of oligomers A and B was designed to contain a SNP site base, and one of oligomers C and D was designed to contain a SNP site base.

  Oligomer A (SEQ ID NO: 10), Oligomer B (SEQ ID NO: 11), Oligomer C (SEQ ID NO: 12), and Oligomer, sandwiching the SNP of the 203rd base of 16S rDNA (SEQ ID NO: 3) as an oligomer for P. pyri discrimination D (SEQ ID NO: 13) was designed.

  As an oligomer for P. mali identification, oligomer S (SEQ ID NO: 14), oligomer B (SEQ ID NO: 15), oligomer C (SEQ ID NO: 16) and oligomer sandwiching the SNP of the 308th base of 16S rDNA (SEQ ID NO: 1) D (SEQ ID NO: 17) was designed.

  Oligomer A (SEQ ID NO: 18), Oligomer B (SEQ ID NO: 19), Oligomer C (SEQ ID NO: 20), and Oligomer as P. prunorum discriminating oligomer sandwiching the 1090 base SNP of 16S rDNA (SEQ ID NO: 2) D (SEQ ID NO: 21) was designed.

  As an oligomer for P. pruni identification, oligomer S (SEQ ID NO: 22), oligomer B (SEQ ID NO: 23), oligomer C (SEQ ID NO: 24) and oligomer sandwiching the SNP of the 462nd base of 16S rDNA (SEQ ID NO: 5) D (SEQ ID NO: 25) was designed.

  The 5 'ends of oligomer A and oligomer D, respectively, were phosphorylated.

[LCR method]
DNA was extracted by known methods from phytoplasma-infected plants infected with P. pyri, P. mali, P. prunorum and P. pruni, respectively.

Known primer sets (P1 / P7 (SEQ ID NO: 26 / SEQ ID NO: 27), fU1 / rU1 (SEQ ID NO: 28) for amplifying sequences common to phytoplasma as a primary screening using DNA extracted from each infected plant as a template / SEQ ID NO: 29) and f0 / r0 (SEQ ID NO: 30 / SEQ ID NO: 31)) were used for PCR. PCR was performed using ExTaq hot start version (TAKARA BIO INC., Shiga, Japan) as a DNA polymerase in a mixed solution containing 100 ng / μl of template DNA and 0.2 μM of a primer-attached buffer. PCR conditions were as follows:
(1) Denature at 96 ° C for 4 minutes;
(2) Denature at 96 ° C for 30 seconds;
(3) Annealing at 55 ° C for 1 minute;
(4) Elongate at 72 ° C for 30 seconds;
(5) Repeat the above cycles (2), (3) and (4) 30 times.
FIG. 1 is a diagram showing the results of electrophoresis of PCR products. As shown in FIG. 1, the presence of PCR-amplified fragments by each primer was confirmed.

Next, the LCR method was performed using oligomers A to D designed for P. pruni using the fragment amplified by the P1 / P7 primer set as a template. LCR uses Taq DNA ligase (New England Biolabs) as a thermostable DNA ligase and mixes with 100 ng / μl template DNA, 0.1 μM of each oligomer and 0.4 μg / μl salmon DNA in a buffer attached to the enzyme. Performed in solution. The conditions for LCR were as follows:
(1) denature at 94 ° C. for 1.5 minutes;
(2) Annealing at 55 ° C for 6 minutes;
(3) Repeat the above cycles (1) and (2) five times;
(4) denature at 91 ° C. for 30 seconds;
(5) Annealing at 55 ° C for 6 minutes;
(1) Repeat the above cycles (4) and (5) 15 times.

  The product after LCR was electrophoresed on 3% Lonza MetaPhor Agarose gel (Takara Bio Inc., Shiga, Japan). FIG. 2 shows the results of electrophoresis of the product after LCR. As shown in FIG. 2, a 96 bp long amplified fragment derived from the oligomer set of P. pruni was obtained only in the plant sample infected with P. pruni.

  From this result, it was shown that the species of phytoplasma infecting plants can be diagnosed accurately and rapidly.

[Example 2]
As a model of a DNA sample of a plant infected with multiple species, DNA from phytoplasma infected plants infected with P. pyri, P. mali and P. prunorum was mixed (sample 1). As another model, DNA from Phytoplasma-infected plants infected with P. pyri, P. mali and P. pruni was mixed (Sample 2).

  Using each mixed infection DNA sample as a template, the LCR method was performed using the oligomer set designed for each of P. pyri, P. mali, P. prunorum and P. pruni. LCR conditions were the same as in Example 1.

  FIG. 3 shows the results of electrophoresis of the product after LCR. As shown in FIG. 3, in sample 1, amplified fragments were observed when the P. pyri, P. mali or P. prunorum oligomer set was used, and when the P. pruni oligomer set was used, the amplified fragment was observed. I was not able to admit. In Sample 2, amplified fragments were observed when the P. pyri, P. mali or P. pruni oligomer set was used, and no amplified fragments were observed when the P. prunorum oligomer set was used. .

  Therefore, by using an oligomer set for identifying each phytoplasma, it is possible to accurately and quickly diagnose a plurality of infected phytoplasma species from a plant sample infected with a plurality of phytoplasmas. Indicated.

[Example 3]
DNA samples from P. pruni-infected plants (P. pruni single-infected DNA), DNA samples from P. prunorum-infected plants (P. prunorum single-infected DNA), and DNA samples from plants infected with both of these phytoplasmas As a model, a DNA sample (P. pruni, P. prunorum mixed infection DNA) prepared by mixing these DNA samples was prepared.

  Using each DNA sample as a template, each oligomer set for discriminating these two types of phytoplasma was used alone or in combination to carry out the LCR method.

  (A), (b) and (c) of FIG. 4 are diagrams showing the results of electrophoresis of the product after LCR. As shown in FIG. 4 (a), when the P. pruni identification oligomer set is used alone and at the same time as the P. prunorum identification oligomer set, as shown in FIG. A 96 bp LCR amplified fragment derived from the identification oligomer set for use was found. LCR amplified fragments derived from the discriminating oligomer set for P. prunorum were not found. The short DNA fragment observed when the identification oligomer set for P. prunorum is used alone and when two oligomer sets are used simultaneously is not an amplified fragment but a 42 bp oligomer that is one of the oligomer sets. It is.

  As shown in FIG. 4 (b), when P. prunorum single-infected DNA is used, the P. prunorum discriminating oligomer set is used alone and simultaneously with the P. pruni discriminating oligomer set. A 63 bp LCR amplified fragment derived from the identification oligomer set for use was found. No LCR amplified fragment derived from P. pruni discriminating oligomer set was found.

  In the mixed infection DNA, as shown in FIG. 4 (c), both a 96 bp LCR amplified fragment derived from the identification oligomer set for P. pruni and a 63 bp LCR amplified fragment derived from the identification oligomer set for P. prunorum were observed. It was. In particular, when the identification oligomer set for P. pruni and the identification oligomer set for P. prunorum were used at the same time, both LCR amplified fragments were observed simultaneously.

  From these results, it was shown that the present invention can accurately detect two or more types of phytoplasma infecting plants simultaneously. Therefore, it was shown that the present invention can simultaneously detect two or more kinds of detection target organisms.

  The present invention can be used for simultaneous detection of two or more organisms. In particular, the present invention can be suitably used for simultaneous detection of a plurality of phytoplasma in import and export quarantine of plants and disease diagnosis in Japan.

Claims (9)

A ligase chain reaction step in which ligase chain reaction (LCR) is performed using a sample to be detected as a template, simultaneously using two or more types of oligomer sets corresponding to each of two or more types of phytoplasma;
Detecting the presence or absence of amplified DNA fragments derived from each oligomer set by the ligase chain reaction,
The oligomer set is composed of four oligomers for ligase chain reaction designed by sandwiching any single nucleotide polymorphism (SNP) of the corresponding phytoplasma ,
The four oligomers are designed to generate amplified DNA fragments in the LCR using the corresponding phytoplasma DNA as a template, but not to amplify in the LCR for template DNA having a different SNP site base,
The phytoplasma is Phytoplasma mali, Phytoplasma prunorum, Phytoplasma pyri, Phytoplasma pruni, Phytoplasma aurantifolia ), Phytoplasma vitis, Phytoplasma castaneae and Phytoplasma phoenicium, and the 16SrX group consisting of Phytoplasma phoenicium and Phytoplasma phoenicium At least two selected from the group consisting of:
The SNP is the 308th, 402th, 560th, 617th, or 1398th base of 16S rDNA (SEQ ID NO: 1) of Phytoplasma mari, 16S rDNA (SEQ ID NO: 2) of Phytoplasma purunoram 178th base, 415th base, 1090th base or 1400th base, Phytoplasma pylori 16S rDNA (SEQ ID NO: 3), 203rd base, 258th base or 678th base, Phytoplasma pylori PD-TW strain 179th base, 396th base or 1224th base of 16S rDNA (SEQ ID NO: 4), 167th base, 182nd base, 195th base, 202th base, 16th base of 16S rDNA (SEQ ID NO: 5) of Phytoplasma pruni 230 bases, 448 bases, 461 bases, 462 bases, 823 bases, 828 bases, 975 bases, 975 bases, 1017 bases, 1218 bases, 1221 bases, 1267 bases, 1270 bases, 1418 bases , 1437 base or 1462 base, 16S rDNA of Phytoplasma olantifolia ( 429th base, 441th base, 442th base, 443th base, 450th base, 453th base, 454th base, 458th base, 459th base, 464th base, 473th base, 572, 575, 582, 583, 588, 593, 594, 613, 619, 627, 629, 630, 650 , 718 base, 945 base, 972 base, 985 base, 986 base, 986 base, 990 base, 996 base, 1014 base, 1080 base, 1174 base, 1226 base, 1240 base, 1st base 1248 bases, 1256 bases or 1258 bases, 186 base, 206 bases, 554 bases, 557 bases, 557 bases, 622 bases, 761 bases, 982 bases of 16S rDNA of Phytoplasma vitis (SEQ ID NO: 7) Base, 1019 base, 1028 base, 1080 base, 1084 base, 1104 base, 1104 base, 1159 base, 1205 base, 1207 base, 1217 base, 1250 base, 1337 base, 1359 base, 1430 salt 1st base, 1443 base, 63rd base, 124th base, 125th base, 151st base, 152nd base, 230th base, 230th base, 246th base, 260th base of 16S rDNA (SEQ ID NO: 8) of Phytoplasma castaneae , 276 base, 461 base, 462 base, 485 base, 577 base, 585 base, 585 base, 595 base, 596 base, 620 base, 629 base, 882 base, 905 base, 907 bases, 987 bases, 996 bases, 1122 bases, 1142 bases, 1142 bases, 1155 bases, 1220 bases, 1328 bases, 1399 bases, 1400 bases, 1401 bases, 1424 bases, 1429 bases , 1431 base or 1452 base, and 336, 377, 414, 445, 573, 590, 590, and 803 bases of 16S rDNA (SEQ ID NO: 9) of Phytoplasma forenisium , 804 base, 964 base, 983 base, 996 base, 1083 base, 1083 base, 1098 base, 1216 base, 1217 base or 1380 base And the 126th base, the 136th base, the 137th base, the 189th base, the 190th base, the 192nd base, the 193rd base, the 205th base, the 209th base, the second base of SEQ ID NO: 3 for identifying the 16SrX group 219 bases, 289 bases, 308 bases, 438 bases, 439 bases, 439 bases, 453 bases, 453 bases, 500 bases, 513 bases, 534 bases, 584 bases, 603 bases, 604 bases , 696 base, 701 base, 807 base, 818 base, 818 base, 821 base, 1049 base, 1093 base, 1097 base, 1100 base, 1114 base, 1241 base, 1298 base, 1st base 1331 bases, the first 1398 bases, Ru is selected from the group consisting of the 1413 base or first 1416 bases,
A method to detect two or more types of phytoplasma simultaneously. The four oligomers are
A first oligomer comprising a sequence complementary to the 5 ′ region of the SNP site in the (+) strand;
A second oligomer consisting of a sequence complementary to the 3 'region of the SNP site in the (+) strand;
A third oligomer comprising a sequence complementary to the 3 ′ region of the SNP site in the (−) strand; and a third sequence comprising a sequence complementary to the 5 ′ region of the SNP site in the (−) strand. Four oligomers,
Either one of the first oligomer and the second oligomer includes a base of the SNP site, and one of the third oligomer and the fourth oligomer includes a base of the SNP site,
The method of claim 1.   3. The method according to claim 1, wherein the detection target sample is a plant-derived DNA sample. The size of the amplified DNA fragment is different for each of the oligomer set, to detect the presence or absence of said amplified DNA fragment by detecting the size of the DNA fragments amplified by the ligase chain reaction in the detection step, according to claim 1 to 3 The method according to any one of the above. At least one of the four oligomers constituting the oligomer set is labeled with a labeling molecule;
Detecting the presence or absence of the amplified DNA fragment by detecting a labeled molecule of the DNA fragment amplified by the ligase chain reaction in the detection step;
The method according to any one of claims 1 to 3 . Includes two or more oligomer sets corresponding to each of two or more phytoplasma,
The oligomer set is composed of four oligomers for ligase chain reaction (LCR) designed by sandwiching any single nucleotide polymorphism (SNP) of the corresponding phytoplasma ,
The four oligomers are designed to generate amplified DNA fragments in the LCR using the corresponding phytoplasma DNA as a template, but not to amplify in the LCR for template DNA having a different SNP site base,
The phytoplasma is Phytoplasma mali, Phytoplasma prunorum, Phytoplasma pyri, Phytoplasma pruni, Phytoplasma aurantifolia ), Phytoplasma vitis, Phytoplasma castaneae and Phytoplasma phoenicium, and the 16SrX group consisting of Phytoplasma phoenicium and Phytoplasma phoenicium At least two selected from the group consisting of:
The SNP is the 308th, 402th, 560th, 617th, or 1398th base of 16S rDNA (SEQ ID NO: 1) of Phytoplasma mari, 16S rDNA (SEQ ID NO: 2) of Phytoplasma purunoram 178th base, 415th base, 1090th base or 1400th base, Phytoplasma pylori 16S rDNA (SEQ ID NO: 3), 203rd base, 258th base or 678th base, Phytoplasma pylori PD-TW strain 179th base, 396th base or 1224th base of 16S rDNA (SEQ ID NO: 4), 167th base, 182nd base, 195th base, 202th base, 16th base of 16S rDNA (SEQ ID NO: 5) of Phytoplasma pruni 230 bases, 448 bases, 461 bases, 462 bases, 823 bases, 828 bases, 975 bases, 975 bases, 1017 bases, 1218 bases, 1221 bases, 1267 bases, 1270 bases, 1418 bases , 1437 base or 1462 base, 16S rDNA of Phytoplasma olantifolia ( 429th base, 441th base, 442th base, 443th base, 450th base, 453th base, 454th base, 458th base, 459th base, 464th base, 473th base, 572, 575, 582, 583, 588, 593, 594, 613, 619, 627, 629, 630, 650 , 718 base, 945 base, 972 base, 985 base, 986 base, 986 base, 990 base, 996 base, 1014 base, 1080 base, 1174 base, 1226 base, 1240 base, 1st base 1248 bases, 1256 bases or 1258 bases, 186 base, 206 bases, 554 bases, 557 bases, 557 bases, 622 bases, 761 bases, 982 bases of 16S rDNA of Phytoplasma vitis (SEQ ID NO: 7) Base, 1019 base, 1028 base, 1080 base, 1084 base, 1104 base, 1104 base, 1159 base, 1205 base, 1207 base, 1217 base, 1250 base, 1337 base, 1359 base, 1430 salt 1st base, 1443 base, 63rd base, 124th base, 125th base, 151st base, 152nd base, 230th base, 230th base, 246th base, 260th base of 16S rDNA (SEQ ID NO: 8) of Phytoplasma castaneae , 276 base, 461 base, 462 base, 485 base, 577 base, 585 base, 585 base, 595 base, 596 base, 620 base, 629 base, 882 base, 905 base, 907 bases, 987 bases, 996 bases, 1122 bases, 1142 bases, 1142 bases, 1155 bases, 1220 bases, 1328 bases, 1399 bases, 1400 bases, 1401 bases, 1424 bases, 1429 bases , 1431 base or 1452 base, and 336, 377, 414, 445, 573, 590, 590, and 803 bases of 16S rDNA (SEQ ID NO: 9) of Phytoplasma forenisium , 804 base, 964 base, 983 base, 996 base, 1083 base, 1083 base, 1098 base, 1216 base, 1217 base or 1380 base And the 126th base, the 136th base, the 137th base, the 189th base, the 190th base, the 192nd base, the 193rd base, the 205th base, the 209th base, the second base of SEQ ID NO: 3 for identifying the 16SrX group 219 bases, 289 bases, 308 bases, 438 bases, 439 bases, 439 bases, 453 bases, 453 bases, 500 bases, 513 bases, 534 bases, 584 bases, 603 bases, 604 bases , 696 base, 701 base, 807 base, 818 base, 818 base, 821 base, 1049 base, 1093 base, 1097 base, 1100 base, 1114 base, 1241 base, 1298 base, 1st base 1331 bases, the first 1398 bases, Ru is selected from the group consisting of the 1413 base or a 1416 base, a kit for simultaneously detecting two or more Fight plasma. The four oligomers are
A first oligomer comprising a sequence complementary to the 5 ′ region of the SNP site in the (+) strand;
A second oligomer consisting of a sequence complementary to the 3 'region of the SNP site in the (+) strand;
A third oligomer comprising a sequence complementary to the 3 ′ region of the SNP site in the (−) strand; and a third sequence comprising a sequence complementary to the 5 ′ region of the SNP site in the (−) strand. Four oligomers,
Either one of the first oligomer and the second oligomer includes a base of the SNP site, and one of the third oligomer and the fourth oligomer includes a base of the SNP site,
The kit according to claim 6 . The kit according to claim 6 or 7 , wherein the detection target sample is a plant-derived DNA sample. The kit according to any one of claims 6 to 8 , further comprising a thermostable DNA ligase.