JP6292659B2 - Method for detecting bovine mastitis-causing microorganism, primer set and assay kit used therefor - Google Patents

Description

  Embodiments of the present invention relate to a method for detecting a bovine mastitis-causing microorganism, a primer set used in the method, and an assay kit.

  Bovine mastitis is a general term for infectious inflammation that occurs in bovine mammary tissue. The direct cause is bacterial and fungal infection. Bovine mastitis is broadly divided into clinical and latent mastitis. Clinical mastitis is accompanied by clinical symptoms. Individuals suffering from clinical mastitis have gross abnormalities such as red and swollen breasts, pain, and / or mixed milk. Individuals with latent mastitis do not have the gross abnormalities seen with clinical mastitis. In individuals with latent mastitis, inflammation in the mammary gland is only revealed after a milk test.

  When dairy cows develop mastitis, dairymen not only incur costs for treatment, but also reduce milk production and quality, renewal costs for strawberries, as well as reduced shipments due to restrictions on shipping and raw milk. It suffers various economic losses due to disposal. According to a trial calculation for domestic clinical mastitis, the loss per animal is 50,000 to 80,000 yen per year, an average of about 1.5 million yen per farm, and 80 billion yen for Japan as a whole. It is said to go up.

  In order to minimize the economic loss due to the spread of infection, it is important to quickly identify all infected individuals, including potentially infected individuals who do not show symptoms, and take appropriate measures.

JP 2002-186871 A JP 2012-60925 A JP 2005-95043 A

  The problem to be solved by the present invention is to provide a method for specifically detecting a bovine mastitis-causing microorganism, a primer set and an assay kit used therefor.

  In order to solve the above-mentioned problems, a method for detecting a bovine mastitis-causing microorganism is performed by amplifying a nucleic acid in a sample by the LAMP method using a specific LAMP amplification primer set, and obtaining the obtained amplification product. Reacting with a specific nucleic acid probe set and determining the presence and / or amount of bovine mastitis-causing microorganism in the sample based on the obtained hybridization signal.

The figure which shows the relationship between the template sequence which should be amplified by the LAMP method according to this embodiment, and a primer. The figure which shows the structure of the amplification product obtained by amplification by the LAMP method according to this embodiment. The schematic diagram which shows an example of the amplification product according to this embodiment. The schematic diagram of the nucleic acid probe fixed base | substrate according to this embodiment. The schematic diagram of the nucleic acid probe fixed base | substrate according to this embodiment.

  Hereinafter, embodiments will be described in detail.

  According to the embodiment, it is possible to specifically detect bovine mastitis-causing microorganisms. First, in order to specifically detect bovine mastitis-causing microorganisms, the following bacteria, mycoplasma, algae and yeast-like fungi may be detected and / or quantified. By specifically detecting these bovine breast-causing microorganisms, it is possible to quickly identify the presence of mastitis in an animal individual.

1. Bovine mastitis-causing microorganism <Staphylococcus> (genus Staphylococcus)
Among staphylococci, Staphyrococcus aureus (SA) is an infectious fungus. Mastitis derived from this bacterium is intractable and has few symptoms in the early stages of infection. Therefore, early detection is difficult, and effective elimination by antibiotic administration cannot be expected. Staphylococcus other than Staphylococcus aureus is called Coagulase-negative staphylococcus (CNS) in the sense that it does not produce coagulase that coagulates plasma, and is an environmental bacterium that is resident in the skin. It has been regarded as a secondary mastitis-causing bacterium so far, but since many cows causing intramammary infections are seen from the covering material, it is a bacteria that cannot be ignored from the viewpoint of strict mastitis control. .

<Streptococcus> (Streptococcus)
Among the streptococci that cause mastitis, the most malignant species is Streptococcus agalactiae (SAG). SAG is so prominent that it can be distinguished from Other Streptococcus (OS) as a whole. SAG is also called non-milky streptococci due to its clinical symptoms. Mastitis caused by this bacterium is characterized by high infectivity, and the number of somatic cells in milk, which indicates the degree of inflammation, remains high for a long time from the beginning of infection. However, the number is gradually decreasing due to the presence of effective ointments that are highly sensitive to antibiotics and used during the dry period. Other than SAG, streptococci as a mastitis-causing microorganism is streptococcus, which is more likely to recur than other streptococci and causes chronic mastitis.

<Corynebacterium genus> (Coprynebacterium genus)
It is a kind of actinomycetes and is a resident bacteria distributed in various places in the human living environment. Causes mastitis that occurs mainly in summer. As a causative bacterium of mastitis, it is contagious and / or environmental, and is transmitted by flies and the like.

<Enterobacteriaceae (E. coli group)> (Enterobacteria family)
Enterobacteriaceae are generally classified as Enterobacteriaceae or coliforms. It is an environmental causative bacterium that exists in the environment such as bovine body surface, feces, and floor coverings, and invades and infects through the mouth of the nipple. It is a fungus that increases when the temperature rises. Individuals infected with this fungus may become latent types that do not exhibit symptoms, may heal spontaneously, or may worsen. When worsened, it can cause acute mastitis with systemic symptoms, which can result in death or disuse. It is said that the cause of the deterioration is a toxin excreted when the bacteria of the Enterobacteriaceae family die.

<Mycoplasma genus> (Mycoplasma genus)
Mycoplasma is not a bacterium or virus, but a medium-sized microorganism. It is difficult to find by ordinary culture tests, and antibiotics are ineffective. Mycoplasmal mastitis is very infectious and has significant economic damage. Infection may not only enter through the mouth of the nipple like other bacteria, but may also infect the mammary gland from the bovine body (eg, from pneumonia) via blood.

<Algae>
Among the algae, the main cause of bovine mastitis is Prototheca zopfii. Prototheca's cell wall has a three-layer structure and has strong hydrolysis resistance. It has strong drug resistance thanks to its strong cell wall. Prototheca mastitis occurs sporadically within a herd of animals, and often results in multiple quarters of infection and is often culled.

<Yeast-like fungus> (genus Candida)
Candida is a family of yeast-like fungi that are insensitive to antibiotics and cause refractory mastitis. It has been reported that it is related to fungal change and opportunistic infections. It is considered that the development of mastitis due to this bacterium is influenced by the environment in which immunity decreases, such as the rearing environment in summer and the stress of milking.

2. Method for detecting bovine mastitis-causing microorganisms A method for specifically detecting these bovine mastitis-causing microorganisms involves amplifying nucleic acids in a sample by a LAMP method using a specific primer set for LAMP amplification. Reacting the obtained amplification product with a specific nucleic acid probe set and determining the presence of a bovine mastitis-causing microorganism in the sample based on the obtained hybridization signal.

  Samples are mammals or mammals other than humans, e.g. cows, goats, mice, rats, guinea pigs, hamsters, ferrets, rhesus monkeys, rabbits, dogs, cats, cows, pigs, sheep, seals and porpoises such as porpoises, bodily fluids, It may be a tissue, cell or stool. Alternatively, the sample may be an environmental substance, for example, any substance present in the environment such as water, lake water, river water, sea water, and soil, for example, a living environment substance of an animal individual that is a source of the sample. Alternatively, the sample may be a substance suspected of containing a microorganism classified as a bovine mastitis-causing microorganism or a gene thereof. For example, a culture medium may be used. Such a substance may be pretreated by means known per se into a state suitable for nucleic acid amplification, and any pretreatment may not be performed. Examples of such pretreatments may include homogenate, denaturation, filtration, nucleic acid extraction, impurity removal and / or purification and the like.

  Also, based on the results of detection of bovine mastitis-causing microorganisms on the sample, an animal individual that is the source of the sample, for example, cow, goat, mouse, rat, guinea pig, hamster, ferret, rhesus, rabbit, dog, cat, Cattle, pigs, sheep, seals, and porpoises are more or less likely to have mastitis, more or less likely to have mastitis, mastitis, good prognosis It is possible to determine whether or not.

3. LAMP amplification The “LAMP method” is a method for amplifying nucleic acids. These are also referred to as isothermal gene amplification methods. When a nucleic acid is amplified using DNA as a template by the LAMP method, the LAMP method uses two types of primers or primers corresponding to four regions of the template nucleic acid, or four types of primers or primers corresponding to the six regions of the template nucleic acid. Is used.

  Here, the relationship between the template nucleic acid and the primer in amplification by the LAMP method will be described with reference to FIG. Amplification by the LAMP method can be started from either single-stranded DNA or double-stranded DNA. However, as an example, FIG. 1 illustrates a case where amplification is started from double-stranded DNA.

  The double-stranded DNA 1 to be amplified includes a first template nucleic acid 2 and a second template nucleic acid 3 that are complementary to each other. The first template nucleic acid 2 has an F3c region, an F2c region, and an F1c region from the 3 'side, and has a B3 region, a B2 region, and a B1 region from the 5' side. The second template nucleic acid 3 has an F3 region, an F2 region, and an F1 region from the 5 'side, and has a B3c region, a B2c region, and a B1c region from the 3' side. “F1 region” and “F1c region”, their sequences “F1 sequence” and “F1c sequence”, “F2 region” and “F2c region”, their sequences “F2 sequence” and “F2c sequence”, “F3 region” and “F3c region”, their sequences “F3 sequence” and “F3c sequence”, “B1 region” and “B1c region”, their sequences “B1 sequence” and “B1c sequence”, “B2 region” and “B2c region”, their sequences “B2 sequence” and “B2c sequence”, “B3 region” and “B3c region”, and their sequences “B3 sequence” and “B3c sequence” These combinations are complementary to each other.

  When this double-stranded DNA 1 is amplified by the LAMP method, a primer set consisting of four primers, ie, FIP primer 4, F3 primer 5, BIP primer 6 and B3 primer is basically used.

  FIP primer 4 has an F1c sequence on the 5 'side and an F2 sequence on the 3' side. F3 primer 5 has an F3 sequence. BIP primer 6 has a B1c sequence on the 5 'side and a B2 sequence on the 3' side. The B3 primer has a B3 sequence.

  When the double-stranded DNA 1 is amplified by the LAMP method using the primer set, from the first template nucleic acid 2 and the second template nucleic acid 3, for example, as shown in FIG. 2 (1) and (2), respectively. Thus, a dumbbell-shaped first amplification product 8 and second amplification product 9 having a stem-and-loop structure are obtained.

  The amplification product 8 has an F1 sequence, F2c sequence, F1c sequence, B1 sequence, B2 sequence and B1c sequence in this order from the 3 'or 5' end. The F1 and F1c sequences, and the B1 and B1c sequences are linked to each other. The portion sandwiched between the F1 sequence and the F1c sequence and including the F2c sequence is single-stranded. The portion sandwiched between the B1 sequence and the B1c sequence and including the B2 sequence is single-stranded. These single-stranded portions are called loop portions because of their shapes. On the other hand, the portion sandwiched between the F1c sequence and the B1 sequence is also single-stranded.

  The amplification product 9 has F1c sequence, F2 sequence, F1 sequence, B1c sequence, B2c sequence, and B1 sequence in this order from the 3 'or 5' end. The F1c and F1 sequences, and the B1 and B1c sequences are linked to each other. The portion sandwiched between the F1c sequence and the F1 sequence and including the F2 sequence is single-stranded. The portion sandwiched between the B1 sequence and the B1c sequence and including the B2c sequence is single-stranded. These single-stranded portions are called loop portions because of their shapes. On the other hand, the portion sandwiched between the F1 sequence and the B1c sequence is also single-stranded.

  When amplification by the LAMP method is performed, an amplification product that repeatedly includes the first amplification product 8 and / or the second amplification product 9 as one unit, and / or the first amplification product 8 and / or the second amplification product, respectively. An amplification product comprising the amplification product 9, an amplification product comprising a portion of the first amplification product 8 and / or the second amplification product 9 comprising at least one loop portion is obtained. The amplification mechanism of amplification by the LAMP method is described in, for example, JP-A-2002-186871.

  The primer set may further include a loop primer in addition to the four types of primers described above. The loop primer may be an LF primer and / or an LB primer. The LF primer is complementary to the region sandwiched between the F1c region and F2c region of the first amplification product 8, or complementary to the region sandwiched between the F1 region and F2 region of the second amplification product 9. It is sufficient to have a simple arrangement. The LB primer is complementary to the region sandwiched between the B1 region and B2 region of the first amplification product 8, or complementary to the region sandwiched between the B1c region and B2c region of the second amplification product 9. It suffices to have a sequence complementary to a simple sequence.

  For example, a LAMP amplification primer set for specifically amplifying mastitis-causing microorganism nucleic acid includes an FIP primer, a BIP primer, an F3 primer, and a B3 primer. The LAMP amplification primer set may further optionally include an LF primer and / or an LB primer.

  For example, the amplification reaction may be performed with one primer set per reaction field. Alternatively, in one reaction field, a plurality of primer sets corresponding to various genotypes may be used. When it is necessary to identify a plurality of mastitis-causing microorganism species, the latter method is more efficient. An “amplification reaction field” is a field in which a reaction is performed. For example, an amplification reaction field is an interior of a container capable of performing a reaction therein, an upper part of a substrate capable of performing a reaction thereon, What is necessary is just the partial area inside the flow path which performs reaction inside. Examples of the container may include, but are not limited to, a tube, a microtube, a beaker, a multiwell plate, a substrate having a reaction channel, and the like.

  In order to comprehensively amplify mastitis-causing microbial nucleic acid, it is more preferable to use a plurality of types of FIP primer, BIP primer, F3 plummer and B3 primer, respectively. In order to amplify a specific species of mastitis-causing microbial nucleic acid, a sequence having nucleotides specific to a specific species may be used. In order to more reliably and comprehensively amplify, it is preferable to design and use primers so that they are universal nucleotides or mixed bases at least for species-specific mutation sites.

  A universal nucleotide is also called a universal base, and is a nucleotide capable of forming a base pair without distinguishing four kinds of natural bases. The universal nucleotide that can be used may be any nucleotide known per se that forms a base pair without distinguishing the four natural bases.

  Examples of universal nucleotides include, but are not limited to, deoxyinosine (dI), Glen Research's 3-nitropyrrole, 5-nitroindole, deoxyribonosyl ( deoxyribofuranosyl; dP), deoxy-5'-dimethoxytrityl-D-ribofuranosyl (dK).

  “Mixed base” refers to the use of a mixture of two or more nucleic acid probes designed as “adenine”, “thymine”, “cytosine”, and “guanine”, respectively, at the desired bases to be mixed bases. Say. Moreover, you may design so that it may substitute with the modified base which can be paired with respect to multiple types of bases. The mixed base may be used as a mixed base for a base at one position in one reaction field, or may be used as a mixed base corresponding to all of the bases at a plurality of positions. For the indication of the mixed base, the base present at the site in question may be indicated using, for example, the following symbols that represent multiple types of bases for the mutation site simultaneously: guanine or adenine “ "r", "y" for thymine or cytosine, "m" for adenine or cytosine, "k" for guanine or thymine, "s" for guanine or cytosine, "w" for adenine or thymine, guanine or “B” for cytosine or thymine, “d” for adenine or guanine or thymine, “h” for adenine or cytosine or thymine, “v” for adenine or guanine or cytosine, adenine or guanine or cytosine or thymine "N" to represent.

  Between each region included in one primer or at the end of the primer, a sequence of about 1 to 100 nucleotides, preferably about 2 to 30 nucleotides may be further included as a spacer, for example. Further, the specific sequence of such a sequence may be a sequence that is not identical to or complementary to the template nucleic acid and each primer sequence, and may be a sequence generally used as a spacer, for example. .

  The length of the loop may be about 30 to 200 nucleotides, preferably 35 to 100 nucleotides, more preferably 43 to 60 nucleotides.

The amplification reaction is not limited to this. For example, the amplification reaction may be performed in a buffer having the following composition:
Reaction solution composition of LAMP method 20 mM Tris-HCL (pH 8.8)
10 mM KCL
8 mM MgSO ₄
10 mM (NH ₄ ) ₂ SO ₄
0.1% Tween20
0.8M Betaine
1.4 mM dNTPs
16U Bst DNA polymerase.

When the reaction is carried out with a reaction solution having such a composition in a total volume of 25 μL, each primer may be brought in at the following concentration;
Primer addition amount FIP primer 40pmoL
BIP primer 40pmoL
F3 primer 5pmoL
B3 primer 5pmoL
If necessary,
LF primer 20pmoL
LB primer 20 pmoL.

  The LAMP method is superior in amplification efficiency and less susceptible to impurities in the sample than the method using PCR.

4). Detection of amplification product By detecting and / or quantifying the amplification product obtained by amplifying a nucleic acid in a sample by the LAPM method as described above, a bovine mastitis-causing microorganism can be specifically detected.

  The detection and / or quantification of the amplification product may be performed by, for example, detecting hybridization with a nucleic acid probe, measuring turbidity, measuring absorbance, or electrophoresis. Preferably, the amplification product may be detected by detecting specific hybridization with the nucleic acid probe. “Specific hybridization” means that the amplification product and the nucleic acid probe hybridize without causing cross-hybridization. Preferably, specific hybridization can detect even slight differences in the presence of single nucleotide polymorphisms (Single Nucleotide PolymorpHisms: SNPs) or mutations.

  One example for amplifying a nucleic acid in a sample by the LAMP method and then detecting the obtained amplification product with a nucleic acid probe set will be described below. Since the amplification product is obtained by specific amplification of the bovine mastitis-causing microorganism, it is possible to specifically detect bovine mastitis-causing microorganism in the sample by specifically detecting the amplification product. Is possible.

  The amplification product 8 and / or the amplification product 9 may be detected using a nucleic acid probe containing a sequence complementary to the target sequence contained therein. That is, the target sequence is a sequence to be hybridized with the nucleic acid probe. The position and sequence of the target sequence in the amplification products 8 and 9 may be determined when designing the primer set.

  For example, the target sequence may be designed such that the target sequence is located in a single stranded portion of the amplification product 8 and / or amplification product 9, preferably in the loop portion. That is, the target sequence is the sequence of the region sandwiched between the F1c region and the B1 region of the amplification product 8 of FIG. 2 (1), the sequence of the loop portion sandwiched between the F1c region and the F1 region, and / or the B1 region. And a sequence of a loop part sandwiched between the B1c region and a part of these sequences. Similarly, the target sequence is the sequence of the region sandwiched between the F1 region and the B1c region of the amplification product 9 in FIG. 2 (2), the sequence of the loop portion sandwiched between the F1 region and the F1c region, and / or B1c. It may be a sequence of a loop portion sandwiched between the region and the B1 region, or a sequence of a part of these sequences. Preferred target sequences include a portion of the loop portion sequence. The region selected as the target sequence may be at least one single-stranded portion, preferably a loop portion.

  One example of the target sequence to be designed for the template nucleic acid sequence will be described with reference to FIG. The target sequence may be the sequence of the FP region, the FPc region, the BP region, or the BPc region shown in FIG. 1, or a partial sequence thereof. For the first template nucleic acid 2, the target sequence may be a region sandwiched between the F1c region and the F2c region, and a sequence of a part of the F2c region or F2c region in the region sandwiched between the F1c region and the F2c region. It may be a region to which is added. Alternatively, the target array may be a region sandwiched between the B1 region and the B2 region, or a region obtained by adding a part of the B2 region or the B2 region to the region sandwiched between the B1 region and the B2 region. Good. For the second template nucleic acid 3, the target sequence may be a region sandwiched between the F1 region and the F2 region, and a part of the F2 region or F2 region is added to the region sandwiched between the F1 region and the F2 region. It may be an area. Alternatively, the target sequence may be a region sandwiched between the B1c region and the B2c region, and is a region obtained by adding a partial sequence of the B2c region or the B2c region to the region sandwiched between the B1c region and the B2c region. May be.

  An amplification product that can be obtained when the primer set is set so that the target sequence is included in the loop portion and a nucleic acid probe containing the target sequence may be hybridized in the reaction field. By detecting the resulting hybridization signal, the presence or absence of the amplified product can be determined. Based on the result, bovine mastitis microorganisms in the sample can be detected.

  The target sequence FP region, FPc region, BP region, and BPc region are respectively in a region sandwiched between F1 region and F2 region, in a region sandwiched between F1c region and F2c region, B1 region and B2 region, FIG. 3 shows an example of the loop portion of the amplification product obtained when designed in the region sandwiched between and the region sandwiched between the B1c region and the B2c region.

  The loop portion shown in FIG. 3A includes an F1c region, an FPc region, an F2c region, and an F1 region in this order from the 5 'end toward the 3' end. The F1c region and the F1 region are complementary to each other and are bonded to each other to form a loop portion. The FPc region is the target sequence. For detection of an amplification product having this loop portion, a nucleic acid probe containing an FP sequence that is a sequence complementary to the FPc sequence of the FPc region is used. The nucleic acid probe hybridizes to the FPc region. The loop portion shown in FIG. 3 (2) includes the F1c region, the F2 region, the FP region, and the F1 region in this order from the 5 'end toward the 3' end. The amplification product containing this is detected by a nucleic acid probe complementary to the FP sequence of the FP region that is the target sequence. The loop portion shown in FIG. 3 (3) includes a B1c region, a BPC region, a B2c region, and a B1 region in this order from the 5 'end toward the 3' end. The amplification product containing this is detected by a nucleic acid probe complementary to the BPc sequence of the BPc region that is the target sequence. The loop portion shown in FIG. 3 (4) includes the B1c region, the B2 region, the BP region, and the B1 region in this order from the 5 'end toward the 3' end. An amplification product containing this is detected by a nucleic acid probe complementary to the BP sequence of the BP region that is the target sequence. Since the loop portion in the amplification product is always maintained in a single-stranded state, it is possible to easily bind the nucleic acid probe to each different target sequence subjected to the denaturation operation.

  The nucleic acid probe may contain additional sequences in addition to the target sequence. The further sequence may be a sequence used as a spacer as necessary, for example, when a nucleic acid probe is immobilized. The nucleic acid probe may also contain other labeling substances that make it detectable.

  The target sequence preferably has a length of about 15 to about 30 bases or about 31 to about 80 bases, and may have a length that does not inhibit the efficiency of the LAMP reaction. The nucleic acid probe preferably has a length of about 10 to about 60 bases or about 12 to about 40 bases.

  It may be a nucleic acid probe for comprehensively detecting nucleic acids derived from mastitis-causing microorganisms, or a nucleic acid probe for specifically detecting a desired species.

  The structure of the nucleic acid probe is not particularly limited, and DNA, RNA, PNA, LNA, methylphosphonate skeleton nucleic acid, and / or other artificial nucleic acid strands can be used. In addition, a chimeric nucleic acid of any of these nucleic acids may be used.

  The nucleic acid probe or nucleic acid probe group may contain a mutated base and / or a mutated sequence as long as the nucleic acid derived from the mastitis-causing microorganism can be specifically detected. “As long as it can be specifically detected” means that a mutation may exist in a site or sequence that is not necessarily required to be identified taxonomically as a mastitis-causing microorganism. For example, in any of the nucleic acid probes described above, one or several bases at any position in the sequence represented by the above SEQ ID NO may be substituted, deleted or added. The nucleic acid probe may vary depending on the genetic polymorphism or mutation held by the strain and / or species of the mastitis-causing microorganism to be detected, and any position of the sequence represented by the above SEQ ID NO: It may be a sequence in which one or several bases are substituted, deleted or added at the site. Amplification products may be detected by simultaneously presenting a plurality of types of nucleic acid probes containing such mutation sites and having different bases at the mutation sites in one reaction field. In that case, 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by the sequence described in any of the SEQ ID NOs specifically described below or a complementary sequence thereof are mixed. It may be prepared as a nucleotide, and 1 to 5, preferably 1 to several nucleotides at any position may be universal nucleotides. Alternatively, it is possible to use a group of nucleic acid probes designed with mixed bases composed of a plurality of nucleic acid probes each containing a base that may be present at the mutation site and having the same sequence as the sequence other than the mutation site. Good.

  The nucleic acid probe may be immobilized on a solid phase, for example, a substrate surface, or may be used in a liquid phase.

  For example, when the nucleic acid probe is used while being immobilized on a substrate, it may be typically used as a nucleic acid probe immobilized on a DNA chip, or may be used as a nucleic acid probe constituting another microarray. . The nucleic acid probe for detecting the amplification product may be changed according to the genetic polymorphism or mutation held by the microorganism strain or species to be detected.

  Any of the nucleic acid probes described above may be used alone or in multiple types in one reaction field. In addition, when the nucleic acid probe is used by being immobilized on the surface of the substrate, one type may be used alone or a plurality of types may be used together in one region for obtaining one signal on the substrate. May be. When used in multiple types, all nucleic acid probes having two, two or more, eg, three, four, or desired sequences may be used together to obtain one signal. Further, any one or more bases constituting these sequences may be designed to be a mixed base.

  Means for detecting hybridization between the nucleic acid probe and the amplification product are not particularly limited to these, but turbidity, visible light, fluorescence, chemiluminescence, electrochemiluminescence, chemiluminescence, fluorescence energy transfer method An optical method such as ESR, or electrical properties such as current, voltage, frequency, conductivity, and resistance may be used.

  A nucleic acid probe-immobilized chip can be used as a means for detecting hybridization between the nucleic acid probe and the amplification product. The nucleic acid probe immobilization chip is an apparatus in which the above-described nucleic acid probe is immobilized on a substrate that is a solid phase. This is generally called a microarray or a DNA chip.

  A non-limiting example of the nucleic acid probe-immobilized chip is shown in FIG. 4 as a schematic diagram. The nucleic acid probe immobilization chip 41 includes an immobilization region 43 on a base 42. The nucleic acid probe 44 is immobilized on the immobilization region 43. Such a nucleic acid probe immobilization chip 41 can be manufactured by a method well known in the art. A person skilled in the art can appropriately change the design of the number and arrangement of the immobilization regions 43 arranged on the base body 42 as necessary. Such a nucleic acid probe-immobilized chip may be suitably used for a detection method using fluorescence. In one immobilization region 43, one type or one or more types of nucleic acid probes or nucleic acid probe sets necessary for obtaining information that needs to be obtained as one independent signal are immobilized.

  A further non-limiting example of a nucleic acid probe immobilization chip is shown in FIG. The nucleic acid probe immobilization chip 51 of FIG. 5 includes an electrode 53 as an immobilization region on a base 52. The nucleic acid probe 54 is immobilized on the electrode 53. The electrode 53 is connected to the pad 55. Electrical information from the electrode 53 is acquired via the pad 55. Such a nucleic acid probe immobilization chip 51 can be manufactured by a method well known in the art. The number of electrodes 53 arranged on the base 52 and the arrangement thereof can be appropriately changed by those skilled in the art as needed. Furthermore, the nucleic acid probe immobilization chip 51 of this example may include a reference electrode and a counter electrode as necessary. One kind or one or more kinds of nucleic acid probes or nucleic acid probe sets necessary for obtaining information necessary to be obtained as one independent signal are immobilized in one immobilization region.

  When the nucleic acid probe is immobilized on a solid phase, an amino group, a thiol group, or a functional group such as biotin may be introduced at the end of the nucleic acid probe described above. Furthermore, a spacer may be introduced between the functional group and the nucleotide. Although the kind of spacer used here is not specifically limited, For example, you may use alkane frame | skeleton, ethylene glycol frame | skeleton, etc. Moreover, a part of the bases of the nucleic acid probe may be substituted with universal nucleotides. Examples of universal nucleotides that can be used include deoxyinosine (dI), Gren Research 3-Nitropyrrole, 5-Nitroindole, deoxyribofuranosyl (dP), deoxy-5'-dimethoxytrityl-D-ribofuranosyl (dK), and the like.

  The substrate for immobilizing the nucleic acid probe is not particularly limited, and may be a resin bead, a magnetic bead, a metal fine particle, a microtiter pout, a glass substrate, a silicon substrate, a resin substrate, an electrode substrate, or the like. .

5. Primer Set and Nucleic Acid Probe LAMP amplification primer set for specifically amplifying bovine mastitis-causing microorganisms includes Staphylococcus genus, Streptococcus genus, Corynebacterium genus (Coprynebacterium genus), It includes primer sets that can specifically amplify Enterobacteriaceae (Enterobacteriaceae), Mycoplasma (Mycoplasma), algae, and yeast-like fungi (Candida). Further, the LAMP amplification primer set may include a primer set for specifying a specific species of the microorganism.

(1) <Staphylococcus genus> (genus Staphylococcus)
S. hominis, S. simulans, S.equorum, and S. intermedius were selected as staphylococci that cause bovine mastitis because they are frequently detected in the mammary glands of individuals with bovine mastitis. . By detecting these microorganisms, staphylococci as bovine mastitis-causing microorganisms can be specifically detected.

(1-1) Staphylococcus genus
The primer set for specifically amplifying Staphylococcus is a primer comprising the polynucleotides represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively. May be a first primer set. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 are sequences for the F3 primer, B3 primer, FIP primer, BIP primer and LB primer, respectively. The F3 primer, B3 primer, FIP primer, BIP primer, and LB primer included in the preferred first primer set may be composed of the polynucleotides indicated by the respective SEQ ID NOs. The first primer set is particularly excellent in specifically amplifying staphylococcal microorganisms.

  Nucleic acid probes for specifically detecting an amplification product obtained by amplification using the first primer set are nucleic acids each comprising a polynucleotide represented by SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 15, respectively. It may be a first nucleic acid probe group including probes. Each nucleic acid probe included in the preferred first nucleic acid probe group may consist of the polynucleotides respectively represented by SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 15. The first nucleic acid probe group is particularly excellent in comprehensive and specific detection of staphylococcal microorganisms.

(2) <Streptococcus> (Enterococcus, Streptococcus, S.agalactiae, S. ubelis)
Since it is a bacterium frequently found in the mammary gland of individuals suffering from bovine mastitis, Streptococcus and Enterococcus were selected as streptococci causing bovine mastitis. Furthermore, S.agalactiae and S.ubelis were respectively selected as the bacterial species having particularly strong pathogenicity. By detecting these microorganisms, streptococci as bovine mastitis-causing microorganisms can be specifically detected, and the symptoms and prognosis of bovine mastitis can be predicted.

  Primer sets for specifically amplifying Streptococcus and specific Streptococcus include the following second primer set, third primer set, fourth primer set and / or fifth primer set: May include.

(2-1) Enterococcus genus
The primer set for specifically amplifying Enterococcus genus comprises a second primer comprising a polynucleotide respectively represented by SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. It may be a primer set. SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 are sequences for the F3 primer, FIP primer, BIP primer, B3 primer and LB primer, respectively. The F3 primer, FIP primer, BIP primer, B3 primer and LB primer included in the preferred second primer set are composed of the polynucleotides indicated by the respective SEQ ID NOs. The second primer set is particularly excellent for specifically amplifying Enterococcus microorganisms.

(2-2) Streptococcus genus
Primer sets for specifically amplifying Streptococcus are primers comprising the polynucleotides respectively represented by SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51. A third primer set may be included. SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51 are respectively F3 primer, first FIP primer, second FIP primer, BIP primer, B3 primer, and LBc primer. Is an array for Preferably, the F3 primer, the first FIP primer, the second FIP primer, the BIP primer, the B3 primer, and the LBc primer included in the third primer set are composed of polynucleotides represented by the respective sequence numbers. Good.

(2-3) S. agalactiae
A primer set for specifically amplifying S. agalactiae is a fourth set comprising primers each comprising a polynucleotide respectively represented by SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69. It may be a primer set. SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69 are sequences for the F3 primer, B3 primer, FIP primer, BIP primer, and LF primer, respectively. Preferably, the F3 primer, the B3 primer, the FIP primer, the BIP primer, and the LF primer included in the fourth primer set may be composed of the polynucleotides represented by the respective sequence numbers.

(2-4) S. uberis
A primer set for specifically amplifying S. uberis is a fifth set comprising primers each comprising a polynucleotide respectively represented by SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84. It may be a primer set. SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84 are sequences for the F3 primer, B3 primer, FIP primer, BIP primer, and LF primer, respectively. Preferably, the F3 primer, the B3 primer, the FIP primer, the BIP primer, and the LF primer included in the fifth primer set may be composed of the polynucleotides represented by the respective sequence numbers.

(2-5) Nucleic acid probe The nucleic acid probe for specifically detecting the amplification product obtained by amplification using the second primer set is a polynucleotide represented by SEQ ID NO: 92 and SEQ ID NO: 95, respectively. The nucleic acid probe may be included. Preferred nucleic acid probes The nucleic acid probes may consist of the polynucleotides respectively represented by SEQ ID NO: 92 and SEQ ID NO: 95.

  A nucleic acid probe for specifically detecting an amplification product obtained by amplification using the third primer set includes a nucleic acid probe that contains the polynucleotides represented by SEQ ID NO: 99 and SEQ ID NO: 101, respectively. Good. Preferred nucleic acid probes may consist of the polynucleotides respectively shown by SEQ ID NO: 99 and SEQ ID NO: 101.

  The nucleic acid probe for specifically detecting the amplification product obtained by the amplification using the fourth primer set may include a nucleic acid probe containing the polynucleotide represented by SEQ ID NO: 106. A preferred nucleic acid probe may consist of the polynucleotide shown by SEQ ID NO: 106.

The nucleic acid probe for specifically detecting the amplification product obtained by amplification using the fifth primer set may include a nucleic acid probe containing the polynucleotide represented by SEQ ID NO: 110. A preferred nucleic acid probe may consist of the polynucleotide represented by SEQ ID NO: 110 .

  In addition, the nucleic acid probe group used for detecting Streptococcus microorganisms includes polynucleotides represented by SEQ ID NO: 92, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 106, and SEQ ID NO: 110, respectively. It may be a second nucleic acid probe group including the nucleic acid probes included in each. Each nucleic acid probe included in the preferred second nucleic acid probe group may consist of the polynucleotides respectively represented by SEQ ID NO: 92, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 106, and SEQ ID NO: 110. . The second nucleic acid probe group is particularly excellent in specifically detecting staphylococcal microorganisms. Of the second group of nucleic acid probes, the nucleic acid probes each comprising the polynucleotides represented by SEQ ID NO: 92, SEQ ID NO: 95, SEQ ID NO: 99, and SEQ ID NO: 101 are comprehensive and specific for Streptococcus microorganisms. Especially good for detecting. In addition, a nucleic acid probe that contains the polynucleotides represented by SEQ ID NO: 106 and SEQ ID NO: 110, respectively, is a particularly useful nucleic acid probe for predicting the prognosis of mastitis. If a detection signal is detected for a nucleic acid probe comprising a polynucleotide represented by SEQ ID NO: 106 and / or a nucleic acid probe comprising a polynucleotide represented by SEQ ID NO: 110, mastitis in the individual that is the source of the sample is, for example, Can be determined to be severe, difficult to treat, and / or good or poor prognosis.

(3) <Enterobacteriaceae (E. coli group)> (Enterobacteriaceae, E. coli, Klebsiella)
Since it is a fungus frequently detected in the mammary gland of individuals with bovine mastitis, E. cloacae, E. fergusonii, E. vulneris, E. coli was selected. By detecting these microorganisms, it is possible to specifically detect microorganisms of the family Enterobacteriaceae as microorganisms causing bovine mastitis. In addition, E. coli and Klebsiella were selected because they are frequently detected in the mammary glands of individuals with bovine mastitis. By specifically detecting E. coli and Klebsiella, it is possible to predict individuals to be followed up with particular attention among coliform group positive individuals.

(3-1) Enterobacteria family
A primer set for amplifying Enterobacteriaceae microorganisms is a sixth primer including primers each containing a polynucleotide respectively represented by SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 132 It can be a set. SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 132 are sequences for the F3 primer, B3 primer, FIP primer, BIP primer, and LB primer, respectively. The F3 primer, the B3 primer, the FIP primer, the BIP primer, and the LB primer included in the preferred sixth primer set may be composed of the polynucleotides represented by the respective sequence numbers. The sixth primer set is particularly excellent in specifically amplifying microorganisms of the family Enterobacteriaceae.

(3-2) E.coli
Primer sets for specifically amplifying E. coli include primers each containing a polynucleotide respectively represented by SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153 and SEQ ID NO: 154. A seventh primer set may be included. SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153 and SEQ ID NO: 154 are respectively the first F3 primer, the second F3 primer, the BIP primer, the LB primer, the FIP primer and the B3 primer. Is an array for The first F3 primer, the second F3 primer, the BIP primer, the LB primer, the FIP primer, and the B3 primer included in the preferred seventh primer set may be composed of the polynucleotides represented by the respective sequence numbers. The seventh primer set is particularly excellent for specifically amplifying E. coli.

(3-3) Klebsiella
The primer set for specifically amplifying Klebsiella comprises primers each containing a polynucleotide respectively represented by SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168. There may be 8 primer sets. SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168 are respectively F3 primer, B3 primer, FIP primer, first BIP primer, LB primer, and second BIP primer. Is an array for The first BIP primer, the LB primer, and the second BIP primer included in the preferred eighth primer set may be composed of the polynucleotides indicated by the respective sequence numbers. The eighth primer set is particularly excellent for specifically amplifying Klebsiella.

(3-4) Nucleic acid probe The nucleic acid probe for specifically detecting the amplification product obtained by the amplification using the sixth primer set may include a nucleic acid probe containing the polynucleotide represented by SEQ ID NO: 170. . A preferred nucleic acid probe may consist of the polynucleotide shown by SEQ ID NO: 170.

  The nucleic acid probe for specifically detecting an amplification product obtained by amplification using the seventh primer set may include a nucleic acid probe containing the polynucleotide represented by SEQ ID NO: 180. A preferred nucleic acid probe may consist of the polynucleotide represented by SEQ ID NO: 180.

  The nucleic acid probe for specifically detecting the amplification product obtained by amplification using the eighth primer set may include a nucleic acid probe containing the polynucleotide represented by SEQ ID NO: 175. A preferred nucleic acid probe may consist of the polynucleotide represented by SEQ ID NO: 175.

  The nucleic acid probe group used for detecting microorganisms of the family Enterobacteriaceae includes a third nucleic acid including a nucleic acid probe that includes the polynucleotides represented by SEQ ID NO: 170, SEQ ID NO: 175, and SEQ ID NO: 180, respectively. It may be a probe group. Each nucleic acid probe included in the preferred third nucleic acid probe group may consist of the polynucleotides respectively represented by SEQ ID NO: 170, SEQ ID NO: 175, and SEQ ID NO: 180. The third nucleic acid probe group is particularly excellent in specifically detecting microorganisms of the family Enterobacteriaceae. Of the third nucleic acid probe group, the nucleic acid probe containing the polynucleotide represented by SEQ ID NO: 170 is particularly excellent for comprehensively and specifically detecting microorganisms of the family Enterobacteriaceae. In addition, a nucleic acid probe containing the polynucleotides represented by SEQ ID NO: 175 and SEQ ID NO: 180, respectively, is a particularly useful nucleic acid probe for predicting the prognosis of mastitis. When a detection signal is detected for a nucleic acid probe comprising a polynucleotide respectively represented by SEQ ID NO: 175 and SEQ ID NO: 180, mastitis in the individual that is the source of the sample is, for example, “the mastitis of the individual is severe It is possible to determine that it is necessary to prevent infection to other individuals, or “in some cases, the collected milk must be discarded”.

(4) <Corynebacterium genus> (Coprynebacterium genus)
Corynebacterium bovis and Corynebacterium pyogenes (Arcanobacterium pyogenes) were selected as microorganisms belonging to the genus Corynebacterium that cause bovine mastitis because they are frequently detected in the mammary glands of individuals affected with bovine mastitis. By detecting these microorganisms, it is possible to specifically detect microorganisms of the genus Corynebacterium as bovine mastitis-causing microorganisms.

(4-1) Corynebacterium bovis
A primer set for amplifying Corynebacterium bovis is a ninth primer set including primers each containing a polynucleotide respectively represented by SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228 and SEQ ID NO: 229. It may be. SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228 and SEQ ID NO: 229 are sequences for the F3 primer, FIP primer, BIP primer, B3 primer and LFc primer, respectively. The F3 primer, the FIP primer, the BIP primer, the B3 primer, and the LFc primer included in the preferred ninth primer set may be composed of the polynucleotides represented by the respective sequence numbers. The ninth primer set is particularly excellent for specifically amplifying Corynebacterium bovis.

(4-2) Corynebacterium pyogenes (Arcanobacterium pyogenes)
Corynebacterium pyogenes is also called Arcanobacterium pyogenes. A primer set for specifically amplifying Corynebacterium pyogenes is a tenth primer including primers each containing a polynucleotide respectively represented by SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, and SEQ ID NO: 234 It can be a set. SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233 and SEQ ID NO: 234 are sequences for the F3 primer, FIP primer, BIP primer, B3 primer and LBc primer, respectively. The F3 primer, FIP primer, BIP primer, B3 primer and LBc primer included in the preferred 10th primer set may be composed of the polynucleotides indicated by the respective SEQ ID NOs. The tenth primer set is particularly excellent for specifically amplifying Corynebacterium pyogenes.

(4-3) Nucleic acid probe The nucleic acid probe for specifically detecting the amplification product obtained by amplification using the ninth primer set may include a nucleic acid probe comprising the polynucleotide represented by SEQ ID NO: 252. . A preferred nucleic acid probe may consist of the polynucleotide represented by SEQ ID NO: 252.

  The nucleic acid probe for specifically detecting the amplification product obtained by amplification using the tenth primer set may include a nucleic acid probe containing the polynucleotide represented by SEQ ID NO: 254. A preferred nucleic acid probe may consist of the polynucleotide set forth in SEQ ID NO: 254.

  In addition, the nucleic acid probe group used for detecting a microorganism belonging to the genus Corynebacterium is a fourth nucleic acid probe group including a nucleic acid probe that includes a polynucleotide represented by each of SEQ ID NO: 252 and SEQ ID NO: 254, respectively. It's okay. Each nucleic acid probe included in the preferred fourth nucleic acid probe group may consist of the polynucleotides respectively shown by SEQ ID NOs: 252 and 254. The fourth nucleic acid probe group is particularly excellent for comprehensively and specifically detecting microorganisms belonging to the genus Corynebacterium.

(5) Mycoplasma genus
Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma canadense, Mycoplasma arginini, and Mycoplasma bovis are selected as the microorganisms of the genus Mycoplasma that cause bovine mastitis because they are frequently detected in the mammary gland of individuals with bovine mastitis. did. By detecting these microorganisms, it is possible to specifically detect microorganisms of the genus Mycoplasma as bovine mastitis-causing microorganisms. Mycoplasma canadense and Mycoplasma arginini have the ability to degrade arginine. If arginine is broken down and absent from the cells, a complete immune response cannot be maintained and mastitis becomes severe. Therefore, identifying an arginine-degrading strain in Mycoplasma will help identify mastitis that can become severe.

(5-1) Mycoplasma genus Primer sets for comprehensive and specific amplification of Mycoplasma genus (Mycoplasma genus) are SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263 may be the first 11 primer set including a primer comprising a polynucleotide shown in the more respectively in SEQ ID NO: 264 and SEQ ID NO: 26 5. SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264 and SEQ ID NO: 265 are respectively the first F3 primer, Second F3 primer, first FIP primer, second FIP primer, first BIP primer, second BIP primer, first B3 primer, second B3 primer, first LFc primer and second The sequence for the LFc primer. A first F3 primer, a second F3 primer, a first FIP primer, a second FIP primer, a first BIP primer, a second BIP primer, and a first B3 primer included in a preferred eleventh primer set The second B3 primer, the first LFc primer, and the second LFc primer may be composed of the polynucleotides represented by the respective SEQ ID NOs. The eleventh primer set is particularly excellent for comprehensively and specifically amplifying Mycoplasma microorganisms.

(5-2) Arginine-degrading mycoplasma Primer sets for specifically amplifying arginine-degrading mycoplasma are SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, and SEQ ID NO: it may be a twelfth primer set including a primer comprising a polynucleotide shown in the more each respectively 30 9. SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308 and SEQ ID NO: 30 9, FIP primer respectively, BIP primer is the sequence for the F3 primer, B3 primer LF primer and LB primers . The FIP primer, the BIP primer, the F3 primer, the B3 primer, the LF primer, and the LB primer included in the preferred twelfth primer set may be composed of the polynucleotides represented by the respective sequence numbers. The twelfth primer set is particularly excellent for specifically amplifying arginine degradable mycoplasma.

(5-3) Nucleic acid probe A nucleic acid probe for specifically detecting an amplification product obtained by amplification using the eleventh primer set is a nucleic acid comprising polynucleotides represented by SEQ ID NO: 310 and SEQ ID NO: 311 respectively. A probe may be included. A preferred nucleic acid probe may be a nucleic acid probe comprising the polynucleotides respectively represented by SEQ ID NO: 310 and SEQ ID NO: 311 .

The nucleic acid probe for specifically detecting the amplification product obtained by amplification using the twelfth primer set may include a nucleic acid probe containing a polynucleotide respectively represented by SEQ ID NO: 314 . A preferred nucleic acid probe may consist of the polynucleotide set forth in SEQ ID NO: 314 .

In addition, the nucleic acid probe group used for detecting Mycoplasma microorganisms is a fifth nucleic acid probe group including nucleic acid probes each containing a polynucleotide represented by SEQ ID NO: 310 , SEQ ID NO: 311 and SEQ ID NO: 314 , respectively. It may be. Each nucleic acid probe included in the preferred fifth nucleic acid probe group may consist of the polynucleotides respectively represented by SEQ ID NO: 310 , SEQ ID NO: 311 and SEQ ID NO: 314 . The fifth nucleic acid probe group is particularly excellent in specifically detecting Mycoplasma microorganisms. Of the fifth nucleic acid probe group, the nucleic acid probe containing the polynucleotides represented by SEQ ID NO: 310 and SEQ ID NO: 311 is particularly excellent for comprehensively and specifically detecting Mycoplasma microorganisms. In addition, the nucleic acid probe containing the polynucleotide represented by SEQ ID NO: 314 is a particularly useful nucleic acid probe for predicting the prognosis of mastitis. If a detection signal is detected for a nucleic acid probe comprising a polynucleotide represented by SEQ ID NO: 314, mastitis in the individual that is the source of the sample is, for example, whether the prognosis is mastitis or not. It is possible to determine whether it is a strong mastitis or whether it is a mastitis that should be considered for disuse of infected individuals.

(6) Algae Prototheca zopfii was selected as an algae that causes bovine mastitis because it is a fungus frequently detected in the mammary gland of individuals suffering from bovine mastitis. By detecting these microorganisms, it is possible to specifically detect algae as bovine mastitis-causing microorganisms.

(6-1) Primer set Prototheca Sophie (Prototheca zopfii) Primer set for comprehensive and specific amplification of the SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, the SEQ ID NO: 321 and SEQ ID NO: 32 2 It may be a thirteenth primer set including a primer containing the polynucleotide shown in each of the above. SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321 and SEQ ID NO: 322 are sequences for the F3 primer, B3 primer, FIP primer, BIP primer and LF primer, respectively. The F3 primer, B3 primer, FIP primer, BIP primer, and LF primer included in the preferred thirteenth primer set may be composed of the polynucleotides indicated by the respective SEQ ID NOs. The thirteenth primer set is particularly excellent for comprehensive and specific amplification of Prototheca zopfii.

(6-2) Nucleic acid probe The nucleic acid probe for specifically detecting the amplification product obtained by amplification using the thirteenth primer set may include a polynucleotide represented by SEQ ID NO: 333 . A preferred nucleic acid probe may be a nucleic acid probe consisting of the polynucleotide set forth in SEQ ID NO: 333 .

Further, the nucleic acid probe used to detect Prototheca Sophie (Prototheca zopfii) can be a sixth nucleic acid probe group comprising a nucleic acid probe comprising a polynucleotide represented by SEQ ID NO: 333, represented by SEQ ID NO: 333 A sixth nucleic acid probe containing the polynucleotide to be prepared may be used.

  The sixth nucleic acid probe group or the sixth nucleic acid probe is particularly excellent in comprehensively and specifically detecting Prototheca zopfii.

(7) <Yeast-like fungi> (Candida genus, Cryptococcus genus)
Candida and Cryptococcus were selected as yeast-like fungi that cause bovine mastitis because they are frequently detected in the mammary glands of individuals with bovine mastitis. By detecting these microorganisms, it is possible to specifically detect yeast-like fungi as bovine mastitis-causing microorganisms.

  The primer set for specifically amplifying a yeast-like fungus may include a 14th primer set and a 15th primer set.

(7-1) Candida genus
Primer sets for specifically amplifying Candida are SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344 , SEQ ID NO: 347 and SEQ ID NO: 34 8 it may be a fourteenth primer set including a primer comprising a polynucleotide of the respective shown in more respectively. SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 347 and SEQ ID NO: 348 are respectively F3 primer, first B3 primer, second Sequences for the B3 primer, the first FIP primer, the second FIP primer, the first BIP primer, the second BIP primer, the first LB primer and the second LB primer. Preferred F14 primer set includes F3 primer, first B3 primer, second B3 primer, first FIP primer, second FIP primer, first BIP primer, second BIP primer, first The LB primer and the second LB primer consist of the polynucleotides represented by the respective SEQ ID NOs. The fourteenth primer set is particularly excellent for specifically amplifying Candida microorganisms.

(7-2) Cryptococcus genus
The primer set for specifically amplifying the Cryptococcus genus, including a primer comprising a polynucleotide shown in the more respectively in SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360 and SEQ ID NO: 36 1 second There may be 15 primer sets. SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360 and SEQ ID NO: 36 1, F3 primer, respectively, B3 primer, FIP primer, a sequence for BIP primer and LB primers. The F3 primer, B3 primer, FIP primer, BIP primer and LB primer included in the preferred 15th primer set are composed of the polynucleotides indicated by the respective SEQ ID NOs. The fifteenth primer set is particularly excellent for specifically amplifying a microorganism belonging to the genus Cryptococcus.

(7-3) Nucleic acid probe Nucleic acid probes for specifically detecting amplification products obtained by amplification using the fourteenth primer set are the polynucleotides represented by SEQ ID NO: 368 and SEQ ID NO: 369 , respectively. May be included.

The nucleic acid probe for specifically detecting the amplification product obtained by amplification using the fifteenth primer set may include the polynucleotide represented by SEQ ID NO: 386 . A preferred nucleic acid probe may be a nucleic acid probe consisting of the polynucleotide set forth in SEQ ID NO: 386 .

In addition, the nucleic acid probe group used for detecting the genus Candida and the Cryptococcus genus is a seventh nucleic acid probe group including a nucleic acid probe that includes the polynucleotides represented by SEQ ID NO: 368 , SEQ ID NO: 369, and SEQ ID NO: 386 , respectively. It may be. Each nucleic acid probe included in the preferred seventh nucleic acid probe group may consist of polynucleotides represented by SEQ ID NO: 368 , SEQ ID NO: 369 and 386 , respectively. The seventh nucleic acid probe group is particularly excellent for comprehensively and specifically detecting microorganisms of the genera Candida and Cryptococcus.

7). Assay Kit According to one embodiment, an assay kit for detecting and / or classifying mastitis-causing microorganisms may be provided. According to a further aspect, an assay kit for performing genotyping and / or species classification of mastitis-causing microorganisms may be provided.

  The assay kit may include the above-described primer or primer set and / or nucleic acid probe or nucleic acid probe group or nucleic acid probe set. Preferably, the assay kit may include the above-described primer or primer set and a nucleic acid probe or nucleic acid probe group or nucleic acid probe set. The assay kit may contain, in addition to the primer or primer set and / or the nucleic acid probe or nucleic acid probe group or nucleic acid probe set, a buffer necessary for amplification by the LAMP method, a buffer necessary for hybridization, and the buffer in the buffer. A container for performing amplification and / or hybridization may be provided. In addition, the nucleic acid probe contained in the assay kit may be provided immobilized on a substrate as described above, or the assay kit may further include a substrate for immobilization.

  The primer set included in the assay kit includes a first primer set, a second primer set, a third primer set, a fourth primer set, a fifth primer set, a sixth primer set, and a seventh primer set. , 8th primer set, 9th primer set, 10th primer set, 11th primer set, 12th primer set, 13th primer set, 14th primer set and 15th primer set Or at least one of these primer sets, or any combination of these primer sets.

  The nucleic acid probe set included in the assay kit may include a nucleic acid probe or a group of nucleic acid probes selected according to the amplification product to be detected. The nucleic acid probe set includes a first nucleic acid probe group, a second nucleic acid probe group, a third nucleic acid probe group, a fourth nucleic acid probe group, a fifth nucleic acid probe group, a sixth nucleic acid probe, and a seventh nucleic acid. A probe group may be included, at least one of these nucleic acid probe groups or nucleic acid probes may be included, and any combination of these nucleic acid probe groups or nucleic acid probes may be included.

  According to this aspect, there is provided a method for detecting microorganisms classified as mastitis-causing microorganisms at a high speed, at a low cost, and at a high speed and classifying genotypes at the family level, genus level or species level.

  By amplifying nucleic acid in a sample using the primer set as described above, it is possible to specifically amplify mastitis-causing microbial nucleic acid simply, inexpensively and at high speed. Using such a primer set, the nucleic acid contained in the sample is amplified by the LAMP method, and the presence or absence and / or the amount of the resulting amplification product is determined, whereby the nucleic acid derived from the mastitis-causing microorganism contained in the sample. Can be detected specifically. Furthermore, the results indicate that the animal source that is the source of the sample is mastitis and / or whether the prognosis of mastitis is good or bad, or mastitis that must isolate the individual. It is possible to make a diagnosis such as whether or not mastitis has to be discarded.

  In the conventional method, the specificity at the time of sequence detection is generally detected by a DNA chip for the target gene product amplified by the PCR method. In this case, since the product generated by PCR is a double strand, when a hybridization reaction is performed with a nucleic acid probe, the complementary strand becomes a competitor to the nucleic acid probe, resulting in low hybridization efficiency and poor detection sensitivity. In such a method, conventionally, in order to make a target a single strand, a method of decomposing or separating a complementary strand is also performed. In such a case, the working cost becomes high or the operation becomes complicated due to the use of enzymes and magnetic beads. Such a problem can also be solved by this embodiment.

  Until now, a method of amplifying a region containing a characteristic sequence by a PCR method and detecting it by an electrophoresis method has been used as a rapid diagnostic method. However, the diagnostic method using the PCR method has various disadvantages, for example, a complicated temperature control device such as a thermal cycler is necessary and there is a problem in simplicity. In addition, in the PCR method, in the unlikely event that complementary strand synthesis is performed by mistake, the product will be amplified as a template and may be erroneously determined. In fact, it is difficult to control specific amplification only by a single base difference at the end of the primer. By using the primer set according to this embodiment, it is possible to more specifically, more accurately, more rapidly, specifically amplify nucleic acid derived from a mastitis-causing microorganism with a wider detection range.

  Research on microbial sac in the environment, etc., requires the development of a method that can detect and rapidly identify the presence of gene sequences characteristic of mastitis-causing microorganisms. Satisfied by the embodiment.

[Example]
Hereinafter, a specific method for detecting nucleic acids of bovine mastitis-causing microorganisms will be described as an example.

[Example 1] Detection of Staphylococci LAMP amplification primers were designed to detect S. hominis, S. simulans, S. equimor, and S. intermedius among staphylococci that cause bovine mastitis. The 16S-rDNA gene was selected as a target region, and as a result of alignment, primers shown in Table 1 were selected as candidates.

As a result of evaluating the characteristics of the LAMP amplification reaction with the rise time of turbidity, the amplification time may be the shortest when using a primer set consisting of the primers represented by SEQ ID NOs: 1, 2, 3, 4 and 5, respectively. I understood. For nucleic acid probes, the candidates shown in Table 2 were designed.

  Detection of amplification products obtained using S. hominis, S. simulans, S. equimor and S. intermedius as templates could be carried out specifically with the nucleic acid probes shown in SEQ ID NOs: 6, 9 and 15, respectively. It was.

[Example 2] Detection of Streptococcus LAMP amplification primers were designed to detect Streptococcus spp. And Enterococcus spp. Among streptococci causing bovine mastitis. The 16S-rDNA gene was selected as a target region, and as a result of alignment, primers shown in Table 3 were selected as candidates.

As a result of evaluating the characteristics of the LAMP amplification reaction with the rise time of turbidity, the Streptococcus genus has the longest amplification time when using the primer set consisting of the primers shown in SEQ ID NOs: 46, 47, 48, 49 and 50, respectively. I found it to be shorter. The candidate shown in Table 4 was designed about the nucleic acid probe. Detection of amplification products from the genus Streptococcus could be performed specifically with the nucleic acid probes represented by SEQ ID NOs: 99 and 101, respectively.

The Enterococcus genus was found to have the shortest amplification time when using a primer set consisting of the primers represented by SEQ ID NOs: 30, 31, 32, 33 and 34, respectively. The candidate shown in Table 5 was designed about the nucleic acid probe. Detection of amplification products from the genus Enterococcus could be performed specifically with the nucleic acid probes shown in SEQ ID NOs: 92 and 95, respectively.

A LAMP amplification primer for detecting S.agalactiae and S.ubelis was designed. The recN gene was selected as a target region, and as a result of alignment, primers shown in Table 6 and Table 7 were selected as candidates.

  As a result of evaluating the characteristics of the LAMP amplification reaction with the rise time of turbidity, S. agalactiae has the shortest amplification time when using the primer sets shown by SEQ ID NOs: 65, 66, 67, 68 and 69, respectively. I understood that. The candidate shown in Table 5 was designed about the nucleic acid probe. Detection of amplification products from S.agalactiae and S.ubelis could be performed specifically with the nucleic acid probe represented by SEQ ID NO: 106.

  S. ubelis was found to have the shortest amplification time when using a primer set consisting of the primers represented by SEQ ID NOs: 80, 81, 82, 83 and 84, respectively. The candidate shown in Table 5 was designed about the nucleic acid probe. Detection of the amplification product from S. ubelis could be performed specifically with the nucleic acid probe represented by SEQ ID NO: 110.

[Example 3] Detection of coliform group LAMP amplification primers for detecting coliform group causing bovine mastitis were designed. The rpoA gene was selected as a target region, and as a result of alignment, primers shown in Table 5 were selected as candidates.

As a result of evaluating the characteristics of the LAMP amplification reaction with the rise time of turbidity, the amplification time may be the shortest when a primer set consisting of the primers represented by SEQ ID NOs: 127, 128, 12, 130 and 132 is used. I understood. The candidate shown in Table 9 was designed about the nucleic acid probe.

  Detection of the amplification product from the coliform group could be performed specifically with the nucleic acid probe represented by SEQ ID NO: 170.

A LAMP amplification primer for detecting E. coli was designed. The uvrCA gene was selected as a target region, and as a result of alignment, primers shown in Table 10 were selected as candidates. As a result of evaluating the characteristics of the LAMP amplification reaction with the rise time of turbidity, the amplification time is the shortest when a primer set consisting of the primers shown in SEQ ID NOs: 163, 164, 165, 166, 167 and 168 is used. I understood that. The candidate shown in Table 9 was designed about the nucleic acid probe.

  Detection of the amplification product from E. coli could be performed specifically with the nucleic acid probe represented by SEQ ID NO: 180.

  A LAMP amplification primer for detecting Klebsiella was designed. The metG gene was selected as a target region, and as a result of alignment, primers shown in Table 10 were selected as candidates. As a result of evaluating the characteristics of the LAMP amplification reaction with the rise time of turbidity, the amplification time is the shortest when the primer set comprising the primers shown in SEQ ID NOs: 149, 150, 151, 152, 153 and 154 is used. I understood that. The candidate shown in Table 9 was designed about the nucleic acid probe. Detection of the amplification product from Klebsiella could be carried out specifically with the nucleic acid probe represented by SEQ ID NO: 175.

[Example 4] Detection of Corynebacterium A LAMP amplification primer for detecting C. bovis causing bovine mastitis was designed. The rpoB gene was selected as a target region, and as a result of alignment, primers shown in Table 11, that is, Table 11-1 and Table 11-2 were selected as candidates.

As a result of evaluating the characteristics of the LAMP amplification reaction with the rise time of turbidity, the amplification time is the shortest when the primer set consisting of the primers shown in SEQ ID NOs: 224, 225, 226, 227 and 228 is used. I understood. The candidate shown in Table 12 was designed about the nucleic acid probe.

  Detection of the amplification product from C. bovis could be performed specifically with the nucleic acid probe represented by SEQ ID NO: 251.

  A LAMP amplification primer for detecting C.pyogenes (ie, A.pyogenes) was designed. The 16S-rDNA gene was selected as a target region, and as a result of alignment, primers shown in Table 11-2 were selected as candidates. As a result of evaluating the characteristics of the LAMP amplification reaction with the rise time of turbidity, the amplification time may be the shortest when using a primer set consisting of the primers represented by SEQ ID NOs: 229, 230, 231, 232 and 233, respectively. I understood. The candidate shown in Table 12 was designed about the nucleic acid probe. Detection of the amplification product from T. pyogenes could be performed specifically with the nucleic acid probe represented by SEQ ID NO: 253.

[Example 5] Detection of mycoplasma LAMP amplification primers were designed to detect M. bovis, M. californicum, M. bovigenitalium, M. canadense, and M. arginini among mycoplasmas causing bovine mastitis. . The 16S-rDNA gene was selected as a target region, and as a result of alignment, primers shown in Table 13, that is, Table 13-1, Table 13-2, and Table 13-3 were selected as candidates.

As a result of evaluating the characteristics of the LAMP amplification reaction using the rise time of turbidity, when a primer set comprising the primers shown in SEQ ID NOs: 256, 257, 258, 259, 260, 261, 262, 263, 264 and 265, respectively, was used It was found that the amplification time was the shortest. For nucleic acid probes, the candidates shown in Table 14 were designed, and specific detection was possible with SEQ ID NOs: 310 and 311.

  For arginine-degrading mycoplasma, the nucleic acid from arginine-degrading mycoplasma was specifically amplified as a template nucleic acid when using a primer set comprising the primers shown in SEQ ID NOs: 304, 305, 306, 307, 308 and 309, respectively. . Detection of the amplification product from arginine degradable mycoplasma could be carried out specifically with the nucleic acid probe represented by SEQ ID NO: 314.

[Example 6] Detection of algae Among the algae causing bovine mastitis, a LAMP amplification primer for detecting Prototheca zopfii was designed. The NL1-NL4 gene was selected as a target region, and as a result of alignment, primers shown in Table 15 were selected as candidates.

As a result of evaluating the characteristics of the LAMP amplification reaction with the rise time of turbidity, the amplification time may be the shortest when a primer set consisting of the primers represented by SEQ ID NOs: 318, 319, 320, 321, and 322 is used. I understood. The candidate shown in Table 16 was designed about the nucleic acid probe.

  Detection of the amplification product from Prototheca zopfii could be performed specifically with the nucleic acid probe represented by SEQ ID NO: 333.

Example 7 Detection of Yeast-Like Fungi LAMP amplification primers were designed to detect the yeast-like fungi Candida and Cryptococcus causing bovine mastitis. The NL1-NL4 gene was selected as a target region, and as a result of alignment, primers shown in Table 17 were selected as candidates.

As a result of evaluating the characteristics of the LAMP amplification reaction with respect to the turbidity for Candida, when using a primer set comprising the primers represented by SEQ ID NOs: 338, 339, 340, 341, 342, 343, 344, 347 and 348, respectively It was found that the amplification time was the shortest. For the probes, the candidates shown in Table 18 were designed.

  Specific detection was possible with SEQ ID NO: 368,369.

  For Cryptococcus, primers shown in Table 17 were selected as candidates as a result of the alignment. The characteristics of the LAMP amplification reaction were evaluated by the rise time of turbidity. As a result, for Cryptococcus, it was found that the amplification time was the shortest when a primer set consisting of the primers represented by SEQ ID NOs: 357, 358, 359, 360 and 361 was used. That is, Cryptococcus was specifically and efficiently amplified by these sequences. The candidates shown in Table 14 were designed as nucleic acid probes for specifically detecting the amplification product from Cryptococcus. Detection of the amplification product from Cryptococcus could be performed specifically with the nucleic acid probe represented by SEQ ID NO: 386.

[Example 8]
Using a field specimen, each test was performed by a bacterial culture method, which is a conventional method for testing bovine mastitis-causing bacteria, and a method using a DNA chip according to the embodiment, and the results were compared.

(1) Purpose of the experiment In order to show what kind of point the DNA chip method according to the embodiment is superior to the bacterial culture method which is a conventional method, analysis was actually performed using a specimen.

(2) Materials The experiment was conducted on 40 cattle raised on A farm in Tokachi district, Hokkaido. One cow has four quarters, and the samples collected from each were measured individually, so the number of tests was the number of heads multiplied by the number of quarters. The milk squeezed for each sample and each quarter was transported to the experimental facility near Tokyo in a refrigerated state. The bacterial culture test was started the next day after collection. The same milk was stored in a refrigerated state for one more day after the start of the culture test, and then subjected to a DNA chip test.

<Bacteria culture method>
Bacterial culture was performed by applying 10 to 50 μL of milk to an appropriate medium and leaving it in a 37 ° C. thermostat. The medium is selected for each expected infectious bacterium, and was selected from enzyme substrate medium “chromoagar orientacion”, blood agar medium, DHL agar medium, mannitol salt medium, and the like. The primary culture performed using these media was performed at 37 ° C., and the culture time required 24 to 72 hours. After culturing, for example, in the case of “chromoagar orientation”, rough bacteria were identified by the color of the colonies that appeared.

Identification criteria Identification criteria were as follows:
Pale yellow: Staphylococcus aureus (SA), Staphylococcus (CNS)
White to blue-green: Streptococcus genus, Enterococcus genus Pink: E.coli
Metal blue: Klebsiella, Enterobacteria
Brown: Proteus
White to colorless: Pseudomonas.

  The roughly identified bacteria were further precisely identified using a BD BBL CRYSTAL kit (Nippon Becton Dickinson). Here, further culture at 37 ° C. was required for 18 to 20 hours.

<DNA chip method>
(1) Nucleic acid extraction method Nucleic acid extraction from milk was performed according to the recommended method by the manufacturer of FAST-ID DNA extraction kit (manufactured by Japan Authentication Service Co., Ltd.). Specifically, in addition to the recommended extraction process, the following process was further added. That is, in addition to the recommended extraction steps, bead beating to destroy solids and fats, and the outer shell of the fungus, and precipitation with ammonium sulfate and acetic acid to further remove proteins and lipids. Processed. The milk used was 200 μL to 400 μL per test. The nucleic acid after extraction was dissolved in 60 μL of TE buffer. The sample after extraction was stored at −20 ° C. until it was used for the subsequent amplification reaction.

(1) LAMP amplification reaction The composition of the LAMP reaction solution used for amplification by the LAMP method was as follows:
LAMP reaction solution composition:
20 mM Tris-HCl (pH 8.8)
10 mM KCl
8 mM MgSO ₄
10 mM (NH ₄ ) ₂ SO ₄
0.1% Tween20
0.8M Betaine
1.4mMeach dNTPs
Bst DNA Polymerase 8 unit.

  Necessary primers were brought into the reaction solution having this composition at the following concentrations.

Amount of primer added:
FIP 40pmoL
BIP 40pmoL
F3 5pmoL
B3 5pmoL
Loop 20 pmoL.

  The reaction was performed on a reaction scale of 25 μL in a separate reaction tube for each bacterial species. The extracted product was rapidly cooled after heat treatment at 95 ° C. for 3 minutes. Thereafter, 2 μL of it was added to each reaction tube, and the reaction was carried out at 61 ° C. for 1 hour. The amplified product was heated at 80 to 95 ° C. for 3 minutes to inactivate the enzyme.

(2) DNA chip measurement The DNA chip is SEQ ID NO: 6, 9, 15, 92, 95, 99, 101, 106, 110, 170, 175, 180, 252, 254, 310, 311, 314, 333, 368, Nucleic acid probes indicated by 369 and 386 were prepared by immobilizing each type on each surface for obtaining one signal on the surface of the glass substrate. Using such a DNA chip, 2 μL of amplification product was added per reaction and measured. The measuring device used was a generator 2C600. The reaction conditions used are shown in Table 29.

(3) Results Of the measurement results, the results of 10 heads are shown in Table 20-1 and Table 20-2.

  Table 20-1 and Table 20-2 show the individual number (individual No.) from the left, each quarter (A, B, C, D), the number of bacteria found in the primary culture (cuf / ml), After that, rough identification results in primary culture were SA (Staphylococcus aureus), SAG (Streptococcus agalactiae), CNS (Staphylococcus), OS (Streptococcus other than SAG), CO (Coliform group), Coryne. Spp ( The column was divided into (genus Corynebacterium), and the detected bacteria were marked with black circles. The “number of somatic cells” on the right side is a numerical value indicating the number of mixed neutrophil multilateral leukocytes, lymphocytes, macrophages and the like present in milk, and is an index indicating the degree of inflammation. Approximately 100,000 cells / mL or less are considered normal quarters. In the next column, the result of precise identification by the BD BBD kit is shown. The numerical value on the right side of the column is a numerical value indicating the reliability of the identification result. Higher values indicate higher reliability. On the far right side of the table, the results of a test performed using the bovine mastitis DNA chip according to the embodiment are shown. A black circle was attached to the bacterial species determined to be positive.

  For example, no. 1, individual no. In the result of 347, the number of somatic cells exceeded 100,000, and it was found that the cell was affected by mastitis. In the culture results, staphylococci are positive, and the species in the staphylococci have been clarified from the results of precise identification using the BD BBD crystal kit. However, when examined with a DNA chip, it was found that Streptococcus spp., Enterococcus spp., And Enterobacteria were positive in addition to staphylococci. In other words, compared to the culture method, using a DNA chip, it was possible to find positive streptococci and coliform bacteria. Similarly, in the result of the B quarter, since the number of somatic cells exceeds 100,000, it was found that this quarter also suffers from mastitis. In the culture result, staphylococci are positive as in the case of the A quarter, and the positive identification name among the staphylococci is also clarified from the precise identification result using the BD BBD crystal kit. However, when examined with a DNA chip, it was found that in addition to staphylococci, Streptococcus spp., Enterococcus spp., Enterobacteriaceae, E. coli, and Candida spp. From these facts, it was found that the ability of the DNA chip to determine a fine mixed bacterial species as positive is superior to the culture method. Similar phenomena were generally observed in other individuals and quarters.

Table 21 summarizes the test results of all 40 quadrants in terms of the difference in results between the culture method and the DNA chip method.

  From the results in Table 21, it can be seen that the positive coincidence rate and the negative coincidence rate shown at the right end of the table show generally high numbers, and the DNA chip test does not show a result greatly different from the culture method. However, in staphylococci, E. coli, and Pseudomonas aeruginosa, which have a low negative concordance rate, many of them were negative in the culture method but positive in the DNA chip, and the superiority of the sensitivity of the DNA chip appears. . In particular, Pseudomonas aeruginosa is difficult to cultivate, and it is predicted that the negative concordance rate will be low for bacterial species that do not necessarily form colonies even if bacteria are present. It was confirmed that the results of the experiment were correct. In addition, even staphylococci and E. coli, which do not have low colony-forming ability, have an activity that can form a colony even if the bacteria are present after about 24 hours in the refrigerated environment until the start of culture. Is not maintained, and the negative coincidence rate is considered to be low. From these facts, it can be said that a DNA chip that compensates for the instability of the culture method and that can be detected with high sensitivity is a useful technique for examining causal mastitis causative bacteria. In addition, in the culture method, it takes 2 to 4 days from the start of the culture to obtain a precise identification result, whereas in the DNA chip method, the result can be obtained in about 3.5 hours. However, it was considered useful from a clinical point of view such as early treatment initiation and individual isolation.

  From these experimental results, it was proved that mastitis-causing microorganisms can be characteristically and rapidly detected and identified by the method according to the embodiment.

Claims (13)

(1) (i) A method for detecting a microorganism belonging to the genus Staphylococcus , comprising primers each containing a polynucleotide represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively. 1 primer set,
(Ii) a second primer for detecting a microorganism belonging to the genus Enterococcus , comprising primers each containing a polynucleotide represented by SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively set,
(Iii) for detecting a microorganism belonging to the genus Streptococcus , comprising primers each containing the polynucleotides represented by SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51 A third primer set,
(Iv) A fourth primer set for detecting S. agalactiae, comprising primers each containing the polynucleotides represented by SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, respectively ,
(V) A fifth primer set for detecting S. uberis, comprising primers each containing the polynucleotides represented by SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively . ,
(Vi) a sixth primer for detecting a microorganism belonging to the family Enterobacteria , comprising primers each containing the polynucleotide represented by SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 132 set,
(Vii) SEQ ID NO: 149, SEQ ID NO: 1 50, SEQ ID NO: 151, SEQ ID NO: 152, consists of primers containing each a polynucleotide shown in the respective by SEQ ID NO: 153 and SEQ ID NO: 154, for the detection of E.coli 7th primer set,
(Viii) an eighth for detecting Klebsiella , comprising primers each comprising a polynucleotide respectively represented by SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168; (9) Primer set (ix) Ninth primer for detecting Corynebacterium bovis, comprising primers each containing the polynucleotide shown by SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228 and SEQ ID NO: 229 set,
(X) a tenth primer set for detecting Corynebacterium pyogenes, comprising primers each comprising a polynucleotide represented by SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, and SEQ ID NO: 234;
(Xi) polynucleotides represented by SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264 and SEQ ID NO: 265, respectively. An eleventh primer set for detecting a microorganism belonging to the genus Mycoplasma , comprising primers contained in each of them ;
(Xii) for detecting arginine-degrading Mycoplasma, comprising primers each comprising the polynucleotides represented by SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308 and SEQ ID NO: 309, respectively To detect Prototheca zopfii, comprising a twelfth primer set, and (xiii) primers each comprising a polynucleotide respectively represented by SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321 and SEQ ID NO: 322 the 13 primer set of,
Amplifying a nucleic acid in a sample by the LAMP method using a primer set for LAMP amplification containing
(2) Amplification product mixture containing the amplification products obtained by all the primer sets of (1) was used as SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 15 (for detecting microorganisms of the genus Staphylococcus) , SEQ ID NO: 92, SEQ ID NO: 95 (for detecting microorganisms of the genus Enterococcus) , SEQ ID NO: 99 and SEQ ID NO: 101 (for detecting microorganisms of the genus Streptococcus) , SEQ ID NO: 106 (for detecting S. agalactiae) , SEQ ID NO: 110 (for detecting S. uberis) , SEQ ID NO: 170 (for detecting microorganisms of the Enterobacteria family) , SEQ ID NO: 175 (for detecting Klebsiella) , SEQ ID NO: 180 (for detecting E. coli) , SEQ ID NO: 252 (for detecting Corynebacterium bovis) , SEQ ID NO: 254 (for detecting Corynebacterium pyogenes) , sequence (for microorganism detection of Mycoplasma spp) No. 310 and SEQ ID NO: 311, SEQ ID NO: 314 (arginine degradable Mycoplasma detection) and SEQ ID NO 333 and reacting with a nucleic acid probe set comprising a nucleic acid probe comprising a polynucleotide into each indicated respectively by (for Prototheca zopfii detection),
(3) determining the presence and / or amount of a bovine mastitis-causing microorganism contained in the sample based on the presence and / or magnitude of the hybridization signal obtained after the reaction of (2);
A method for detecting bovine mastitis-causing microorganisms.   The LAMP amplification primer set is
  (Xiv) Primers comprising the polynucleotides respectively represented by SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 347 and SEQ ID NO: 348 A fourteenth primer set for detecting microorganisms of the genus Candida, and
  (Xv) a fifteenth primer for detecting a microorganism belonging to the genus Cryptococcus, comprising primers each containing a polynucleotide represented by SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, and SEQ ID NO: 361, respectively set
Further including
  The nucleic acid probe set further includes SEQ ID NO: 368 and SEQ ID NO: 369 (for detecting microorganisms of the genus Candida) and SEQ ID NO: 386 (for detecting microorganisms of the genus Cryptococcus)
The method of claim 1. 3. The method according to claim 1 or 2 , wherein all of the primers are composed of polynucleotides respectively represented by each of the SEQ ID NOs, and all of the nucleic acid probes are respectively represented by each of the SEQ ID NOs. A method comprising a polynucleotide. The method according to any one of claims 1 to 3, wherein the hybridization signal obtained in (3) has any nucleic acid probe included in the nucleic acid probe set of (2). Classifying the bovine mastitis-causing microorganism contained in the sample based on whether it is a result of the reaction in the method. The method according to any one of claims 1 to 4 , wherein the nucleic acid probe set is immobilized on a substrate. A method for diagnosing whether or not an animal individual other than a human being, which is a source of the sample, has mastitis based on the result obtained by the method according to any one of claims 1 to 5 . (I) a first primer set comprising primers each comprising a polynucleotide represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5;
(Ii) a second primer set comprising primers each comprising a polynucleotide represented by SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34,
(Iii) a third primer set comprising primers each comprising a polynucleotide represented by SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51,
(Iv) a fourth primer set comprising primers each comprising a polynucleotide represented by SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69,
(V) a fifth primer set comprising primers each comprising a polynucleotide respectively represented by SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84;
(Vi) a sixth primer set comprising primers each comprising a polynucleotide represented by SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 132,
(Vii) SEQ ID NO: 149, SEQ ID NO: 1 50, SEQ ID NO: 151, SEQ ID NO: 152, seventh primer set consisting of primers containing each a polynucleotide shown in the respective by SEQ ID NO: 153 and SEQ ID NO: 154,
(Viii) an eighth primer set comprising primers each comprising a polynucleotide represented by SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168,
(Ix) a ninth primer set comprising primers each containing a polynucleotide represented by SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, and SEQ ID NO: 229,
(X) a tenth primer set comprising primers each comprising a polynucleotide represented by SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, and SEQ ID NO: 234;
(Xi) polynucleotides represented by SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264 and SEQ ID NO: 265, respectively. An eleventh primer set comprising primers contained in each;
(Xii) a twelfth primer set comprising primers each comprising a polynucleotide respectively represented by SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, and SEQ ID NO: 309, and (xiii) In order to detect bovine mastitis-causing microorganisms, comprising a thirteenth primer set consisting of primers each comprising a polynucleotide respectively represented by SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321 and SEQ ID NO: 322 LAMP amplification primer set.   (Xiv) Primers comprising the polynucleotides respectively represented by SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 347 and SEQ ID NO: 348 A fourteenth primer set for detecting microorganisms of the genus Candida, and
  (Xv) a fifteenth primer for detecting a microorganism belonging to the genus Cryptococcus, comprising primers each containing a polynucleotide represented by SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, and SEQ ID NO: 361, respectively set
The primer set for LAMP amplification according to claim 7, further comprising: The primer set for LAMP amplification according to claim 7 or 8 , wherein all of the primers are composed of polynucleotides respectively represented by the SEQ ID NOs. The LAMP amplification primer set according to claim 7 ,
Sequence number 6, Sequence number 9, Sequence number 15, Sequence number 92, Sequence number 95, Sequence number 99, Sequence number 101, Sequence number 106, Sequence number 110, Sequence number 170, Sequence number 175, Sequence number 180, Sequence number 252, SEQ ID NO: 254, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 315 and SEQ ID NO: 33 3 more each containing a nucleic acid probe comprising each a polynucleotide shown, the nucleic acid probe for detecting milk mastitis causative agent And an assay kit for detecting a bovine mastitis-causing microorganism.   The primer set for LAMP amplification according to claim 8,
  Sequence number 6, Sequence number 9, Sequence number 15, Sequence number 92, Sequence number 95, Sequence number 99, Sequence number 101, Sequence number 106, Sequence number 110, Sequence number 170, Sequence number 175, Sequence number 180, Sequence number 252, SEQ ID NO: 254, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 315, SEQ ID NO: 333, SEQ ID NO: 368, SEQ ID NO: 369, and a bovine breast comprising a nucleic acid probe each comprising a polynucleotide respectively represented by SEQ ID NO: 386 Nucleic acid probe sets for detecting flame-causing microorganisms and
An assay kit for detecting a bovine mastitis-causing microorganism. 12. The assay kit according to claim 10 or 11 , wherein all of the nucleic acid probes are composed of polynucleotides respectively represented by the respective SEQ ID NOs. The assay kit according to any one of claims 10 to 12 , wherein the nucleic acid probe set is immobilized on a substrate.

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