JP5787503B2 - Primer set for mycoplasma, assay kit and method using the same - Google Patents

Description

  Embodiments of the invention relate to amplification and / or detection means for mycoplasma.

  Mycoplasma (Mycoplasma) is a genus of eubacteria and is classified into the family Mycoplasmataceae and genus Mycoplasma. Currently, about 100 species are known. There is no peptidoglycan cell wall found in common eubacteria. Therefore, the cell shape is irregular and plastic, and penicillin and cephem drugs are ineffective. In natural conditions, it parasitizes certain eukaryotes. Most are difficult to grow on synthetic media and often require the addition of multiple growth factors.

  Since it does not have a cell wall, the shape of the cell is plastic, and passing through a 0.22 μm filter sterilization filter is also a unique feature of this microorganism. Therefore, even if filter sterilization is performed, mycoplasma cannot be removed from the cell culture medium, which causes a problem due to contamination.

  In general, contamination of bacteria and fungi is easy to detect contamination due to discoloration of culture medium or denaturation of cultured cells. On the other hand, it is difficult for the mycoplasma contamination to detect the alteration of the medium at the visual and optical microscope level. In addition, since mycoplasma often coexists with cultured cells, it is easy to miss contamination. As the influence of mycoplasma contamination, the growth inhibition of cultured cells due to the nutrient consumption of the medium, the influence on the metabolic pathway by the direct action of mycoplasma, and the influence on gene expression have been confirmed. Therefore, in order to correctly evaluate the experimental results using cells, it is necessary to carefully confirm that there is no mycoplasma contamination. It is also necessary to estimate the species in order to identify the contamination route.

  Many mycoplasmas parasitize various vertebrates, including humans. This parasitism is well known to exhibit pathogenicity such as arthropathy and pneumonia. For example, in humans, it causes atypical pneumonia and presents cold-like symptoms such as irritating cough without headache, headache, sore throat, malaise and fever. In cattle, infection with microorganisms belonging to the Mycoplasma family causes pneumonia, mastitis, arthritis, miscarriage, and the like. In particular, mycoplasmal mastitis in dairy cows is highly contagious and refractory. This is a major threat in dairy management. Once mycoplasma mastitis develops and becomes clinical mastitis, it is very difficult to cure. Therefore, it is important to quickly identify all mycoplasma-infected individuals, including potentially infected individuals who do not show symptoms, and take appropriate measures in order to minimize economic losses due to spread of infection.

JP 2004-350092 A JP 2009-131174 A

  An object of the present invention is to provide a means for specifically amplifying and / or detecting mycoplasma.

  The above object is achieved by the following embodiments.

(A) A nucleic acid primer set for LAMP amplification for specifically amplifying nucleic acid derived from mycoplasma, wherein the nucleic acid primer set for LAMP amplification comprises a FIP primer, a BIP primer, an F3 primer and a B3 primer,
FIP primers were SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120 and their complementary sequences, and at least one selected from the group consisting of polynucleotides each represented by any one of the aforementioned sequences containing mutations to the extent that the nucleic acid derived from mycoplasma can be specifically amplified At least one FIP primer comprising a polynucleotide to be
BIP primers were SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 81, SEQ ID NO: 89, SEQ ID NO: 97, SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 121 and their complementary sequences, and mycoplasma-derived nucleic acids are mutated within a specific amplification range At least one BIP primer comprising a polynucleotide selected from the group consisting of polynucleotides each represented by any of the sequences comprising:
F3 primer is SEQ ID NO: 9, SEQ ID NO: 28, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 79, SEQ ID NO: 87, SEQ ID NO: 95, SEQ ID NO: 103, SEQ ID NO: 111, SEQ ID NO: 119 and their complements. At least one kind of F3 primer comprising a sequence and at least one polynucleotide selected from the group consisting of polynucleotides each represented by any of the above-mentioned sequences containing a mutation within a range in which a nucleic acid derived from mycoplasma can be specifically amplified Yes,
The B3 primer was SEQ ID NO: 10, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 114, SEQ ID NO: 122 and its complementary sequence, and at least one polynucleotide comprising at least one polynucleotide selected from the group consisting of polynucleotides each represented by any one of the above sequences containing mutations within a range in which nucleic acid derived from mycoplasma can be specifically amplified A primer set of F3 primers;
(B) an assay kit for detecting and / or classifying mycoplasma,
The primer set according to (A),
A nucleic acid probe for specifically detecting mycoplasma from an amplification product obtained by amplification by the primer set,
(1) SEQ ID NO: 127 and SEQ ID NO: 128;
(2) SEQ ID NO: 131, SEQ ID NO: 132 and SEQ ID NO: 133;
(3) SEQ ID NO: 136 and SEQ ID NO: 139;
(4) SEQ ID NO: 137 and SEQ ID NO: 138;
(5) SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150;
(6) SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102 SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 125 and SEQ ID NO: 126; and (7) a complementary sequence of the sequences described in (1) to (6) above;
And (C) a method for specifically amplifying and / or classifying a nucleic acid derived from mycoplasma, comprising a nucleic acid probe selected from the group of nucleic acid probes each consisting of a polynucleotide represented by A method comprising amplifying a sample using the primer set described in A);
It is.

The schematic diagram for demonstrating the amplification method by the LAMP method according to this aspect. The schematic diagram for demonstrating the amplification product obtained using the LAMP method according to this aspect. The schematic diagram which shows an example of the amplification product according to this aspect. The schematic diagram of the nucleic acid probe fixed base | substrate according to this aspect. The schematic diagram of the nucleic acid probe fixed base | substrate according to this aspect.

1. Primer According to one embodiment of the present invention, a nucleic acid primer set for LAMP and RT-LAMP amplification for specifically amplifying nucleic acid derived from mycoplasma is provided.

  The “LAMP method” and the “RT-LAMP method” are nucleic acid amplification methods. These are also referred to as isothermal gene amplification methods. Nucleic acids are amplified using DNA as a template by the LAMP method. The RT-LAMP method amplifies a nucleic acid using RNA as a template by simultaneously performing a reverse transcription reaction and a LAMP reaction.

  The LAMP method is different from the PCR method in that two or four regions or four or six regions of primers are used. The LAMP method is superior in amplification efficiency to the method using PCR and is not easily affected by impurities in the sample. Therefore, by using the primer according to the embodiment, it is possible to detect microorganisms classified into a trace amount of Mycoplasma by simple sample pretreatment.

  Here, primer design in the LAMP method and amplification products obtained by amplification will be described with reference to FIG. 1 and FIG. FIG. 1 shows the double-stranded DNA to be detected. A total of four types of primer sequences, that is, FIP primer, F3 primer, BIP primer, and B3 primer are set. The FIP primer and the BIP primer each contain two regions (FIP is F1c and F2, and BIP is B2 and B1c). The F3 primer contains the F3 region. The B3 primer contains the B3 region. Here, the F3, F2, F1, B1c, B2c, and B3c regions are regions set in this order from the 5 ′ to 3 ′ direction of the single-stranded DNA of the double-stranded DNA. The B3, B2, B1, F1c, F2c, and F3c regions are regions set in this order from the complementary strand to the 5 'to 3' direction of the single-stranded DNA. At this time, B3 and B3c, B2 and B2c, B1 and B1c, F1c and F1, F2c and F2, and F3c and F3 are complementary strands.

  When LAMP amplification is performed using four types of primers composed of these regions, that is, FIP primer, F3 primer, BIP primer, and B3 primer, from each strand of the double-stranded DNA of FIG. A dumbbell-shaped stem-and-loop amplification product as shown is obtained. The description of the amplification mechanism is omitted, but if necessary, for example, Japanese Patent Laid-Open No. 2002-186871 may be referred to. In addition to these four types of primers, a loop primer may be used in combination. The loop primer may be an LPf primer and / or an LPb primer.

  The RT-LAMP method is a method in which an enzyme having reverse transcription activity is added to the LAMP method reaction solution and the LAMP amplification method is performed while performing the reverse transcription reaction. The primer used may be the same primer as in the LAMP method. Here, when “LAMP method” is described, it may be understood that it is a reference to both the LAMP method and the RT-LAMP method.

  In this embodiment, the primer is designed so that the target sequence is located in the single-stranded loop portion. A “target sequence” is a sequence to be hybridized to a nucleic acid probe. As shown in FIG. 1, the target array (for example, any one of FPc, FP, BP, and BPc in FIG. 1) is between the regions F1 and F2 (this region may include the F2 region), and the region F2c Between F1c (this region may include F2c region), between regions B1 and B2 (this region may include B2 region), and / or between regions B2c and B1c (this region is B2c Six primer regions are set so as to correspond to any of the regions (which may include a region). The target sequence may be designed to be located at any part of the single-stranded loop portion formed between the respective regions. Here, the F2 region itself may be included in the loop portion between the primer regions F1 and F2. Four types of primers are prepared based on the six primer regions thus set, and RT-LAMP amplification is performed using these primers. Thereby, a part of the LAMP amplification product as shown in FIG. 3 is obtained. In this LAMP amplification product, the target sequences FPc, FP, BP, and BPc are located in a single-stranded loop in the dumbbell structure of the amplification product.

  On the other hand, since the primer regions F1c and F1 and B1c and B1 originally have mutually complementary sequences, self-hybridization occurs with each other to form a double strand. Thus, the target sequence contained in the amplification product exists in a single-stranded state as shown in FIG. Therefore, specific hybridization with a nucleic acid probe complementary to each target sequence (for example, a nucleic acid having a sequence of FP, FPc, BP, BPc) is possible without performing a denaturation operation, as shown in the figure. . Detection of the amplification product can also be performed by using such a nucleic acid probe. Here, “specific hybridization” means that when a single nucleotide polymorphism (Single Nucleotide PolymorpHisms: SNPs) or mutation is present, even a slight difference thereof can be detected.

  As described above, the primer set of this embodiment is designed based on the F1, F2, F3, B1c, B2c, and B3c regions.

An example of a nucleic acid primer set for LAMP amplification for specifically amplifying nucleic acid derived from mycoplasma, the nucleic acid primer set for LAMP amplification comprises FIP primer, BIP primer, F3 primer and B3 primer,
FIP primers were SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120 and their complementary sequences, and at least one selected from the group consisting of polynucleotides each represented by any one of the aforementioned sequences containing mutations to the extent that the nucleic acid derived from mycoplasma can be specifically amplified At least one FIP primer comprising a polynucleotide to be
BIP primers were SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 81, SEQ ID NO: 89, SEQ ID NO: 97, SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 121 and their complementary sequences, and mycoplasma-derived nucleic acids are mutated within a specific amplification range At least one BIP primer comprising a polynucleotide selected from the group consisting of polynucleotides each represented by any of the sequences comprising:
F3 primer is SEQ ID NO: 9, SEQ ID NO: 28, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 79, SEQ ID NO: 87, SEQ ID NO: 95, SEQ ID NO: 103, SEQ ID NO: 111, SEQ ID NO: 119 and their complements. At least one kind of F3 primer comprising a sequence and at least one polynucleotide selected from the group consisting of polynucleotides each represented by any of the above-mentioned sequences containing a mutation within a range in which a nucleic acid derived from mycoplasma can be specifically amplified Yes,
The B3 primer was SEQ ID NO: 10, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 114, SEQ ID NO: 122 and its complementary sequence, and at least one polynucleotide comprising at least one polynucleotide selected from the group consisting of polynucleotides each represented by any one of the above sequences containing mutations within a range in which nucleic acid derived from mycoplasma can be specifically amplified It may be a primer set which is F3 primer.

Further examples of the LAMP amplification nucleic acid primer set for specifically amplifying nucleic acid derived from Mycoplasma, the LAMP amplification nucleic acid primer set comprises FIP primer, BIP primer, F3 primer and B3 primer,
(1) The FIP primer is a polynucleotide represented by SEQ ID NO: 1 and / or its complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 6 and / or its complementary sequence, and the F3 primer is a sequence A primer set of No. 9 and / or its complementary sequence, wherein said B3 primer is a polynucleotide of SEQ ID No. 10 and / or its complementary sequence, and / or (2) said FIP primer is a sequence The polynucleotide shown by No. 29 and / or its complementary sequence, the BIP primer is the polynucleotide shown by SEQ ID No. 30 and / or its complementary sequence, and the F3 primer is SEQ ID No. 28 and / or its complementary sequence Indicated by It may be a primer set which is a renucleotide and is a primer set in which the B3 primer is a polynucleotide represented by SEQ ID NO: 31 and / or its complementary sequence.

A nucleic acid primer set for LAMP amplification for specifically amplifying nucleic acid derived from mycoplasma, the nucleic acid primer set for LAMP amplification comprises FIP primer, BIP primer, F3 primer and B3 primer, and further comprises a loop primer,
The loop primer is an LPf primer and / or an LPb primer;
The LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 83, SEQ ID NO: 91, SEQ ID NO: 99, SEQ ID NO: 107, SEQ ID NO: 115 and SEQ ID NO: 123 and at least one kind of LPf primer comprising at least one polynucleotide selected from the group consisting of polynucleotides respectively represented by the complementary sequences thereof, wherein the LPb primer has the sequence SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84, SEQ ID NO: 92, SEQ ID NO: 100, SEQ ID NO: 108, SEQ ID NO: 116, SEQ ID NO: 124 and their complementary sequences, respectively. At least one selected from the group consisting of polynucleotides It may be a primer set comprising at least one LPb primer comprising a polynucleotide.

An example of a nucleic acid primer set for LAMP amplification for specifically amplifying nucleic acid derived from mycoplasma, the nucleic acid primer set for LAMP amplification comprises FIP primer, BIP primer, F3 primer and B3 primer,
(1) At least one type of FIP primer wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. The BIP primer includes at least one BIP primer that is at least one selected from the group consisting of polynucleotides represented by SEQ ID NO: 6 and SEQ ID NO: 8 and their complementary sequences, and the F3 primer is At least one F3 primer, which is a polynucleotide selected from the group consisting of the polynucleotides represented by SEQ ID NO: 9 and its complementary sequence, respectively, wherein the B3 primer is SEQ ID NO: 10 and its complementary sequence At least one B3 primer that is at least one polynucleotide selected from the group consisting of the polynucleotides indicated by the above, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 and at least one LPf primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by a complementary sequence, wherein the LPb primer comprises SEQ ID NO: 18 and a polynucleotide each represented by its complementary sequence. A primer set comprising at least one LPb primer that is at least one polynucleotide selected from the group;
(2) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is a polynucleotide selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, and a polynucleotide respectively represented by its complementary sequence, The F3 primer includes at least one F3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides represented by SEQ ID NO: 9 and its complementary sequence, and the B3 primer includes SEQ ID NO: 2 And at least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by its complementary sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: A primer set comprising at least one LPf primer that is at least one polynucleotide selected from the group consisting of polynucleotides represented by 16 and SEQ ID NO: 17 and the complementary sequences, respectively;
(3) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is a polynucleotide selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, and a polynucleotide respectively represented by its complementary sequence, The F3 primer includes at least one F3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides represented by SEQ ID NO: 9 and its complementary sequence, and the B3 primer includes SEQ ID NO: 2 And at least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by its complementary sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and at least one LPf primer that is at least one polynucleotide selected from the group consisting of polynucleotides respectively represented by complementary sequences, wherein the LPb primer is represented by SEQ ID NO: 18 and its complementary sequence, respectively. A primer set comprising at least one LPb primer that is at least one polynucleotide selected from the group consisting of:
(4) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is a polynucleotide selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, and a polynucleotide respectively represented by its complementary sequence, The F3 primer includes at least one F3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides represented by SEQ ID NO: 9 and its complementary sequence, and the B3 primer includes SEQ ID NO: 2 And at least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by its complementary sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and at least one LPf primer that is at least one polynucleotide selected from the group consisting of polynucleotides respectively represented by complementary sequences, wherein the LPb primer is represented by SEQ ID NO: 22 and its complementary sequence, respectively. A primer set comprising at least one LPb primer that is at least one polynucleotide selected from the group consisting of:
(5) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is a polynucleotide selected from the group consisting of the polynucleotides represented by SEQ ID NO: 24 and SEQ ID NO: 25 and their complementary sequences, respectively, and the F3 primer is Comprising at least one F3 primer that is at least one polynucleotide selected from the group consisting of the polynucleotides respectively represented by SEQ ID NO: 9 and its complementary sequence, wherein the B3 primer comprises SEQ ID NO: 23 and its phase At least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by a sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and a sequence A primer set comprising at least one LPf primer that is at least one polynucleotide selected from the group consisting of the polynucleotide number 17 and the polynucleotide each represented by a complementary sequence;
(6) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is a polynucleotide selected from the group consisting of the polynucleotides represented by SEQ ID NO: 24 and SEQ ID NO: 25 and their complementary sequences, respectively, and the F3 primer is Comprising at least one F3 primer that is at least one polynucleotide selected from the group consisting of the polynucleotides respectively represented by SEQ ID NO: 9 and its complementary sequence, wherein the B3 primer comprises SEQ ID NO: 23 and its phase At least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by a sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and a sequence Polynucleotides comprising at least one LPf primer which is at least one polynucleotide selected from the group consisting of No. 17 and a polynucleotide each represented by a complementary sequence, wherein the LPb primer is represented by SEQ ID No. 18 and its complementary sequence, respectively. A primer set comprising at least one LPb primer that is at least one polynucleotide selected from the group consisting of:
(7) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is at least one polynucleotide selected from the group consisting of the polynucleotides represented by SEQ ID NO: 26 and SEQ ID NO: 27 and their complementary sequences, respectively, and the F3 primer is Comprising at least one F3 primer that is at least one polynucleotide selected from the group consisting of the polynucleotides respectively represented by SEQ ID NO: 9 and its complementary sequence, wherein the B3 primer comprises SEQ ID NO: 23 and its phase At least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by a sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and a sequence No. 17 and a primer set comprising at least one LPf primer which is a polynucleotide selected from the group consisting of the polynucleotides respectively represented by complementary sequences; or (8) the FIP primer is SEQ ID NO: 1, SEQ ID NO: 3 , SEQ ID NO: 4 and SEQ ID NO: 5, and at least one FIP primer which is a polynucleotide selected from the group consisting of polynucleotides respectively represented by complementary sequences, wherein the BIP primer is a sequence No. 26 and SEQ ID NO: 27 and at least one BIP primer which is a polynucleotide selected from the group consisting of polynucleotides respectively represented by the complementary sequences thereof, wherein the F3 primer is represented by SEQ ID NO: 9 and its complementary sequences At least one F3 primer that is at least one polynucleotide selected from the group consisting of each of the polynucleotides shown, wherein the B3 primer is at least one from the group consisting of the polynucleotides respectively represented by SEQ ID NO: 23 and its complementary sequence Comprising at least one B3 primer which is a selected polynucleotide, wherein said LPf primer comprises SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 and complementary sequences At least one LPf primer that is at least one polynucleotide selected from the group consisting of the polynucleotides shown, wherein the LPb primer is from the group consisting of the polynucleotides respectively represented by SEQ ID NO: 18 and its complementary sequence A primer set comprising at least one LPb primer that is at least one selected polynucleotide;
May be a primer set.

Further examples of the LAMP amplification nucleic acid primer set for specifically amplifying nucleic acid derived from Mycoplasma, the LAMP amplification nucleic acid primer set comprises FIP primer, BIP primer, F3 primer and B3 primer,
(1) The FIP primer is a polynucleotide represented by SEQ ID NO: 56 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 57 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 55 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide respectively represented by SEQ ID NO: 58 and / or a complementary sequence, and the LPf primer is represented by SEQ ID NO: 59 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 60 and / or a complementary sequence;
(2) The FIP primer is a polynucleotide represented by SEQ ID NO: 64 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 65 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 53 and / or a polynucleotide represented by a complementary sequence, said B3 primer is a polynucleotide represented by SEQ ID NO: 56 and / or a complementary sequence, respectively, and said LPf primer is represented by SEQ ID NO: 67 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 68 and / or a complementary sequence;
(3) The FIP primer is a polynucleotide represented by SEQ ID NO: 72 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 73 and / or a complementary sequence, respectively, and the F3 primer is a sequence 71 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide represented by SEQ ID NO: 74 and / or a complementary sequence, respectively, and the LPf primer is a sequence represented by SEQ ID NO: 75 and / or a complementary sequence. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 76 and / or a complementary sequence;
(4) The FIP primer is a polynucleotide represented by SEQ ID NO: 80 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 81 and / or a complementary sequence, respectively, and the F3 primer is a sequence 79 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide represented by SEQ ID NO: 82 and / or a complementary sequence, respectively, and the LPf primer is a sequence represented by SEQ ID NO: 83 and / or a complementary sequence. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 84 and / or complementary sequence;
(5) The FIP primer is a polynucleotide represented by SEQ ID NO: 88 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 89 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 87 and / or a polynucleotide represented by a complementary sequence, said B3 primer is a polynucleotide represented by SEQ ID NO: 90 and / or a complementary sequence, respectively, and said LPf primer is represented by SEQ ID NO: 91 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 92 and / or a complementary sequence;
(6) The FIP primer is a polynucleotide represented by SEQ ID NO: 96 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 97 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 95 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide respectively represented by SEQ ID NO: 98 and / or a complementary sequence, and the LPf primer is represented by SEQ ID NO: 99 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 100 and / or a complementary sequence;
(7) The FIP primer is a polynucleotide represented by SEQ ID NO: 104 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 105 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 103 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide respectively represented by SEQ ID NO: 106 and / or a complementary sequence, and the LPf primer is represented by SEQ ID NO: 107 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 108 and / or a complementary sequence;
(8) The FIP primer is a polynucleotide represented by SEQ ID NO: 112 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 113 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 111 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide respectively represented by SEQ ID NO: 114 and / or a complementary sequence, and the LPf primer is represented by SEQ ID NO: 115 and / or a complementary sequence, respectively. A primer set each of which is a polynucleotide indicated and wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 116 and / or a complementary sequence; and / or (9) said The FIP primer is a polynucleotide indicated by SEQ ID NO: 120 and / or a complementary sequence, respectively, the BIP primer is a polynucleotide indicated by SEQ ID NO: 121 and / or a complementary sequence, respectively, and the F3 primer is SEQ ID NO: 119 and / or Or a polynucleotide represented by a complementary sequence, the B3 primer is a polynucleotide represented by SEQ ID NO: 122 and / or a complementary sequence, respectively, and the LPf primer is a polynucleotide represented by SEQ ID NO: 123 and / or a complementary sequence, respectively. A primer set that is a nucleotide and wherein the LPb primer is a polynucleotide respectively represented by SEQ ID NO: 124 and / or a complementary sequence;
May be a primer set.

  In the example of the primer set, the sequence containing a mutation within a range capable of specifically amplifying the mycoplasma-derived nucleic acid is any of the polynucleotides represented by the sequence described in any of the above SEQ ID NOs or a complementary sequence thereof. It refers to a sequence having mutations such as substitution, deletion and / or insertion in 1 to 5, preferably 1 to several nucleotides at a position. In the example of the primer, any mutation may be included in a part of at least one primer as long as the nucleic acid derived from mycoplasma can be specifically amplified. The specifically amplifiable range is a range in which a mutation exists within a range that is not necessarily required for taxonomic identification as mycoplasma.

  A plurality of sequences containing such mutations may be simultaneously present in one amplification 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 above SEQ ID NOs or a complementary sequence thereof may be prepared as a mixed nucleotide. Well, 1 to 5 nucleotides, preferably 1 to several nucleotides at any position may be universal nucleotides.

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

  Here, a sequence of about 1 to 100 nucleotides, preferably about 2 to 30 nucleotides (for example, a sequence used as a spacer) may be included between each sequence of the primer or on the terminal side thereof. The length of the primer may be about 30 to 200 nucleotides, preferably 35 to 100 nucleotides, and more preferably 43 to 60 nucleotides.

  In the example of the primer set, each of the FIP primer, the BIP primer, the F3 plummer, and the B3 primer may be composed of one kind of sequence, and each may contain a plurality of kinds of sequences. When LPf primer and / or LPb primer is further used in these primer sets, each of LPf primer and LPb primer may be composed of one kind of sequence, and may contain plural kinds of sequences.

  When amplification by a LAMP method or RT-LAMP method is performed on a specimen sample containing nucleic acid derived from mycoplasma using a primer using the region as described above, as described in FIG. A loop structure is formed, and a mycoplasma-derived target sequence contained within the single-stranded loop structure is obtained.

  In this embodiment, when the primer set as described above is applied to a sample containing nucleic acid derived from mycoplasma, the nucleic acid corresponding to the mycoplasma target sequence is specifically amplified. In addition, by detecting the presence of the amplification product obtained, it is possible to detect the presence of nucleic acid derived from mycoplasma. Detection of mycoplasma in a sample can be performed by performing an amplification reaction on the sample to be detected using the primer set.

  The method of specifically amplifying nucleic acid derived from mycoplasma is a method characterized by amplifying a sample using any of the above primer sets.

  As the conditions for the amplification reaction, any conditions known per se may be used. Moreover, what is necessary is just to determine with the said sample containing the nucleic acid derived from mycoplasma (it is also called "mycoplasma nucleic acid") based on the result of performing the said amplification reaction. Such determination can detect and classify the presence of mycoplasma nucleic acid in the sample.

  For example, the amplification reaction may be performed with one primer set per reaction field, for example, with one primer set per tube, or with a plurality of primer sets corresponding to various genotypes in one reaction field (for example, one tube). May be. When it is necessary to specify a plurality of mycoplasma species, the latter method is more efficient. The amplification reaction field may be, for example, a container capable of performing the reaction therein, and may be, for example, a tube, a microtube, a beaker, a multiwell plate, or the like, but is not limited thereto.

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

  In addition, a plurality of sequences having different base types may be represented by a single sequence for the mutation part. In that case, the base of the mutation part in question should just contain the symbol by 1 base display which shows several bases. The meanings of the bases indicated by these symbols are those commonly used in the art. That is, “r” is guanine or adenine, “y” is thymine or cytosine, “m” is adenine or cytosine, “k” is guanine or thymine, “s” is guanine or cytosine, “w” is adenine or thymine, “B” is guanine or cytosine or thymine, “d” is adenine or guanine or thymine, “h” is adenine or cytosine or thymine, “v” is adenine or guanine or cytosine, “n” is adenine or guanine or cytosine or It is thymine.

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 RT-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
16 U Bst DNA polymerase
0.625U AMV Reverse transcriptase.

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 5 pmoL.

  Here, “sample” refers to body fluids such as humans, mice, rats, guinea pigs, hamsters, ferrets, rhesus monkeys, rabbits, dogs, cats, cows, pigs, sheep, seals and porpoises, birds, and other body fluids, tissues, and cells. , Feces and any substance present in the environment such as water, lake water, river water, sea water and soil. Alternatively, the sample may be a substance suspected of containing a microorganism classified into the genus Mycoplasma 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 pretreatment include denaturation, filtration, nucleic acid extraction, impurity removal and the like.

  By performing nucleic acid amplification using the primer set as described above, it is possible to specifically amplify mycoplasma nucleic acid simply, inexpensively and at high speed. Utilizing this, the nucleic acid contained in the sample is subjected to an RT-LAMP or LAMP amplification reaction using the primer or primer set, and the presence or absence of an amplification product resulting from the amplification reaction is determined, whereby the mycoplasma contained in the sample It is possible to specifically detect the nucleic acid derived from it.

  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, as a rapid diagnosis method, a method in which a region containing a characteristic sequence is amplified by polymerase chain reaction (PCR) and detected by electrophoresis is known. However, the method using PCR has disadvantages such as requiring a complicated temperature control device such as a thermal cycler and 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 amplify nucleic acid derived from mycoplasma specifically, more simply, more accurately, more rapidly, and in a wider detection range.

  In conventional human and animal clinical diagnostic settings, cell-based research, epidemiological research, and other various fields, there is a development of methods that can detect and quickly identify the presence of gene sequences characteristic of mycoplasma. Although required, such a requirement is also satisfied by this aspect.

2. Nucleic acid probes This aspect further provides nucleic acid probes and nucleic acid probe sets for detecting and / or classifying nucleic acids derived from mycoplasma amplified by the above-described primers.

  The nucleic acid probe may be a nucleic acid probe for comprehensively detecting nucleic acids derived from mycoplasma, or may be a nucleic acid probe for specifically detecting a desired species. Such a nucleic acid probe should just be set so that it may couple | bond with the area | region of the single-stranded loop part of the polynucleotide which has the stem and loop structure amplified by the above-mentioned primer, for example. In addition, the primer preferably has a length of about 10 to 60 bases or about 12 to 40 bases.

  For example, preferred nucleic acid probes include a stem-and-loop structure of primer regions F1 and F2 as shown in FIG. 1 (this region may include the F2 region), and between primer regions F2c and F1c. (This region may include the F2c region), between the primer regions B1 and B2 (this region may include the B2 region), and / or between the primer regions B2c and B1c (this region may include the B2c region). May be selected to correspond to any position).

An example of a nucleic acid probe for specifically detecting mycoplasma from an amplification product obtained by amplification with the primer set is as follows:
(1) SEQ ID NO: 127 and SEQ ID NO: 128;
(2) SEQ ID NO: 131, SEQ ID NO: 132 and SEQ ID NO: 133;
(3) SEQ ID NO: 136 and SEQ ID NO: 139;
(4) SEQ ID NO: 137 and SEQ ID NO: 138;
(5) SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150;
(6) SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102 SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 125 and SEQ ID NO: 126; and (7) a complementary sequence of the sequences described in (1) to (6) above;
At least one nucleic acid probe selected from the group of nucleic acid probes each containing a polynucleotide represented by.

  Examples of nucleic acid probes for detecting and / or classifying specific species of mycoplasmas are SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150, and at least one kind from a nucleic acid probe group comprising polynucleotides respectively represented by their complementary sequences A nucleic acid probe comprising a selected sequence.

  In the example of the nucleic acid probe or the nucleic acid probe set, a mutation may be included as long as the nucleic acid derived from mycoplasma can be specifically detected. The specifically detectable range is a range in which a mutation exists within a range that is not necessarily required for taxonomic identification as mycoplasma. For example, each of the above-described nucleic acid probes may be composed of a sequence in which one or several bases at any position in the sequence represented by the above SEQ ID No. are substituted, deleted, or inserted. The nucleic acid probe is changed according to the genetic polymorphism or mutation held by the mycoplasma strain and / or species to be detected, and one or a number of bases at any position in the sequence indicated by the above SEQ ID NO. It may consist of a sequence in which individual substitutions, deletions or bases are inserted. A plurality of sequences containing such mutations may be simultaneously present 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 above SEQ ID NOs or a complementary sequence thereof may be prepared as a mixed nucleotide. Well, 1 to 5 nucleotides, preferably 1 to several nucleotides at any position may be universal nucleotides.

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

  Here, a sequence of about 1 to 100 nucleotides, preferably about 2 to 30 nucleotides (for example, a sequence used as a spacer) may be included between each sequence of the primer or on the terminal side thereof. The length of the primer may be about 30 to 200 nucleotides, preferably 35 to 100 nucleotides, and more preferably 43 to 60 nucleotides.

  Any nucleic acid probe and nucleic acid probe set may be used to detect the amplification product derived from the above-mentioned mycoplasma as desired. The nucleic acid probe may be immobilized on the surface of a solid phase substrate or used in a liquid phase.

  When used in a solid phase substrate, it may typically be used as a nucleic acid probe constituting 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. Moreover, when using it fix | immobilizing on the solid-phase base | substrate surface, it may be used by 1 type in 1 area | region for obtaining 1 signal in the said base | substrate, or may be used by multiple types. When used in multiple types, two, two or more, or all nucleic acid probes containing the respective sequences may be used together. Further, any one or more bases constituting these sequences may be designed as a mixed base. Here, the “mixed base” is a mixture of two or more nucleic acid probes designed as “adenine”, “thymine”, “cytosine”, and “guanine” as bases at desired positions to be mixed bases. A nucleic acid probe set. 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.

  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 chains can be used. In addition, a chimeric nucleic acid of any of these nucleic acids may be used.

  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-immobilized 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 immobilization chip is shown in FIG. 4 as a schematic diagram. The nucleic acid probe immobilization chip 1 includes an immobilization region 3 on a base 2. The nucleic acid probe 4 is immobilized on the immobilization region 3. Such a nucleic acid probe immobilization chip 1 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 the arrangement of the immobilization regions 3 arranged on the base 2 as necessary. Such a nucleic acid probe-immobilized chip may be suitably used for a detection method using fluorescence. In one immobilization region 3, 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 11 in FIG. 5 includes an electrode 13 as an immobilization region on a base 12. The nucleic acid probe 14 is immobilized on the electrode 13. The electrode 13 is connected to the pad 15. Electrical information from the electrode 13 is acquired via the pad 15. Such a nucleic acid probe immobilization chip 11 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 of electrodes 13 arranged on the substrate 12 and the arrangement thereof as necessary. Furthermore, the nucleic acid probe-immobilized chip 11 of this example may include a reference electrode and a counter electrode as necessary. In one immobilization region, 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.

  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 in this embodiment include deoxyinosine (dI), Gren Research 3-Nitropyrrole, 5-Nitroindole, deoxyribofuranosyl (dP), deoxy-5'-dimethoxytrityl-D-ribofuranosyl (dK), etc. including.

  The surface for immobilizing the nucleic acid probe used in this embodiment is not particularly limited, but resin beads, magnetic beads, metal fine particles, microtiter plates, glass substrates, silicon substrates, resin substrates, electrode substrates, etc. May be used.

  The substrate material used in this embodiment is not particularly limited. For example, inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used. In addition, polyethylene, ethylene, polypropylene, polyisobutylene, polymethyl methacrylate, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, Organic materials such as polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, polysulfone may be used. .

  For example, when using electrical properties, it is preferable to place the electrode on the substrate. The electrode is not particularly limited. For example, simple metals such as gold, gold alloy, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium, tungsten, and alloys thereof, or graphite, glassy Carbon such as carbon, or oxides or compounds thereof may be used. Furthermore, various semiconductor devices such as semiconductor compounds such as silicon oxide, CCD, FET, and CMOS may be used.

  The extraction method is not particularly limited, and a liquid-liquid extraction method such as a phenol-chloroform method or a solid-liquid extraction method using a carrier can be used. Further, a commercially available nucleic acid extraction method QIAamp (manufactured by QIAGEN), (manufactured by Co., Ltd.) or the like can be used. Next, RT-LAMP amplification is performed using the extracted nucleic acid component as a template, and then a hybridization reaction is performed with the gene detection electrode. The reaction solution is carried out in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In this solution, it is possible to add dextran sulfate as a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant, and the like. The extracted and amplified nucleic acid component is added to this and then donated to the hybridization reaction with the nucleic acid probe. During the reaction, the reaction rate can be increased by an operation such as stirring or shaking. The reaction temperature is in the range of 10 ° C to 90 ° C, and the reaction time is about 1 minute to overnight. For washing after the hybridization reaction, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 is used.

  Extracted and amplified sample nucleic acid is labeled in advance with fluorescent dyes such as FITC, Cy3, Cy5, rhodamine, enzymes such as biotin, hapten, oxidase and phosphatase, and electrochemically active substances such as ferrocene and quinones. Alternatively, detection may be performed by using a second probe labeled with the aforementioned substance.

  When detection is performed using an electrochemically active nucleic acid binding substance, detection is performed according to the following procedure. After the substrate is washed, a nucleic acid binding substance that selectively binds to the double-stranded portion formed on the electrode surface may be allowed to act to perform electrochemical measurement. Although the nucleic acid binding substance used here is not particularly limited, for example, Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator, polyintercalator, etc. May be used. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene or viologen. The concentration of the nucleic acid binding substance varies depending on the type, but is generally used in the range of 1 ng / ml to 1 mg / ml. At this time, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 is used. After the electrode is reacted with the nucleic acid binding substance, electrochemical measurement is performed. The electrochemical measurement is performed with a three-electrode type, that is, a reference electrode, a counter electrode, and a working electrode, or a two-electrode type, that is, a counter electrode and a working electrode. In the measurement, a potential higher than the potential at which the nucleic acid binding substance reacts electrochemically is applied, and the reaction current value derived from the nucleic acid binding substance is measured. At this time, the potential may be swept at a constant speed, applied with a pulse, or a constant potential may be applied. For the measurement, current and voltage are controlled by using a potentiostat, a digital multimeter, a function generator or the like. Based on the obtained current value, the concentration of the target gene is calculated from the calibration curve. A gene detection apparatus using a gene detection electrode includes a gene extraction unit, a gene reaction unit, a nucleic acid binding substance reaction unit, an electrochemical measurement unit, a washing unit, and the like.

3. Assay Kit According to one embodiment, an assay kit for detecting and / or classifying mycoplasma may be provided. According to a further embodiment, an assay kit for genotyping and / or species classification of mycoplasma may be provided.

  Such an assay kit may comprise a primer or primer set as described above and a nucleic acid probe or nucleic acid probe set. The assay kit may contain, in addition to the primer or primer set and the nucleic acid probe or nucleic acid probe set, a buffer necessary for LAMP amplification and / or RT-LAMP amplification, a buffer necessary for hybridization, and 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, and the assay kit may further include a substrate for immobilization.

  The reaction between the amplification product and the nucleic acid probe may be carried out under any known condition that allows hybridization using any of the above-described nucleic acid probes.

  The LAMP product has a characteristic complex higher-order structure. Such a publicly known structure is known to cause a physical obstacle with a substrate to which a nucleic acid probe is bound during a hybridization reaction. Such obstacles adversely affect hybridization efficiency (see, for example, Japanese Patent Application Laid-Open No. 2005-95043). In order to avoid such adverse effects, the nucleic acid probe is different from the position of the conventional target sequence so that a target sequence derived from a microorganism classified into the genus Mycoplasma is located in a single-stranded loop portion. The primer is designed.

  According to this aspect, a method for detecting microorganisms classified into the Mycoplasma genus at high speed and at a high speed and at a high speed and classifying the genotype at the genus level or species level is provided.

4). Method for detecting and / or classifying mycoplasma According to a further aspect, a method for detecting and / or classifying mycoplasma is provided. The detection method is a method of detecting microorganisms classified into mycoplasma and genotyping at the genus level or species level by using the above-described primer, nucleic acid probe, and amplification method.

  For example, a step of amplifying RT-LAMP or LAMP of a nucleic acid contained in a sample using any of the above primer sets, reacting an amplification product generated by the amplification with any of the above nucleic acid probes, The method may comprise detecting and / or classifying mycoplasma present in the sample from the result of the reaction.

  Also, for example, using any of the above primer sets, performing a RT-LAMP or LAMP amplification reaction on the sample, reacting the amplification product generated by the amplification reaction with any of the above nucleic acid probes, It may be a method comprising determining the presence or absence of mycoplasma in the sample by detecting the presence or absence of the resulting hybridization and / or classifying the mycoplasma in the sample.

Also, for example, a method for detecting the infection and presence of microorganisms classified as mycoplasmas
Performing an amplification reaction on a sample using at least one primer set arbitrarily selected from the primers according to the above-described embodiments;
It may be a method characterized by determining whether or not mycoplasma exists in the sample based on the presence or absence of an amplification product generated by the amplification reaction.

  The sample used for detection may be pretreated by means known per se, depending on its state and the components contained. Such pretreatment may be, for example, homogenate, nucleic acid extraction and / or purification.

  The amplification reaction may be LAMP amplification or RT-LAMP amplification. Amplification may be performed under any known conditions that can amplify the nucleic acid to be amplified using any of the primers described above. The presence or absence of the amplification product may be determined by detecting white turbidity due to the amplification product, or may be performed by detecting hybridization occurring between the amplification product and any of the above-described nucleic acid primers.

  The reaction between the amplification product and the nucleic acid probe may be carried out under any known condition that allows hybridization using any of the above-described nucleic acid probes.

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

  In addition, as described above, up to now, as a rapid diagnosis method, DNA is prepared from viral RNA by reverse transcriptase, and then a region containing a characteristic sequence in the DNA sequence is amplified by polymerase chain reaction (PCR), A method for detecting by electrophoresis (RT-PCR method) is known. However, the method using PCR has disadvantages such as requiring a complicated temperature control device such as a thermal cycler and a problem in simplicity. Also, in the PCR method, if complementary strand synthesis is performed by mistake, the product will be amplified as a template and may be erroneously determined. Actually, it is difficult to control specific amplification only by one base difference at the end of the primer. As a method for raising the specificity at the time of sequence detection, there is generally a technique of detecting a target gene product amplified by a PCR method with a DNA chip, but the product generated by PCR is double-stranded. Therefore, when a hybridization reaction is carried out with a nucleic acid probe, the problem is that the complementary strand becomes a competitor to the nucleic acid probe and the hybridization efficiency is low and the detection sensitivity is poor. According to this embodiment, it is possible to solve such problems caused by the PCR method.

  In addition, there are conventional methods such as decomposing or separating complementary strands to make the target single-stranded, but these methods are all expensive due to the use of enzymes, magnetic beads, etc. The complexity of the operation is a problem. Such a problem can also be solved by this embodiment.

[Example]
Hereinafter, the nucleic acid detection method according to this embodiment will be specifically described with reference to examples.

  In the microbial species constituting the genus Mycoplasma, as a result of microbial genome analysis, a partial region of the gene sequence encoding 16s-ribosomal RNA is highly conserved while retaining a unique sequence for each microbial species. I found. Therefore, first, six species of the genus Mycoplasma, that is, M.argininim, M.orale, M.hyorhinis, M.bovis, M.fermentans, and M.pulmonis were selected as representative rows of the genus Mycoplasma. These species are known to be mixed with bovine, human and horse serum, and have been reported to be infected in various cultured cells. Using these as detection targets, an optimal primer / probe design gene sequence was designed to detect the gene region. Various experiments described below were performed based on these sequences. Accordingly, preferred primer sets and nucleic acid probes were selected. It was confirmed that the selected primer set and nucleic acid probe had favorable characteristics with respect to the detection signal amount and detection specificity when the target nucleic acid was finally detected.

  Furthermore, for other species of the genus Mycoplasma, namely M. bovirhinis, M. bovigenitalium, M. arginini, Malkalescens, M. californicum, M. canadense, M. haemofelis, Candidatus Mycoplasma haemominutum and M. felis / Probe design Primers and nucleic acid probes were designed based on the sequence of the gene sequence region corresponding to the gene sequence. Each of the obtained primer and nucleic acid probe was confirmed for each nucleic acid detection property. They were confirmed to have desirable detection properties. Details are described below.

[Example 1]
Selection of primer set Six species from the genus Mycoplasma were selected, namely M.argininim, M.orale, M.hyorhinis, M.bovis, M.fermentans and M.pulmonis. For representative sequences of these species (ie Accession No .; AF125581, AY796060, AF258792, U02968, M24289 and AF125582), an alignment of the 16s-rRNA region was made. Primers were designed as follows with reference to the LAMP primer design method recommended by Eiken Chemical for areas with high storage stability on alignment. That is, a region common to the Mycoplasma genus and a region that can be identified are selected, and F1, F2, B1, and B2 are arranged so as to be assigned to two loops generated by LAMP amplification. Among the six species, LAMP amplification was performed using a plasmid (10 ³ copies / reaction) obtained by cloning a part of M.pulmonis genomic DNA as a template. Amplification was performed using Realop-30 (manufactured by Moritex) at 63 ° C. for 90 minutes using the LAMP reaction solution composition having the composition described below. The used primers are the FIP primer, BIP primer, F3 primer and B3 primer shown in Table 1-1 and Table 1-2.

Table 2 shows the turbidity rise time when amplification is performed using these.

From these results, two types of MpCS-1 and MpS-1 showed rise in turbidity from the 10 primer sets used (Table 2). Of these two types, MpS-1, which is a primer set with a high amplification rate, was selected. Using this primer, LAMP amplification was carried out as described later by adding further primers corresponding to 5 types other than the loop primer and M. pulmonis. The primer combinations used are listed in Table 3.

  The amount of primer to be added was assigned to the total primer amount according to the mutation ratio.

<LAMP reaction solution composition (1 ×)>
FIP primer 40 pmol
BIP primer 40 pmol
F3 primer 5 pmol
B3 primer 5 pmol
(Lfc / Lb 20 pmol)
2 x Reaction Mixture 12.5μL
8 U / μl Bst DNA polymerase 1μL
Template 1μL
Distilled water Up to 25 μL.

<Reaction mixture (reaction concentration)>
Tris-HCl (pH8.8) 20 mM
KCL 10 mM
MgSO ₄ 8 mM
(NH ₄ ) SO ₄ 10 mM
Tween-20 0.1%
Betaine 0.8 M
dNTPs 1.4 mM each.

Selection of Loop Primer A loop primer was designed and synthesized according to a conventional method for the primer set as a candidate above. LAMP amplification was performed in the same manner as described above by adding 20 pmol of loop primer to the above reaction composition. The mold used was treated at 95 ° C. for 5 minutes before addition. The loop primer described in the sequence shown in Table 1 was used. Amplification results are shown in Table 4 together with primer combinations.

  From the results in Table 4, a loop primer for each primer set used in the subsequent experiments was selected.

[Example 2]
There is a species-specific mutation in the set site of the amplification primer of Mycoplasma. A unique sequence for each such species, ie, a primer corresponding to the mutation was prepared. Using this, the allocation according to the mutation ratio was performed. Furthermore, the total primer amount was changed in several steps. From the results, conditions were set for efficiently amplifying nucleic acids derived from six species of Mycoplasma sp. Specifically, genomic DNAs of the above six microbial species were prepared, and optimum conditions for the total primer amount and enzyme amount were searched using each genomic DNA as a template. As a result, it was confirmed that all species could be amplified in 20 minutes by using a genome equivalent to 10 ³ copies / reaction for 6 types of genomes in a composition with double the total amount of primers and double the amount of enzymes. . This condition was used in the following tests.

In addition, the amplification cross-reactivity was investigated about Acholeplasma laidrawii which is a related species of the genus Mycoplasma when the above primer set was used. Cross amplification was shown at 10 ⁶ copies / reaction or more. Therefore, as described later, the possibility of installing Acholeplasma laidrawii discriminating nucleic acid probe on a DNA chip was examined.

Table 5 shows primer sets selected as preferable from the above examination.

[Example 3]
Sequence design of nucleic acid probe A probe for detecting the LAMP amplification product amplified by the primer set was designed. First, a plurality of candidate sequences were designed. These candidate sequences were designed such that when the LAMP amplification product and the nucleic acid probe were hybridized, they hybridized in the single-stranded nucleic acid region of the LAMP amplification product. Candidate sequences were prepared as synthetic nucleic acids with a 3 ′ terminal thiol modification.

The amplification product of mycoplasma is designed so that the sequence that differs depending on the species is located in the F loop, and the common genus sequence is located in the B loop. Therefore, a nucleic acid probe for discriminating between M. pulmonis and related species A. laidrawii is included in the F-loop of the loop-and-stem structure as an amplification product, and a nucleic acid for commonly detecting the genus in the B-loop. The sequence of the nucleic acid probe was designed to include the probe. Candidate sequences for nucleic acid probes are listed in Table 6.

[Example 4]
Nucleic acid probe selection
An example of probe selection when a Mycoplasma pulmonis specimen is used is shown below.

  Using the nucleic acid probe candidate sequences described in Example 3, a nucleic acid probe-immobilized chip (also referred to as “nucleic acid probe-immobilized electrode”) as shown in FIG. 6 was prepared. Synthetic nucleic acid (3 ′ end is thiol-modified) composed of each nucleic acid probe candidate sequence was spotted on the gold electrode arranged on the substrate 10 for each type of the sequence. Thereby, the nucleic acid probe was immobilized on one electrode per sequence. Also, negative control probes were spotted against additional electrodes. Thereafter, the substrate was washed with ultrapure water and air-dried to produce a current detection type nucleic acid probe-immobilized chip. The prepared nucleic acid chip was immersed in the LAMP amplification product obtained in Example 2 to which 2 × SSC salt was added. This was allowed to stand at 35 ° C. for 60 minutes to carry out a hybridization reaction. As the LAMP amplification product, a product obtained by amplifying a plasmid having a standard strain sequence as a template was used. Furthermore, this electrode was immersed for 15 minutes in a phosphate buffer containing 50 μM Hoechst 33258 as an intercalating agent. Thereafter, the oxidation current response of Hoechst 33258 molecules was measured. Table 6 shows an example of the result of current measurement when the Mycoplasma pulmonis specimen is used (see Table 6).

  A plurality of nucleic acid probes immobilized on the electrode were prepared in consideration of mutations existing in the region. An optimal nucleic acid probe showing an increase of 30 nA or more and a good nucleic acid probe showing an increase of 20-30 nA were obtained. Based on the above results, probe sequences to be mounted on the Mycoplasma genus and Mycoplasma pulmonis detection nucleic acid chip were selected.

In addition, the optimal nucleic acid probe selected above and a probe sequence designed at the same position on the genome are used in the same manner as described above, and amplification of LAMP from five Mycoplasma microorganisms other than Mycoplasma pulmonis and A. laidrawii The product was reacted. As a result, for these samples, an increase in current of 30 nA or more was obtained from any probe sequence when the nucleic acid probe and the sample nucleic acid test were in full match (Tables 7-1 and 7-2). .

[Example 5]
For other Mycoplasma genus microorganisms, gene regions corresponding to the primer / probe design gene sequences selected in Examples 1 to 6 were specified. The microorganisms used were M. bovirhinis, M. bovigenitalium, M. arginini, Malkalescens, M. californicum, M. canadense, M. haemofelis, Candidatus Mycoplasma haemominutum, M. felis. Accession No .; NR — 025986, AY121109, GQ409971, NR — 025984, NR — 029166, EU925158, AY529632, AY529634, NR029174. Based on these primer / probe design gene sequences, primers and nucleic acid probes corresponding to each microbial species were designed and synthesized. Those primers and nucleic acid probes are shown in Table 8-1, Table 8-2, Table 8-3, Table 8-4, and Table 8-5.

Six kinds of the above-mentioned representative Mycoplasma genera and the detection characteristics for the Mycoplasma genera including these were confirmed. As a result, it was confirmed that the constructed primer and nucleic acid probe can detect microbial species classified into the genus Mycoplasma with a specific and sufficient signal amount. Tables 9-1, 9-2, 9-3 and 9-4 show the detection results when using each primer and the nucleic acid probe for species discrimination.

[Example 6]
<Selection of primer set that does not cause non-specific amplification>
Primers composed of oligonucleotides of SEQ ID NO: 1 to SEQ ID NO: 27 shown in Table 10-1 and Table 10-2 were prepared.

LAMP amplification was performed using these primers as a primer set. The rise time of turbidity when a plasmid (10 ³ copies / reaction) in which a part of Mycoplasma pulmonis was cloned was used as a template, and the rise time of nonspecific amplification turbidity when none of the templates were contained were measured. The results and the composition of the primer set used are shown in Table 11-1 and Table 11-2.

  As a result, except for the primer set 104-1, the rise time of the specific amplified turbidity was shorter than the rise time of the nonspecific amplified turbidity, which was a good primer set. It was suggested that the primer sets 102-1, 102-2, and 103-1 were better primer sets without the rise of nonspecific amplification turbidity. Furthermore, primer set 102-2 has a turbidity rise time of 30 minutes or less when Mycoplasma pulmonis is used as a template, and primer set 102-2 has a rise in nonspecific amplification turbidity when no template is contained. I couldn't see it. Therefore, it was suggested that the primer set 102-2 is the best among these primer sets.

  As described above, a primer capable of specifically amplifying the Mycoplasma genus, a nucleic acid probe capable of specifically detecting Mycoplasma, and a nucleic acid probe capable of performing good species discrimination have been provided. These primers had desirable amplification characteristics, ie amplification specificity and good amplification efficiency. In addition, these nucleic acid probes had desirable detection characteristics, that is, detection signal amount and detection specificity.
In the following, the embodiments described in the scope of the claims of the present application are appended.
[1] A nucleic acid primer set for LAMP amplification for specifically amplifying nucleic acid derived from mycoplasma, the nucleic acid primer set for LAMP amplification comprising a FIP primer, a BIP primer, an F3 primer, and a B3 primer,
FIP primers were SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120 and their complementary sequences, and at least one selected from the group consisting of polynucleotides each represented by any one of the aforementioned sequences containing mutations to the extent that the nucleic acid derived from mycoplasma can be specifically amplified At least one FIP primer comprising a polynucleotide to be
BIP primers were SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 81, SEQ ID NO: 89, SEQ ID NO: 97, SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 121 and their complementary sequences, and mycoplasma-derived nucleic acids are mutated within a specific amplification range At least one BIP primer comprising a polynucleotide selected from the group consisting of polynucleotides each represented by any of the sequences comprising:
F3 primer is SEQ ID NO: 9, SEQ ID NO: 28, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 79, SEQ ID NO: 87, SEQ ID NO: 95, SEQ ID NO: 103, SEQ ID NO: 111, SEQ ID NO: 119 and their complements. At least one kind of F3 primer comprising a sequence and at least one polynucleotide selected from the group consisting of polynucleotides each represented by any of the above-mentioned sequences containing a mutation within a range in which a nucleic acid derived from mycoplasma can be specifically amplified Yes,
The B3 primer was SEQ ID NO: 10, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 114, SEQ ID NO: 122 and its complementary sequence, and at least one polynucleotide comprising at least one polynucleotide selected from the group consisting of polynucleotides each represented by any one of the above sequences containing mutations within a range in which nucleic acid derived from mycoplasma can be specifically amplified F3 primer
Primer set.
[2] The primer set according to [1],
(1) The FIP primer is a polynucleotide represented by SEQ ID NO: 1 and / or its complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 6 and / or its complementary sequence, and the F3 primer is a sequence A primer set of No. 9 and / or its complementary sequence, wherein said B3 primer is a polynucleotide of SEQ ID No. 10 and / or its complementary sequence, and / or
(2) The FIP primer is a polynucleotide represented by SEQ ID NO: 29 and / or its complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 30 and / or its complementary sequence, and the F3 primer is a sequence No. 28 and / or a polynucleotide represented by a complementary sequence thereof, and the primer set wherein the B3 primer is a polynucleotide represented by SEQ ID NO: 31 and / or a complementary sequence thereof
Primer set.
[3] The primer set according to [1], further including a loop primer,
The loop primer comprises an LPf primer and / or an LPb primer;
The LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 83, SEQ ID NO: 91, SEQ ID NO: 99, SEQ ID NO: 107, SEQ ID NO: 115 and SEQ ID NO: 123 and at least one kind of LPf primer comprising at least one polynucleotide selected from the group consisting of polynucleotides respectively represented by the complementary sequences thereof, wherein the LPb primer has the sequence SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84, SEQ ID NO: 92, SEQ ID NO: 100, SEQ ID NO: 108, SEQ ID NO: 1
16 and a primer set comprising at least one LPb primer comprising at least one polynucleotide selected from the group consisting of polynucleotides represented by SEQ ID NO: 124 and their complementary sequences.
[4] The primer set according to [3],
(1) At least one type of FIP primer wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. The BIP primer includes at least one BIP primer that is at least one selected from the group consisting of polynucleotides represented by SEQ ID NO: 6 and SEQ ID NO: 8 and their complementary sequences, and the F3 primer is At least one F3 primer, which is a polynucleotide selected from the group consisting of the polynucleotides represented by SEQ ID NO: 9 and its complementary sequence, respectively, wherein the B3 primer is SEQ ID NO: 10 and its complementary sequence At least one B3 primer that is at least one polynucleotide selected from the group consisting of the polynucleotides indicated by the above, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 and at least one LPf primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by a complementary sequence, wherein the LPb primer comprises SEQ ID NO: 18 and a polynucleotide each represented by its complementary sequence. A primer set comprising at least one LPb primer that is at least one polynucleotide selected from the group;
(2) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is a polynucleotide selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, and a polynucleotide respectively represented by its complementary sequence, The F3 primer includes at least one F3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides represented by SEQ ID NO: 9 and its complementary sequence, and the B3 primer includes SEQ ID NO: 2 And at least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by its complementary sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: A primer set comprising at least one LPf primer that is at least one polynucleotide selected from the group consisting of polynucleotides represented by 16 and SEQ ID NO: 17 and the complementary sequences, respectively;
(3) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is a polynucleotide selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, and a polynucleotide respectively represented by its complementary sequence, The F3 primer includes at least one F3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides represented by SEQ ID NO: 9 and its complementary sequence, and the B3 primer includes SEQ ID NO: 2 And at least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by its complementary sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and at least one LPf primer that is at least one polynucleotide selected from the group consisting of polynucleotides respectively represented by complementary sequences, wherein the LPb primer is represented by SEQ ID NO: 18 and its complementary sequence, respectively. A primer set comprising at least one LPb primer that is at least one polynucleotide selected from the group consisting of:
(4) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is a polynucleotide selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, and a polynucleotide respectively represented by its complementary sequence, The F3 primer includes at least one F3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides represented by SEQ ID NO: 9 and its complementary sequence, and the B3 primer includes SEQ ID NO: 2 And at least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by its complementary sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and at least one LPf primer that is at least one polynucleotide selected from the group consisting of polynucleotides respectively represented by complementary sequences, wherein the LPb primer is represented by SEQ ID NO: 22 and its complementary sequence, respectively. A primer set comprising at least one LPb primer that is at least one polynucleotide selected from the group consisting of:
(5) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is a polynucleotide selected from the group consisting of the polynucleotides represented by SEQ ID NO: 24 and SEQ ID NO: 25 and their complementary sequences, respectively, and the F3 primer is Comprising at least one F3 primer that is at least one polynucleotide selected from the group consisting of the polynucleotides respectively represented by SEQ ID NO: 9 and its complementary sequence, wherein the B3 primer comprises SEQ ID NO: 23 and its phase At least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by a sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and a sequence A primer set comprising at least one LPf primer that is at least one polynucleotide selected from the group consisting of the polynucleotide number 17 and the polynucleotide each represented by a complementary sequence;
(6) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is a polynucleotide selected from the group consisting of the polynucleotides represented by SEQ ID NO: 24 and SEQ ID NO: 25 and their complementary sequences, respectively, and the F3 primer is Comprising at least one F3 primer that is at least one polynucleotide selected from the group consisting of the polynucleotides respectively represented by SEQ ID NO: 9 and its complementary sequence, wherein the B3 primer comprises SEQ ID NO: 23 and its phase At least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by a sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and a sequence Polynucleotides comprising at least one LPf primer which is at least one polynucleotide selected from the group consisting of No. 17 and a polynucleotide each represented by a complementary sequence, wherein the LPb primer is represented by SEQ ID No. 18 and its complementary sequence, respectively. A primer set comprising at least one LPb primer that is at least one polynucleotide selected from the group consisting of:
(7) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is at least one polynucleotide selected from the group consisting of the polynucleotides represented by SEQ ID NO: 26 and SEQ ID NO: 27 and their complementary sequences, respectively, and the F3 primer is Comprising at least one F3 primer that is at least one polynucleotide selected from the group consisting of the polynucleotides respectively represented by SEQ ID NO: 9 and its complementary sequence, wherein the B3 primer comprises SEQ ID NO: 23 and its phase At least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by a sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and a sequence A primer set comprising at least one LPf primer that is at least one polynucleotide selected from the group consisting of the polynucleotides each indicated by number 17 and the complementary sequence; or
(8) At least one type of FIP primer, wherein the FIP primer is a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and a polynucleotide respectively represented by a complementary sequence. Wherein the BIP primer is at least one BIP primer that is at least one polynucleotide selected from the group consisting of the polynucleotides represented by SEQ ID NO: 26 and SEQ ID NO: 27 and their complementary sequences, respectively, and the F3 primer is Comprising at least one F3 primer that is at least one polynucleotide selected from the group consisting of the polynucleotides respectively represented by SEQ ID NO: 9 and its complementary sequence, wherein the B3 primer comprises SEQ ID NO: 23 and its phase At least one B3 primer that is at least one polynucleotide selected from the group consisting of polynucleotides each represented by a sequence, wherein the LPf primer is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and a sequence Polynucleotides comprising at least one LPf primer which is at least one polynucleotide selected from the group consisting of No. 17 and a polynucleotide each represented by a complementary sequence, wherein the LPb primer is represented by SEQ ID No. 18 and its complementary sequence, respectively. A primer set comprising at least one LPb primer that is at least one polynucleotide selected from the group consisting of:
Primer set.
[5] The primer set according to [3],
(1) The FIP primer is a polynucleotide represented by SEQ ID NO: 56 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 57 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 55 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide respectively represented by SEQ ID NO: 58 and / or a complementary sequence, and the LPf primer is represented by SEQ ID NO: 59 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 60 and / or a complementary sequence;
(2) The FIP primer is a polynucleotide represented by SEQ ID NO: 64 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 65 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 53 and / or a polynucleotide represented by a complementary sequence, said B3 primer is a polynucleotide represented by SEQ ID NO: 56 and / or a complementary sequence, respectively, and said LPf primer is represented by SEQ ID NO: 67 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 68 and / or a complementary sequence;
(3) The FIP primer is a polynucleotide represented by SEQ ID NO: 72 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 73 and / or a complementary sequence, respectively, and the F3 primer is a sequence 71 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide represented by SEQ ID NO: 74 and / or a complementary sequence, respectively, and the LPf primer is a sequence represented by SEQ ID NO: 75 and / or a complementary sequence. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 76 and / or a complementary sequence;
(4) The FIP primer is a polynucleotide represented by SEQ ID NO: 80 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 81 and / or a complementary sequence, respectively, and the F3 primer is a sequence 79 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide represented by SEQ ID NO: 82 and / or a complementary sequence, respectively, and the LPf primer is a sequence represented by SEQ ID NO: 83 and / or a complementary sequence. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 84 and / or complementary sequence;
(5) The FIP primer is a polynucleotide represented by SEQ ID NO: 88 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 89 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 87 and / or a polynucleotide represented by a complementary sequence, said B3 primer is a polynucleotide represented by SEQ ID NO: 90 and / or a complementary sequence, respectively, and said LPf primer is represented by SEQ ID NO: 91 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 92 and / or a complementary sequence;
(6) The FIP primer is a polynucleotide represented by SEQ ID NO: 96 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 97 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 95 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide respectively represented by SEQ ID NO: 98 and / or a complementary sequence, and the LPf primer is represented by SEQ ID NO: 99 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 100 and / or a complementary sequence;
(7) The FIP primer is a polynucleotide represented by SEQ ID NO: 104 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 105 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 103 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide respectively represented by SEQ ID NO: 106 and / or a complementary sequence, and the LPf primer is represented by SEQ ID NO: 107 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, wherein said LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 108 and / or a complementary sequence;
(8) The FIP primer is a polynucleotide represented by SEQ ID NO: 112 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 113 and / or a complementary sequence, respectively, and the F3 primer is a sequence No. 111 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide respectively represented by SEQ ID NO: 114 and / or a complementary sequence, and the LPf primer is represented by SEQ ID NO: 115 and / or a complementary sequence, respectively. A primer set, each of the polynucleotides indicated, and wherein the LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 116 and / or a complementary sequence; and / or
(9) The FIP primer is a polynucleotide represented by SEQ ID NO: 120 and / or a complementary sequence, the BIP primer is a polynucleotide represented by SEQ ID NO: 121 and / or a complementary sequence, respectively, and the F3 primer is a sequence 119 and / or a polynucleotide respectively represented by a complementary sequence, the B3 primer is a polynucleotide represented by SEQ ID NO: 122 and / or a complementary sequence, respectively, and the LPf primer is a sequence represented by SEQ ID NO: 123 and / or a complementary sequence. A primer set, each of the polynucleotides indicated, wherein the LPb primer is a polynucleotide respectively indicated by SEQ ID NO: 124 and / or a complementary sequence;
Primer set.
[6] An assay kit for detecting and / or classifying mycoplasma,
The primer set according to any one of [1] to [5],
A nucleic acid probe for specifically detecting mycoplasma from an amplification product obtained by amplification by the primer set,
(1) SEQ ID NO: 127 and SEQ ID NO: 128;
(2) SEQ ID NO: 131, SEQ ID NO: 132 and SEQ ID NO: 133;
(3) SEQ ID NO: 136 and SEQ ID NO: 139;
(4) SEQ ID NO: 137 and SEQ ID NO: 138;
(5) SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150;
(6) SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102 SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 125 and SEQ ID NO: 126; and
(7) a complementary sequence of the sequence described in (1) to (6) above;
An assay kit comprising at least one nucleic acid probe selected from the group of nucleic acid probes each comprising a polynucleotide represented by
[7] An assay kit for detecting and / or classifying mycoplasma,
The primer set according to any one of [1] to [5],
A nucleic acid probe for detecting a desired species of mycoplasma from an amplification product obtained by amplification using the primer set, which is SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141 SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150, and their respective complementary sequences. A nucleic acid probe comprising a sequence selected from at least one nucleic acid probe group;
An assay kit comprising:
[8] A method for specifically amplifying a nucleic acid derived from mycoplasma, wherein the sample is amplified using the primer set according to any one of [1] to [5].
[9] A method for detecting mycoplasma,
Performing an amplification reaction on the sample using the primer set according to any one of [1] to [5],
Determining whether mycoplasma is present in the sample based on the presence or absence of an amplification product generated by the amplification reaction;
A method characterized by.
[10] A method for detecting and / or classifying mycoplasma,
Performing an amplification reaction on the sample using the primer set according to any one of [1] to [5],
A reaction product generated by the amplification;
(1) SEQ ID NO: 127 and SEQ ID NO: 128;
(2) SEQ ID NO: 131, SEQ ID NO: 132 and SEQ ID NO: 133;
(3) SEQ ID NO: 136 and SEQ ID NO: 139;
(4) SEQ ID NO: 137 and SEQ ID NO: 138;
(5) SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150;
(6) SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102 SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 125 and SEQ ID NO: 126; and
(7) a complementary sequence of the sequence described in (1) to (6) above;
Reacting a nucleic acid probe group selected from at least one nucleic acid probe group consisting of polynucleotides respectively represented by
Detecting and / or classifying mycoplasma present in the sample from the results of the reaction;
A method characterized by.
[11] The method according to [10], wherein the nucleic acid probe group is fixed to a substrate.

1. 1. Nucleic acid probe immobilization chip Substrate 3. Immobilization area 4. Nucleic acid probe 11. Nucleic acid probe immobilization chip 12. Substrate 13. Electrode (ie immobilization region) 14. Nucleic acid probe 15. Pat

Claims (7)

A nucleic acid primer set for LAMP amplification for specifically amplifying nucleic acid derived from mycoplasma, wherein the nucleic acid primer set for LAMP amplification is FIP primer, BIP primer, F3 primer and B3 primer, and LPf primer and / or LPb primer Including
(1), the FIP primer SEQ ID NO: 1, SEQ ID NO: 3, a plurality of FIP primer or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 4 and SEQ ID NO: 5 a plurality of a polynucleotide shown respectively includes an FIP primer, comprising a plurality of BIP primer is a polynucleotide represented each by a plurality of BIP primers or their complementary sequence is a polynucleotide shown more to each of the BIP primer SEQ ID NO: 6 and SEQ ID NO: 8, wherein F3 primer comprises F3 primer is a polynucleotide shown Ri by the SEQ ID NO: 9 or a complementary sequence thereof, wherein the B3 primer is a polynucleotide and the B3 primer is shown Ri by the SEQ ID NO: 10 or a complementary sequence thereof, before LPf primer SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, by SEQ ID NO: 16 and SEQ ID NO: 1 7 more of a plurality of LPf primers or polynucleotides respectively represented by their complementary sequence is a polynucleotide shown respectively comprises LPf primer, a primer set comprising a LPb primer is a polynucleotide the LPb primer is shown Ri by the SEQ ID NO: 18 or a complementary sequence thereof;
(2) the FIP primer SEQ ID NO: 1, SEQ ID NO: 3, a plurality of FIP primer is a polynucleotide shown respectively by SEQ ID NO: 4 and SEQ ID NO: 5 or more FIP respectively polynucleotide shown by their complementary sequences includes a primer, a plurality of BIP primer is a polynucleotide represented each by a plurality of BIP primers or their complementary sequence is a polynucleotide shown more to each of the BIP primer SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 wherein, include F3 primer is a polynucleotide the F3 primer is shown Ri by the SEQ ID NO: 9 or a complementary sequence thereof, B3-flop is a polynucleotide and the B3 primer is shown Ri by the SEQ ID NO: 10 or its complementary sequence Includes a timer, said LPf primer SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, respectively polynucleotide shown by SEQ ID NO: 16 and SEQ ID NO: 17 multiple LPf primers or polynucleotides respectively represented by their complementary sequences A primer set comprising a plurality of LPf primers ;
(3), the FIP primer SEQ ID NO: 1, SEQ ID NO: 3, a plurality of FIP primer or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 4 and SEQ ID NO: 5 a plurality of a polynucleotide shown respectively includes an FIP primer, a plurality of BIP primer is a polynucleotide represented each by a plurality of BIP primers or their complementary sequence is a polynucleotide shown respectively by the BIP primer SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 wherein, include F3 primer is a polynucleotide the F3 primer is shown Ri by the SEQ ID NO: 9 or a complementary sequence thereof, B3-flop is a polynucleotide and the B3 primer is shown Ri by the SEQ ID NO: 10 or its complementary sequence Includes a timer, poly wherein LPf primer respectively represented by the plurality of LPf primers or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 includes a plurality of LPf primers are nucleotides, a primer set comprising a LPb primer is a polynucleotide the LPb primer is shown Ri by the SEQ ID NO: 18 or a complementary sequence thereof;
(4), the FIP primer SEQ ID NO: 1, SEQ ID NO: 3, a plurality of FIP primer or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 4 and SEQ ID NO: 5 a plurality of a polynucleotide shown respectively includes an FIP primer, a plurality of BIP primer is a polynucleotide the BIP primer are indicated respectively by a plurality of BIP primers or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 comprises the F3 primer SEQ ID NO: 9 or include F3 primer is a polynucleotide shown Ri by the complementary sequence thereof, B3-flop is a polynucleotide and the B3 primer is shown Ri by the SEQ ID NO: 10 or its complementary sequence Includes a timer, poly wherein LPf primer respectively represented by the plurality of LPf primers or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 includes a plurality of LPf primers are nucleotides, a primer set comprising a LPb primer is a polynucleotide the LPb primer is shown Ri by the SEQ ID NO: 22 or a complementary sequence thereof;
(5), the FIP primer SEQ ID NO: 1, SEQ ID NO: 3, a plurality of FIP primer or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 4 and SEQ ID NO: 5 a plurality of a polynucleotide shown respectively A plurality of BIP primers , each of which is a polynucleotide respectively represented by SEQ ID NO: 24 and SEQ ID NO: 25 or a complementary sequence thereof; include F3 primer of a polynucleotide primer is set forth Ri by the SEQ ID NO: 9 or a complementary sequence thereof, containing a B3 primer is a polynucleotide and the B3 primer is shown Ri by the SEQ ID NO: 10 or its complementary sequence The LPf primer SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 is the polynucleotide shown respectively by a plurality of LPf primers or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 16 and SEQ ID NO: 17 A primer set comprising a plurality of LPf primers ;
(6), the FIP primer SEQ ID NO: 1, SEQ ID NO: 3, a plurality of FIP primer or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 4 and SEQ ID NO: 5 a plurality of a polynucleotide shown respectively includes an FIP primer, comprising a plurality of BIP primer is a polynucleotide represented each by a plurality of BIP primers or their complementary sequence is a polynucleotide shown more to each of the BIP primer SEQ ID NO: 24 and SEQ ID NO: 25, wherein include F3 primer is a polynucleotide F3 primer is shown Ri by the SEQ ID NO: 9 or a complementary sequence thereof, wherein the B3 primer is a polynucleotide and the B3 primer is shown Ri by the SEQ ID NO: 10 or its complementary sequence Wherein LPf primer SEQ ID NO: 12, a plurality a polynucleotide shown SEQ ID NO: 14, SEQ ID NO: 15, respectively by a plurality of LPf primers or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 16 and SEQ ID NO: 17 the include LPf primer, the LPb primer set primers containing LPb primer is a polynucleotide shown Ri by the SEQ ID NO: 18 or a complementary sequence thereof; or (7) wherein the FIP primer SEQ ID NO: 1, SEQ ID NO: 3, SEQ includes a plurality of FIP primer is a polynucleotide shown respectively by a plurality of FIP primer or their complementary sequence is a polynucleotide shown more respectively to the numbers 4 and SEQ ID NO: 5, the BIP primer SEQ ID NO: 2 And a plurality of BIP primer is a polynucleotide represented each by a plurality of BIP primers or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 27, the F3 primer SEQ ID NO: 9 or a complementary sequence thereof Ri include F3 primer is a polynucleotide shown, includes a B3 primer is a polynucleotide and the B3 primer is shown Ri by the SEQ ID NO: 10 or a complementary sequence thereof, wherein LPf primer SEQ ID NO: 12, SEQ ID NO: 14, SEQ No. 15, includes a plurality of LPf primer is a polynucleotide shown respectively by a plurality of LPf primers or their complementary sequence is a polynucleotide shown more in SEQ ID NO: 16 and SEQ ID NO: 17, wherein LPb ply A primer set comprising a LPb primer is a polynucleotide mer is shown Ri by the SEQ ID NO: 18 or a complementary sequence thereof;
Primer set. An assay kit for detecting and / or classifying mycoplasma, comprising:
A primer set according to claim 1 ;
A nucleic acid probe for specifically detecting mycoplasma from an amplification product obtained by amplification by the primer set,
(1) SEQ ID NO: 127 and SEQ ID NO: 128;
(2) SEQ ID NO: 131, SEQ ID NO: 132 and SEQ ID NO: 133;
(3) SEQ ID NO: 136 and SEQ ID NO: 139;
(4) SEQ ID NO: 137 and SEQ ID NO: 138;
(5) SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150;
(6) SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102 SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 125 and SEQ ID NO: 126; and (7) a complementary sequence of the sequences described in (1) to (6) above;
An assay kit comprising at least one nucleic acid probe selected from the group of nucleic acid probes each comprising a polynucleotide represented by An assay kit for detecting and / or classifying mycoplasma, comprising:
A primer set according to claim 1 ;
A nucleic acid probe for detecting a desired species of mycoplasma from an amplification product obtained by amplification using the primer set, which is SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141 SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150, and their respective complementary sequences. A nucleic acid probe comprising a sequence selected from at least one nucleic acid probe group;
An assay kit comprising: A method for specifically amplifying nucleic acid derived from mycoplasma, wherein the sample is amplified using the primer set according to claim 1 . A method for detecting mycoplasma comprising:
Amplifying the sample using the primer set according to claim 1 ;
Determining whether or not mycoplasma is present in the sample based on the presence or absence of an amplification product generated by the amplification reaction. A method for detecting and / or classifying mycoplasma comprising:
Amplifying the sample using the primer set according to claim 1 ;
A reaction product generated by the amplification;
(1) SEQ ID NO: 127 and SEQ ID NO: 128;
(2) SEQ ID NO: 131, SEQ ID NO: 132 and SEQ ID NO: 133;
(3) SEQ ID NO: 136 and SEQ ID NO: 139;
(4) SEQ ID NO: 137 and SEQ ID NO: 138;
(5) SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150;
(6) SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102 SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 125 and SEQ ID NO: 126; and (7) a complementary sequence of the sequences described in (1) to (6) above;
Reacting a nucleic acid probe group selected from at least one nucleic acid probe group consisting of polynucleotides respectively represented by
Detecting and / or classifying mycoplasma present in the sample from the result of the reaction. The method according to claim 6 , wherein the nucleic acid probe group is fixed to a substrate.

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