JP3449961B2 - Pathogen detection by multi-primer PCR - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting pathogenic organisms that infect animals, and more particularly, PCR of microsporidia infected with animals or insect pathogenic organisms infected with insects.
Detection method.

[0002]

2. Description of the Related Art Currently, as pathogenic organisms for animals,
Various things such as viruses, bacteria, fungi, parasites and protozoa are known. Microsporida, which is one of the above pathogenic organisms, is a protozoan belonging to Microspora (Microsporida) and is known to parasitize a wide range of animal cells from protists to mammals. Moreover, it is known that the parasitic microsporidia cause various diseases in its host. For example, Nosema bombycis , which is a type of microsporidian parasite, mainly parasitizes silkworms and causes fine particle disease. Therefore, it is necessary to assay whether or not useful animals such as silkworms are infected with microspores as quickly as possible.

Conventionally, in general, in order to detect a pathogenic organism in the case of an infectious disease for which the pathogenicity cannot be determined, the pathogenic organism is predicted from the symptom, and a test for detecting each is conducted individually. Therefore, it was necessary to perform a plurality of tests for each individual, but as a method for such a test, a method using an antibody against a pathogen to be detected, a method of culturing cells of a pathogenic bacterium, etc. are mainly used. Therefore, it took a lot of time to test a large number of individuals.

[0004] In particular, since the assay of microspores that infect silkworms is performed by a method using a specific antibody that reacts with the spores of each microspore, it is necessary for a skilled technician to perform the assay. In addition, it could not be detected when spores were not formed, and it was very difficult to assay eggs with a low infection amount. Therefore, it would be convenient if a method capable of easily detecting a microsporidia was developed even by an unskilled person.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for detecting a microsporidian which is easy to operate, can be detected visually, and can be carried out by one operation.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a plurality of pathogenic organisms specific to a plurality of pathogenic organisms to be detected by the multi-primer PCR method are used. It has been found that these pathogenic organisms can be detected simultaneously by using a primer pair, and the present invention has been completed.

That is, the present invention comprises at least two primer pairs capable of amplifying a partial region of the genomic DNA of each of a plurality of types of microsporidia to be detected, using a nucleic acid existing in the body of a test animal as a template. Provided is a method capable of simultaneously detecting the plurality of types of microsporidia in the subject animal, which comprises performing amplification treatment using a mixed primer. This method of the present invention is hereinafter referred to as "first method of the present invention".

[0008] In the above method, the plurality of species of microspores to be detected are preferably Nosema bombysis, Bailimorpha sp. M11, Bailimorpha sp. M12,
Baimorpha Necatrix and Plastoforas
p. PSD is at least two species of microspores, more preferably Nosema bombysis, Baimorpha sp. M11, Baimorpha sp. M12, Baimorpha necatrix and Plastophora sp. PSD.
Is.

The mixed primer is preferably
(I) a primer pair consisting of an oligonucleotide containing the base sequence represented by SEQ ID NO: 1 and an oligonucleotide containing the base sequence represented by SEQ ID NO: 2, (ii) an oligonucleotide containing the base sequence represented by SEQ ID NO: 3 and a sequence A primer pair consisting of an oligonucleotide containing the base sequence represented by No. 4, (iii) a primer pair consisting of an oligonucleotide containing the base sequence represented by SEQ ID No. 5 and an oligonucleotide containing the base sequence represented by SEQ ID No. 6, and (Iv) SEQ ID NO: 7
Containing at least two primer pairs selected from the group consisting of an oligonucleotide containing the nucleotide sequence represented by and an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 8, and more preferably SEQ ID NO: 1 It includes an oligonucleotide containing the nucleotide sequence represented by 8. Further, the test animal is preferably an insect.

Further, the present invention comprises at least two primer pairs capable of amplifying a partial region of the genomic DNA of each of a plurality of insect pathogenic organisms to be detected, using a nucleic acid existing in the test insect body as a template. Provided is a method capable of simultaneously detecting the plurality of insect pathogenic organisms in the test insect body, which comprises performing amplification treatment using a mixed primer. This method of the present invention is hereinafter referred to as "second method of the present invention".
Say.

In the above method, it is preferable that the entomopathogenic organism is a microsporidia. In this case, the plurality of microsporidia to be detected is preferably selected from the group consisting of Nosema bombysis, Baimorpha sp. M11, Baimorpha sp. M12, Baimorpha necatrix and Plastophora sp. PSD. At least two types of microsporidia, more preferably Nosema bombysis, Bailimorpha sp. M11, Bailimorpha sp. M12, Bailimorpha necatrix and Plastophora sp. PSD.

The mixed primer is preferably
(I) a primer pair consisting of an oligonucleotide containing the base sequence represented by SEQ ID NO: 1 and an oligonucleotide containing the base sequence represented by SEQ ID NO: 2, (ii) an oligonucleotide containing the base sequence represented by SEQ ID NO: 3 and a sequence A primer pair consisting of an oligonucleotide containing the base sequence represented by No. 4, (iii) a primer pair consisting of an oligonucleotide containing the base sequence represented by SEQ ID No. 5 and an oligonucleotide containing the base sequence represented by SEQ ID No. 6, and (Iv) SEQ ID NO: 7
Containing at least two primer pairs selected from the group consisting of an oligonucleotide containing the nucleotide sequence represented by and an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 8, and more preferably SEQ ID NO: 1 It includes an oligonucleotide containing the nucleotide sequence represented by 8.

Further, the present invention provides Nosema bombysis,
Baimorpha sp. M11, Baimorpha sp. M12, Baimorpha necatrix and Plastophora sp. PS
Provided is a primer set for detecting a microsporidia, which comprises at least two primer pairs selected from the group consisting of primer pairs capable of amplifying a partial region of genomic DNA of D.

The above-mentioned primer set is preferably
(I) a primer pair consisting of an oligonucleotide containing the base sequence represented by SEQ ID NO: 1 and an oligonucleotide containing the base sequence represented by SEQ ID NO: 2, (ii) an oligonucleotide containing the base sequence represented by SEQ ID NO: 3 and a sequence A primer pair consisting of an oligonucleotide containing the base sequence represented by No. 4, (iii) a primer pair consisting of an oligonucleotide containing the base sequence represented by SEQ ID No. 5 and an oligonucleotide containing the base sequence represented by SEQ ID No. 6, and (Iv) SEQ ID NO: 7
Containing at least two primer pairs selected from the group consisting of an oligonucleotide containing the nucleotide sequence represented by and an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 8, and more preferably SEQ ID NO: 1 It includes an oligonucleotide containing the nucleotide sequence represented by 8.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. [First Method of the Present Invention] The present invention relates to a method capable of simultaneously detecting a plurality of types of microsporidia present in the body of a test animal,
This detection is performed using a nucleic acid present in the test animal as a template, and an amplification treatment using a mixed primer containing a primer pair capable of amplifying a partial region of the genomic DNA of each of a plurality of types of microsporidia to be detected. It can be carried out. Here, the mixed primer contains at least two kinds of primer pairs.

[0016] In the method of the first aspect of the present invention, the plurality of species of microspores to be detected are
It is not particularly limited as long as it is a protist that belongs to ra), but is preferably a protist that belongs to Microsporida, and more preferably, Nosema bombisis ( Nosema).
bombycis ), Baimorpha sp. M11 ( Vairimorpha sp.
M11), Bairimorpha sp. M12 ( Vairimorpha sp. M1
2), Bairimorufa-necatrix (Vairimorpha nec
atrix ) and Plastophora sp. PSD ( Pleistophora s
p. PSD) at least two species of microspores selected from the group consisting of Nosema bombysis, Bailimorpha sp. M11, Bailimorpha sp. M12, Bailimorpha necatrix and Plastophora sp. PSD. The Nosema bombysis includes both virulent and attenuated strains.

The primers contained in the mixed primer used in the method of the first aspect of the present invention can be designed by comparing the sequences of DNAs derived from a plurality of microsporidian insects to be detected with each other. DN used for sequence comparison
The region A may be any gene and is not particularly limited. For example, mitochondrial heat shock protein 70 (HSP70) gene, peptide elongation factor 1α
Examples include genes and small subunit rRNA genes. The nucleotide sequence of DNA used for sequence comparison may be a known nucleotide sequence and can be obtained from a database such as GenBank. A person skilled in the art can easily search and obtain an appropriate sequence from the sequences registered in the database according to the type of the microsporidia to be detected. Examples of the base sequence thus obtained include those shown in Table 1 below.

[0018]

[Table 1]

In order to design a primer for each microsporidia to be detected based on the above sequence comparison,
First, the obtained plurality of base sequences are aligned.
The alignment can be performed using analysis software, for example, ClustalX (freeware). All the parameters of the software at that time may remain the initial values, or may be changed as necessary. Those skilled in the art can easily change such parameters. Next, based on such an alignment (result of sequence comparison), the following criteria: (1) As a general rule, one kind of amplification product is formed for one kind of microsporidia; (2) Primer Design so that the mixed primers do not amplify undesired sites when mixed (change the primer sequence if undesired sites are amplified); (3) Target sequence to be amplified Need not necessarily be the same gene, but may differ from organism to organism; (4) design primers so that the products to be amplified can be separated by electrophoresis; it can. At this time, the number of bases of each primer is not particularly limited, but is preferably 15 bases or more, more preferably 20 bases or more.

The size of the DNA fragment amplified by each primer pair is not particularly limited as long as it can be detected by ordinary electrophoresis, but is preferably 100 bases to 3000 bases, more preferably 100 bases. ~ 2000 bases. Here, when the size of the DNA fragment amplified by each primer pair is dispersed to such an extent that the bands of each DNA fragment are clearly separated from each other by electrophoresis, the above method simultaneously detects each microsporidia. Not only can it be identified. However, such identification does not necessarily have to distinguish all the microsporidia to be detected. That is, the size of the amplified fragment derived from each of the microspores that need not be distinguished does not have to be different to the extent that they can be clearly separated by electrophoresis. The base sequence of the primer designed as described above is not limited to a specific sequence, and examples thereof include those containing the base sequences shown in Table 2 below.

[0021]

[Table 2]

In Table 2 above, NBEF35F and NBEF957R
Is a primer designed on the basis of the Nosema bombysis elongation factor 1α gene sequence, and can detect both attenuated and strongly virulent lines of Nosema bombysis. M1
1-96F and M11-822R are primers designed based on the small subunit rRNA gene sequence of Bilimorpha sp. M11, and can detect Bilimorpha sp. M11 only. V70-176F and V70-1898R are Baimorpha s
It is a primer designed based on the HSP70 gene sequence of p. M12, and can detect Bilimorpha sp. M12 and Bilimorpha necatrix (foreign species).
PSDF1 is a primer designed based on the consensus sequence of the microsporidian small subunit rRNA gene.
Is the small subunit rRN of Plaestora sp. PSD
It is a primer designed based on the A gene sequence. PSDF
1 and PSDR450 can detect only Plaestora sp. PSD. The last number in the name of each primer indicates the base number of the 3'terminal base of each primer in the sequence that is the basis of the design of that primer. Since these primers have very high specificity, they do not react with microspores and other pathogenic organisms other than those shown as the detection targets. The oligonucleotide (primer) designed as described above can be easily prepared by a method known to those skilled in the art, such as chemical synthesis.

The mixed primer used in the first method of the present invention contains two or more kinds of primer pairs designed as described above depending on the type of microsporidia to be detected. For example, when the primers listed in Table 2 above are used, two or more of all primer pairs can be mixed to form a mixed primer, depending on the type of microsporidia to be detected. Preferably, all of these primers are mixed to form a mixed primer.

The amplification treatment used in the first method of the present invention is not particularly limited, but is preferably polymerase chain reaction (PCR). Here, the template used for the amplification treatment is a nucleic acid existing in the body of the test animal, and preferably D
It is NA. Such nucleic acid can be extracted from the test animal by a method known to those skilled in the art. The above-mentioned mixed primer is used as the primer. Those skilled in the art can appropriately set the template, the primer, the reagents necessary for the amplification treatment, the treatment conditions, and the like. For example, P
When performing CR, in addition to the template and primer, Tris
-HCI, KCl, MgCl2, dNTPs, Taq DNA polymerase and other reagents can be mixed to form a PCR reaction solution,
Such reagents are also commercially available as a PCR kit. Then, the PCR reaction solution thus prepared is put into an apparatus such as a thermal cycler and a temperature cycle reaction is carried out to carry out an amplification treatment.

The detection of the amplification product obtained by the amplification treatment can be carried out by a known method capable of separating by the size (base length) of the product, preferably an electrophoresis method such as agarose gel electrophoresis. By such a method, it is possible to investigate whether or not an amplified fragment having a size predicted from each primer pair used in the amplification treatment is detected, and thus, the nucleic acid existing in the test animal body is detected by the mixed primer. It can be investigated whether or not it is amplified by. Here, a person skilled in the art can easily predict the size of an amplified fragment from a primer pair. As described above, if each primer pair contained in the mixed primer gives DNA fragments of different sizes from each other, not only the detection of microsporidia but also its identification (specific detection of various microspores) ) Can also be done. For example, the base length of the amplified fragment predicted from each primer pair shown in Table 2 is 943 bases for NBEF35F and NBEF957R, and 7 for M11-96F and M11-822R.
52 bases, 1750 for V70-176F and V70-1898R
There are 450 bases for PSDF1 and PSDR450.

The animal to which the method of the first aspect of the present invention can be applied, that is, the test animal is not particularly limited as long as it can be infected with microsporidia, but is preferably mammals, fish or insects. , And more preferably Bombix mori . The test animal may be in any stage of development and may be an egg, but is preferably an adult or egg, more preferably an adult stage. In the method of the present invention, it is necessary to obtain the nucleic acid from the test animal, and the nucleic acid extraction from the test animal as described above is a method known to those skilled in the art, such as trituration using liquid nitrogen and the phenol method. Alternatively, a commercially available kit can be used. When the test animal is a human, a nucleic acid sample can be obtained from a tissue, cell or the like of a subject by a method known to those skilled in the art.

[Second Method of the Present Invention] The present invention also relates to a method for simultaneously detecting a plurality of insect pathogenic organisms present in a test insect body, the detection comprising nucleic acid present in the test insect body. Can be used as a template and a mixed primer containing a primer pair capable of amplifying a partial region of the genome of each of a plurality of insect pathogenic organisms to be detected can be used for amplification. Here, the mixed primer contains at least two kinds of primer pairs.

In the second method of the present invention, let's detect.
Said multiple species of entomopathogenic organisms
And cause pathological conditions such as diseases and disorders
It is sufficient if it is not particularly limited. Such insect pathogens
Examples of organisms include viruses, chlamydia, mycobacterium
Plasma, bacteria, fungi, rickettsia, protozoa (protozoa
Insects), parasites, etc., but preferably silkworm bodies
Pathogenic organisms that enter inside, more preferably microspores
It Microspora, belonging to the genus Microspora
It is not limited to a protist that can
A protist that belongs to Microsporida,
More preferably, Nosema bombysis (Nosemabombyci
s), Baimorpha sp. M11 (Vairimorpha sp. M1
1), Bailimorpha sp. M12 (Vairimorpha sp. M1
2), Baimorpha Necatrix (Vairimorpha nec
atrix), Plaest Fora sp. PSD (Pleistophora sp.
 PSD) etc. In Nosema Bombisis,
Both strongly virulent and attenuated strains are included.

The primers contained in the mixed primer used in the second method of the present invention can be designed by mutually comparing the sequences of DNAs or RNAs derived from a plurality of insect pathogenic organisms to be detected. The DNA or RNA base sequence used for sequence comparison may be a known base sequence, and can be obtained from a database such as GenBank, for example. A person skilled in the art can easily search and obtain an appropriate sequence from the sequences registered in the database according to the type of insect pathogenic organism to be detected. When the detection target is a microsporidia, examples of the base sequence thus obtained include those shown in Table 1 above.

The design of a primer based on the above sequence comparison can be carried out in the same manner according to the method described for the method of the first present invention. When the target to be detected is a microsporidia, examples of the primer thus designed include those shown in Table 2 above.

The mixed primer used in the second method of the present invention contains two or more primer pairs designed as described above depending on the type of insect pathogenic organism to be detected. For example, when the target to be detected is a microsporia, and when the primers described in Table 2 are used, two or more of all primer pairs are mixed depending on the type of the microsporia to be detected. It is possible to prepare a mixed primer by mixing, but preferably, all of these primers are mixed to form a mixed primer.

The amplification treatment used in the method of the second aspect of the present invention and the detection of the amplification product obtained by the amplification treatment can be carried out in the same manner according to the method described for the method of the first aspect of the present invention. When an insect pathogenic organism having genomic RNA is to be detected, RT-PCR can be performed instead of ordinary PCR. This RT-PCR
Is obtained by obtaining a single-stranded cDNA from the template RNA using a reverse primer and reverse transcriptase, degrading the RNA using RNase, and then performing a normal PCR using the single-stranded cDNA as a template. This is a method for obtaining double-stranded cDNA. Here, as the reverse transcriptase, known ones such as Moloney murine leukemia virus-derived reverse transcriptase can be used. Others, RT-PCR
Those skilled in the art can easily select and set the reagents, reaction conditions and the like used in.

The animal to which the method of the second aspect of the present invention can be applied, that is, the test insect is not particularly limited, but is preferably silkworm ( Bombix mori ). The test insect may be in any stage of development and may be an egg, but is preferably an adult or egg, and more preferably an adult stage. In the second method of the present invention, it is necessary to obtain the nucleic acid from the test insect. However, nucleic acid extraction from the test insect as described above can be performed by a person skilled in the art such as trituration using liquid nitrogen and the phenol method. It can be carried out by a known method, or can be carried out using a commercially available kit.

[Primer Set of the Present Invention] The present invention also provides a primer set for using the method of the present invention,
That is, Nosema Bombisis, Baimorpha sp. M1
1. For detection of microsporidia containing at least two primer pairs selected from the group consisting of primer pairs capable of amplifying a partial region of genomic DNA of Bilimorpha sp. M12, Bilimorpha necatrix and Plastophora sp. The primer set preferably includes all of the primer pairs.

The primer pair contained in the primer set of the present invention can be designed and prepared as described above. Further, specific examples of the primer pair thus prepared are as shown in Table 2 above. Therefore, the primer set of the present invention preferably contains at least two kinds of the primer sets described in Table 2 above, and more preferably all (oligonucleotides containing the nucleotide sequences represented by SEQ ID NOS: 1 to 8). )including.

Furthermore, reagents other than the primers used in the method of the present invention are added to the primer set of the present invention,
It can also be used as a kit for detecting microsporidia. Such reagents include DNA extraction reagents such as Tris-HCl, EDTA, SDS, proteinase K, RNaseA, phenol, sodium acetate and ethanol, Tris-HCl, KCl and MgCl.
2. Various dNTPs, PCR reagents such as Taq DNA polymerase, TE buffer, 40% sucrose Dye, electrophoresis reagents such as agarose gel, and the like, but are not limited thereto.

[0037]

EXAMPLES The present invention will be described in more detail with reference to the following examples. However, these examples are for the purpose of explanation, and do not limit the technical scope of the present invention. [Example 1] Design and Preparation of Detection Primers Nucleotide sequences derived from various microspores registered in a database are compared, and several regions specific to each microspore to be detected are selected at several locations. did. Here, the nucleotide sequences used for sequence comparison are as shown in Table 3 below, and these sequences were compared using analysis software ClustalX (freeware). The software parameters are
All were left at their initial values. In addition, a part of the comparison diagram for sequence comparison is shown in FIG.

[0038]

[Table 3]

Next, the size of the product amplified by PCR when each selected region is used as a primer is estimated, and even if these amplified products are mixed, they can be separated / identified by electrophoresis. The combinations of sequences that amplify products of various sizes were selected and the primers shown in Table 4 below were designed. These primers were synthesized according to a conventional method.

[0040]

[Table 4]

[Preparation Example 1] Preparation of DNA sample for use as template for PCR (1) DNA extraction from Bombyx mori infected with each microsporidia and uninfected Bombyx mori A single Bombyx mori was placed in a mortar, and liquid nitrogen was added thereto for grinding. The obtained powder was placed in a 50 ml centrifuge tube, and 32 mM Tris-HCl (pH 8.
0), 8mM EDTA (pH8.0), 3.2% SDS, 32μg / ml Proteinas
e K, 160 μg / ml RNase A reaction system at 37 ℃
Reacted for hours. After the reaction, perform phenol extraction to 1/10
The amount of 3M sodium acetate (pH 5.2) was added, and double the amount of ethanol was added to recover the DNA.

(2) Extraction of DNA from spores of each microsporidian 10 ml of spores was taken in a 1.5 ml tube, and STE buffer (10
It was washed with mM Tris-HCl pH8.0, 1 mM EDTA pH8.0, 10 mM NaCl). After that, 50 mg of glass beads (SIGMA
, G-8772) and 200 μl of STE buffer were added, and the cells were disrupted for 30 seconds using a vortex. After the treatment, heat treatment was carried out at 95 ° C. for 5 minutes, and 150 μl of supernatant was collected. 1/10 volume of 3M sodium acetate (pH5.2) and
2.5 times the amount of 99.5% ethanol was added to carry out ethanol precipitation. After centrifugation (10,000 xg, 4 ° C, 10 minutes), 7
Rinse with 0% ethanol and collect 20 μl of the collected precipitate.
Suspended in TE buffer.

[Example 2] Specificity Experiment of Primers for Detecting Microspores Using each of the primers prepared in Example 1, PCR was performed using a DNA sample from the microspores of spores to be amplified by each primer as a template. Then, amplification of the target product was confirmed. Further, in order to confirm whether these primers form an amplification product when the genome of a microsporidate other than the target microspores is used as a template, each primer is used for the microspores other than the detection target. Mix with DNA sample from
CR was performed. Furthermore, it was examined whether or not these primers amplify a DNA fragment derived from the silkworm moth genome.

That is, Nosema bombysis virulent system (particulate disease pathogen) and attenuated system, Bailimorpha sp. M1
1. Baimorpha sp. M12, Baimorpha necatrix (foreign species), Plastophora sp. PSD, and DNA samples from Nosema fulnacaris (foreign species) and DNA samples from Bombyx mori, and 4 in Table 4 above. PC in all combinations with each primer pair of the species
R was done.

The PCR reaction solution had a final concentration of 10 mM Tris-HCl.
(pH8.9), 50mM KCl, 1.5mM MgCl2, 0.2mM each dNTP, 2.5U
A total of 100 μl of a solution containing nit Taq DNA polymerase (manufactured by Sawady Technology), 0.5 μM each of homologous primer and complementary primer, and further containing 10 ng of template DNA was prepared. For PCR, use Astec PC800 Thermal Cycler, heat treatment at 94 ℃ for 2 minutes, and then at 94 ℃ for 30 seconds at −55 ℃ for 30 seconds.
The reaction was carried out at −72 ° C. for 30 seconds under the condition of 35 cycles.

2.5 μl of the reaction solution after PCR was collected and
TE buffer and 1 μl of 40% sucrose dye (0.03% BP
B, 50 mM EDTA, pH 8.0, 40% sucrose) was added to prepare a sample for electrophoresis. The electrophoresis was performed at 100 V for 30 minutes using a 2% agarose gel (Sigma, Type-II medium EEO A-6877) and an electrophoresis apparatus Mupid 21 (Cosmo Bio).

The results of electrophoresis are shown in FIG. In FIG. 2, panel A shows primers NBEF35F and NBEF957R for detection of Nosema bombysis virulent and attenuated strains.
Shows the result of PCR using, and Panel B shows primers M11-96F and M1 for detection of Bilimorpha sp. M11.
The result of PCR using 1-822R is shown, Panel C shows the result of PCR using primers V70-176F and V70-1898R for detecting Bilimorpha sp. M12 and Baimorpha necatrix, and Panel D shows Plastophora sp. Primers PSDF1 and PSDR that detect PSD
The result of PCR using 450 is shown.

According to these results, each primer amplifies a specific product only when the DNA sample from the target microsporidia is used as a template, and the DNA from other microspores is amplified.
No amplification of the product was observed when the sample was used as the template. Furthermore, no amplification product derived from the Bombyx mori genome was detected using any of the primers.

[Example 3] Detection of microsporidia by mixed primers A plurality of primers specific to each microsporidia were mixed to examine whether or not a site other than the target gene was amplified. In addition, it was examined whether such a mixed primer amplifies a DNA fragment derived from the host silkworm moth genome. In other words, Nosema bombysis virulent strain (particulate disease pathogen)
And attenuated strains, Baimorpha sp. M11, Baimorpha sp. M12, Bilimorpha necatrix (foreign species), and Plastophora sp.
PCR was performed using a sample and a DNA sample from Bombyx mori as a template and mixed primers in which the four primer pairs shown in Table 4 were mixed.

The PCR reaction solution had a final concentration of 10 mM Tris-HCl.
(pH8.9), 50mM KCl, 1.5mM MgCl2, 0.2mM each dNTP, 2.5U
A total of 100 μl of a solution containing nit Taq DNA polymerase (manufactured by Sawady Technology) and 0.5 μM of each primer and 10 ng of template DNA was prepared. For PCR, heat treatment at 94 ° C for 2 minutes using Astec PC800 Thermal Cycler, followed by reaction at 94 ° C for 30 seconds-55 ° C for 30 seconds-72 ° C for 30 seconds
It was performed under the condition of 35 cycles.

2.5 μl of the reaction solution after PCR was collected and
TE buffer and 1 μl of 40% sucrose dye (0.03% BP
B, 50 mM EDTA, pH 8.0, 40% sucrose) was added to prepare a sample for electrophoresis. The electrophoresis was performed at 100 V for 30 minutes using a 2% agarose gel (Sigma, Type-II medium EEO A-6877) and an electrophoresis apparatus Mupid 21 (Cosmo Bio).

The results of electrophoresis are shown in FIG. In FIG. 3, the four arrows on the left side are, as shown there,
Primers (NBEF35F and NBEF957R) for detecting NB ( Nosema bombycis ), M11 ( Vairimorpha sp. M1)
Primers (M11-96F and M11-822R) that detect 1), M1
Primer for detecting 2 ( Vairimorpha sp. M12) (V70-
176F and V70-1898R), and PSD ( Pleistophora sp. PS
The estimated mobility of the PCR product amplified by the primers (PSDF1 and PSDR450) for detecting D) is shown. According to FIG. 3, each primer amplifies a product of the expected size,
No amplification of other products was confirmed. In addition, the above mixed primer did not amplify a DNA fragment derived from the silkworm moth genome.

[Example 4] Detection / discrimination of each microsporidia in the state of being infected with the host In order to investigate whether or not the microsporidia can be detected even in the state of being infected with the host, the microspore was infected with the microspores. PCR was performed using a DNA sample from an adult Bombyx mori as a template. That is,
Nosema bombysis virulent strain (particulate disease pathogen) and attenuated strain, Bailimorpha sp. M11, Bailimorpha sp. M1
2. PCR was carried out in the same manner as in Example 3 except that each DNA sample from each Bombyx mori nematotricus (foreign species) and each Bombyx mori adults infected with Plastophora sp. PSD was used as a template.

The results of electrophoresis are shown in FIG. According to FIG. 4, the results of the above PCR show that D from spores of each microsporidian
It showed no difference compared to using the NA sample as a template. Therefore, it was found that each microspore worm can be detected in a distinguishable form from the DNA sample from Bombyx mori which was infected with the microspore worm by the multi-primer PCR using the mixed primer as described above.

[Example 5] Effect of Microsporidia Infection Period It was examined whether the detection accuracy is affected by the growth stage of Bombyx mori. That is, Nosema bombysis virulent strain (particulate disease pathogen) and attenuated strain, Bailimorpha sp. M1
1, Baimorpha sp. M12, as well as Plaestora
p. PSD respectively infected silkworm (1st, 2nd, 3rd, 3rd
Ages, 4th and 5th age pupae, and adult)
PCR was performed in the same manner as in Example 3 except that the sample was used as the template. As a result, it was revealed that the growth of Bombyx mori does not directly affect the detection result.

[Example 6] Reaction of mixed primer against non-target organisms Genomes of mixed primers including all the primers shown in Table 4 other than the microsporidian organisms and capable of growing in the same environment as the microsporidian organisms. In order to investigate whether or not to amplify the DNA fragment derived from Bombyx mori (BmNP)
V), white tine fungus Boberia bassiana ( B. bassian
a ), Bacillus thuringiensis ( B. thuringiensi
s ) and PCR were performed in the same manner as in Example 3 except that a DNA sample prepared from E. coli was used as a template.

Of these results, the results for Beauveria bassiana and Bacillus thuringiensis are shown in FIG. As can be seen from FIG. 5, the mixed primer did not amplify the DNA fragments derived from these two kinds of organisms. In addition, DNA fragments derived from BmNPV and E. coli were not amplified.

[Example 7] Detection and identification of microsporelings when microsporelings were co-infected Even if two types of microsporelings to be detected were infected, these were detected from adult silkworm moths. It was investigated whether microspores could be detected in a distinguishable form. That is, Nosema
DNA samples from Bombysis and Plastophora sp.
PCR was performed in the same manner as in Example 3 except that the DNA sample from PSD was mixed and used as a template. At this time, these DN
Experiments in which PCR products are mixed after performing separate PCR using each of the A samples as templates, and electrophoresis is performed, and experiments in which a mixture of these DNA samples is further added with a DNA sample from Bombyx mori as a template At the same time. The result is shown in FIG. As can be seen from FIG. 6, the products specific to the two types of microsporidium infecting were amplified, and the infection of the two types of microsporidia was confirmed by electrophoresis.

[0059]

Industrial Applicability According to the present invention, a plurality of pathogens can be detected by a single test using one tube per individual. In addition, the pathogen can be easily identified by setting such that DNA fragments of different sizes are amplified for each pathogenic organism. Furthermore, the present invention can be used for emergency diagnosis in the medical field as well as in the agricultural field. Further, in this detection method, it is not necessary to limit the type of pathogen to one type, so that it is possible to compare different organism groups such as viruses and bacteria.

In the case of the silkworm particle disease assay, microsporidia can be detected without depending on the growth state or the infectious dose, so that it is possible to detect microspores in the state where spores have not been formed, which was difficult in the past. It is also possible to easily test eggs. In addition, because the assay does not depend on traits, microsporidia and other pathogens are not mistaken.

[0061]

[Sequence list]                                SEQUENCE LISTING <110> Director-General of National Institute of Sericultural       and Entomological Science       Japan Science and Technology Corporation <120> Method for Identifying Pathogenic organisms using       multi-primer PCR <130> P99-0623 <160> 8 <170> PatentIn Ver. 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 1 tggcgctgtt gataagagat t 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 2 aatttagcaa cacaagcctt at 22 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 3 tactttattt aatgtacatt tgaaaa 26 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 4 ctcgaattag aaaattctct caa 23 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 5 caaatgacag ggaaagaaat aagttcca 28 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 6 ttaaatattt tgtgctatag cttactc 27 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 7 caccaggttg attctgcctg acg 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 8 gctccgcctc tctttccgtc tcc 23

[0062]

[Sequence list free text] SEQ ID Nos. 1 to 8: primers

[Brief description of drawings]

FIG. 1 is a diagram showing an alignment of partial DNA sequences derived from six types of microsporidia and E. coli.

FIG. 2 is an electrophoretic photograph showing the detection results of various microspores when four types of primer pairs were used alone.

FIG. 3 is an electrophoretic photograph showing the detection results of various microspores when a mixed primer consisting of four types of primer pairs was used.

FIG. 4 is an electrophoretic photograph showing the results of detecting Microsporia in a DNA sample obtained from Bombyx mori which has been infected with various Microspores.

FIG. 5: DNA obtained from organisms other than Microsporia (Boberia baciana and Bacillus thuringiensis)
It is an electrophoretic photograph which shows the result of PCR which uses a sample as a template and the mixed primer for detecting Microsporia.

FIG. 6 is an electrophoretic photograph showing the results of detecting Microsporia from a DNA sample obtained from Bombyx mori which was co-infected with two types of Microsporia.

Front page continued (72) Inventor Masayuki Yonemura 2-3-2-708-607 Azuma Tsukuba, Ibaraki Prefecture (56) Reference JP-A-8-252099 (JP, A) Japanese Silkworm Magazine, Vol. 66, No. 4 (1997), p. 242-252 Abstracts of the General Meeting of the American Society for Microbiology, Vol. 94th (1994), p. 529 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C12Q 1/00-1/70 CA / REGISTRY (STN) JISST file (JOIS ) BIOSIS / WPI (DIALOG) PubMed

Claims (8)

(57) [Claims] 1. A mixed primer containing at least two kinds of primer pairs capable of amplifying a partial region of genomic DNA of each of a plurality of types of microsporidia to be detected, using a nucleic acid existing in the body of a test animal as a template. A method capable of simultaneously detecting the plurality of types of microsporidia in the subject animal, which comprises performing an amplification treatment , wherein the mixed primer comprises:
The following (i) to (iv): (i) an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO : 1
Includes nucleotide and the nucleotide sequence represented by SEQ ID NO: 2
A primer pair consisting of an oligonucleotide, (ii) an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 3
Includes nucleotide and the nucleotide sequence represented by SEQ ID NO: 4
A primer pair consisting of an oligonucleotide, (iii) an oligo containing the nucleotide sequence represented by SEQ ID NO: 5
Includes nucleotides and the base sequence represented by SEQ ID
Primer pair consisting of an oligonucleotide, and (iv) an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 7.
Includes nucleotide and the nucleotide sequence represented by SEQ ID NO: 8
Consisting of a primer pair consisting of oligonucleotides
Containing at least two primer pairs selected from the group
The above method. 2. At least the microsporidia of the plurality of species to be detected are selected from the group consisting of Nosema bombysis, Baimorpha sp. M11, Baimorpha sp. M12, Baimorpha necatrix and Plastophora sp. PSD. The method according to claim 1, which is two kinds of microsporidia. 3. The method according to claim 2, wherein the plurality of species of microsporidia to be detected are Nosema bombysis, Bailimorpha sp. M11, Bailimorpha sp. M12, Bailimorpha necatrix and Plastophora sp. PSD. . 4. A nucleic acid existing in the body of a test animal is used as a template.
The sequence consisting of the base sequences represented by SEQ ID NOS: 1-8.
Amplification using mixed primers containing gonucleotides
The microspores of the plurality of species in the body of the subject, including
A method capable of simultaneously detecting insects. 5. The method according to claim 1, wherein the test animal is an insect. 6. The insect is Bombix mori.
The method according to claim 5. . 7. A primer pair comprising (i) an oligonucleotide containing the base sequence represented by SEQ ID NO: 1 and an oligonucleotide containing the base sequence represented by SEQ ID NO: 2, and (ii) a base sequence represented by SEQ ID NO: 3. A primer pair comprising an oligonucleotide containing and an oligonucleotide containing the base sequence represented by SEQ ID NO: 4,
(Iii) a primer pair consisting of an oligonucleotide containing the base sequence represented by SEQ ID NO: 5 and an oligonucleotide containing the base sequence represented by SEQ ID NO: 6, and (iv) an oligonucleotide containing the base sequence represented by SEQ ID NO: 7 A microsporia detection primer set comprising at least two primer pairs selected from the group consisting of a primer pair consisting of an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 8. 8. The primer set for detecting Microsporidae according to claim 7, which comprises an oligonucleotide containing the nucleotide sequences represented by SEQ ID NOs: 1 to 8.