JP2001231575A - Method for detecting pathogenic microbe by multiprimer pcr technique - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a plural species of pathogenic microbes by a single test alone using one tube per individual object, and to provide a method for easily identifying such pathogenic microbes as well by setting conditions so as to amplify DNA fragments differing in size for the corresponding respective pathogenic microbes. SOLUTION: This method for simultaneously detecting plural species of microspora in a test animal body comprises the following practice: a amplification treatment is performed, using a nucleic acid present in the test animal as template, with a mixed primer comprising at least two primer pairs capable of amplifying the partial domains of the respective genomic DNAs of a plural species of microspora to be detected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting pathogenic organisms infected with animals, and more particularly, to PCR of microsporidians infected with animals or insect pathogens infected with insects.
The detection method.

[0002]

2. Description of the Related Art At present, pathogenic organisms for animals include:
Various things such as viruses, bacteria, fungi, parasites and protozoa are known. Microsporida, one of the above pathogens, is a protozoan belonging to Microspora (Microsporidia), and is known to infest a wide range of animal cells from protozoa to mammals. In addition, it is known that parasitized microsporidians cause various diseases in their hosts. For example, Nosema bombycis , a kind of microsporidian, mainly infests silkworms and causes microbial disease. Therefore, it is necessary to test as quickly as possible whether useful animals such as silkworms are infected with microsporidians.

[0003] Conventionally, in general, in order to detect a pathogenic organism in the case of an infectious disease in which the pathogen cannot be determined, the pathogenic organism is predicted from its symptoms, and tests for detecting each are performed individually. Therefore, it was necessary to perform a plurality of tests for each individual, but as such a test method, a method using an antibody against the pathogen to be detected, a method of culturing cells of the pathogenic bacterium, and the like are mainly used. Therefore, it took a lot of time to test a large number of individuals.

[0004] In particular, since the test for microsporidians infecting silkworms is performed by a method using a specific antibody that reacts with the spores of each microsporidian, a skilled technician must perform the test. In addition, when spores were not formed, the spores could not be detected, and it was very difficult to test eggs with a small amount of infection. Therefore, it would be convenient if a method capable of easily detecting microsporidians even if not an expert was developed.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for detecting microsporidians which can be easily operated, can be visually detected, and can be performed by a single operation.

[0006]

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

That is, the present invention includes at least two primer pairs capable of amplifying partial regions of genomic DNA of each of a plurality of microsporidians to be detected, using a nucleic acid present in the test animal as a template. It is intended to provide a method for simultaneously detecting the plurality of microsporidians in the test animal, which comprises performing an amplification treatment using a mixed primer. This method of the present invention is hereinafter referred to as “first method of the present invention”.

In the above method, the plurality of microsporidians to be detected are preferably Nosema bombysis, Bilimorpha sp. M11, Bilimorpha sp. M12,
Bilimorpha Necatrix and Prestora
p. PSD, more preferably at least two microsporidians selected from the group consisting of Nosema bombissis, Bilimorpha sp. M11, Bilimorpha sp. M12, Bilimorpha necatrix, and Plestophora sp. PSD.
It is.

[0009] The mixed primer is preferably
(I) a primer pair consisting of an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 and an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2; (ii) an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3; A primer pair consisting of 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) SEQ ID NO: 7
And at least two primer pairs selected from the group consisting of an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8 and an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8, more preferably SEQ ID NO: 1 And an oligonucleotide containing the base sequence represented by 8. Further, it is preferable that the test animal is an insect.

Further, the present invention includes at least two primer pairs capable of amplifying partial regions of genomic DNAs of a plurality of species of insect pathogens to be detected, using a nucleic acid present in a test insect as a template. It is intended to provide a method for simultaneously detecting the plurality of insect pathogens in the test insect, which comprises performing an amplification treatment using a mixed primer. This method of the present invention is hereinafter referred to as “second method of the present invention”.
That.

In the above method, it is preferable that the entomopathogen is a microsporidian. In this case, the plurality of microsporidians to be detected are preferably selected from the group consisting of Nosema bombysis, Bilimorpha sp.M11, Bilimorpha sp.M12, Bilimorpha necatrix, and Prestora sp.PSD. At least two kinds of microsporidians, more preferably Nosema bombis, Bilimorpha sp. M11, Bilimorpha sp. M12, Bilimorpha necatrix, and Prestora sp. PSD.

[0012] The mixed primer is preferably
(I) a primer pair consisting of an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 and an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2; (ii) an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3; A primer pair consisting of 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) SEQ ID NO: 7
And at least two primer pairs selected from the group consisting of an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8 and an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8, more preferably SEQ ID NO: 1 And an oligonucleotide containing the base sequence represented by 8.

[0013] The present invention further provides a Nosema bombisis,
Bilimorpha sp. M11, Bilimorpha sp. M12, Bilimorpha Necatrix and Plestfora sp. PS
The present invention provides a primer set for detecting microsporidians, comprising at least two types of primer pairs selected from the group consisting of primer pairs capable of amplifying a partial region of genomic DNA of D.

[0014] The above primer set is preferably
(I) a primer pair consisting of an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 and an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2; (ii) an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3; A primer pair consisting of 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) SEQ ID NO: 7
And at least two primer pairs selected from the group consisting of an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8 and an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8, more preferably SEQ ID NO: 1 And an oligonucleotide containing the base sequence represented by 8.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. [Method of the First Invention] The present invention relates to a method capable of simultaneously detecting a plurality of microsporidians present in a test animal,
This detection is performed by using a nucleic acid present in the test animal as a template, and performing an amplification treatment using a mixed primer containing a primer pair capable of amplifying a partial region of genomic DNA of each of a plurality of species of microsporidians to be detected. Can be implemented. Here, the mixed primer contains at least two primer pairs.

In the first method of the present invention, the plurality of microsporidians to be detected are selected from the group consisting of Microsporidium (Microspodia).
ra), but is not particularly limited, and is preferably a protist belonging to Microsporida, more preferably Nosema bombysis ( Nosema
bombycis ), Varimorpha sp. M11 ( Vairimorpha sp.
M11), Bairimorufa sp. M12 (Vairimorpha sp. M1
2), Bairimorufa-necatrix (Vairimorpha nec
atrix ) and Plestfora sp. PSD ( Pleistophora s
p. PSD), most preferably Nosema bombysis, Bilimorpha sp. M11, Bilimorpha sp. M12, Bilimorpha necatrix and Prestora sp. PSD. The Nosema bombisis includes both highly virulent and attenuated strains.

The primer contained in the mixed primer used in the first method of the present invention can be designed by comparing the sequences of DNAs derived from a plurality of microsporidians to be detected with each other. DN used for sequence comparison
The region of A may be any gene, and is not particularly limited. For example, mitochondrial heat shock protein 70 (HSP70) gene, peptide elongation factor 1α
Gene, small subunit rRNA gene and the like. The base sequence of the DNA used for the sequence comparison may be a known sequence, and can be obtained from, for example, 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 microsporidium to be detected. Examples of the base sequence thus obtained include those shown in Table 1 below.

[0018]

[Table 1]

To design primers for each microsporidium to be detected based on the above sequence comparison,
First, alignment of the obtained plurality of base sequences is performed.
The alignment can be performed using analysis software, for example, ClustalX (freeware). At this time, all software parameters may be left as initial values, or may be changed as needed. Such parameter changes can be easily made by those skilled in the art. Next, based on such an alignment (result of the sequence comparison), the following criteria are used: (1) In principle, one kind of amplification product is formed for each kind of microsporidia; Design so that the mixed primers do not amplify the unintended site when mixed (the primer sequence is changed if the unintended site is amplified); (3) The target sequence to be amplified Does not necessarily have to be the same gene and may vary from organism to organism; (4) designing 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, and is preferably 100 to 3000 bases, more preferably 100 bases. ２０００2000 bases. Here, if 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, simultaneous detection of each microsporidian by the above-described method is possible. In addition, identification can be performed. However, such identification need not necessarily distinguish all microsporidians to be detected. In other words, the microsporidians that do not need to be distinguished need not have different sizes of amplified fragments derived from each other to the extent that they are clearly separated by electrophoresis. The nucleotide sequence of the primer designed as described above is not limited to a specific sequence, but includes, for example, those containing the nucleotide sequence shown in Table 2 below.

[0021]

[Table 2]

In Table 2 above, NBEF35F and NBEF957R
Is a primer designed based on the Nosema bombis elongation factor 1α gene sequence, and can detect both attenuated and highly virulent Nosema bombisis strains. M1
1-96F and M11-822R are primers designed based on the small subunit rRNA gene sequence of Bilimorpha sp. M11, and can detect only Bilimorpha sp. M11. V70-176F and V70-1898R are bilimorphas
A primer designed based on the HSP70 gene sequence of p.M12, which 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, PSDR450
Is a small subunit rRN of the playstfora sp. PSD
This is a primer designed based on the A gene sequence. PSDF
1 and PSDR 450 can detect only the prestophora sp. PSD. The number at the end of the name of each primer indicates the base number of the 3 'terminal base of each primer in the sequence on which the primer was designed. These primers have very high specificity and do not react with microsporidians and other pathogenic organisms other than those indicated as detection targets. The oligonucleotide (primer) designed as described above can be easily prepared by a method known to those skilled in the art, for example, chemical synthesis.

The mixed primer used in the first method of the present invention contains two or more primer pairs designed as described above according to the type of microsporidium to be detected. For example, when using the primers described in Table 2 above, two or more of all primer pairs can be mixed to form a mixed primer, depending on the type of microsporidium 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 a polymerase chain reaction (PCR). Here, the template used for the amplification treatment is a nucleic acid present in the test animal, and is preferably D
NA. Such a nucleic acid can be extracted from a test animal by a method known to those skilled in the art. As the primer, the above-mentioned mixed primer is used. Those skilled in the art can appropriately set the template, the primer, the reagents required for the amplification treatment, the processing conditions, and the like. For example, P
When performing CR, in addition to the template and primer, Tris
-HCl, KCl, MgCl2, each dNTP, reagents such as Taq DNA polymerase can be mixed to make a PCR reaction solution,
Such reagents are also commercially available as PCR kits. Next, the amplification reaction can be performed by introducing the PCR reaction solution thus prepared into a device such as a thermal cycler and performing a temperature cycle reaction.

The amplification product obtained by the amplification treatment can be detected by a known method capable of separating according to the size (base length) of the product, preferably by an electrophoresis method such as agarose gel electrophoresis. By such a method, it is possible to examine whether or not an amplified fragment of the size predicted from each primer pair used for the amplification treatment is detected, whereby the nucleic acid present in the subject animal is It can be checked whether or not it is amplified. Here, prediction of the size of the amplified fragment from the primer pair can be easily performed by those skilled in the art. As described above, if each primer pair contained in the mixed primers gives DNA fragments of mutually different sizes, not only detection of microsporidia but also identification (specific detection of various microsporidians) ) Can also be performed. 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 bases for M11-96F and M11-822R.
52 bases, 1750 for V70-176F and V70-1898R
The bases, PSDF1 and PSDR450, are 450 bases.

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 microsporidians, and is preferably a mammal, fish or insect. And more preferably a silkworm ( Bombix mori ). The test animal may be in any stage of development or may be an egg, but is preferably an adult or an egg, more preferably an adult. In the method of the present invention, it is necessary to obtain a nucleic acid from a test animal. The nucleic acid extraction from the test animal as described above can be performed by a method known to those skilled in the art, such as trituration using liquid nitrogen and a phenol method. And a commercially available kit. When the test animal is a human, a nucleic acid sample can be obtained from tissues, cells, and the like from 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 species of insect pathogens present in a test insect, the method comprising detecting nucleic acids present in the test insect. And using as a template a mixed primer containing a primer pair capable of amplifying a partial region of the genome of each of a plurality of species of insect pathogens to be detected. Here, the mixed primer contains at least two primer pairs.

In the second method of the present invention, the plurality of species of insect pathogenic organisms to be detected are not particularly limited as long as they enter the insect body and cause a pathological condition such as a disease or disorder. . Such insect pathogens include, for example, viruses, chlamydia, mycoplasma, bacteria, fungi, rickettsia, protozoa (protozoa), parasites, and the like. It is a sporeworm. The microsporidian is not particularly limited as long as it is a protist belonging to the phylum Microspora, and is preferably a protist belonging to the microsporids (Microsporida),
More preferably, Nosemabombyci
s), Bairimorufa sp. M11 (Vairimorpha sp. M1
1), Bairimorufa sp. M12 (Vairimorpha sp. M1
2), Bairimorufa-necatrix (Vairimorpha nec
atrix ), Pleistophora sp. PSD ( Pleistophora sp.
PSD) and the like. The Nosema Bombisis includes:
Both highly virulent and attenuated strains are included.

The primer contained in the mixed primer used in the second method of the present invention can be designed by comparing sequences of DNAs or RNAs derived from a plurality of insect pathogens to be detected. The nucleotide sequence of DNA or RNA used for the sequence comparison may be a known 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 insect pathogen to be detected. When the detection target is a microsporidian, the base sequence thus obtained may include, for example, those shown in Table 1 above.

The design of primers based on the above sequence comparison can be performed in the same manner according to the method described for the method of the first present invention. When the detection target is a microsporidian, examples of the primer designed in this manner include those shown in Table 2 above.

The mixed primer used in the second method of the present invention includes two or more primer pairs designed as described above according to the kind of insect pathogen to be detected. For example, when the detection target is a microsporidian and the primers described in Table 2 are used, two or more of all primer pairs are mixed according to the type of the microsporidian to be detected. To obtain a mixed primer. Preferably, all of these primers are mixed to obtain a mixed primer.

The amplification process used in the second method of the present invention and the detection of the amplification product obtained by the amplification process can be performed in the same manner according to the method described in the first method of the present invention. When an insect pathogen 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 a template RNA using a reverse primer and a reverse transcriptase, degrading the RNA using RNase, and then performing a normal PCR using the single-stranded cDNA as a template. This is a technique for obtaining double-stranded cDNA. Here, as the reverse transcriptase, a known reverse transcriptase such as Moloney murine leukemia virus-derived reverse transcriptase can be used. Other, RT-PCR
The reagents, reaction conditions and the like used in can be easily selected and set by those skilled in the art.

The animal to which the second method of the present invention can be applied, ie, the test insect, is not particularly limited, but is preferably a silkworm ( Bombix mori ). The test insect may be in any stage of growth or may be an egg, but is preferably an adult or an egg, and more preferably an adult. In the second method of the present invention, it is necessary to obtain a nucleic acid from a test insect. However, nucleic acid extraction from the test insect as described above requires a person skilled in the art such as grinding using liquid nitrogen and a phenol method. It can be performed by a known method, or can be performed 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, bilimorpha sp. M1
1, for detecting microsporidians, comprising at least two types of 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 Pleistrophora sp. PSD. Regarding the primer set, it preferably includes all of the primer pairs.

The above-mentioned primer pairs included in the primer set of the present invention can be designed and prepared as described above. Specific examples of the primer pairs thus prepared are as shown in Table 2 above. Therefore, the primer set of the present invention preferably contains at least two of the primer sets shown in Table 2 above, and more preferably all of them (the oligonucleotides containing the base sequence 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,
A kit for detecting microsporidians can also be used. Examples of such reagents include reagents for DNA extraction 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]

The present invention will be described more specifically with reference to the following examples. However, these examples are for 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 microsporidians registered in the database are compared, and several regions specific only to each microsporidium to be detected are selected. did. Here, the nucleotide sequences used for the sequence comparison are as described in Table 3 below, and the comparison of these sequences was performed using ClustalX (freeware) which is analysis software. The parameters of the software are:
All were left at the initial values. In addition, FIG. 1 shows a part of a comparison diagram obtained by sequence comparison.

[0038]

[Table 3]

Next, when each selected region is used as a primer, the size of the product to be amplified by PCR is estimated, and even if these amplification products are mixed, separation and identification can be performed by electrophoresis. Combinations of sequences that amplify products of various sizes were selected, and 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 to be used as template for PCR (1) Extraction of DNA from infected and uninfected silkworms of each microsporidium Bombyx mori infected or uninfected with microsporidians One silkworm was placed in a mortar and ground with liquid nitrogen. Take the resulting powder in a 50 ml centrifuge tube, 32 mM Tris-HCl (pH 8.
0), 8mM EDTA (pH8.0), 3.2% SDS, 32μg / ml Proteinas
e K, 160 µg / ml RNase A in a reaction system at 37 ° C.
Allowed to react for hours. After the reaction, perform phenol extraction, and
An amount of 3M sodium acetate (pH 5.2) was added, and a 2-fold amount of ethanol was added to recover the DNA.

(2) Extraction of DNA from spores of each microsporidian 10 mg of spores were placed in a 1.5 ml tube and placed in an STE buffer (10
Washed with mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 10 mM NaCl). Then, add 50mg glass beads (SIGMA
, G-8772) and 200 μl of STE buffer, and crushed using a vortex for 30 seconds. After the treatment, heat treatment was performed at 95 ° C. for 5 minutes, and 150 μl of the supernatant was collected. 1/10 volume of 3M sodium acetate (pH 5.2) and
A 2.5-fold amount of 99.5% ethanol was added to perform ethanol precipitation. After centrifugation (10,000 xg, 4 ° C, 10 minutes), 7
Rinse with 0% ethanol and collect 20 μl of the collected precipitate
In TE buffer.

[Example 2] Specificity test of each microsporidian detection primer Using each primer prepared in Example 1, PCR was performed using a DNA sample from microsporidian spores to be amplified with each primer as a template. Was performed to confirm the amplification of the target product. In addition, in order to confirm whether or not these primers form an amplification product when the genome of a microsporidium other than the target microsporidian is used as a template, each primer was used to determine whether or not a microsporidium other than the detection target was used. Mixed with the DNA sample from
CR was performed. Furthermore, it was examined whether or not these primers amplify a DNA fragment derived from the silkworm genome.

That is, Nosema bombis virulent strain (pathogen of microbial disease) and attenuated strain, Bilimorpha sp. M1
1. Bilimorpha sp. M12, Bilimorpha necatrix (foreign species), pristophora sp. PSD, DNA samples from Nosema flunacaris (foreign species) and DNA samples from silkworm, and 4 shown in Table 4 above PC in all combinations with each primer pair of the species
R was performed.

The PCR reaction solution was a final concentration of 10 mM Tris-HCl.
(pH8.9), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 2.5 U
A total of 100 μl of a solution containing nit Taq DNA polymerase (manufactured by Sawady Technology), homologous primers and complementary primers of 0.5 μM each, and further containing 10 ng of template DNA was prepared. PCR was performed using an Astec PC800 thermal cycler at 94 ° C for 2 minutes, followed by 94 ° C for 30 seconds and -55 ° C for 30 seconds.
The reaction was carried out at -72 ° C for 30 seconds for 35 cycles.

[0048] 2.5 µl of the reaction solution after the PCR is collected and
TE buffer and 1 μl of 40% sucrose dye (0.03% BP
B, 50 mM EDTA, pH 8.0, 40% sucrose) to prepare a sample for electrophoresis. 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).

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

According to these results, each primer amplifies a specific product only when a DNA sample from the target microsporidian is used as a template, and DNAs from other microsporidians are amplified.
When the sample was used as a template, no amplification of the product was observed. Furthermore, no amplification product derived from the silkworm genome was detected using any of the primers.

Example 3 Detection of Microsporidians Using Mixed Primers A plurality of types of primers specific to each microsporidian were mixed to determine whether or not a site other than the target gene was amplified. Further, it was examined whether or not such a mixed primer amplifies a DNA fragment derived from the host silkworm genome. That is, Nosema bombis virulent strain (pathogen of microbial disease)
M11, bilimorpha sp. M12, bilimorpha necatrix (foreign species), and attenuated strains, and pristostora sp. PSD
Using the sample and the DNA sample from Bombyx mori as a template, PCR was performed using mixed primers obtained by mixing the four primer pairs shown in Table 4 above.

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

[0051] 2.5 µl of the reaction solution after the PCR is collected, and an equal volume
TE buffer and 1 μl of 40% sucrose dye (0.03% BP
B, 50 mM EDTA, pH 8.0, 40% sucrose) to prepare a sample for electrophoresis. 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).

FIG. 3 shows the results of the electrophoresis. In FIG. 3, the four arrows on the left, as shown there,
Primers (NBEF35F and NBEF957R) for detecting NB ( Nosema bombycis ), M11 ( Vairimorpha sp. M1)
1) Primers for detecting (M11-96F and M11-822R), M1
2 ( Vairimorpha sp. M12) primer (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 the expected size product,
No amplification of other products was confirmed. Moreover, the mixed primer did not amplify a DNA fragment derived from the silkworm genome.

[Example 4] Detection and discrimination experiment of each microsporidium in a state infected with a host In order to examine whether microsporidians can be detected even in a state infected with a host, microsporidians were infected. PCR was performed using a DNA sample from Bombyx mori as a template. That is,
Nosema bombis virulent strain (pathogen of microbial disease) and attenuated strain, bilimorpha sp. M11, bilimorpha sp. M1
2. PCR was performed in the same manner as in Example 3, except that each DNA sample from each silkworm adult infected with Bilimorpha necatrix (foreign species) and Pleistphora sp. PSD was used as a template.

FIG. 4 shows the results of the electrophoresis. According to FIG. 4, the results of the above PCR show that the D.S.
No difference from the case where the NA sample was used as a template was shown. Therefore, it was found that each of the microsporidians can be detected in a discriminable manner from the DNA sample from the silkworm, Bombyx mori, infected with the microsporidians by the multi-primer PCR using the mixed primers as described above.

Example 5 Influence by Microsporidian Infection Timing Whether the detection accuracy was affected by the growth stage of Bombyx mori was examined. Nosema bombis virulent strain (pathogen of microbial disease) and attenuated strain, bilimorpha sp. M1
1, Bailimorpha sp. M12, as well as Plestfora
B. silkworms (1st, 2nd, 3rd)
Age, 4th and 5th pupa, and adult DNA)
PCR was performed in the same manner as in Example 3 except that the sample was used as a template. The results showed that silkworm growth did not directly affect the detection results.

Example 6 Reaction of Mixed Primers with Non-Target Organisms The genome of a mixed primer containing all the primers shown in Table 4 is an organism other than microsporidians and can grow in the same environment as microsporidians. In order to examine whether or not to amplify the DNA fragment derived from silkworm nuclear polyhedrosis virus (BmNP),
V), Beauveria basiana ( B. bassian)
a), Bacillus thuringiensis (B. thuringiensi
s ) and PCR was performed in the same manner as in Example 3 except that DNA samples prepared from E. coli were used as templates.

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

[Example 7] Detection and identification of microsporidia when microsporidia was co-infected Even if two types of microsporidia to be detected were infected, they were not detected from Bombyx mori. We examined whether microsporidians could be detected in a discernable manner. That is, Nosema
DNA sample from Bombysis and prestophora sp.
PCR was carried out in the same manner as in Example 3 except that a DNA sample from the PSD was mixed and used as a template. At this time, these DNs
An experiment in which separate PCR was performed using each of the A samples as templates, followed by mixing PCR products and electrophoresis, and an experiment in which a mixture of these DNA samples and a DNA sample from Bombyx mori was used as a template Was performed at the same time. FIG. 6 shows the result. As can be seen from FIG. 6, products specific to the two types of microsporidians infected were amplified, respectively, and infection of the two types of microsporidians was confirmed by electrophoresis.

[0059]

According to the present invention, a plurality of pathogens can be detected by only one test using one tube per individual. In addition, by setting such that DNA fragments of different sizes are amplified for each pathogenic organism, the pathogen can be easily identified. Further, the present invention can also be used for emergency diagnosis in medical sites other than the agricultural field. Furthermore, in this detection method, it is not necessary to limit the type of the pathogen to one type, so that it is possible to compare a heterogeneous population such as a virus and a bacterium.

In the case of the silkworm microbial disease assay, microsporidians can be detected without depending on the growth state and the amount of infection, and thus it is also possible to perform detection in a state in which spores have not been formed, which has been conventionally difficult. Yes, and eggs can be easily tested. In addition, since the assay does not depend on the trait, the microsporidians 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 aagttc ca 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 the drawings]

FIG. 1 is a view showing alignment of DNA partial sequences derived from six kinds of microsporidians and Escherichia coli (E. coli).

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

FIG. 3 is an electrophoretic photograph showing detection results of various microsporidians when a mixed primer composed of four types of primer pairs is used.

FIG. 4 is an electrophoretic photograph showing the detection results of microsporidians from a DNA sample obtained from a silkworm, which has been infected with various microsporidians.

FIG. 5: DNA obtained from organisms other than microsporidians (Boweria basiana and Bacillus thuringiensis)
It is an electrophoresis photograph showing the result of PCR using a sample as a template and a mixed primer for microsporidian detection.

FIG. 6 is an electrophoretic photograph showing the results of detection of microsporidia from a DNA sample obtained from a silkworm that has been co-infected with two types of microsporidians.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Masayuki Yonemura 2-3-2-708-607 Azuma, Tsuzuba-shi, Ibaraki F-term (reference) 4B024 AA10 AA13 CA01 CA11 CA20 HA11 4B063 QA01 QA18 QA19 QQ02 QQ05 QQ42 QQ52 QR08 QR32 QR35 QR40 QR42 QR62 QS16 QS25 QX01

Claims (15)

[Claims] 1. A mixed primer comprising at least two primer pairs capable of amplifying a partial region of genomic DNA of each of a plurality of microsporidians to be detected, using a nucleic acid present in a test animal as a template. A method capable of simultaneously detecting the plurality of microsporidians in the subject animal, which comprises performing amplification treatment. 2. The method according to claim 1, wherein the plurality of microsporidians to be detected are at least one selected from the group consisting of Nosema bombis, Bilimorpha sp. M11, Bilimorpha sp. M12, Bilimorpha necatrix, and Prestora sp. The method according to claim 1, which is two kinds of microsporidians. 3. The method according to claim 2, wherein the plurality of microsporidians to be detected are Nosema bombysis, Bilimorpha sp. M11, Bilimorpha sp. M12, Bilimorpha necatrix, and Prestora sp. PSD. . 4. The mixed primer comprises: (i) a primer pair consisting of an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 and an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2; A primer pair consisting of an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 4 and an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 4, (iii) an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 5 and a nucleotide sequence represented by SEQ ID NO: 6 And (iv) at least one selected from the group consisting of an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7 and a primer pair comprising an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8 Including two primer pairs, The method of Motomeko 1, wherein the. 5. The mixed primer according to any one of SEQ ID NOs: 1 to 8
5. The method according to claim 4, comprising an oligonucleotide comprising a base sequence represented by the formula: 6. The method according to claim 1, wherein the test animal is an insect. 7. A mixed primer containing at least two primer pairs capable of amplifying a partial region of each genomic DNA of a plurality of insect pathogens to be detected, using a nucleic acid present in the test insect as a template. A method capable of simultaneously detecting said plurality of insect pathogenic organisms in said test insect body, comprising performing amplification treatment by amplifying the same. 8. The method according to claim 7, wherein the entomopathogen is a microsporidian. 9. The method of claim 9, wherein the plurality of microsporidia species to be detected are at least selected from the group consisting of Nosema bombis, Bilimorpha sp. M11, Bilimorpha sp. M12, Bilimorpha necatrix, and Prestora sp. The method according to claim 8, which is two kinds of microsporidians. 10. The plurality of microsporidians to be detected are Nosema bombis, Bilimorpha sp. M11,
10. The method of claim 9, wherein the method is Bilimorpha sp. M12, Bilimorpha necatrix and pristostora sp. PSD. 11. The primer pair comprising: (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; A primer pair consisting of an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 4 and an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 4, (iii) an oligonucleotide containing the nucleotide sequence represented by SEQ ID NO: 5 and a nucleotide sequence represented by SEQ ID NO: 6 And (iv) at least one selected from the group consisting of an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7 and a primer pair comprising an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8 Includes two primer pairs The method of claim 7 wherein. 12. The mixed primer according to any one of SEQ ID NOs: 1 to
The method according to claim 11, comprising an oligonucleotide containing the base sequence represented by 8. 13. Nosema bombisis, bilimorpha
sp. M11, bilimorpha sp. M12, bilimorpha necatrix and pristostora sp.
A comprising at least two types of primer pairs selected from the group consisting of primer pairs capable of amplifying the partial region of A.
Primer set for microsporidian detection. 14. A primer pair comprising (i) an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 and an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2; (ii) a primer pair comprising the nucleotide sequence represented by SEQ ID NO: 3 A primer pair comprising an oligonucleotide comprising the oligonucleotide and the oligonucleotide comprising the base sequence represented by SEQ ID NO: 4,
(Iii) a primer pair consisting of an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5 and an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 6, and (iv) an oligonucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7 14. The microsporidian detection primer set according to claim 13, comprising at least two types of primer pairs selected from the group consisting of primer pairs consisting of oligonucleotides containing the nucleotide sequence represented by SEQ ID NO: 8. 15. The primer set for detecting microsporidians according to claim 14, which comprises an oligonucleotide containing the nucleotide sequence represented by SEQ ID NOS: 1 to 8.