JPH0361487A - Detection method for swine epidemic mycoplasma pneumonia - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for detecting Mycoplasma hyopneumoniae, a bacterium that causes swine epidemic pneumonia.

[Conventional technology]

Porcine epidemic pneumonia (Mycoplas pneumonia)
maPneumoniae of 5w1ne+r below
MPsJ) is Mycoplasma hyopneumoniae.
) (hereinafter referred to as rM, hpJ). The main disease is characterized by a chronic course, and the mortality rate is low. However, the morbidity rate is extremely high, and the economic losses caused to the pig farming industry, such as decreased feed efficiency, are enormous.

According to a survey by the Ministry of Agriculture, Forestry and Fisheries, more than half of the pigs shipped to Japan's stockyards now have lung lesions caused by this disease, and it has become clear that the disease is widespread nationwide.

Regarding diagnostic methods for MPS, a serum diagnostic method using complement fixation reaction, BLISA method, etc., or a method based on macroscopic findings of lung lesions has been reported. There is also a method of separating M and hp from lesions, but M and hp have strict nutritional requirements and require a long period of time to grow in an artificial medium. For this reason, it conventionally required at least two weeks to one month to isolate and identify M and hp from Mpsg-stained pigs.

[Problem to be solved by the invention]

An object of the present invention is to provide a method for detecting M, hp from lesions in a short period of time.

[Means to solve the problem]

The object of the present invention is to have the following formulas (I), (n), (I), (I
V) or nucleotides of the base sequence shown by (V),
Recombinant DNA containing these as part of it.

Or DNA containing these as part of DNA
This is achieved by a DNA hybridization method using an A probe.

Shishi IjLTAALU’1 Ui’ltA’l’AA’1r UTATTTTTG(:[’ The present invention will be explained in detail below.

Ribosomal RNA (hereinafter referred to as r rRNA) is 1
There are over 10,000 copies per bacterial cell, and it is considered to be very effective in terms of detection sensitivity. However, since rRNA is one of the genes that has been relatively well conserved during the evolution of living organisms, its specificity is lower than that of antigen genes.

For example, Escherichia coli and Bacillus subtilis are said to have 70=80% homology.

By the way, regarding the rRNA of M and hp, a group in West Germany has determined the base sequence of 165 rRNA [C, T
a5chke et al, Nucl, Ac1ds
Re5115, 3918 (1987) “Nucle
otide 5equence of the16S
rRNA of Mycoplasma hyopne
Umoniae": l. Using this data, the present inventors have identified the 16 species of other organisms that have been determined to date.
Compared with the base sequence of SrRNA, M, hp 16S r
We searched for regions unique to RNA.

As a result, we discovered several regions thought to be specific to M, hp, selected oligonucleotides considered to be useful as DNA probes from these regions, chemically synthesized them, and detected IVlhp as DNA probes. As a result, they discovered that scales can detect M and hp with extremely high sensitivity, and have completed the present invention.

(, to) M, Search for unique regions in hp 16 S rRNA JOIS-F Gen 13ank and EMBL DNA
Using a database, we searched for 163 rRNA base sequences of various organisms that have been reported to date.

All the obtained base sequences and Ivf, h shown in Figure 1
The base sequence of p 16 S rRNA was compared using computer software for gene analysis GENIAS (Mitsui Knowledge Development).

As a result, in FIG. 1, base No. 180-24
3 places from 0.590 to 680.990-1040 are M,
It was found that this region can be considered as a hp-specific region.

(B) Oligonucleotides to be chemically synthesized that are considered useful as DNA probes in the oligonucleotide synthesis method were determined with the following points in mind.

[1) Do not form a higher-order structure within the molecule.

(2) It has no homology with other regions of 16S rRNA.

(3) No repeating structure within the molecule.

(4) The composition ratio of the base should not vary significantly.

Two types of oligonucleotides (probes 1 and 2) with a length of 30 bases that meet these conditions and are considered suitable as DNA probes were developed. The base sequences of probes 1 and 2 are represented by formulas (IV) and (V) above. Note that this oligonucleotide is a reverse complementary strand of 16S rRNA. That is, probe 1 is M, hp 165
rRNA base numbers 203-232, and probe 2 corresponds to the reverse complementary strand of base numbers 610-639.

(C) “DNA using P-labeled synthetic oligonucleotide
Detection of M and hp by probe method The 5' end of a synthetic oligonucleotide was labeled with 32p, and detection of M and hp by DNA probe method was attempted.

1) Preparation of ``P-labeled synthetic oligonucleotide 32pg labeling was performed by phosphorylating the 5' end of the platform oligonucleotide using T4 polynucleotide kinase and rr-''Pl ATP. The specific radioactivity of the obtained oligonucleotide was approximately 4 X 10' c,p,m,
/pmole.

2) DNA probe method Since the above probe binds to rRNA and is also an oligonucleotide probe, the reaction does not occur under harsh hybridization conditions.

Therefore, we investigated various conditions such as culture solution treatment methods, hybridization methods, and membrane selection.

As a result, it became clear that the following method was appropriate.

Add 20% dithiothreitol or 20% dithiothreitol to 100 μl of test solution.
Add 100 μl of %N-acetyl-L-cysteine and
Treat at 7°C for 30 minutes to lyse.

In addition to lysing bacteria, this treatment can reduce the viscosity of highly viscous samples such as pig nasal secretions. This sample is spotted onto a membrane (Biotrace RP manufactured by Gelman, etc.). 80℃
After drying for 2 hours at 60 °C, prehybridization solution (: 5
15M Sodium Citrate>, 5 x Denholdt
'S solution (0.02% BSA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 0.2% 5DS
(sodium dodecyl sulfate), 200 μg/ml yeast t
Soak in RNA:] for 1 hour. Next, add 60°C hybridization solution [2 x 10gc, p, m, 710mI! No. 3
2P-marked oligonucleotide] for 2 hours.

After washing twice with 5xSSC-0.2% SDS solution at 60°C for 2 minutes each time and air drying, autoradiography is performed at -70°C for 16 hours using an X-ray film and an intensifying screen.

M, hp and M, hr (Mycoplas+na hy
Orhinis) was cultured for 2 days and used as a stock solution, and this stock solution was used as sample 1, sample 5.10.50.
Samples 2, 3, 4, 5 and 6 were prepared using the 00 times diluted solution, and each 100 μm of the solution was prepared according to the above method.
M and hp were detected using probes 1 and 2. The results are shown in Figure 2. (c) shows the results when probe 1 and probe 2 were used in amounts of I x 106c, p, m, /10-, respectively. In all cases (a), (b), and (c), M and hp could be detected in the 1000-fold diluted solution. The number of bacteria in the stock culture solution of M and hp is 1.
06-10'cfu/m1. Therefore, 1
It can be seen that a bacterial count of 02 to 10' can be sufficiently detected.

Furthermore, M and hr, which are most closely related to M and hp, did not react at all even with the stock culture solution.

Furthermore, both of the two types of oligonucleotides have specificity for M and hp, and by mixing the two, detection sensitivity can be further increased.

〔Effect of the invention〕

The method for detecting M and hp using the DNA of the present invention as a probe can be performed in a shorter time (about 2 days) than conventional separation detection methods. Furthermore, when the sample is treated with dithiothreitol or N-acetyl-L-steine, it can also be applied to samples such as highly viscous nasal secretions.

The nasal secretions of M, hp-infected pigs contain 10' to 10' cfu/
- Since it is thought that M.hp.- is present, it is also possible to directly diagnose M.hp. infected pigs using nasal discharge samples.

[Brief explanation of drawings]

FIG. 1 shows the entire base sequence of 16S rRNA of M and hp. FIG. 2 shows M measurements performed using probes 1 and 2 of the present invention. The results of the hp detection test are shown.

Claims (1)

[Scope of Claims] (1) A DNA having a nucleotide base sequence selected from the group consisting of the following formulas (I) to (V). (I) CCCCATTTC^1T^0TGGTGAAGC^2
T^0TGAAGGCTC^3C^0TTTGAATA
A^4A^0AATTCATGC^5A^0AATTT
TTAT^6C^0A(II) CCGCTAACC^1T^0CTTCATAAT^2
T^0CTATTTTGC^3C^0AGTATCTA
A^4A^0GCGGACTAA^5A^0GTTGA
GCTT^6T^0AGCATTTAA^7C^0TT
TAAAACTT^8A^0ACAAAAAAC^9C^
0T(III) GTGATCTCG^1T^0TAGCCTCGG^2
C^0TATATCTCT^3A^0TAGTTTTG
C^4G^0AGAATGTCA^5A^0G(IV) CTTGGTGAA^1G^0CTTGAAGGC^2
T^0CCTTTGAAT^3A^0 (V) CGGACTAAA^1G^0TTGAGCTTT^2
A^0GCATTTAAC^3^0T (2) Claim (1
) A recombinant DNA containing the nucleotide base sequence described in (a) as a part thereof. (3) A synthetic DNA comprising the nucleotide base sequence according to claim (1) as a part thereof. (4) Any one of claims (1), (2), and (3)
A method for detecting Mycoplasma swine epidemic pneumonia (Mycoplasma hyopneumoniae) by a DNA hybridization method using DNA containing the nucleotide base sequence described in Section 1 as a probe.