JPH0763375B2 - Reagent for genetic diagnosis of tsutsugamushi disease - Google Patents

Description

TECHNICAL FIELD The present invention relates to a reagent for gene diagnosis of tsutsugamushi disease, and more specifically, to a reagent that can be used for rapid diagnosis of tsutsugamushi disease caused by ricketts tsutsugamushi and a rickettsia DNA having low type specificity. Reagents for

[Prior arts and problems to be solved by the invention]

For diagnosis of tsutsugamushi disease patients, which have been occurring frequently not only in Japan but in Asian countries since the 1980s, immunological antigen-antibody reaction such as fluorescent antibody method, enzyme immunoassay method, complement fixation reaction, etc. was used. Methods using proteins have been mainly used. In addition, for the detection of rickettsia, which is a pathogen, a method in which a living mouse is used and it takes 1 to 3 months to separate and determine is generally performed.

By the way, the pathogen Rickettsia
The gene sequence of tsia tsutsugamushi) is
It is determined for the portion encoding the 56K protein of the Gilliam strain, which is one of the three standard strains.

Therefore, if a method for genetically diagnosing helminth disease using a part of this sequence is established, rapid diagnosis will be possible, and this will have a great impact on this field.

[Means for Solving the Problems]

As described above, many immunological techniques have been used to elucidate the actual condition of tsutsugamushi disease, but this method requires at least one week after infection for diagnosing the patient. Sensitive detection of rickettsiae, a pathogen that infects ticks (tsutsugamushi), a vector of this disease, is the most important preventive measure.

However, the above-mentioned conventional methods are technically limited and require many days to reach a conclusion. Therefore, there has been a strong demand for the development of a technique for efficiently and rapidly detecting a pathogen rickettsia.

It is an object of the present invention to provide a reagent for gene diagnosis of tsutsugamushi disease that meets such a demand.

The present invention relates to Gilliam, Karp, Kato of Rickettsia
Complementarity of the 5'ends 622 to 699 of the 2.3Kb HindIII fragment of the Gilliam strain of Rickettsia guttus, which encodes the 56K protein and has high homology to the DNA of each of the standard 3 strains of Kawasaki, kuroki and Shimokoshi. A pair of primers having the following base sequences necessary for synthesizing DNA Primer 1: ATAGAATTGGGTGAGGAAGGAGGATTAGAG Primer 2: ACCAGTAATCATTCCTCCAACGATTCCAAC and Rickettsia Atsushi Beetle DNA produced by allowing the above-mentioned complementary DNA to act as a template and the DNA synthase.
To a probe that specifically binds to.

The present invention will be described in detail below.

Selection of primers with high homology to the DNA of the other Rickettsia's chiggers (Karp, Kato, Kawasaki, kuroki, Shimokoshi) than the Gilliam gene HindIII fragment encoding the 56K protein of Gilliam strain determined by Ohashi et al., 2.3 Multiple sequences were selected from the Kb DNA sequence, and the GC content was calculated by a computer, and the 30'mer from the 622nd to 651st and the 699th to 670th positions from the 5'end of the sequence portion which seems to have the highest annealing temperature, respectively. The (base) DNA was synthesized.

This is a pair of primers having the above-mentioned base sequence.

Detection and determination of Rickettsia chigger beetle using a primer An attempt was made to detect cultured Rickettsia chigger DNA using the primer synthesized above, and it was confirmed that the theoretical value of 78b DNA was synthesized. By using a DNA amplification device with this primer, 3
Pathogens can be detected in ~ 4 hours. On the other hand, in the same Rickettsia genus, spotted fever rickettsia, rash fever rickettsia, Proteus bacterium, Shigella bacterium, Escherichia coli, etc., there was no DNA synthesized by this primer. In addition, since no cross-reactivity was observed with other bacteria, it can be used for the differential diagnosis of tsutsugamushi disease and other diseases.

It is known that rickettsia exists in the blood of patients with tsutsugamushi disease in the form of coexistence with antibodies, and by using the reagent of the present invention, rickettsia in blood can be directly detected. .

Preparation of probe The purified Gilliam strain digested with the restriction enzyme HindIII was separated by agarose gel electrophoresis, and the fragment was taken out according to a conventional method. Using this as a template and the artificially prepared primer and DNA synthesizing enzyme described above, ³² P is labeled to Rickettsia prickly beetle DNA by a commercially available primer labeling system. In addition, by using a synthetic base labeled with biotin or digoxigenin in advance in this probe in a similar manner, it is possible to produce a DNA probe that specifically binds to Rickettsia scrubella with high sensitivity without using a radioisotope. it can.

Diagnosis of tsutsugamushi disease using probe. Rickettsia tsutsugamushi DN on nylon membrane immobilized by dot hybridization and Southern blot hybridization using labeled DNA probe.
A could be detected with high sensitivity.

〔Example〕

 Next, the present invention will be described in detail with reference to examples.

Example 1 1) Preparation of Rickettsia chigger DNA from blood of tsutsugamushi disease patient Separation of lymphocytes 5 ml of patient blood was subjected to density gradient centrifugation with Ficoll to obtain a lymphocyte fraction. This lymphocyte fraction was washed twice with phosphate buffer.

Extraction of DNA Buffer for DNA extraction (2% SDS, 10mg / μl Proteinase K, 10
Add 500 μl of mM EDTA, 100 mM sodium acetate) to 56 ℃
And reacted for 2 hours.

Phenol extraction Add equal amount of phenol / chloroform / isoamyl (25/24/1) to the above reaction mixture, shake well, and then 10,000 rpm
After centrifugation for 5 minutes, a supernatant was obtained. This operation was repeated twice. Then an equal volume of chloroform / isoamyl (24/1)
Was added and treated in the same manner.

Ethanol precipitation To the above supernatant, add 1/20 volume of 3M sodium acetate and -70
Add twice the amount of ethanol cooled to ℃ and mix well,
After standing still at 70 ° C for 10 minutes, centrifugation was performed at 10,000 rpm for 30 minutes to obtain a precipitate. Then, after drying this, 50 μl of TE buffer (10
The sample was suspended in mM Tris-hydrochloric acid (pH 8.0), 1 mM EDTA).

2) Detection of Rickettsia chigger beetle DNA by polymerase chain reaction (PCR) Preparation sample 10 μl Purified water 53.5 μl PCR buffer ^* 10 μl 1.25 mM dNPT 16 μl Primer 1 (0.0025 0D / μl) 5 μl Primer 2 (0.0025 0D / μl) ) 5 μl Thermostable polymerase 0.5 μl ^* 100 mM Tris-hydrochloric acid (pH 8.3), 500 mM KCl, 15 mM MgCl ₂ ,
0.01% Gelatin The above samples were mixed and allowed to react for 30 cycles with one cycle consisting of 94 ° C for 30 seconds, 57 ° C for 2 minutes and 70 ° C for 2 minutes.

A part of the DNA grown by the PCR method was subjected to 2% agarose electrophoresis, stained with ethidium bromide, and then fluorescently colored by irradiation of ultraviolet rays to confirm the migration position, and then judged. If negative, thermostable polymerase 0.5 after the above reaction
μl was added and the reaction was further carried out for 30 salicles. In this way, it was possible to detect Rickettsia chigger DNA.

Example 2 Production of digoxigenin-labeled probe using PCR method DNA was extracted from the purified Rickettsia scrubbill Gilliam strain and used as a sample.

Sample 10 μl Purified water 59.5 μl PCR buffer ^* 10 μl Primer 1 (0.0025 0D / μl) 5 μl Primer 1 (0.0025 0D / μl) 5 μl Thermostable polymerase 0.5 μl ^* 100 mM Tris-HCl (pH8.3), 500 mM KCl, 15 mM MgCl ₂ ,
0.01% Gelatin The above samples were mixed and reacted for 30 cycles with 94 ° C for 30 seconds, 57 ° C for 2 minutes, and 70 ° C for 2 minutes as one cycle. This reaction solution was used as a probe.

Example 3 Detection of Rickettsia chigger DNA by dot hybridization method Rickettsia chigger beetle prepared in 1) of Example 1
The DNA sample was treated at 95 ° C. for 10 minutes for denaturation, quickly cooled in an ice bath, applied to a nylon membrane, air-dried well, and then the DNA on the nylon membrane was fixed by ultraviolet rays. The membrane was placed in a shield bag and the hybridization solution (5xSSC (20xSSC: 3M sodium chloride, 0.3M sodium citrate, pH 7.0), 0.5% blocking reagent, 0.1% sodium N-lauroylsarcosinate, 0.02% SDC) was added. ) Is added and prehybridization is performed at 68 ° C. for 4 hours,
Mix the solution from time to time. Next, the probe prepared in Example 2 was immediately heat-denatured at 95 ° C. for 10 minutes, added to the hybridization solution, and replaced with the solution in the shield bag. The shield bag was reacted at 68 ° C. for 18 hours and mixed occasionally. Membranes were washed twice with 2x SSC, 0.1% SDC for 5 minutes at room temperature. The membrane was then washed twice at 68 ° C. with 0.1 × SSC, 0.1% SDC for 5 minutes twice.

The membrane was washed with buffer solution 1 (100 mM Tris-hydrochloric acid, 150 mM sodium chloride, pH 7.5) for 1 minute. Then Buffer 2 (0.2
The reaction was performed for 30 minutes with the% blocking reagent / buffer solution 1).
This was washed again with buffer solution 1. Next, the labeled antibody (anti-digoxigenin antibody) was diluted to 150 mU / ml with buffer solution 1, and the membrane was reacted for 30 minutes. Then, the unreacted labeled antibody was removed by washing twice with Buffer 1 for 15 minutes. Then, the membrane was washed with buffer solution 3 (100 mM Tris-HCl, 100 mM sodium chloride, 50 mM).
Equilibrated with mM magnesium chloride, pH 9.5 for 2 minutes.

Then, in the dark, the color developing solution (45 μl nitroblue tetrazolium salt solution, 35 μl 5-bromo-4-chloro-3-indolyl phosphate solution / 10 ml buffer 3) was reacted with the membrane in a shield bag. Buffer 4 (10 mM Tris-HCl, 1 mM EDTA, pH 8) was used to stop the color development, and the membrane was washed for 5 minutes. The presence or absence of the desired DNA was determined by the color developed on the nylon membrane.

Example 4 Diagnosis of Patients by Southern Blot Hybridization Rickettsia scrub vulgaris prepared in 1) of Example 1
Enzyme reaction solution (50 mM Tris-hydrochloric acid, 10 mM magnesium chloride, 50 mM sodium chloride) 2 μl, 10 UHindII in 10 μl of DNA sample
Add 1 μl of I and 7 μl of purified water, react at 37 ℃ for 2 hours, then 1%
Perform agarose gel electrophoresis. Then, the agarose gel was transferred to an appropriate container and immersed in a depurination solution (250 mM hydrochloric acid). Next, the depurinating solution was discarded, washed with purified water three times, and the DNA of the agarose gel was transferred to a nylon membrane.
Then, the nylon membrane was immersed in 0.5N sodium hydroxide, 1.5M sodium chloride solution for 30 seconds, and then neutralized with 0.5M Tris-hydrochloric acid (pH7.4), 1.5M sodium chloride solution for 2 minutes,
It was air-dried well, and then the DNA on the nylon membrane was fixed with ultraviolet light. The membrane was placed in a sealed bag and the hybridization solution (5xSSC, 0.5% blocking reagent, 0.1%
% N-lauroyl sodium sarcosinate, 0.02% SDS)
Was added, prehybridization was performed at 68 ° C. for 4 hours, and the solutions were occasionally mixed.

Next, the probe prepared in the example was immediately heat-denatured at 95 ° C. for 10 minutes, added to the hybridization solution, and replaced with the solution in the shield bag. Shield back at 68 ℃
The mixture was reacted for 18 hours and mixed occasionally. 2xSSC, 0.1% S at room temperature
The membrane was washed twice with DS for 5 minutes. Then likewise
The membrane was washed twice at 68 ° C. with 0.1 × SSC, 0.1% SDS for 5 minutes twice.

The membrane was washed with buffer solution 1 (100 mM Tris-hydrochloric acid, 150 mM sodium chloride, pH 7.5) for 1 minute. Then Buffer 2 (0.2
The reaction was performed for 30 minutes with the% blocking reagent / buffer solution 1).
This was washed again with buffer solution 1. Next, the labeled antibody (anti-digoxigenin antibody) was diluted to 150 mU / ml with buffer solution 1, and the membrane was reacted for 30 minutes. Then, the unreacted labeled antibody was removed by washing twice with Buffer 1 for 15 minutes. Then, the membrane was washed with buffer solution 3 (100 mM Tris-HCl, 100 mM sodium chloride, 50 mM).
Equilibrated with mM magnesium chloride, pH 9.5 for 2 minutes.

Then, in the dark, the membrane was reacted with a color developing solution (45 μl nitroblue tetrazolium salt solution, 35 μl 5-bromo-4-chloro3-3 indolyl phosphate solution / 10 ml buffer 3) in a shield bag. Buffer 4 (10 mM
The membrane was washed with Tris-hydrochloric acid, 1 mM EDTA, pH8) for 5 minutes. The presence or absence of the desired DNA was judged from the color developed on the nylon membrane.

〔The invention's effect〕

According to the present invention, the genetic technology can be applied to the diagnosis of tsutsugamushi patients whose actual infections have not yet been elucidated, and therefore accurate and rapid diagnosis is possible. Furthermore, by using the reagent of the present invention, the pathogen Rickettsia, which is infected with a tick (tsutsugamushi), which is a vector of this disease, can be detected with high sensitivity, and the present invention is also useful in terms of preventive measures.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ken Tamura 5-13-2, Kamiieicho, Niigata City, Niigata Prefecture Niigata Technical College Niigata Pharmaceutical University (72) Inventor Norio Ohashi, Niigata City, Niigata Prefecture 5-13-2 Shineicho Niigata Technical College Niigata Pharmaceutical University (56) References Infect. Immun. , (1987) 55P. 1156-1162

Claims (2)

[Claims] 1. Gilliam, Karp, Kat of Rickettsia scrubbing beetle
The 5'end of the 2.3 Kb HindIII fragment of the Gilliam strain of Rickettsia scrubella, which encodes a 56K protein and has high homology to the DNA of each of the standard 3 strain of Kawasaki, kuroki, and Shimokoshi, from the 622nd to the 699th. A pair of primers having the following base sequences necessary for synthesizing complementary DNA. Primer 1: ATAGAATTGGGTGAGGAAGGAGGATTAGAG Primer 2: ACCAGTAATCATTCCTCCAACGATTCCAAC 2. A probe which binds specifically to Rickettsia staghorn DNA produced by reacting the primer of claim 1 with a DNA synthase using the complementary DNA of claim 1 as a template.