JPH0436200A - Method for detecting mycobacterium tuberculosis - Google Patents

Abstract

PURPOSE:To quickly detect Mycobacterium tuberculosis for clinical examination in high sensitivity by subjecting a mixed liquid of the first and second DNA primers having specific base sequences, DNA polymerase and aqueous liquid sample to be examined to DNA amplification, then subjecting the resultant reaction liquid to DNA examination. CONSTITUTION:A mixed liquid containing the first DNA primer containing an oligonucleotide part consisting of a base sequence expressed by formula I, the second DNA primer containing an oligonucleotide part consisting of a base sequence expressed by formula II, DNA polymerase and aqueous liquid sample to be examined is subjected to DNA amplification and the resultant reaction liquid is subjected to DNA amplification by a polymerase chain reaction method and successively the resultant treated liquid is subjected to DNA examination to detect the Mycobacterium tuberculosis.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to human tuberculosis Mycobacterium tuberculosis (Mycobacterium tuberculosis).
erculosis).

[Prior Art] Mycobacterium tuberculosis is the pathogenic microorganism of human tuberculosis, and its testing is clinically extremely important. Mycobacterium tuberculosis is highly pathogenic to humans and causes tuberculous lesions in almost all organs and tissues.

In particular, pulmonary tuberculosis is overwhelmingly common. Mycobacteria (Mycobacteria)
In addition to the aforementioned Mycobacterium tuberculosis, examples of microorganisms belonging to the genus Bacteria include atypical acid-fast bacteria such as Mycobacterium bovis (i.e., Mycobacterium bovis; M.
or Mycobacterium avium (i.e., Mycobacterium avium; M.

avium) is known. These atypical mycobacteria are less pathogenic but often affect humans, especially the human lungs.
However, because the clinical symptoms are very similar to those of pulmonary tuberculosis, it is extremely important to differentiate between them. Therefore, identifying Mycobacterium tuberculosis and separating it from atypical mycobacteria in a short time is of particular importance in carrying out appropriate chemotherapy.

Traditionally, culture methods have been widely used as a method for detecting Mycobacterium tuberculosis.9 For example, in Japanese Patent Publication No. 7723/1987, Mycobacterium tuberculosis was inoculated into Ogawa medium and cultured, and Mycobacterium tuberculosis human and Mycobacterium bovis were detected. A technique for identifying the two is described. However, according to these conventional culture methods, a period of 3 to 8 weeks is required for culturing, and furthermore, a period of 2 to 4 weeks is required for final identification. Therefore, it was inconvenient for early diagnosis.

On the other hand, as an early diagnosis method, JP-A-63-180859
The publication describes a measurement method using a monoclonal antibody specific for Mycobacterium tuberculosis produced by a hybridoma cell line. However, in this method, pretreatment of the test sample was complicated. In addition, 125-labeled D
Hybridization methods using NA probes have also been developed (e.g., Diagn, Micro-bio
l, Infect, Dis, 1987;8:69-78
). However, since this method uses radioactive materials, it requires special equipment, and furthermore, it has not always been possible to directly detect and identify Mycobacterium tuberculosis from clinical samples.

[Problems to be Solved by the Invention] As a result of various studies to realize the early detection of Mycobacterium tuberculosis contained in clinical samples, the present inventor has discovered that using specific DNA primers, It has been discovered that by amplifying the Mycobacterium tuberculosis DNA in a sample, Mycobacterium tuberculosis can be specifically, highly sensitively, and rapidly detected and identified. Therefore, an object of the present invention is to provide a means for specifically, highly sensitively, and rapidly detecting and identifying Mycobacterium tuberculosis.

[Means for Solving the Problems] According to the present invention, the above-mentioned object is to provide a first DNA primer containing an oligonucleotide portion consisting of a base sequence represented by the formula % formula % (3); (3) A mixture containing a second DNA primer containing an oligonucleotide portion consisting of the base sequence represented by, DNA polymerase, and an aqueous liquid test sample is subjected to a DNA amplification step, and then the obtained Mycobacterium tuberculosis, which is characterized by subjecting the reaction solution to a DNA testing process.
cterium tuberculosis).

In this specification, the following symbols are used to simplify the description.

A: Adenine C: Cytosine G Nigeanine T: Thymine DNA: Deoxyribonucleic acid tRNA: Transcriptional ribonucleic acid dATP: Deoxyadenosine triphosphate dCTP: Deoxycytidine triphosphate dGTP: Deoxyguanosine triphosphate dTTP: Deoxythymisine trisinate acid dNT
P: deoxynucleoside triphosphate bp: base pair In the present invention, PCR (polymerase chain
n reaction) method can be used as the DNA amplification step. PCR method can be used (S
c 1ence, 239:487-491.1988
>. In the PCR method, amplification cycles are repeated using two types of DNA primers for ten strands and one strand, and a thermostable DNA polymerase, sandwiching the DNA region to be amplified.

First and second DNs that can be used in the method of the present invention
Primer A is a 19kD protein that is a specific protein of Mycobacterium tuberculosis DNA.
They are an oligonucleotide of approximately 16 to 30 bases on the 5'' side upstream of the DNA sequence encoding the a protein, and an oligonucleotide of approximately 16 to 30 bases on the 3' side.Specifically, as the first DNA primer, the base Array (5')
An oligonucleotide containing at least a hexadecanucleotide moiety consisting of -CACGGCGAT'GGAGT-(3'), preferably the base sequence (5') -AGCACGGCGATTTGGAGTC
An oligonucleotide containing at least an eicosanucleotide moiety consisting of G-(3°) can be used.

In addition, in the method of the present invention, as the second DNA primer,
Specifically, the base sequence (5') -CGTTTTTCGGCGGTATC-(3
An oligonucleotide containing at least a hexadecanucleotide moiety consisting of the base sequence (5')-ATCGTTTTCGGCGGTATCT
An oligonucleotide containing at least an eicosanucleotide moiety consisting of G-(3') can be used.

Using oligonucleotides with less than 16 bases as the first and second DNA primers is not preferable because the specificity is not sufficient, and using oligonucleotides with more than 30 bases increases the possibility of self-polymerization. So I don't like it.

Each base constituting the first and second DNA primers used in the method of the present invention may be modified in any manner known to those skilled in the art (eg, biotinylation, labeling with a luminescent substance).

The first and second DNA primers used in the present invention are prepared using a known DNA synthesis method (for example, manufactured by Applied Biosystems) using an ordinary automatic DNA synthesizer (for example, manufactured by Applied Biosystems).
For example, it can be prepared by the phosphoamitite method).

In the present invention, thermostable DNA is used as the DNA polymerase.
Polymerases, especially thermostable DNA polymerases that can maintain activity at temperatures up to about 95°C, e.g.
Commercially available Taq polymerase can be used.

The liquid test sample used in the method of the present invention is not particularly limited as long as it is a sample suspected of containing Mycobacterium tuberculosis.

For example, sputum, gastric aspirate, bronchoalveolar lavage fluid, stool, urine,
Cerebrospinal fluid or blood can be used. Before applying the DNA amplification process to these liquid test samples,
Preferably, an extraction treatment (for example a phenol/chloroform treatment) is carried out.

In the method of the present invention, a mixed solution containing the first and second DNA primers, DNA polymerase, and a liquid test sample is used. The amounts of the first and second DNA primers and DNA polymerase to be used vary depending on the type of liquid test sample, but can be easily determined within the range that allows the DNA amplification step by PCR method to be performed.

The mixture may optionally contain a buffer (eg, Tris-HCl buffer), a stabilizer (eg, gelatin), or a salt (eg, sodium chloride).

In the method of the present invention, the amplification cycle of the PCR method is performed using the above-mentioned mixed solution. The amplification cycle consists of (i) a DNA denaturation step (about 0°C to about 95°C, about 10 seconds to about 2 seconds);
(ii) an annealing step between the single-stranded DNA and the primer (approximately 37° C. to approximately 70° C., approximately 30 seconds to approximately 3 minutes), and (iii) a DNA synthesis step using DNA polymerase (
at about 65°C to about 80°C for about 30 seconds to about 5 minutes). With each cycle, the DNA is amplified twice, n
After the cycle, it is amplified 2n times. In the present invention, the above amplification cycle is carried out 10 to 60 times, preferably 20 times.
Repeat ~40 times. In the last cycle, the process (
In step iii), extend the heating time to about 5 to 10 minutes and add the DNA.
Preferably, the synthesis is complete.

If Mycobacterium tuberculosis is present in the test sample, approximately 50 to 1000 bp of DNA is added after the amplification cycle is completed.
is synthesized in large quantities. This DNA is detected by the next DNA testing process.

As the DNA testing step, a method using gel electrophoresis and ethidium bromide staining, a dot hybridization method, a base sequencing method using a dideoxy method, etc. can be used. When performing gel electrophoresis, for example, submarine electrophoresis using agarose gel as a carrier or slab electrophoresis using acrylamide can be used.

When performing the dot hybridization method,
Non-radioactive probes (eg, biotinylated probes or chemiluminescent probes) can be used.

Furthermore, when using a base sequencing method using the ditheoxy method, a DNA autohuman quencher using a fluorescent label (Abride Biosystems) can be used.

[Function] In the method of the present invention, a 20-base oligonucleotide starting from position 981 of the sequence encoding the 19 kDa protein of Mycobacterium tuberculosis DNA is used as the first primer, and as the second probe, In particular, since a 20-base oligonucleotide starting from position 1300 of the sequence encoding the 19 kDa protein of Mycobacterium tuberculosis DNA is used, only when Mycobacterium tuberculosis is present in the test sample, can the DNA of Mycobacterium tuberculosis be detected. 320b corresponding to part of the gene encoding the 19kDa protein of
The p portion is specifically amplified and synthesized in large quantities within a short period of time. Therefore, the presence of Mycobacterium tuberculosis in the test sample can be detected very specifically. Furthermore, even if the amount of Mycobacterium tuberculosis in the test sample is minute, DNA can be amplified and synthesized, resulting in high sensitivity. Moreover, the total time required for the amplification step and inspection step in the method of the present invention is about 4 to
It takes 8 hours, which is extremely fast.

[Real Color Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples, but these are not intended to limit the scope of the present invention.

1 primer, i.e. (5°)-AGCACGGCGATTTGGAGTCG
-(3°) and the second primer, i.e. (5')-ATCGTTTTCGGCGGTATCT
An oligonucleotide having a base sequence of 380A-
Synthesis was performed using a DNA synthesizer (Applied Biosystems) under trityl-one conditions. The column was cut out with a 27% ammonia aqueous solution and heated at 55°C overnight. 2 ml of the obtained solution was diluted with 1 ml of ion-exchanged water, and the diluted solution was diluted with 0PC (01i gouc 1 eot
i depurification column
) Cartridge (Applied Biosystems) 1
Added at a rate of ~2 drops/sec. The eluate was washed three times with 5 ml of diluted ammonia aqueous solution, twice with 5 ml of ion-exchanged water, and washed with 2% T.
2 times with 5 ml of FA solution and then 5 ml of ion-exchanged water
Washed twice with The adsorbed nucleotides were eluted three times with 1 ml of 20% acetonitrile solution to obtain purified oligonucleotides. Using the thus obtained first primer (hereinafter referred to as Primer 1) and second primer (hereinafter referred to as Primer 2), the following Examples 2 to 4 were carried out.
3 solid colors.

Manu 2: As shown in Table 1 below, the persimmons used in various types of fresh balls contain mycobacteria (Mycobacteria).
Examine whether the DNA of various bacteria belonging to the genus Bacteria, Clam-positive bacteria, Clam-positive bacteria, and fungi can be amplified by the primers prepared in Example 1 above.

Prepare 100 μl of a reaction solution consisting of the following components in a 500 μl Eppendorf tube.

Component liquid volume Final concentration H2O 53.5 Reaction buffer 10 1 concentration (10 times concentration) Each dNTP mixture 16 200 μM 1
．． 25mM Brimer 1 5 1.0μM Brimer 2 5 1.0μM Analyte D
NA 10 1ng/assay
T DNA polymerase 0.5 2.5Ta
q As a DNA polymerase, AmpliTaq
DNA polymerase (Cetus) was used.

Next, the reaction solution was soaked in mineral oil (Si gmaCal).
M-3516) was covered with 100 μl. The obtained sample was subjected to DNA Thermal Cyc 1 e r (P
Erk in-E 1mer Cetus Inc.). A three-step amplification cycle was repeated 37 times: (i) 94°C for 1 minute, (ii) 65°C for 2 minutes, and (iii) 72°C for 2 minutes. In addition, in the 37th and final cycle, step (iii) was performed for 5 minutes.

10μ each of the reaction solution obtained by the DNA amplification procedure described above.
1 was electrophoresed on a 2% agarose gel containing ethidium bromide (0.5 μg/ml). In Table 1, “
As shown in the "Amplification" column, a DNA band was observed at 320 bp in Mycobacterium tuberculosis and Mycobacterium bovis, but no DNA band was observed in other microorganisms. Note that, as it is known that Mycobacterium tuberculosis and Mycobacterium bovis are very closely related species, the method of the present invention results in positive results for both. However, since the incidence of Mycobacterium bovis infection is extremely low and only occurs in immunocompromised humans, testing positive for both has virtually no effect on the purpose of clinical diagnosis. Table 1 below shows the microorganisms used in the test and the amplification results.

[Margin below] M, tuberculosis M, bovis M, avium M, fortuitum M, gordonae M, intracellulare M, kansasii M, marinum M, 5crof uraceum M , smegmatis M, Actiomyces naeslundi Actio
myces propionica Bacillus
alvei Bacillus cereus Bacillus coagulans Table 1 ATCC27294 ATCC19274 ATCC11739 ATCC19542 ATCC14470 ATCC15984 ATCC12478 ATCC927 AT CC19275 ATCC14468 + + ATCC12104 ATCC6344 ATCC14579 ATCC23498 Bacillus megateriumBacill
us polymyxa Bacillus pumilus Bacillus subtilis Corynobacterium erose Corynobacterium diphtheriae Lactobacillus acidophilus Lactobacillus brevis Lacto
bacillus fermentum Lactobacillus jenseniiATC
C14581 ATCC7061 ATCC7051 ATCC27094 ATCC4356 ATCC14869 ATCC14869 ATCC25258 LACTOBACILLUS PLANTARUM ATCC1491
7Staphylococcusaureus AT
CC6538 Achromobacter xylosoxidans Acinetobacter calcoacetics RIMDOlolool Aeromonas caviae ATCC15468 Aeromonas caviae Enterobacter colacae Esher
ichia cc city ATCC7966 ATCC13047 ATCC12814 Morganella morganii ATCC
8076Proteus m1rabilis
IFO3849Proteus vulgaris
ATCC13315Serratia marce
sceneVibrio a1ginolyticu
s Candida albicans ATCC14756 ATCC17749 1 of Mycobacterium tuberculosis (strain used in Example 2) on 1% Ogawa medium
Suspend the loop in 1 ml of saline and perform a dilution series (
1x, 10-3x, 10-4x, 10-5x, 10-6
A 1/10 volume portion of each dilution series was inoculated onto the medium and the number of colonies was counted.

On the other hand, DNA was extracted from each dilution series described above using phenol/chloroform, and 1/10 of the extracted DNA was subjected to the DNA propagation step and DNA testing step described in Example 2 above. The results are shown in Figure 1A. In Figure 1A, the upper row (
Dilution) shows the dilution level, the middle row (PCR) shows the results of the method of the present invention, and the bottom row (Colonie) shows the results of the method of the present invention.
s) indicates the number of colonies in the culture method. As is clear from FIG. 1A, it can be seen that according to the method of the present invention, DNA can be amplified and detected from a 10-6 times diluted solution. In contrast, the culture method is 10-6 times or 10-
Positive for 7-fold dilution. That is, the method of the present invention has substantially the same degree of stability as the culture method with respect to pre-cultured Mycobacterium tuberculosis.

(i) Testing according to the method of the present invention of human type (49 people) at birth 0. Sputum and bronchoalveolar lavage fluid were collected from humans (49 people), liquefied with a 2% aqueous sodium hydroxide solution, and centrifuged (
2000×g for 10 minutes).

The obtained pellet was washed with 10 mM Tris buffer (pH 8,0) containing 1 mM EDTA, and then washed with 10 mM Tris buffer (pH 8,0) containing 1% sodium dodecyl sulfate.
DN in phenol/chloroform.
Extract A. The obtained DNA was digested with yeast tRNA (10
μg) and precipitated with ethanol. The resulting precipitate was dissolved in 10mM) Lys buffer (pH 8,0) IOC)μ
The DNA amplification operation and DNA testing operation described in Example 2 above were performed on 10 μm of the solution.

For comparison purposes, for each of the same clinical samples as above, (i
Do not perform tests by i) microscopic observation and (iii) culture method.

(ii) 2. That is, the same sputum and bronchoalveolar lavage fluid as above were fixed on glass slides, and Zie 1
-Nee 1sen) method and microscope (500x magnification)
The judgment was made by observing.

1ii) By P' 4% of the same sputum and bronchoalveolar lavage fluid as before the examination was twice the volume.
The mixture was mixed with an aqueous sodium hydroxide solution at room temperature for 15 minutes, and 100 μm of the mixture was inoculated into 1% Ogawa medium and cultured at 37° C. for 8 weeks. Zie 1-Nee
Mycobacterium tuberculosis was identified using the niacin test (Niacin Test Kobayashi; manufactured by Kobayashi Pharmaceutical Co., Ltd.).

The results of (i), (ii), and (iii) above are shown in Figure 2. In Figure 2, "column 1" is a sample that was positive in all of the microscopic examination, culture method, and method of the present invention, "Column 2" is a sample for which the microscopic examination was negative and both the culture method and the method of the present invention were positive, and "column 3" is a sample for which the microscopic examination and the culture method were negative and only the method of the present invention was positive. , and "column 4" is a sample that is negative in both the microscopic examination, the culture method, and the method of the present invention.Second
As is clear from the figure, the method of the present invention shows a positive result (a band appears at 320 bp) even for samples that are negative in microscopic observation and culture tests.

In addition, Table 2 below shows the comparison results of culture method (iii) and method (i) of the present invention. In Table 2, (+) indicates positive, (-) indicates negative, and the numerical value is the number of specimens. shows.

Table 2 Method of the present invention (+) ' (+) 14 1 Method of the present invention (+) As is clear from Table 2, samples positive by the culture method (15
All specimens) were also positive using the method of the present invention. Furthermore, of the samples (34 samples) that were negative by the culture method, 9 samples were positive by the method of the present invention. Therefore, it can be seen that for clinical samples, the method of the present invention is more effective than the culture method.

5: Human type 0 The procedure described in Example 3 above was repeated, but instead of using pre-cultured Mycobacterium tuberculosis, sputum from a patient with Mycobacterium tuberculosis was used. That is, 1 ml of sputum from a Mycobacterium tuberculosis patient was diluted with Telj buffer, and dilution series (1x, 10-3x, 10-
4x, 10-5x, 10-6x, 10-7x, and 1
0-8 times) were prepared. The results are shown in Figure 1B. In FIG. 1B, the lower row (Dilution) shows the dilution, the middle row (PCR) shows the results of the method of the present invention, and the lower row (Colonies) shows the number of colonies in the culture method. As is clear from FIG. 1B, it can be seen that according to the method of the present invention, DNA can be amplified and detected from a 10-6 times diluted solution. In contrast, in the culture method, 10
-Positive for dilutions up to 4 times. That is, the method of the present invention has a 100 times higher sensitivity than the culture method with respect to samples from patients with Mycobacterium tuberculosis.

[Effects of the Invention] According to the method of the present invention, the presence of Mycobacterium tuberculosis in a test sample can be detected specifically, highly sensitively, and rapidly.

[Brief explanation of the drawing]

FIG. 1A is an explanatory diagram showing the results of testing precultured Mycobacterium tuberculosis by the method of the present invention and the conventional culture method, and is a photograph in place of a drawing. FIG. 1B is an explanatory diagram showing the results of testing the sputum of Mycobacterium tuberculosis patients using the method of the present invention and the conventional culture method, and is a photograph in place of a drawing. FIG. 2 is an explanatory diagram showing the results of amplifying various samples by the method of the present invention, and is a photograph in place of a drawing. Figure 1 Cofor++6s 1 (110"tO' 34 C010n i e S wa 0 et al Figure procedure amendment writing method) % formula % 1, Indication of the incident Patent application No. 142581 of 1990 2, Name of the invention Human tuberculosis Bacteria detection method 3゜Relationship with the case of the person making the amendment Patent applicant name Yatron Co., Ltd. 4゜

Claims (1)

[Claims] (1) Formula (5')-CACGGCGATTTGGAGT-(3'
) a first DNA primer containing an oligonucleotide portion consisting of the base sequence represented by the formula (5')-C
A mixture containing a second DNA primer containing an oligonucleotide portion consisting of a base sequence represented by GTTTTCGGCGGTATC-(3'), a DNA polymerase, and an aqueous liquid test sample is subjected to a DNA amplification step,
Subsequently, the resulting reaction solution is subjected to a DNA testing process to test Mycobacterium tuberculosis (Mycobacterium tuberculosis).
umtuberculosis) detection method.