JP2966478B2 - Mycobacterial microorganism detection method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for detecting a microorganism belonging to the genus Mycobacteria and a primer set.

[Background Art] Mycobacterium tuberculosis is a pathogenic microorganism of tuberculosis in humans, and its test is extremely important clinically. Mycobacterium tuberculosis shows strong pathogenicity to humans,
Causes tuberculous lesions in almost all organs and tissues.
In particular, pulmonary tuberculosis predominates. Mycobact
As microorganisms belonging to the genus eria, in addition to the above-mentioned M. tuberculosis, atypical mycobacteria such as M. bovis (ie, Mycobacterium bovis; M. bovis) or M. avium (ie, M. tuberculosis) Bacterium avium (M.avium) is known. These atypical mycobacteria are poorly pathogenic but often affect humans, especially human lungs. However, its clinical manifestations are very similar to those of pulmonary tuberculosis, and its differentiation is extremely important. Therefore, performing identification of Mycobacterium tuberculosis and differentiation from atypical mycobacteria in a short time is particularly important in performing accurate chemotherapy.

Conventionally, a culture method has been widely used as a method for detecting Mycobacterium tuberculosis. For example, Japanese Patent Publication No. 51-7723 describes a technique of inoculating M. tuberculosis into Ogawa medium and culturing the M. tuberculosis to discriminate between M. tuberculosis and M. bovis. However, according to these conventional culturing methods, culturing requires a period of 3 to 8 weeks, and final identification requires 2 to 4 weeks. Therefore, it was inconvenient for early diagnosis.

On the other hand, as an early diagnosis method, JP-A-63-180859 describes a measurement method in which a monoclonal antibody specific to M. tuberculosis is produced by a hybridoma cell line and the monoclonal antibody is used. However, in this method, the pretreatment of the test sample was complicated. In addition, a hybridization method using an I ^125- labeled DNA probe specific to various Mycobacterium tuberculosis has been developed (for example, Diagn. Microbiol. Infect. Dis. 1987; 8: 69-7).
8). However, this method requires special equipment because it uses a radioactive substance, and it is not always possible to directly detect and identify M. tuberculosis from clinical samples.

[Problem to be Solved by the Invention] The present inventors have conducted various studies in order to realize early detection and differentiation of M. tuberculosis bacteria and atypical mycobacteria contained in clinical samples. Using specific DNA primers, amplify the DNA of mycobacteria (ie, Mycobacterium tuberculosis and atypical mycobacteria) in clinical samples, treat the amplified DNA with restriction enzymes, and analyze the treatment pattern By doing so, it has been found that acid-fast bacilli (ie, Mycobacterium tuberculosis and atypical acid-fast bacilli) can be specifically and sensitively and rapidly detected and differentiated. Accordingly, an object of the present invention is to provide a means for specifically and rapidly detecting and separating acid-fast bacteria with high sensitivity.

[Means for Solving the Problems] The above object is achieved by the present invention, A first DNA primer containing an oligonucleotide portion consisting of a base sequence represented by A second DNA primer containing an oligonucleotide portion consisting of a base sequence represented by
Achieved by a method for detecting a microorganism belonging to the genus Mycobacteria, which comprises subjecting a mixture containing an aqueous liquid test sample to a DNA amplification step and subjecting the resulting reaction solution to a DNA fractionation inspection step. be able to.

The present invention also relates to the formula A first DNA primer containing an oligonucleotide portion consisting of a base sequence represented by And a second DNA primer containing an oligonucleotide portion having the nucleotide sequence represented by the following formula:

In the present specification, the following symbols are used for simplifying the description.

A: adenine C: cytosine G: guanine T: thymine DNA: deoxyribonucleic acid tRNA: transcribed ribonucleic acid dATP: deoxyadenosine triphosphate dCTP: deoxycytidine triphosphate dGTP: deoxyguanosine triphosphate dTTP: deoxythymidine triphosphate dNTP : Deoxynucleoside triphosphate bp: base pair The microorganism that can be detected by the method of the present invention is a microorganism belonging to the genus Mycobacteria. These are called mycobacteria, and specifically, M. tuberculosis (M. tuberculosi)
s), M. bovis, M. avis (M.aviu)
m), M. fortuitum
um), Mycobacterium chelonii (M.chelonei),
Mycobacterium gordonae (M. gordonae), Mycobacterium intracellulare (M. intracellular)
e), M. kansasii,
M. marinum, M. scrofuraceum, M. smegmatis, M. szulgai or Mycobacterium gastri (M.gastri). Of particular interest are M. tuberculosis, M. bovis, M. avium, M. intracellulare, and M. kansasii.

In the present invention, a PCR (polymerase chain reaction) method can be used as the DNA amplification step. By using the PCR method, it is possible to automatically amplify only the target DNA region up to about 1 million times from a small amount of cellular DNA (Scien
ce, 239: 487-491, 1988). In the PCR method, an amplification cycle is repeated using two types of DNA primers for a + strand and a − strand and a heat-resistant DNA polymerase across a DNA region to be amplified.

First and second DN that can be used in the method of the present invention
The A primer is a specific protein that is almost common to microorganisms belonging to the genus Mycobacteria, particularly M. tuberculosis DNA, M. avium DNA, M. intracellulare DNA, and M. kansasii DNA, and is a specific protein, Dina J (dna J) An oligonucleotide having 20 to 30 bases 5 ′ to the upstream and a 20 to 30 base oligonucleotide 3 ′ to the DNA sequence (positions 1393 to 1360) encoding the protein. Specifically, as the first DNA primer, An oligonucleotide containing at least an eicosanucleotide moiety consisting of

In the method of the present invention, as the second DNA primer,
In particular, An oligonucleotide containing at least an eicosanucleotide moiety consisting of

It is not preferable to use an oligonucleotide having more than 30 bases as the first and second DNA primers because the possibility of self-polymerization increases.

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

The first and second DNA primers used in the present invention can be obtained by a known DNA synthesis method (for example, a phosphoramidite method) using a normal DNA automatic synthesizer (for example, an automatic synthesizer manufactured by Applied Biosystems). Can be prepared by

In the present invention, a thermostable DNA polymerase, particularly a thermostable DNA polymerase capable of maintaining its activity at a temperature up to about 95 ° C., for example, a commercially available Taq polymerase can be used as the DNA polymerase.

The liquid test sample used in the method of the present invention is a microorganism belonging to the genus Mycobacteria, that is, mycobacteria (M. tuberculosis and atypical mycobacteria, such as M. avium, M. intracellulare and M. intracellulare). The sample is not particularly limited as long as it is suspected of containing KS. For example, sputum, gastric aspirate, bronchoalveolar lavage, stool, urine, cerebrospinal fluid or blood can be used. For these liquid test samples, DNA
It is preferable to perform a DNA extraction treatment (for example, a phenol / chloroform treatment) before the amplification step.

In the method of the present invention, a mixed solution containing the above-mentioned first and second DNA primers, DNA polymerase and a liquid test sample is used. First and second DNA primers and D
The amount of NA polymerase to be used varies depending on the type of the liquid test sample, but can be easily determined as long as the DNA amplification step by the PCR method can be performed.

The mixture can optionally include a buffer (eg, Tris-HCl buffer), a stabilizer (eg, gelatin), or salts (eg, sodium chloride).

In the method of the present invention, an amplification cycle of the PCR method is performed using the above-mentioned mixture. The amplification cycle includes (i) a DNA denaturation step (about 90 ° C. to about 95 ° C. for about 10 seconds to
(Ii) a step of annealing single-stranded DNA to a primer (at about 37 ° C to about 70 ° C for about 30 seconds to about 3 minutes); and (iii) a step of synthesizing DNA with a DNA polymerase (about 65 minutes). ° C ~
(At about 80 ° C. for about 30 seconds to about 5 minutes). The DNA is doubled every cycle, and n
After the cycle, it is amplified by 2 ⁿ times. In the present invention,
The above amplification cycle is repeated 10 to 60 times, preferably 20 to 40 times. In the last cycle, it is preferred to extend the heating time in step (iii) to about 5 to 10 minutes so that the DNA synthesis is completely performed.

Microorganisms belonging to the genus Mycobacteria, especially Mycobacterium tuberculosis, Mycobacterium avium, M. intracellulare or M. kansasii in the test sample, after the end of the amplification cycle, about 50-1000bp. Is synthesized in large quantities. This DNA is treated by the following restriction enzyme treatment step.

In the restriction enzyme treatment step of the method of the present invention, the DNA amplified in the above step is treated with one or more (in particular, two) II
Treat with type restriction endonuclease. The DNA portion that serves as a template for amplification includes M. tuberculosis, M. avium, and M. avium.
It has different base sequence portions depending on the type of intracellulare or M. kansasii, respectively. Therefore, when a specific restriction endonuclease that recognizes the different base sequence portion is used, the template DNA (that is, the amplified DNA) is used.
Depending on the type of digestion, various types of digestion may or may not be performed. As the restriction endonuclease, any restriction endonuclease can be used as long as it can recognize the above-mentioned different nucleotide sequence portion, and in particular, 4 to 14 bases (preferably 6 bases)
A restriction endonuclease that recognizes the palindrome is used. These restriction endonucleases include, for example,
Sma I, Nae I, Acc III or Hinf I. The above restriction endonucleases can be used in combination, and for example, a combination of Sma I and Nae I is preferable.
Acc III can be used alone.

In the restriction enzyme treatment step of the method of the present invention, a buffer solution is added to the reaction solution obtained by the PCR amplification step, the above-described restriction enzyme is further added, and a temperature suitable for the enzyme reaction (for example,
C.) for a period of time during which the digestion with the enzyme is sufficiently carried out (for example, 30 minutes to 2 hours).

The treatment solution containing the DNA thus treated is analyzed by a DNA fractionation inspection step. That is, the mode of digestion can be ascertained from the size of the DNA fraction contained in the treatment solution, and the type of amplified DNA (ie, template DNA) before treatment can be analyzed from the mode of digestion.

As the DNA fractionation inspection step, a method using gel electrophoresis and ethidium bromide staining, a dot hybridization method, a base sequence determination method using the dideoxy method, and the like can be used. When performing gel electrophoresis, for example, submarine electrophoresis using an agarose gel as a carrier, or slab electrophoresis using acrylamide can be used.

When performing the dot hybridization method, a non-radioactive probe (for example, a biotinylated probe or a probe labeled with a chemiluminescent substance) can be used.

Furthermore, when a nucleotide sequence determination method by the dideoxy method is used, a DNA autosequencer using a fluorescent label (Applied Biosystems) can be used.

[Examples] Hereinafter, the present invention will be described more specifically with reference to Examples, but these do not limit the scope of the present invention.

Example 1: Synthesis of primers Oligos having the base sequences of the first primer, (5 ')-GGGTGACGCGGCATGGCCCA- (3') and the second primer, (5 ')-CGGGTTTCGTCGTACTCCTT- (3') Nucleotide to 380A-DNA
It was synthesized under the condition of trityl-one using a synthesizer (Applied Biosystems). The column was cut with a 27% aqueous ammonia solution and heated at 55 ° C. overnight. 2 ml of the obtained solution is diluted with 1 ml of ion-exchanged water, and the diluted solution is diluted with OPC (O
ligonucleotide purification column) cartridge (Applied Biosystems) at a rate of 1-2 drops / sec. Elute the eluate with 5 ml of diluted ammonia solution 3
2 times with 5 ml of deionized water, 2 times with 5 ml of 2% TFA solution,
Then, it was further washed twice with 5 ml of ion-exchanged water. The adsorbed nucleotide was eluted three times with 1 ml of a 20% acetonitrile solution to obtain a purified oligonucleotide. 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 and 4 were carried out.

Example 2: Amplification of DNA of various microorganisms (1) M. tuberculosis (ATCC27294), M. avium (ATCC15769), M. intracellulare (ATCC15984) and M. intracellulare (ATCC15984) DNA was extracted from each culture of Kansasi (M. kansasii; ATCC12478) with phenol-chloroform and amplified with the following reaction mixture using the primers prepared in Example 1 above. 500 μl
In an Eppendorf tube, 100 μl of a reaction solution comprising the following components was prepared.

As Taq DNA polymerase, AmpliTaq DNA polymerase (Citas) was used.

Next, the reaction solution was treated with mineral oil (Sigma Cal M-35).
16) Cover with 100 μl. DNA Thermal C
ycler (Perkin-Elmer Cetus). (I) 94
An amplification cycle consisting of three steps of (1) C. for 1 minute, (ii) 65 ° C. for 2 minutes and (iii) 72 ° C. for 2 minutes was repeated 37 times. In the 37th last cycle, step (iii)
For 5 minutes.

10 μl each of the reaction solution obtained by the above DNA amplification operation
Was electrophoresed on a 2% agarose gel containing ethidium bromide (0.5 μg / ml). First result
This is shown in the left column (undigested column) in the figure. In the left column (undigested column) in FIG. 1, the upper column indicates the type of microorganism, the numerical value on the right (236) indicates the size of base pairs, and the lower column (undigested) indicates that no restriction enzyme is used. It is shown. As is clear from the left column (undigested column) in FIG. 1, the DNAs of all four mycobacteria were amplified and showed a band at 236 bp.

(2) The same operation as described in the above Example 2 (1) is repeated, except that M. bovis;
CC19274), M. fortuitum (ATCC195)
42), M. marinum (ATCC927), and M.
Gastri (M. gastri; ATCC15754) was used. As in the results of Example 2, the DNAs of all four mycobacteria were amplified and 236 bp
Shows the band.

Example 3: Comparison of digestion patterns of various microorganism DNAs (1) 8 μl of the DNA amplification reaction solution obtained in Example 2 (1)
And 1 μl of a dilution (× 10) buffer solution (55 mM-MgCl ₂ , 70 mM 2-mercaptoethanol).
(Boehringer Mannheim) (10 U / μl) was added and left at 37 ° C. for 1 hour. Example 2
Electrophoresis was performed under the same conditions as in (1). The results are shown in the center column (Smal column) in FIG.

The same treatment was carried out using Nae I (Boehringer Mannheim) (10 U / μl) instead of the restriction enzyme Sma I, and the treated solution was subjected to electrophoresis. The results are shown in the right column (Nael column) of FIG.

The center column (Smal column) in FIG. 1 and the right column (Na
In el column), the upper row shows the type of microorganism, the numerical value on the right (such as 236) means the size of base pairs, and the lower row shows the type of restriction enzyme used.

The center column (Smal column) in FIG. 1 and the right column (Na
Table 1 below summarizes the results in column el).

As described above, since the four types of acid-fast bacterium DNAs show different digestion patterns, they can be distinguished from each other.

(2) The same operation as described in the above Example 3 (1) is repeated, except that Acc III (Takara Shuzo Co., Ltd.) is used as a restriction enzyme.
And Hinf I (Boehringer Mannheim) were used. The results are shown in Table 2 below.

(3) The same operation as in Example 3 (1) and Example 3 (2) was performed using the DNA amplification reaction solution obtained in Example 2 (2). The results are shown in Tables 3 and 4 below.

Example 4: Separation and identification of mycobacteria in biological samples (i) Test by the method of the present invention Sputum was collected from each infected patient of M. tuberculosis, M. avium, M. intracellulare and M. kansasii, 2
Liquefied with 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 500 μl of 10 mM Tris buffer (pH 8.0) containing 1% sodium dodecyl sulfate was used.
And the DNA was extracted with phenol / chloroform. The obtained DNA was prepared in the presence of yeast tRNA (10 μg).
Precipitated with ethanol. The resulting precipitate was dissolved in 100 μl of 10 mM Tris buffer (pH 8.0), and 10 μl of the solution was used for the DNA amplification operation described in Example 2 and Example 3
The restriction enzyme treatment and DNA fractionation test operation described in 1) were performed.

(Ii) Inspection by culture method For the purpose of comparison, the same clinical samples as described above were examined by the culture method.

The same sputum was mixed with twice the amount of a 4% aqueous sodium hydroxide solution at room temperature for 15 minutes, and 100 μl of the mixture was inoculated into 1% Ogawa medium and cultured at 37 ° C. for 8 weeks. When the acid-fast bacterium was selected by the Ziel-Neelsen method and the acid-fast bacterium was identified by the niacin test (Niacin Test Kobayashi; manufactured by Kobayashi Pharmaceutical Co., Ltd.), the same results as in the above (i) were obtained.

[Effects of the Invention] According to the method of the present invention, the presence and type of various acid-fast bacteria in a test sample can be detected specifically, with high sensitivity, and quickly.

[Brief description of the drawings]

FIG. 1 shows the results of amplification of four types of acid-fast bacterium DNA (left column).
FIG. 9 is an explanatory view showing the results of treating mycobacterial DNAs of various species with the restriction enzyme Sma I (middle column) and the results of treating the mycobacterial DNAs of four species with the restriction enzyme Nae I (right column). This is an alternative photo.

────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Akiyuki Okubo 1-14-1 Gakuendai, Miyashiro-machi, Minamisaitama-gun, Saitama (72) Inventor Shunichi Takewaki 2-10-21-106 Satsukigaoka, Chiba-shi, Chiba 58) Field surveyed (Int.Cl. ⁶ , DB name) C12Q 1/68 GenBank / EMBL / DDBJ WPI (DIALOG) BIOSIS (DIALOG)

Claims (2)

(57) [Claims] (1) Expression A first DNA primer containing an oligonucleotide portion consisting of a base sequence represented by A second DNA primer containing an oligonucleotide portion consisting of a base sequence represented by
A method for detecting a microorganism belonging to the genus Mycobacteria, comprising subjecting a mixture containing an aqueous liquid test sample to a DNA amplification step, and subjecting the resulting reaction solution to a DNA fractionation inspection step. (2) A first DNA primer containing an oligonucleotide portion consisting of a base sequence represented by And a second DNA primer containing an oligonucleotide portion consisting of the nucleotide sequence represented by