JPH1132792A - Assaying reagent for acid-fact bacterium number and method therefor and test of drug sensitivity for acid-fast bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject reagent capable of conducting a drug sensitivity test for acid-fast bacteria such as tubercule bacillus in a short term by including a component emitting light when reacted with ATP. SOLUTION: This reagent for assaying the number of acid-fast bacteria is obtained by including a component emitting light when reacted with ATP (e.g. luciferin, luciferase, magnesium ion). This reagent is added to a specimen containing acid-fast bacteria to determine the resulting luminescence, based on which the aimed number of the acid-fast bacteria is calculated. It is preferable in terms of sensitivity improvement that, prior to adding the reagent, the specimen is heat-treated, or, before heat treatment, an ATP extractive reagent is added to the specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a reagent for measuring the number of mycobacteria in a sample, a method and an apparatus thereof, and a method and an apparatus for testing the sensitivity of a drug to mycobacteria.

[0002]

2. Description of the Related Art In Japan, the number of mycobacterial infections such as Mycobacterium tuberculosis infections caused by Mycobacterium tuberculosis has been steadily decreasing due to development of therapeutic agents and improvement of nutritional status. In recent years, the decrease has been slowing down, and many patients are still newly registered. Therefore, there is a need to develop a therapeutic agent that is more effective against such mycobacteria and to develop a rapid and accurate drug sensitivity test method. In Japan, drug sensitivity tests against acid-fast bacilli mainly use a fixed concentration method using an Ogawa medium containing chicken eggs as a main component. In this fixed concentration method, the inoculum is inoculated into a test medium containing a target drug and a control medium not containing the target drug, and after culturing, the mycobacterial susceptibility to the target drug is determined by comparing the numbers of both acid-fast bacteria. That is.

[0003]

However, the acid-fast bacterium grows very slowly as compared with other bacteria, and therefore usually requires a culture period of 2 to 4 weeks before it can be determined by this method. . Therefore, in order to perform a drug susceptibility test in a short period of time, a technique for measuring the number of acid-fast bacteria capable of capturing the time-dependent change in the number of acid-fast bacteria in culture is required.

[0004]

Means for Solving the Problems The present inventors have found that an acid-fast bacterium counting reagent containing a component that emits light upon reacting with ATP can solve the above-mentioned problems. Here, for example, the components are luciferin, luciferase and magnesium ion.

Further, the present inventors have solved the above-mentioned problem by a method for measuring the number of acid-fast bacteria, which comprises a step of adding the reagent for counting the number of acid-fast bacteria to a sample containing acid-fast bacteria and measuring the amount of luminescence. I found what I could do. In particular, it is preferable to heat-treat the sample containing the acid-fast bacterium before adding the reagent, and it is more preferable to add the ATP extraction reagent to the sample before the heat treatment.

[0006] Further, in the method for testing the drug susceptibility of acid-fast bacterium, the present inventors added a drug to a sample containing the acid-fast bacterium, cultured the sample for a certain period of time, and added the reagent for measuring the number of acid-fast bacterium. A first step of measuring the amount of luminescence by performing the following steps: culturing the same sample as in the first step without adding the drug, and culturing for the same time as the first step, and then adding the reagent to measure the luminescence amount;
And a method including a third step of judging whether or not the drug is sensitive to the acid-fast bacterium based on the luminescence amounts obtained in the first step and the second step can solve the above-described problem. Was found. Here, it is preferable to heat-treat the acid-fast bacterium-containing sample before adding the reagent in the first step and the second step, and it is more preferable to add an ATP extraction reagent to the sample before the heat treatment. Further, it is preferable that the determination in the third step is made based on the magnitude of the ratio of the light emission amounts obtained in the first step and the second step.

[0007] Furthermore, the inventors of the present invention provide a method for testing the acid susceptibility of an acid-fast bacterium, which comprises adding a drug to a sample containing the acid-fast bacterium and culturing the sample. Step A of measuring the amount of luminescence by adding a reagent; Step B of culturing the same sample as in Step A without adding the agent, and then adding the reagent over time to measure the amount of luminescence; and Step A
And a method including a step C of judging whether or not the drug is sensitive to the acid-fast bacterium based on the time-dependent change in the amount of luminescence obtained in the step B has been found to solve the above problem. . Here, it is preferable to heat-treat the acid-fast bacterium-containing sample before adding the reagent in step A and step B, and it is more preferable to add an ATP extraction reagent to the sample before the heat treatment. Further, it is preferable that the determination in the step C is made based on the increase or decrease of the change with time of the ratio of the light emission amounts obtained in the steps A and B.

Further, the present inventors have found that an apparatus for performing the above-mentioned method for measuring the number of mycobacteria and the method for testing the drug sensitivity of mycobacteria can solve the above problems.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The reagent for determining the number of acid-fast bacteria according to the present invention contains a component which emits light when reacted with ATP. Such components are, for example, luciferin, luciferase and magnesium ion, and a reagent containing these components is commercially available from Kikkoman Corporation under the trade name Lucifer-LU Plus. When this reagent is used, light is emitted by the following reaction mechanism.

[0010]

(Equation 1)

The reagent for measuring the number of acid-fast bacteria according to the present invention may contain components such as a buffer and a stabilizer. For example, H
EPES buffer, EDTA and the like can be mentioned.

The concentration of each component in the reagent for measuring the amount of acid-fast bacterium is appropriately determined by those skilled in the art according to the type and concentration of the acid-fast bacterium.

The method for determining the number of acid- fast bacilli comprises the steps of (1) adding the above reagent to a sample that may contain acid-fast bacilli, (2) measuring the amount of luminescence, And (3) calculating the number of acid-fast bacteria by conventional means based on the measured light emission amount. Regarding the reagent adding step (1), what kind of sample is used (for example, whether or not a diluted stock solution is used),
Those skilled in the art appropriately determine how much sample to use, what concentration of reagent to add and how much.
The light emission amount measuring step (2) is preferably performed at 27 ° C.
The luminescence amount is measured in the vicinity, and the luminescence amount is usually measured with a Lumi counter. In the step (3) of calculating the number of acid-fast bacilli, the amount of luminescence is proportional to the amount of ATP, so that the amount of ATP can be calculated by proportional calculation, and based on the amount of ATP per target acid-fast bacterium. Next, the number of bacteria can be determined from the calculated ATP amount.

From the viewpoint of improving the sensitivity, it is preferable to introduce a step (0) of heating the sample prior to the step (1) of adding the reagent. By this step, the cell wall of the acid-fast bacterium in the sample is destroyed, and ATP in the acid-fast bacterium can be efficiently extracted. In order to further improve the sensitivity, it is preferable to introduce a step of adding the ATP extraction reagent to the (0) ′ sample and heating the sample prior to the reagent adding step (1). By performing the heating in the presence of the ATP extraction reagent, an effect of further improving the ATP extraction efficiency can be obtained. The ATP extraction reagent is not particularly limited, and a known reagent,
For example, a mixed solution of ethanol and ammonia, methanol, ethanol, a surfactant (such as Triton X100),
ATP extraction reagents such as trichloroacetic acid and perchloric acid can be used. Further, commercially available kits such as Lucifer-L
You may use the ATP extraction reagent attached to U plus (made by Kikkoman). In addition, various conditions such as a heating temperature, a heating time, and a reagent concentration are appropriately selected according to the state of the sample, the capability of the measurement device, and the like. However, regarding the heating temperature, 35
It is preferred to choose a temperature above ℃. For example, for reference, Table 1 shows the results obtained by changing the heating temperature and heating time for the same sample and measuring the luminescence amount in accordance with the procedure shown in FIG.
Shown in

[0015]

[Table 1]

From this table, as compared with non-heating (0 minutes),
For example, it can be seen that when heated at 100 ° C. for 2 minutes, a light emission amount of 5 to 6 times is obtained. At the time of measurement, the light emission amount is 10,000
It is preferably 0 RLU or more.
It is preferable to set the temperature and the heating time so that the light emission amount is equal to or more than the dotted line portion in FIG. The heating is preferably performed at a temperature of 35 ° C. or more, and particularly when a microplate is used, it is necessary to set the temperature at or below the heat resistance temperature of the microplate (about 75 ° C. in a normal case). In this case, heating at 60 ° C. for 3 minutes is preferable.

Test Method for Drug Sensitivity of Mycobacteria The method for testing drug sensitivity of mycobacteria according to the present invention is an application of the above-described method for measuring the number of mycobacteria. This drug susceptibility test method comprises the following steps: (1) a first step of adding a drug to a sample containing the acid-fast bacterium, culturing it for a certain period of time, and then adding the reagent for measuring the number of acid-fast bacilli to measure the amount of luminescence. (2) a second step of culturing for the same time as the first step without adding the drug to the same sample as the first step, and then adding the reagent to measure the amount of luminescence; and (3) the first step and A third step of determining whether or not the drug is sensitive to the acid-fast bacterium based on the amount of luminescence obtained in the second step. Here, in the present specification, during the third step (and step C to be described later), the presence or absence of susceptibility of the acid-fast bacterium is determined using a parameter called RLU _ratio shown below. This is an example of the step C) described below, and is not limited thereto.

[0018]

(Equation 2)

Here, the sensitivity (S) or the resistance (R) is determined based on the value of the RLU _ratio . For example, the following RL
If the U _ratio value is 0.5 or less, it is S, and if it exceeds 0.5, it is R.

The test bacteria used in the drug sensitivity test method are as follows:
There are fresh bacteria, precultured bacteria, cryopreserved bacteria, etc., as can be seen from FIG. 3, since cryopreserved bacteria take time to grow, fresh bacteria and precultured bacteria are preferred from the viewpoint of rapid diagnosis. . Here, a fresh bacterium refers to a culture of Ogawa medium for 2 to 3 weeks.
This is a bacterium that is suspended in ycobroth (liquid medium), adjusted for a bacterial solution, and then cultured. Preculture (Mycobroth)
Pre-cultured bacteria) is defined as
This is a bacterium that is prepared after preparing a bacterial solution cultured for 5 to 7 days after suspension. The cryopreserved bacteria (Ogawa -80 ° C stored bacteria) are bacteria that have been cultured in Ogawa medium culture bacteria at -80 ° C, suspended and adjusted for bacterial solution.

The RLU _ratio value can change depending on the culturing time. In some cases, even if the RLU _ratio is initially resistant, it may become sensitive with the passage of time. Culture time is required. The culturing time varies depending on the type and concentration of the test bacterium, the type and concentration of the drug, and the like. However, the culturing time is longer than the longest time among the R⇒S transition times (determination reference arrival times) required in various cases. Is set, there is no need to consider the difference between these conditions. Therefore, according to the procedure shown in FIG. 1, the R → S transition time was examined for some cases in which the type and concentration of the acid-fast bacterium and the type and concentration of the drug were changed, and the results are shown in Tables 2 and 3.

[0022]

[Table 2]

[0023]

[Table 3]

As can be seen from Tables 2 and 3, the R-to-S transition time (the day when the RLU _ratio value becomes 0.5 or less) in these cases was 3 to 7 days. Therefore, based on these results, the culture time may be set to 7 days or more. However, the cultivation time of 7 days is a time based solely on the above results. When a susceptibility test is performed on a new test bacterium or a drug, etc., the above R⇒S transition time measurement test is performed. The culture time may be set separately based on the result.

[0025] Alternatively, without waiting for the RLU _ratio value of 0.5 or less, measured RLU _ratio were taken over time sample from the medium, it may be compared over time RLU _ratio value. In this case, when the RLU _ratio value decreases over time, it can be determined that the drug is sensitive. For example, according to the procedure shown in FIG. 1, in order to determine the presence or absence of the following acid-fast bacteria susceptibility to the following drugs,
The RLU _ratio value was measured over time. Table 4 shows the results.

[0026]

[Table 4]

From Table 4, it can be seen that a decrease in the RLU _ratio value over time was observed in all samples when sensitivity was exhibited. For example, H37RvATCC35822 showed R for EB on both day 5 and day 7, but 7
On the 9th day, the RLU _ratio value became 0.5 or less, indicating sensitivity. From this, in the above case,
It is possible to determine the sensitivity of the test bacterium to the test drug only from the RLU _ratio values on the fifth and seventh days.

[0028]

EXAMPLE A clinical isolate having a known resistance pattern was cultured according to the procedure shown in FIG. 4 and a sensitivity test for various drugs was performed. Sampling was performed over time, and the amount of luminescence of each sample was measured using a Lumitester K-200 manufactured by Kikkoman Corporation according to the procedure of FIG. 1 to obtain an RLU _ratio value. Table 5 shows the results. As a result, according to the test method of the present invention, it was found that the same result as the conventional test method was obtained in a short time.

[0029]

[Table 5]

[Brief description of the drawings]

FIG. 1 is a diagram showing an RLU measurement procedure.

FIG. 2 is a diagram showing a relationship between a heating condition of a sample and a light emission amount.

FIG. 3 is a diagram showing the relationship between the number of days of culture of a sample and the amount of luminescence.

FIG. 4 is a view showing a culture procedure.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazunori Tanno 3333-26 Asayama, Kamitena, Takahagi-shi, Ibaraki Prefecture Within Kyokuto Pharmaceutical Industries Co., Ltd.

Claims (14)

[Claims] 1. A reagent for measuring the number of acid-fast bacteria, which comprises a component that emits light upon reacting with ATP. 2. The reagent according to claim 1, wherein said components are luciferin, luciferase and magnesium ion. 3. A method for measuring the number of acid-fast bacteria, comprising the step of adding the reagent according to claim 1 or 2 to a sample containing the acid-fast bacteria and measuring the amount of luminescence. 4. The method according to claim 3, wherein the sample containing the acid-fast bacterium is heated before adding the reagent. 5. The method for measuring the number of acid-fast bacteria according to claim 4, wherein an ATP extraction reagent is added to the sample before the heating. 6. A method for testing a drug susceptibility of an acid-fast bacterium, comprising: adding a drug to a sample containing the acid-fast bacterium; culturing the sample for a certain period of time; A first step of measuring; a second step of culturing the same sample as in the first step without adding the drug to the same step as the first step, and then adding the reagent to measure the amount of luminescence; and A method comprising a third step of judging whether or not the drug is sensitive to the acid-fast bacterium based on the amount of luminescence obtained in the two steps. 7. The method according to claim 6, wherein in the first step and the second step, heat treatment is performed before adding the reagent. 8. The method according to claim 7, wherein an ATP extraction reagent is added to the sample before the heat treatment. 9. The method according to claim 6, wherein the determination in the third step is made based on the magnitude of the ratio of the amount of light emission obtained in the first step and the second step. . 10. A method for testing drug sensitivity of an acid-fast bacterium, comprising: adding a drug to a sample containing the acid-fast bacterium; culturing the sample;
Step A of measuring the amount of luminescence by adding the reagent according to claim 1 or 2 over time; culturing the same sample as in step A without adding the agent, and then adding the reagent over time. A step B of measuring the amount of luminescence; and a step C of judging whether or not the drug is sensitive to the acid-fast bacterium based on the change over time of the amount of luminescence obtained in steps A and B. . 11. The method according to claim 10, wherein in steps A and B, heat treatment is performed before adding the reagent. 12. The method according to claim 11, wherein an ATP extraction reagent is added to the sample before the heat treatment. 13. The method according to claim 10, wherein the determination in the step C is made on the basis of an increase or decrease in a change with time of the ratio of the luminescence amounts obtained in the steps A and B. . 14. Apparatus for performing the method according to claim 3.