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Method of effecting lysis of acid-fast bacteria and method of performing gene amplification or detection therewith

a technology of acid-fast bacteria and lysis method, which is applied in the field of lysing acid-fast bacteria, can solve the problems of insufficient lysis method, complicated operation, and longer treatment period, and achieve the effects of easy and fast lysing, safe operation, and low risk

Inactive Publication Date: 2005-08-18
ARKRAY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0004] Therefore, with the foregoing in mind, it is an object of the present invention to provide a method of lysing acid-fast bacteria easily and in a short time without using any special device or special reagent.
[0007] According to the first lysis method of the present invention, a gene can be extracted sufficiently from an acid-fast bacterium by merely heating the acid-fast bacterium in a solution containing a non-ionic detergent, for example, at 96° C. for 10 minutes. Thus, a method of amplifying or detecting the gene to be performed subsequently can be carried out easily. Furthermore, since the heating temperature is below the boiling point of the liquid, the method has the following advantages, for example. Since the bumping of the liquid is prevented, there is reduced concern that a sample might be scattered. Moreover, since the temperature can be controlled easily, a special heater is not necessary.
[0009] According to the first and second lysis methods of the present invention, acid-fast bacteria can be lysed easily and in a short time without using any special reagent such as a chaotropic reagent or any special device such as an ultrasonic device. Besides, since the first and second lysis methods are chemical methods, they can be carried out safely with little risk that a sample might be scattered. Moreover, according to the first and the second lysis methods of the present invention, the gene extracted may be subjected to a gene amplification process or a gene detection process as it is without being purified. It is to be noted that the first and the second lysis methods of the present invention are applicable not only to a method of performing gene amplification or gene detection but also to other fields such as gene manipulation, for example.

Problems solved by technology

However, since tubercule bacilli have a high cell-wall lipid content, such conventional lysis methods cannot extract genes sufficiently.
Besides, this may result in a longer treatment period and a more complicated operation.

Method used

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  • Method of effecting lysis of acid-fast bacteria and method of performing gene amplification or detection therewith
  • Method of effecting lysis of acid-fast bacteria and method of performing gene amplification or detection therewith
  • Method of effecting lysis of acid-fast bacteria and method of performing gene amplification or detection therewith

Examples

Experimental program
Comparison scheme
Effect test

example 1-1

(Example 1-1, Comparative Example 1)

[0036] A clinical isolate of a tubercule bacillus was cultured in a product named MycoBroth (Kyokuto Pharmaceutical Industrial Co., Ltd.) at 37° C. until a turbidity corresponding to #1 of the McFarland turbidity standard was obtained. Then, the culture was diluted with phosphate buffer (pH 6.8) so as to achieve a series of 10-fold dilutions (102-fold to 105-fold), thus preparing test solutions containing the tubercule bacillus. Subsequently, 100 μl of the test solutions with the above-described concentrations were poured into screw capped tubes, respectively, and then centrifuged (10000 g, 15 minutes) to prepare pellets. The pellets obtained from the respective test solutions were used as samples to be subjected to a lysis reaction. On the other hand, a product named Triton X-100 (Nacalai Tesque, Inc.) was dissolved in TE buffer (10 mM EDTA and 25 mM Tris-HCl, pH 8.0) so that its concentration became 3 wt % to prepare a lysis reagent solution, an...

example 1-2

(Example 1-2, Comparative Example 2)

[0040] A clinical isolate of a tubercule bacillus was cultured in a product named MycoBroth (Kyokuto Pharmaceutical Industrial Co., Ltd.) at 37° C. until a turbidity corresponding to #1 of the McFarland turbidity standard was obtained. Then, the culture was diluted with a non-tuberculous sputum that had been homogenized with a product named SUPTAZYME (Kyokuto Pharmaceutical Industrial Co., Ltd.) so as to achieve a series of 10-fold dilutions (100-fold to 1010-fold). The resultant diluents were used as samples. On the other hand, a product named Triton X-100 (Nacalai Tesque, Inc.) was dissolved in TE buffer (10 mM EDTA and 25 mM Tris-HCl, pH 8.0) so that its concentration became 3 wt % to prepare a lysis reagent solution, and the lysis reagent solution was sterilized by high-pressure steam in an autoclave.

[0041] Then, 50 μl of the above-described lysis reagent solution was added to the respective samples (100 μl), and a lysis treatment was carried...

example 1-3

(Example 1-3, Comparative Example 3)

[0044] 90 sputum specimens collected from patients were homogenized and sterilized by the NALC-NaOH method (“New Guideline for Tubercle Bacillus Test 2000” edited by The Japanese Society for Tuberculosis). Then, 100 μl of each of the treated sputum specimens was centrifuged at 13,000 g for 10 minutes. After removing the supernatant, the precipitate (pellet) was collected. On the other hand, a product named Triton X-100 (Nacalai Tesque, Inc.) was dissolved in TE buffer (10 mM EDTA and 25 mM Tris-HCl, pH 8.0) so that its concentration became 1 wt % to prepare a lysis reagent solution, and the lysis reagent solution was used after being sterilized by high-pressure steam in an autoclave. Specifically, 50 μl of the sterilized lysis reagent solution was added to the pellets obtained from the respective specimens to suspend the pellets. Then, a lysis treatment was carried out by heating the suspensions at 96° C. for 10 minutes in a heating block. On the ...

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Abstract

A method of effecting lysis of acid-fast bacteria, comprising heating acid-fast bacteria in a liquid containing a non-ionic surfactant at a temperature of below the boiling point of the liquid. This method enables accomplishing secure lysis of acid-fast bacteria in a simple manner within a short period of time without the use of special apparatus and agent and enables extracting genes. The heating is preferably conducted at 96° C. for 10 min. As the nonionic surfactant, use can be made of a d-sorbitol fatty acid ester, a polyoxyethylene glycol sorbitan alkyl ester, a polyoxyethylene glycol p-t-octylphenyl ether or the like. The pH value of the liquid is preferably 8, and the liquid preferably contains EDTA. It is also preferred that before the heating, the acid-fast bacteria be treated with lipase.

Description

TECHNICAL FIELD [0001] The present invention relates to a method of lysing acid-fast bacteria and to a method of performing gene amplification or gene detection using the same. BACKGROUND ART [0002] Tuberculosis still is a serious bacterial disease worldwide, and not only treatment methods but also diagnostic methods therefor are extremely important. A final diagnosis as to the tuberculosis infection is made by carrying out a culture method. However, since tubercule bacilli have an extremely slow growth rate, the establishment of a preliminary diagnostic method to be performed prior to the culture method has been desired. As such a preliminary diagnostic method, a method using a polymerase chain reaction (PCR) method is attracting attention. In this method, a primer specific to a gene of a tubercule bacillus is used to amplify a gene of the tubercule bacillus, if any, so that it can be detected, thereby enabling the presence or absence of the tubercule bacillus to be determined. [00...

Claims

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Application Information

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IPC IPC(8): C12N1/06C12N15/10
CPCC12Q1/6806C12N15/1003C12N1/06C12Q1/6844
Inventor KAMATA, TATSUOIZUMIZAWA, YUJI
Owner ARKRAY INC
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