JP2001204470A - Method for promptly screening bacterium gene and kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gene screening process for the genes in the malignant cells of bacterial that can promptly perform identification of bacteria utilizing DNA detection and screening of the genes in the malignant cells of a bacterium by applying the PCR(polymerase chain reaction) technique and provide the kit for the gene screening. SOLUTION: The gene screening solution that essentially comprises a DNA polymerase that can realize high-speed PCR with high sensitivity, a buffer solution for PCR that can carry out the PCR in no need of extraction of DNA from the bacterial cells and a primer for detecting the bacterial genes is used and the PCR operation is applied to the gene screening solution whereby the urinary tract infection and digestive tract infections, the tumor diagnosis or detection of bacteria in foods can be promptly and directly diagnosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method and kit for rapidly testing bacterial genes using stool, urine, or food as a sample by applying the PCR method.
More specifically, bacterial species identification using bacterial genes, PCR is applied using DNA detection without bacterial culture, and stool or urine is used as a sample to quickly detect bacterial malignant cell genes or foods as samples. The present invention relates to a method and a kit for rapidly testing bacterial genes.

[0002]

2. Description of the Related Art Diagnosis of infectious diseases such as gastrointestinal tract infection and urinary tract infection is performed by using stool or urine as a specimen, separating and culturing the bacteria from the specimen, and then examining the colonies obtained by microscopy and staining. PC
The confirmation is performed by a method such as the R method.

In the diagnosis of gastrointestinal or urinary tract malignant tumors, collected stool or urine is used as a specimen, and cells are collected from the specimen by centrifugation or the like. Has been determined.

[0004] In the case of food poisoning originating from foods, bacteria are separated and cultured from the collected foods, and the resulting bacterial colonies are confirmed by methods such as microscopy, staining, and PCR. Have been done.

[0005]

The gastrointestinal tract or urinary tract infection is collected from a patient's stool or urine as a specimen, the bacteria are separated and cultured, and the resulting bacterial colonies are examined by microscopy, staining, PCR, etc. The diagnostic method confirmed by the method not only requires 1 to 7 days for the test, but also requires a high level of specialized knowledge and technology for the isolation and cultivation of bacteria, and does not allow outpatients to wait and determine the disease state. Was.

[0006] A method has also been developed in which the PCR method is applied directly to a sample without performing the separation and culture of bacteria requiring high specialized knowledge and technology. However, even in this method, centrifugation is performed on the stool or urine of the sample. Since a process such as separation and heat denaturation is performed, and a PCR method is performed after extracting a gene from the bacterium, it requires at least 3 to 4 hours, which is not a quick diagnosis.

[0007] Diagnosis of a gastrointestinal tract or urinary tract malignant tumor is performed by collecting cells from a specimen by a method such as centrifugation, and then performing microscopic examination to determine the morphological properties of the cells. Required not only time but also extremely specialized skills, and diagnosis required skill.

[0008] From the above, the stool or urine collected from a patient can be used as a sample to rapidly diagnose gastrointestinal or urinary tract infections and malignant tumors in a very short time and without requiring advanced technical skills. The development of a diagnostic method was awaited.

In the case of food poisoning, determination of the source and identification of bacteria are urgent issues, and development of a technique for accurately determining bacteria in an extremely short time has been awaited.

[0010]

Means for Solving the Problems In order to solve the problem of rapid diagnosis or rapid identification of food poisoning bacteria, the present inventors have conducted intensive studies, and as a result, a small amount of bacterial genes can be quickly detected by applying the PCR method. As a result, they have developed a revolutionary test method to help diagnose infectious diseases and malignant tumors and a test method for rapidly identifying bacteria in foods.

The present invention relates to a highly sensitive and highly reactive DN.
A polymerase, primers for detecting bacterial genes, and PCR that enables PCR without extracting bacterial DNA
8-Methoxypropyla with buffer as an essential component
After adding len to make a test solution and performing an ultraviolet ray treatment, stool or urine collected from a patient is directly added to the test solution and PCR is performed to detect a bacterial gene. By omitting the extraction and quantification of DNA from the cells, not only is there no artificial loss of the gene, but also the result does not fluctuate due to the skill of the operator.

In the present invention, when bacteria are detected from a specimen, Escherichia coli DNA contaminated in DNA polymerase is used.
8-Methoxysorbor to test solution for the purpose of removing
The psoralen / ultraviolet ray treatment is performed for about 2 minutes using an ultraviolet lamp with the addition of alen, followed by PCR. However, there is no need to perform the psoralen / ultraviolet ray treatment except for detecting a gene derived from bacteria.

As a DNA polymerase for performing high-sensitivity, high-speed PCR, which is one of the features of the present invention, TAKARA z-Taq DNA polymerase manufactured by Takara Shuzo Co., Ltd. is useful. As a PCR buffer solution that enables PCR without performing the above, SHIMADZU Ampdir manufactured by Shimadzu Corporation
ect is useful, but the invention is not so limited.

Since the present invention can be directly subjected to the PCR method without extracting a gene from a specimen, the determination can be made within one hour from the start of the test.

Further, in the present invention, a PCR primer specific to a target bacterium is set, and the determination is made based on whether or not the primer reacts with the primer. The diagnosis can be made accurately.

When the present invention is compared with a conventional diagnosis method using a bacterial gene, the conventional method requires 1 to 3 days for separating and culturing bacteria collected from stool or urine, and 2 days for DNA extraction. DNA quantification took 10 to 20 minutes, PCR took 2 to 3 hours, and electrophoresis took 10 to 15 minutes.

[0017] In the case of a bacterial test in a food in which a food that has caused food poisoning is used as a specimen, the same time is required as in the case of using stool or urine as a specimen.

On the other hand, in the present invention, 2 minutes for psoralen / ultraviolet ray treatment, 30 to 40 minutes for PCR, and 1 minute for electrophoresis
The time can be greatly reduced to 0 to 15 minutes.

After completion of the PCR, the electrophoresis is omitted, and the PCR
It is also possible to judge the product by directly staining it with a dye such as ethidium bromide or SYBR green and colorimetrically. It is also possible to make a simultaneous determination during the execution of PCR by combining a real-time quantitative PCR system such as that manufactured by PerkinElmer.

Further, when detecting bacteria, high sensitivity is required.
A psoralen / ultraviolet ray treatment is required to remove Escherichia coli DNA contaminating z-Taq DNA polymerase, which is a DNA polymerase used to enable high-speed PCR. Is unnecessary, so that a quicker inspection is possible.

In order to accurately and promptly determine the type of the detected bacteria, the exact sequence of the bacteria can be determined by examining the nucleotide sequence of the gene amplified by PCR using a nucleotide sequencing method or a DNA chip. Bacterial species can be identified.

[0022]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to the examples.

[0023]

Example 1 The following solutions were prepared in sterilized plastic tubes. Sterile distilled water 23 μl Ampdirect A solution 10 μl Ampdirect I solution 10 μl DNTP (10 mM) 4 μl 8-Methoxypropylen solution (2.5 mg / ml DMSO) 0.5 μl z-Taq DNA polymerase (5 U / μl) From a distance of 1 cm to 2
Irradiate for minutes. Next, 1 μl of each of a sense primer and an antisense primer was added as a universal primer (10 pmol / μl) for bacterial detection to prepare a test solution.

About 2 to 10 times the volume of physiological saline, Tris-EDTA buffer or sodium-phosphate-EDTA buffer, etc. is added to the collected stool and stirred, and the mixture is allowed to stand, centrifuge, filter paper, or the like. After removing impurities by a method such as filtration using a filter, about 1 μl thereof was added to the test solution to prepare a test sample. The test sample was placed in a thermal cycler and a 98 ° C / 1 second, 68 ° C / 10 second PC
The R protocol was run 30 times.

When urine is used as a sample, the precipitate is collected by centrifugation, and the precipitate is directly or about 2 to 10 times the volume of physiological saline, Tris-EDTA buffer or sodium-phosphate-EDTA buffer. After dilution with a liquid or the like, the same operation as in the case where stool was used as a sample was performed using about 1 μl of the solution.

8 μl of the test sample subjected to PCR was collected, and 100 V was applied to a 1% agarose gel containing ethidium bromide.
/ 15 minutes electrophoresis was performed. The gel after the electrophoresis was irradiated with ultraviolet light, the band of the gene stained with ethidium bromide was observed, and the appearance of the band was determined to be positive.

[0027]

Example 2 A 2 to 10 times volume of physiological saline was added to the collected food, stirred and crushed with a mixer, and impurities were removed by a static method, a centrifugal method, or a filtration method to obtain a sample. About 1 μl was added to the test solution prepared in Example 1 to prepare a test sample.

[0028]

Example 3 In order to identify the species of bacteria, the nucleotide sequence of the bacteria was determined by directly using the PCR product amplified by the method of the present invention in a sequencing reaction. In addition, a sequencing primer was added to the PCR solution of the method of the present invention, so that the amplification reaction and the sequencing reaction were performed simultaneously, thereby making it possible to reduce the required time.

[0029]

Example 4 In order to identify bacterial species using a DNA chip, a necessary number of probes specific to each bacterial species were used in DNs.
The PCR product immobilized on the A chip and amplified by the method of the present invention was subjected to a hybridization reaction on the DNA chip. PC
The probe hybridized with the R product was identified as a bacterial species.

[0030]

According to the present invention, stool, urine, or food is used as a specimen, and endogenous or exogenous cells or bacterial genes present therein are detected rapidly without the extraction operation. The present invention has made it possible to rapidly and reliably perform diagnosis of gastrointestinal tract infection or urinary tract infection, diagnosis of gastrointestinal tract or urinary tract malignancy, or bacterial testing of food-mediated food poisoning.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Joji Oshima 1785-2 Kuboshima, Kumagaya-shi, Saitama F-term (reference) 4B024 AA13 AA20 HA20 4B063 QA01 QA13 QA18 QQ03 QQ42 QQ52 QR08 QR62 QS02 QS25 QX01

Claims (4)

[Claims] A stool or urine is used as a sample, and the sample is directly
DNA polymerase that enables high sensitivity and high speed PCR,
PCR is carried out by adding 8-methoxypsoralen as an essential component, a primer for detecting bacterial genes and a PCR buffer capable of performing PCR without extracting DNA, and adding the resultant to a test solution obtained by ultraviolet treatment. A rapid and direct bacterial genetic test method and kit using the PCR method. 2. The rapid and direct bacterial genetic test method and kit according to claim 1, wherein the sample is a food. 3. The method of claim 1, wherein the diagnosis of gastrointestinal or urinary tract infection or gastrointestinal or urinary tract malignancy is rapidly performed. kit. 4. A rapid and direct bacterial malignant cell, characterized by examining the nucleotide sequence of the gene amplified by the PCR using a nucleotide sequencing method or a DNA chip to determine a detailed type of bacteria. Genetic testing methods and kits.