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Method for detecting bacterium belonging to genus geosmithia

A detection method and technology of fungi, applied in the field of detection of fungi belonging to the genus Geosmithia, can solve the problems of lack of elucidation of specific gene regions, difficulty in specific and rapid detection, etc.

Inactive Publication Date: 2013-12-04
KAO CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above method does not elucidate specific gene regions of fungi belonging to the genus Geosmithia
Therefore, there is a problem that it is difficult to specifically and rapidly detect fungi belonging to the genus Geosmithia

Method used

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  • Method for detecting bacterium belonging to genus geosmithia
  • Method for detecting bacterium belonging to genus geosmithia
  • Method for detecting bacterium belonging to genus geosmithia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Analysis of specific nucleotide sequences of fungi belonging to the genus Geosmithia

[0077] The base sequences of various β-tubulin genes of the genus Geosmithia were determined by the following method. Use GenTorukun TM (manufactured by Takara Bio Co., Ltd.), DNA was extracted from cells of the genus Geosmithia cultured on a potato dextrose agar slant medium at 25° C. in the dark for 7 days. For PCR amplification of the target site, PuRe TaqTM Ready-To-GoPCR Beads (manufactured by GE Health Care UK LTD) were used, and Bt2a (5'-GGTAACCAAATCGGTGCTGCTTTC-3': SEQ ID NO: 6), Bt2b (5'-ACCCTCAGTGTAGTGACCCTTGGC -3': sequence number 7) (Glass and Donaldson, Appl Environ Microbiol 61: 1323-1330, 1995). The amplification conditions are as follows: denaturation temperature is 95°C, annealing temperature is 59°C, extension temperature is 72°C, and 35 cycles are implemented. The PCR product was purified using Auto SegTM G-50 (manufactured by Amersham Pharmacia Biotech...

Embodiment 2

[0079] Example 2 Detection of fungi belonging to the genus Geosmithia by the PCR method

[0080] (1) Preparation of primers

[0081]Among the specific nucleotide sequence regions (base sequence numbers 1 to 3) obtained above for fungi belonging to the genus Geosmithia, starting from a particularly high specificity region belonging to fungi belonging to the genus Geosmithia at the 3' end side, the following conditions are met: Partial regions of 4 conditions were studied: 1) the base sequence of the inherent gene is about 10 bases continuous, 2) the GC content of the oligonucleotide is about 30% to 80%, 3) the oligonucleotide The possibility of acid self-annealing is low. 4) The Tm value of the oligonucleotide is about 55-65°C. Based on the base sequence of this part of the region, 5 sets of primer pairs were designed, and the DNA extracted from various bacteria was used as a template to study the effectiveness of Geosmithia detection by PCR reaction. That is, studies were co...

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Abstract

A method of detecting a fungus belonging to genus Geosmithia, including identifying a fungus belonging to genus Geosmithia using a nucleic acid represented by the nucleotide sequence defined in the following (a) or (b): (a) a partial nucleotide sequence of ²-tubulin gene shown in any one of SEQ ID NOS: 1 to 3, or a complementary sequence thereof; (b) a nucleotide sequence including deletion, substitution, insertion or addition of one or several nucleotide(s) in the nucleotide sequence shown in any one of SEQ ID NOS: 1 to 3, or a complementary sequence thereof.

Description

technical field [0001] The present invention relates to a method for detecting fungi belonging to the genus Geosmithia. Background technique [0002] Fungi belonging to the genus Geosmithia are widely distributed in nature, multiply on crops such as vegetables and fruits, and contaminate foods and beverages made from these crops. However, since fungi belonging to the genus Geosmithia form chlamydospores with high heat resistance, even if the usual sterilization treatment process for other fungi is performed, such as heat sterilization of acidic beverages, etc., fungi belonging to the genus Geosmithia Fungi can also survive and reproduce, thus becoming the cause of mildew. Therefore, fungi belonging to the genus Geosmithia are being alerted as important harmful fungi that cause serious quality accidents. In order to prevent accidents caused by fungi belonging to the genus Geosmithia in foods and beverages and their raw materials, detection and identification of fungi belong...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09
CPCC12Q1/6895
Inventor 细谷幸一中山素一德田一矢口贵志弘佑介
Owner KAO CORP
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