Infectious etiologic agent detection probe and probe set, carreir, and genetic screening method

A technology for pathogen detection and probe sets, applied in microorganism-based methods, biochemical equipment and methods, and resistance to vector-borne diseases, etc., can solve problems such as difficulty in designing primers for PCR amplification reactions.

Inactive Publication Date: 2004-10-27
CANON KK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] However, even if the gene sequences of various bacteria are partially clarified, the full length of 16srRNA cannot be clarified, and the design of primers for PCR amplification reactions is not easy.

Method used

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  • Infectious etiologic agent detection probe and probe set, carreir, and genetic screening method
  • Infectious etiologic agent detection probe and probe set, carreir, and genetic screening method
  • Infectious etiologic agent detection probe and probe set, carreir, and genetic screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Detection of microorganisms using 1-step PCR method

[0037] [1. Preparation of probe DNA]

[0038] Tables 1 to 10 show the nucleic acid sequences designed as probes for the detection of the above-mentioned 10 kinds of pathogens. Specifically, the base sequences of the probes shown below were selected from genomes encoding 16s rRNA of various bacteria. These probe nucleotide sequence sets designed are very specific for this bacterium, and there is no deviation in the nucleotide sequence of each probe, and it is expected that sufficient hybridization sensitivity can be obtained. In addition, it is not necessarily limited to being completely identical to each probe base sequence shown in Tables 1 to 10. Probe base sequences having a length of about 20 to 30 bases including each probe base sequence are also included in the probe base sequences shown in each table. Complementary sequences (complementary strands) to the nucleotide sequences shown in the above ta...

Embodiment 2

[0128] Example 2 Detecting microorganisms with 2-step PCR method

[0129] In the same manner as in Example 1, probe DNA, PCR primers for sample amplification, genomic DNA of each pathogen, and DNA microarray were prepared, and the following experiments were performed.

[0130] [1. Specimen amplification and labeling (PCR amplification & fluorescent labeling)]

[0131] Amplification (Ist PCR) and labeling (2nd PCR) reactions of microbial genes used as samples are shown below.

[0132] [2. Composition of amplification reaction solution: 1st PCR]

[0133] Premixed PCR Reagent (TAKARA ExTaq) 25μl

[0134] Template genomic DNA 2μl (10ng)

[0135] Forward primer mix 2μl (20pmol / each tube)

[0136] Reverse primer mix 2μl (20pmol / each tube)

[0137] Water 19μl

[0138] Total 50μl

[0139] The reaction solution with the above composition was subjected to the amplification reaction using a commercially available thermal cycler according to the following protocol.

[0140] 95°C 1...

Embodiment 3

[0179] Example 3 Detecting microorganisms with 2-step PCR method

[0180] In the same manner as in Examples 1 and 2, probe DNA, PCR primers for sample amplification, genomic DNA of each pathogen, and DNA microarray were prepared, and the following experiments were performed.

[0181] [1. Amplification and labeling of the sample (using fluorescently labeled PCR primers)]

[0182] Amplification (Ist PCR) and labeling (2nd PCR) reactions of microbial genes used as samples are shown below.

[0183] [2. Composition of amplification reaction solution: 1st PCR]

[0184] AmpliTaq Gold LD (50U / μl) 0.5μl

[0185] template genomic DNA variable

[0186] dNTP mis(2.5mM / each) 4.0μl

[0187] ×10 PCR buffer 5.0μl

[0188] 25mM MgCl 2 7.0μl

[0189] Forward primer mix (10μM / each) 0.25μl

[0190] Reverse primer mix (10μM / each) 0.25μl

[0191] water variable

[0192] Total 50μl

[0193] The reaction solution with the above composition was subjected to an amplificat...

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Abstract

An infectious etiologic agent detection probe set which detects an infectious etiologic agent gene, includes a plurality of kinds of probes including oligonucleotide having base sequences selected from each of a plurality of groups selected from a first group including base sequences of SEQ ID Nos. 1 to 14 and complementary sequences thereof, a second group including base sequences of SEQ ID Nos. 15 to 24 and complementary sequences thereof, a third group including base sequences of SEQ ID Nos. 25 to 36 and complementary sequences thereof, a fourth group including base sequences of SEQ ID Nos. 37 to 47 and complementary sequences thereof, a fifth group including base sequences of SEQ ID Nos. 48 to 57 and complementary sequences thereof, a sixth group including base sequences of SEQ ID Nos. 58 to 68 and complementary sequences thereof, a seventh group including base sequences of SEQ ID Nos. 69 to 77 and complementary sequences thereof, an eighth group including base sequences of SEQ ID Nos. 78 to 85 and complementary sequences thereof, a ninth group including base sequences of SEQ ID Nos. 86 to 97 and complementary sequences thereof, and a 10th group including base sequences of SEQ ID Nos. 98 to 106 and complementary sequences thereof.

Description

technical field [0001] The present invention relates to the detection and / or identification of infectious disease pathogens. In particular, inventions related to probes, probe sets, vectors, and genetic testing methods derived from infectious disease pathogens useful for detection and identification of infectious pathogens. [0002] Also, it is an invention related to PCR amplification processing of infectious disease pathogens suitable for detection and / or identification of infectious disease pathogens. technical background [0003] In recent years, gene expression analysis using a DNA chip (or also called a DNA microarray, similarly as follows) has been pioneered in various fields of creating new drugs. The DNA chip is to react the DNA of different specimens with the DNA microarray equipped with various genomes (probes), compare the amount of various genes in each specimen, and analyze the genes (high expression levels) that exist in large quantities...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C07H21/04C12M1/00C12M1/34C12N15/09C12Q1/02C12Q1/70G01N33/569G01N37/00
CPCC12Q1/6837C12Q1/689Y02A50/30C12R2001/19C12Q1/686
Inventor 山本伸子小仓真哉川口正浩塚田护吉井裕人铃木智博石井美绘福井寿文
Owner CANON KK
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