Probe, probe set, probe-immobilized carrier, and genetic testing method

• US 8,148,063 B2
• Filed: 03/30/2007
• Issued: 04/03/2012
• Est. Priority Date: 03/31/2006
• Status: Expired due to Fees

• Current Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Original Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Inventors: Fukui, Toshifumi, Kuribayashi, Hideto
• Primary Examiner(s): SALMON, KATHERINE D
• Application Number:
  US12/295,579
• Publication Number:
  US 20090220972A1
• Time in Patent Office: 1,831 Days
• Field of Search: None
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

A nucleic acid probe for classification of pathogenic bacterial species is capable of collectively detecting bacterial strains of the same species and differentially detecting them from other bacterial species. Any one of the base sequences of SEQ ID NOS. 49 to 51 or a combination of at least two of them is used for detecting the gene of an infectious disease pathogenic bacterium.

Claims

1. A probe set for detecting a gene of infectious disease pathogenic bacterium, Eggerthella lenta, comprising three probes consisting of the following base sequences (1) to (3) respectively:

(1) GGAAAGCCCAGACGGCAAGGGA (SEQ ID NO.

     49) or the complementary sequence thereof;

(2) CCTCTCAAGCGGGATCTCTAATCCGA (SEQ ID NO.

     50) or the complementary sequence thereof; and

(3) TGCCCCATGTTGCCAGCATTAGG (SEQ ID NO.

     51) or the complementary sequence thereof.

2. A probe-immobilized carrier, wherein the probe set of claim 1 is arranged on a solid-phase carrier.

3. A probe-immobilized carrier of claim 2, wherein the probe-immobilized carrier further comprises at least one probe having any one of the base sequences of SEQ ID NOS. 35 to 48 and 52 to 67 immobilized at a position spaced from the probe set.

4. A kit for detecting a gene of Eggerthella lenta, comprising:

the probe set according to claim 1; and

a reagent for detecting a reaction between the probe set and a target nucleic acid.

5. A kit according to claim 4, wherein the reagent comprises a primer set for amplifying the gene of Eggerthella lenta, wherein the primer set comprises:

an oligonucleotide comprising a base sequence of 5′

gcggcgtgcctaacacatgcaag 3′

(SEQ ID NO;

     12); and

at least one of the oligonucleotides from the group consisting of;

an oligonucleotide comprising a base sequence of 5′

atccagccgcaccttccgatac 3′

(SEQ ID NO;

     22) and an oligonucleotide comprising a base sequence of 5′

atccagccgcaccttccggtac 3′

(SEQ ID NO;

     25).

6. A gene detection kit, comprising:

the probe-immobilized carrier of claim 3; and

a reagent for detecting a reaction between the probe set and a target nucleic acid,

wherein the reagent comprises a primer set, wherein the primer set comprises an oligonucleotide comprising a base sequence of 5′

gcggcgtgcctaacacatgcaag 3′

(SEQ ID NO;

     12); and

at least one of the oligonucleotides from the group consisting of;

an oligonucleotide comprising a base sequence of 5′

atccagccgcaccttccgatac 3′

(SEQ ID NO;

     22) and an oligonucleotide comprising a base sequence of 5′

atccagccgcaccttccggtac 3′

(SEQ ID NO;

     25).

10. A probe set according to claim 1, wherein each of the three probes shows a hybridization intensity weak enough to avoid cross-hybridization with the 165 rRNA coding region of any bacterium selected from Anaerococcus prevotii, Bacteroides fragilis, Bacteroides thetaiotaomicron, Clostridium difficile, Clostridium perfringens, Fusobacterium necrophorum, Peptoniphilus asaccharolyticus, Porphyromonas asaccharolytica, Porphyromonas gingivalis, Enterococcus avium, and Providencia rettgeri.

11. A probe set according to claim 1, wherein each of the three probes shows a hybridization intensity weak enough to avoid cross-hybridization with the 165 rRNA coding region of any bacterium selected from Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Streptococcus pneumoniae, Haemophilus influenzae, Enterobacter cloacae, Enterococcus faecalis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Streptococcus sanguinis, Enterococcus avium, Enterococcus faecium, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Stenotrophomonas maltophilia, Acinetobacter baumannii, Acinetobaeter calcoaceticus, Achromobacter xylosoxidans, Vibrio vulnificus, Salmonella choleraesuis, Citrobacter freundii, Klebsiella oxytoca, Enterobacter aerogenes, Hafnia alvei, Serratia liquefaciens, Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia rettgeri, Aeromonas hydrophila, Aeromonas sobria, Gardnerella vaginalis, Corynebacteriun diphtheriae, Legionella pneumophila, Bacillus cereus, Bacillus subtilis, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium chelonae, Nocardia asteroids, Bacteroides fragilis, Bacteroides thetaiotaomicron, Clostridium difficile, Clostridium perfrigens, Fusobacterium necrophorum, Fusobacterium nucleatum, Lactobacillus acidophilus, Anaerococcus prevotii, Peptoniphilus asaccharolyticus, Porphyromonas asaccharolytica, Porphyromonas gingivalis, Prevotella denticola, and Propionibacterium acnes.

12. A method of detecting a gene of Eggerthella lenta in an analyte by using a probe-immobilized carrier, comprising the steps of:

(i) reacting the analyte with the probe set on the probe-immobilized carrier of claim 2; and

(ii) detecting the presence or absence of a hybridization reaction of the probe set on the probe-immobilized carrier with a nucleic acid in the analyte, or detecting the strength of a hybridization reaction of the probe set on the probe-immobilized carrier with the nucleic acid in the analyte, wherein the presence of the hybridization reaction or the strength of the hybridization reaction indicates the presence of a gene of Eggerthella lenta in the analyte.

13. A method according to claim 12, further comprising the step of carrying out PCR amplification of the nucleic acid in the analyte by using a primer set, wherein the primer set comprises:

an oligonucleotide comprising a base sequence of 5′

gcggcgtgcctaacacatgcaag 3′

(SEQ ID NO;

     12); and

at least one of the oligonucleotides from the group consisting of;

an oligonucleotide comprising a base sequence of 5′

atccagccgcaccttccgatac 3′

(SEQ ID NO;

     22) and an oligonucleotide comprising a base sequence of 5′

atccagccgcaccttccggtac 3′

(SEQ ID NO;

     25).

7. A probe set for detecting a gene of infectious disease pathogenic bacterium, Eggerthella lenta, comprising the following three probes (A) to (C), wherein the probe set does not comprise any other probes to detect the gene of Eggerthella lenta:

(A) a probe consisting of a base sequence of

8. A probe-immobilized carrier, comprising the probe set of claim 7, wherein the three probes are arranged on a solid-phase carrier at intervals from each other.

9. A probe-immobilized carrier according to claim 8, wherein the probe-immobilized carrier further comprises at least one probe having any one of the base sequences of SEQ ID NOS. 35 to 48 and 52 to 67 immobilized at a position spaced from the three probes.