PROBE, PROBE SET, PROBE-IMMOBILIZED CARRIER, AND GENETIC TESTING METHOD

• US 20090305260A1
• Filed: 03/30/2007
• Published: 12/10/2009
• Est. Priority Date: 03/31/2006
• Status: Active Grant

• Current Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Original Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Inventors: Kuribayashi, Hideto, Fukui, Toshifumi
• Granted Patent: US 8,207,319 B2
• Time in Patent Office: Days
• 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. 59 to 61 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 for detecting a gene of infectious disease pathogenic bacterium, Porphyromonas gingivalis, capable of hybridizing with the 16S rRNA coding region of Porphyromonas gingivalis, said probe showing a hybridization intensity weak enough to avoid cross-hybridization with the 16S rRNA coding region of Porphyromonas asaccharolytica, said probe having any one of the following base sequences (1) to (4):

(1) TTATAGCTGTAAGATAGGCATGCGTCCC (SEQ ID NO.

     59) or a complementary sequence thereof;

(2) AACGGGCGATACGAGTATTGCATTGA (SEQ ID NO.

     60) or a complementary sequence thereof;

(3) ATATACCGTCAAGCTTCCACAGCGA (SEQ ID NO.

     61) or a complementary sequence thereof; and

(4) a modified sequence prepared such that any one of the sequences of SEQ ID NOS. 59 to 61 and the complementary sequences thereof is subjected to base deletion, substitution, or addition as far as the modified sequence retains a function as the probe.

2. A probe-immobilized carrier, wherein at least one probe according to claim 1 is arranged on a solid-phase carrier.

3. A probe-immobilized carrier according to claim 2, wherein the probe-immobilized carrier comprises at least one probe having any one of the base sequences of SEQ ID NOS. 35 to 67 as mentioned in the specification immobilized at a position spaced from the probe according to claim 1.

4. A kit for detecting a gene of Porphyromonas gingivalis, comprising:

a probe according to claim 1; and

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

5. A kit according to claim 4, wherein the reagent contains a primer for amplifying the gene of Porphyromonas gingivalis, and the primer includes:

(18) an oligonucleotide having a base sequence of 5′

gcgataggcttaacacatgcaag 3′

(SEQ ID NO;

     18); and

at least one of

(22) an oligonucleotide having a base sequence of 5′

atccagccgcaccttccgatac 3′

(SEQ ID NO;

     22) and

(28) an oligonucleotide having a base sequence of 5′

atccagccgcacgttcccgtac 3′

(SEQ ID NO;

     28).

6. A gene detection kit, comprising:

a probe-immobilized carrier according to claim 3; and

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

wherein the reagent contains a primer including at least one oligonucleotide selected from the following items (1) to (21) and at least one oligonucleotide selected from the following items (22) to (28);

(1) an oligonucleotide having a base sequence of 5′

gcggcgtgcctaatacatgcaag 3′

(SEQ ID NO;

     1);

(2) an oligonucleotide having a base sequence of 5′

gcggcaggcctaacacatgcaag 3′

(SEQ ID NO;

     2);

(3) an oligonucleotide having a base sequence of 5′

gcggcaggcttaacacatgcaag 3′

(SEQ ID NO;

     3);

(4) an oligonucleotide having a base sequence of 5′

gcggtaggcctaacacatgcaag 3′

(SEQ ID NO;

     4);

(5) an oligonucleotide having a base sequence of 5′

gcggcgtgcttaacacatgcaag 3′

(SEQ ID NO;

     5);

(6) an oligonucleotide having a base sequence of 5′

gcgggatgccttacacatgcaag 3′

(SEQ ID NO;

     6);

(7) an oligonucleotide having a base sequence of 5′

gcggcatgccttacacatgcaag 3′

(SEQ ID NO;

     7);

(8) an oligonucleotide having a base sequence of 5′

gcggcatgcttaacacatgcaag 3′

(SEQ ID NO;

     8);

(9) an oligonucleotide having a base sequence of 5′

gcggcgtgcttaatacatgcaag 3′

(SEQ ID NO;

     9);

(10) an oligonucleotide having a base sequence of 5′

gcggcaggcctaatacatgcaag 3′

(SEQ ID NO;

     10);

(11) an oligonucleotide having a base sequence of 5′

gcgggatgctttacacatgcaag 3′

(SEQ ID NO;

     11);

(12) an oligonucleotide having a base sequence of 5′

gcggcgtgcctaacacatgcaag 3′

(SEQ ID NO;

     12);

(13) an oligonucleotide having a base sequence of 5′

gcggcgtgcataacacatgcaag 3′

(SEQ ID NO;

     13);

(14) an oligonucleotide having a base sequence of 5′

gcggcatgcctaacacatgcaag 3′

(SEQ ID NO;

     14);

(15) an oligonucleotide having a base sequence of 5′

gcggcgcgcctaacacatgcaag 3′

(SEQ ID NO;

     15);

(16) an oligonucleotide having a base sequence of 5′

gcggcgcgcttaacacatgcaag 3′

(SEQ ID NO;

     16);

(17) an oligonucleotide having a base sequence of 5′

gcgtcatgcctaacacatgcaag 3′

(SEQ ID NO;

     17);

(18) an oligonucleotide having a base sequence of 5′

gcgataggcttaacacatgcaag 3′

(SEQ ID NO;

     18);

(19) an oligonucleotide having a base sequence of 5′

gcgacaggcttaacacatgcaag 3′

(SEQ ID NO;

     19);

(20) an oligonucleotide having a base sequence of 5′

gctacaggcttaacacatgcaag 3′

(SEQ ID NO;

     20);

(21) an oligonucleotide having a base sequence of 5′

acagaatgcttaacacatgcaag 3′

(SEQ ID NO;

     21);

(22) an oligonucleotide having a base sequence of 5′

atccagccgcaccttccgatac 3′

(SEQ ID NO;

     22);

(23) an oligonucleotide having a base sequence of 5′

atccaaccgcaggttcccctac 3′

(SEQ ID NO;

     23);

(24) an oligonucleotide having a base sequence of 5′

atccagccgcaggttcccctac 3′

(SEQ ID NO;

     24);

(25) an oligonucleotide having a base sequence of 5′

atccagccgcaccttccggtac 3′

(SEQ ID NO;

     25);

(26) an oligonucleotide having a base sequence of 5′

atccagcgccaggttcccctag 3′

(SEQ ID NO;

     26);

(27) an oligonucleotide having a base sequence of 5′

atccagccgcaggttctcctac 3′

(SEQ ID NO;

     27); and

(28) an oligonucleotide having a base sequence of 5′

atccagccgcacgttcccgtac 3′

(SEQ ID NO;

     28).

10. A method of detecting a gene of Porphyromonas gingivalis in an analyte by using a probe-immobilized carrier, comprising the steps of:

(i) reacting the analyte with a probe-immobilized carrier according to claim 2; and

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

11. A method according to claim 10, further comprising the step of carrying out PCR amplification of the target nucleic acid in the analyte by using a primer including the following oligonucleotides:

(18) an oligonucleotide having a base sequence of 5′

gcgataggcttaacacatgcaag 3′

(SEQ ID NO;

     18); and

at least one of

(22) an oligonucleotide having a base sequence of 5′

atccagccgcaccttccgatac 3′

(SEQ ID NO;

     22) and

(28) an oligonucleotide having a base sequence of 5′

atccagccgcacgttcccgtac 3′

(SEQ ID NO;

     28).

12. A probe according to claim 1, wherein said probe shows a hybridization intensity weak enough to avoid cross-hybridization with the 16S rRNA coding region of any bacterium selected from Anaerococcus prevotii, Bacteroides fragilis, Bacteroides thetaiotaomicron, Clostridium difficile, Clostridium perfringens, Eggerthella lenta, Fusobacterium necrophorum, Peptoniphilus asaccharolyticus, Porphyromonas asaccharolytica, Enterococcus avium, and Providencia rettgeri.

13. A probe according to claim 1, wherein said probe shows a hybridization intensity weak enough to avoid cross-hybridization with the 16S 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, Stenotrophoimonas maltophilia, Acinetobacter baumannii, Acinetobacter 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, Corynebacterium diphtheriae, Legionella pneumophila, Bacillus cereus, Bacillus subtilis, Mycobacterium kansasil, Mycobacterium intracellulare, Mycobacterium chelonae, Nocardia asteroids, Bacteroides fragilis, Bacteroides thetaiotaomicron, Clostridium difficile, Clostridium perfrigens, Eggerthella lenta, Fusobacterium necrophorum, Fusobacterium nucleatum, Lactobacillus acidophilus, Anaerococcus prevotii, Peptoniphilus asaccharolyticus, Porphyromonas asaccharolytica, Prevotella denticola, and Propionibacterium acnes.

7. A probe set for detecting a gene of infectious disease pathogenic bacterium, Porphyromonas gingivalis, including at least two probes selected from the following items (A) to (L):

(A) a probe having a base sequence represented by TTATAGCTGTAAGATAGGCATGCGTCCC (SEQ ID NO.

     59);

(B) a probe having a base sequence represented by AACGGGCGATACGAGTATTGCATTGA (SEQ ID NO.

     60);

(C) a probe having a base sequence represented by ATATACCGTCAAGCTTCCACAGCGA (SEQ ID NO.

     61);

(D) a probe having a complementary sequence of the base sequence represented by SEQ ID NO. 59;

(E) a probe having a complementary sequence of the base sequence represented by SEQ ID NO. 60;

(F) a probe having a complementary sequence of the base sequence represented by SEQ ID NO. 61;

(G) a probe having a modified sequence obtained by base deletion, substitution, or addition on the base sequence represented by SEQ ID NO. 59 as far as it retains the function of a probe for detecting the gene of Porphyromonas gingivalis;

(H) a probe having a modified sequence obtained by base deletion, substitution, or addition on the base sequence represented by SEQ ID NO. 60 as far as it retains the function of a probe for detecting the gene of Porphyromonas gingivalis;

(I) a probe having a modified sequence obtained by base deletion, substitution, or addition on the base sequence represented by SEQ ID NO. 61 as far as it retains the function of a probe for detecting the gene of Porphyromonas gingivalis;

(J) a probe having a modified sequence obtained by base deletion, substitution, or addition on the complementary sequence of the base sequence represented by SEQ ID NO. 59 as far as it retains the function of a probe for detecting the gene of Porphyromonas gingivalis;

(K) a probe having a modified sequence obtained by base deletion, substitution, or addition on the complementary sequence of the base sequence represented by SEQ ID NO. 60 as far as it retains the function of a probe for detecting the gene of Porphyromonas gingivalis; and

(L) a probe having a modified sequence obtained by base deletion, substitution, or addition on the complementary sequence of the base sequence represented by SEQ ID NO. 61 as far as it retains the function of a probe for detecting the gene of Porphyromonas gingivalis.

8. A probe-immobilized carrier, comprising a plurality of probes constituting the probe set according to claim 7, wherein the plurality of 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 comprises at least one probe having any one of the base sequences of SEQ ID NOS. 35 to 67 as mentioned in the specification immobilized at a position spaced from the plurality of probes.