IP Library › Granted Patent US 8,440,808

Granted Patent B2

US 8,440,808 · App. 11/793,223 · Granted May 14, 2013

Nucleic acid fragments and specific detection method by molecular identification of different bacteria species of the genus

Inventors: Bernard La Scola (Rousset, FR); Didier Raoult (Marseilles, FR)

Assignees: Universite d'Aix-Marseille; Centre National de la Recherche Scientifique (CNRS)

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Quick Facts

Patent No.: US 8,440,808
App. No.: 11/793,223
Granted: May 14, 2013
Kind: B2

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Abstract

RpoB gene sequences of various species of Acinetobacter bacteria, and a method of detection by molecular identification of various species of Acinetobacter bacteria using rpoB gene sequences.

Claims (142)

1. A mixture of oligonucleotides comprising:

(a) an equimolar mixture of each different oligonucleotide defined by a first single sequence consisting of at least 18 consecutive nucleotides of a nucleotide sequence set forth in SEQ ID NOS: 1-5 and 8,

where:

D represents A, G, or T,

Y represents C or T,

B represents C, G, or T,

R represents A or G, and

M represents A or C; or

(b) an equimolar mixture of the full-length complementary sequences of the different oligonucleotides set forth in (a).

2. The mixture of oligonucleotides according to claim 1 , wherein the first single sequence consists of the full-length sequence set forth in SEQ ID NO: 1.

3. A combination of primers comprising:

a 5′ primer comprising the mixture of oligonucleotides according to claim 1 , wherein the first single sequence consists of at least 18 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1; and

a 3′ primer comprising a mixture of oligonucleotides selected from the group consisting of:

(i) an equimolar mixture of each different oligonucleotide defined by a second single sequence consisting of at least 12 consecutive nucleotides of SEQ ID NO:2, where M represents A or C, R represents A or G, and Y represents C or T; and

(ii) an equimolar mixture of the full-length complementary sequences of the different oligonucleotides set forth in (i).

4. A method for determining whether a bacterium belonging to the Acinetobacter genus is present in a sample, the method comprising:

(1) contacting the sample with the combination of primers according to claim 3 ;

(2) amplifying the nucleic acids present in the sample by an enzymatic polymerization reaction; and

(3) determining the presence of amplification product, wherein the presence of amplification product indicates the presence of a bacterium belonging to the Acinetobacter genus in the sample.

5. The method according to claim 4 , further comprising:

(4) determining the presence or absence in the sample of at least one Acinetobacter species selected from the group consisting of A. calcoaceticus (genomic species 1), A. baumannii (genomic species 2), genomic species 3 , A. haemolyticus (genomic species 4), A. junii (genomic species 5), genomic species 6 , A. johnsonii (genomic species 7), A. lwoffii (genomic species 8), genomic species 9, genomic species 10, genomic species 11 , A. radioresistens (genomic species 12), genomic species 13, genomic species 16 , A. schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri , and A. parvus , respectively comprising the rpoB sequences set forth in SEQ ID NOS: 9-32, by carrying out the following steps comprising:

(4a) sequencing the amplification product, and

(4b) comparing the sequence of the amplification product obtained in (4a) with at least one sequence selected from the group consisting of SEQ ID NOS: 33-56, which describe portions of the rpoB gene specific to, respectively, A. calcoaceticus (genomic species 1), A. baumannii (genomic species 2), genomic species 3 , A. haemolyticus (genomic species 4), A. junii (genomic species 5), genomic species 6 , A. johnsonii (genomic species 7), A. lwoffii (genomic species 8), genomic species 9, genomic species 10, genomic species 11 , A. radioresistens (genomic species 12), genomic species 13, genomic species 16 , A schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri , and A. parvus.

6. The method according to claim 4 , further comprising:

wherein formation of a hybridization complex between the species probe and the amplification product is indicative of the presence in the sample of the Acinetobacter species corresponding to the species probe, and the absence of a hybridization complex indicates that the species of Acinetobacter corresponding to the species probe is not present in the sample.

7. A method for detecting whether a species of Acinetobacter selected from the group consisting of: A. calcoaceticus (genomic species 1), A. baumannii (genomic species 2), genomic species 3 , A. haemolyticus (genomic species 4), A. junii (genomic species 5), genomic species 6 , A. johnsonii (genomic species 7), A. lwoffii (genomic species 8), genomic species 9, genomic species 10, genomic species 11 , A. radioresistens (genomic species 12), genomic species 13, genomic species 16 , A schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri , and A. parvus , respectively comprising the rpoB sequences set forth in SEQ ID NOS: 9-32, is present in a sample, comprising:

(1) contacting the sample with the combination of primers according to claim 3 to obtain a first amplification product; and

(2) identifying the Acinetobacter species from which the first amplification product was obtained by:

sequencing the first amplification product and comparing the sequence of the first amplification product to SEQ ID NOS: 33-56, which describe portions of the rpoB gene specific to, respectively, A. calcoaceticus (genomic species 1), A. baumannii (genomic species 2), genomic species 3 , A. haemolyticus (genomic species 4), A. junii (genomic species 5), genomic species 6 , A. johnsonii (genomic species 7), A. lwoffii (genomic species 8), genomic species 9, genomic species 10, genomic species 11 , A. radioresistens (genomic species 12), genomic species 13, genomic species 16 , A schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri , and A. parvus ; or

contacting the first amplification product with a species probe consisting of one of SEQ ID NOS: 33-56, which describe portions of the rpoB gene specific to, respectively, A. calcoaceticus (genomic species 1), A. baumannii (genomic species 2), genomic species 3 , A. haemolyticus (genomic species 4), A. junii (genomic species 5), genomic species 6 , A. johnsonii (genomic species 7), A. lwoffii (genomic species 8), genomic species 9, genomic species 10, genomic species 11 , A. radioresistens (genomic species 12), genomic species 13, genomic species 16 , A schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri , and A. parvus , wherein formation of a hybridization complex between the species probe and the first amplification product is indicative of the presence in the sample of the species of Acinetobacter corresponding to the species probe and the absence of a hybridization complex indicates that the species of Acinetobacter corresponding to the species probe is not present in the sample; and

if the presence of A. grimontii or A. junii , respectively comprising the rpoB sequences set forth in SEQ ID NO: 28 and SEQ ID NO: 13, is determined, the following steps (3) and (4) are carried out:

(3) obtaining a second amplification product by contacting the sample with:

a 5′ primer comprising a mixture of each different oligonucleotide defined by a third single sequence consisting of at least 12 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 3, or the complementary sequences thereof, where R represents A or G, and B represents C, G, or T; and

a 3′ primer comprising a mixture of each different oligonucleotide defined by a fourth single sequence consisting of at least 12 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 4, or the complementary sequences thereof, where R represents A or G, B represents C, G, or T, and Y represents C or T; and

(4) determining whether the second amplification product obtained in (3) corresponds to any of the sequences set forth in SEQ ID NOS: 77-100; or

if the presence of A. baylii or genomic species 11, respectively comprising the rpoB the sequences set forth in SEQ ID NO: 25 and SEQ ID NO: 19 is determined, the following steps (5) and (6) are carried out:

(5) obtaining a second amplification product by contacting the sample with:

a 5′ primer comprising at least 12 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 7, or the complementary sequence thereof; and a 3′ primer comprising a mixture of each different oligonucleotide defined by a third single sequence consisting of at least 12 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 8, or the complementary sequences thereof, where R represents A or G; and

(6) determining whether the second amplification product obtained in (5) corresponds to any of the sequences set forth in SEQ ID NOS: 165-188.

8. The combination of primers according to claim 3 , wherein the second single sequence consists of at least 18 consecutive nucleotides of the sequence set forth in SEQ ID NO: 2.

9. A method for detecting whether a species of Acinetobacter selected from the group consisting of: A. calcoaceticus (genomic species 1), A. baumannii (genomic species 2), genomic species 3 , A. haemolyticus (genomic species 4), A. junii (genomic species 5), genomic species 6 , A. johnsonii (genomic species 7), A. lwoffii (genomic species 8), genomic species 9, genomic species 10, genomic species 11 , A. radioresistens (genomic species 12), genomic species 13, genomic species 16 , A. schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri , and A. parvus , respectively comprising the rpoB sequences set forth in SEQ ID NOS: 9-32, is present in a sample, comprising:

(1) contacting the sample with the combination of primers according to claim 3 to obtain a first amplification product; and

(2) identifying the species from which the first amplification product was obtained by:

carrying out at least a second amplification wherein the following steps (3) and (4) are carried out:

(3) obtaining a second amplification product by contacting the sample with 5′ and 3′ primers selected from the group consisting of:

(i) a 5′ primer comprising a mixture of each different oligonucleotide defined by a third single sequence consisting of at least 12 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 3, or the complementary sequences thereof, where R represents A or G, and B represents C, G, or T; and

(ii) a 5′ primer comprising a mixture of each different oligonucleotide defined by a third single sequence consisting of at least 12 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 5, or the complementary sequences thereof, where D represents A, G, or T, M represents A or C, and R represents A or G; and

a 3′ primer comprising at least 12 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 6, or the complementary sequence thereof; and

(iii) a 5′ primer comprising at least 12 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 7, or the complementary sequence thereof; and

a 3′ primer comprising a mixture of each different oligonucleotide defined by a third single sequence consisting of at least 12 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 8, or the complementary sequences thereof, where R represents A or G; and

(4) determining whether the second amplification product obtained in (3) corresponds to any of the sequences set forth in:

SEQ ID NOS: 77-100, if the 5′ and 3′ primers corresponding to SEQ ID NOS: 3 and 4 are selected in (3);

SEQ ID NOS: 121-144, if the 5′ and 3′ primers corresponding to SEQ ID NOS: 5 and 6 are selected in (3); or

SEQ ID NOS: 165-188, if the 5′ and 3′ primers corresponding to SEQ ID NOS: 7 and 8 are selected in (3).

10. A method for detecting whether a species of Acinetobacter selected from the group consisting of: A. calcoaceticus (genomic species 1), A. baumannii (genomic species 2), genomic species 3 , A. haemolyticus (genomic species 4), A. junii (genomic species 5), genomic species 6 , A. johnsonii (genomic species 7), A. lwoffii (genomic species 8), genomic species 9, genomic species 10, genomic species 11 , A. radioresistens (genomic species 12), genomic species 13, genomic species 16 , A. schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri , and A. parvus , respectively comprising the rpoB sequences set forth in SEQ ID NOS: 9-32, is present in a sample, comprising:

(1) contacting the sample with the combination of primers according to claim 3 to obtain a first amplification product; and

(2) identifying the species from which the first amplification product was obtained by:

if the presence of A. baylii or genomic species 11, respectively comprising the rpoB sequences set forth in SEQ ID NO: 25 and SEQ ID NO: 19 is determined, the following steps (3) and (4) are carried out:

(3) obtaining a second amplification product by contacting the sample with:

a 5′ primer comprising at least 12 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 7, or the complementary sequence thereof; and

(4) determining whether the second amplification product obtained in (3) corresponds to any of the sequences set forth in SEQ ID NOS: 165-188.

11. A method for detecting whether a species of Acinetobacter selected from the group consisting of: A. calcoaceticus (genomic species 1), A. baumannii (genomic species 2), genomic species 3 , A. haemolyticus (genomic species 4), A. junii (genomic species 5), genomic species 6 , A. johnsonii (genomic species 7), A. lwoffii (genomic species 8), genomic species 9, genomic species 10, genomic species 11 , A. radioresistens (genomic species 12), genomic species 13, genomic species 16 , A. schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri , and A. parvus , respectively comprising the rpoB sequences set forth in SEQ ID NOS: 9-32, is present in a sample, comprising:

(1) obtaining a first amplification product by contacting the sample with the combination of primers according to claim 8 , wherein the first single sequence consists of the full-length sequence set forth in SEQ ID NO: 1 and the second single sequence consists of the full-length sequence set forth in SEQ ID NO: 2; and

(2) identifying the species from which the first amplification product was obtained by:

if the presence of A. grimontii or A. junii , respectively comprising the rpoB the sequences set forth in SEQ ID NO: 28 and SEQ ID NO: 13 is determined, the following steps (3) and (4) are carried out:

(3) obtaining a second amplification product by contacting the sample with:

a 5′ primer comprising a mixture of each different oligonucleotide defined by a third single sequence consisting of the full-length sequence set forth in SEQ ID NO: 3, or the complementary sequences thereof, where R represents A or G, and B represents C, G, or T; and

a 3′ primer comprising a mixture of each different oligonucleotide defined by a fourth single sequence consisting of the full-length sequence set forth in SEQ ID NO: 4, or the complementary sequences thereof, where R represents A or G, B represents C, G, or T, and Y represents C or T; and

(4) determining whether the second amplification product obtained in (3) corresponds to any of the sequences set forth in SEQ ID NOS: 77-100; or

if the presence of A. baylii or genomic species 11, respectively comprising the rpoB sequences set forth in SEQ ID NO: 25 or SEQ ID NO: 19 is determined, the following steps (5) and (6) are carried out:

(5) obtaining a second amplification product by contacting the sample with:

a 5′ primer consisting of the full-length sequence set forth in SEQ ID NO: 7, or the complementary sequence thereof; and

a 3′ primer comprising a mixture of each different oligonucleotide defined by a third single sequence consisting of the full-length sequence set forth in SEQ ID NO: 8, or the complementary sequences thereof, where R represents A or G; and

(6) determining whether the second amplification product obtained in (5) corresponds to any of the sequences set forth in SEQ ID NOS: 165-188.

12. A method for detecting whether a species of Acinetobacter selected from the group consisting of: A. calcoaceticus (genomic species 1), A. baumannii (genomic species 2), genomic species 3 , A. haemolyticus (genomic species 4), A. junii (genomic species 5), genomic species 6 , A. johnsonii (genomic species 7), A. lwoffii (genomic species 8), genomic species 9, genomic species 10, genomic species 11 , A. radioresistens (genomic species 12), genomic species 13, genomic species 16 , A. schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri , and A. parvus , respectively comprising the rpoB sequences set forth in SEQ ID NOS: 9-32, is present in a sample, comprising:

(2) identifying the species from which the first amplification product was obtained by:

carrying out at least a second amplification wherein the following steps (3) and (4) are carried out:

(3) obtaining a second amplification product by contacting the sample with 5′ and 3′ primers selected from the group consisting of:

(i) a 5′ primer comprising a mixture of each different oligonucleotide defined by a third single sequence consisting of the full-length sequence set forth in SEQ ID NO: 3, or the complementary sequences thereof, where R represents A or G, and B represents C, G, or T; and

(ii) a 5′ primer comprising a mixture of each different oligonucleotide defined by a third single sequence consisting of the full-length sequence set forth in SEQ ID NO: 5, or the complementary sequences thereof, where D represents A, G, or T, M represents A or C, and R represents A or G; and

a 3′ primer consisting of the full-length sequence set forth in SEQ ID NO: 6, or the complementary sequence thereof; and

(iii) a 5′ primer consisting of the full-length sequence set forth in SEQ ID NO: 7, or the complementary sequence thereof; and

(4) determining whether the second amplification product obtained in (3) corresponds to any of the sequences set forth in:

SEQ ID NOS: 77-100, if the 5′ and 3′ primers corresponding to SEQ ID NOS: 3 and 4 are selected in (3);

SEQ ID NOS: 121-144, if the 5′ and 3′ primers corresponding to SEQ ID NOS: 5 and 6 are selected in (3); or

SEQ ID NOS: 165-188, if the 5′ and 3′ primers corresponding to SEQ ID NOS: 7 and 8 are selected in (3).

13. A method for detecting whether a species of Acinetobacter selected from the group consisting of: A. calcoaceticus (genomic species 1), A. baumannii (genomic species 2), genomic species 3 , A. haemolyticus (genomic species 4), A. junii (genomic species 5), genomic species 6 , A. johnsonii (genomic species 7), A. lwoffii (genomic species 8), genomic species 9, genomic species 10, genomic species 11 , A. radioresistens (genomic species 12), genomic species 13, genomic species 16 , A. schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri , and A. parvus , respectively comprising the rpoB sequences set forth in SEQ ID NOS: 9-32, is present in a sample, comprising:

(2) identifying the species from which the first amplification product was obtained by:

if the presence of A. baylii or genomic species 11, respectively comprising the rpoB sequences set forth in SEQ ID NO: 28 or SEQ ID NO: 19 is determined, the following steps (3) and (4) are carried out:

(3) obtaining a second amplification product by contacting the sample with:

a 5′ primer consisting of the full-length sequence set forth in SEQ ID NO: 7, or the complementary sequence thereof; and

(4) determining whether the second amplification product obtained in (3) corresponds to any of the sequences set forth in SEQ ID NOS: 165-188.

14. A combination of primers, comprising:

a first pair of primers comprising the combination of primers set forth in claim 8 ; and

a second pair of primers selected from the group consisting of:

(i) a 5′ primer comprising an equimolar mixture of each different oligonucleotide defined by a third single sequence consisting of at least 18 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 3, or the complementary sequences thereof, where R represents A or G, and B represents C, G, or T; and

a 3′ primer comprising an equimolar mixture of each different oligonucleotide defined by a fourth single sequence consisting of at least 18 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 4, or the complementary sequences thereof, where R represents A or G, B represents C, G, or T, and Y represents C or T,

(ii) a 5′ primer comprising an equimolar mixture of each different oligonucleotide defined by a third single sequence consisting of at least 18 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO:5, or the complementary sequences thereof, where D represents A, G, or T, M represents A or C, and R represents A or G; and

a 3′ primer comprising at least 18 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 6, or the complementary sequence thereof; and

(iii) a 5′ primer comprising at least 18 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 7, or the complementary sequence thereof; and

a 3′ primer comprising an equimolar mixture of each different oligonucleotide defined by a third single sequence consisting of at least 18 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 8, or the complementary sequences thereof, where R represents A or G.

15. The mixture of oligonucleotides according to claim 1 , wherein the first single sequence consists of the full-length sequence set forth in SEQ ID NO: 2.

16. The mixture of oligonucleotides according to claim 1 , wherein the first single sequence consists of the full-length sequence set forth in SEQ ID NO: 3.

17. The mixture of oligonucleotides according to claim 1 , wherein the first single sequence consists of the full-length sequence set forth in SEQ ID NO: 4.

18. The mixture of oligonucleotides according to claim 1 , wherein the first single sequence consists of the full-length sequence set forth in SEQ ID NO: 5.

19. The mixture of oligonucleotides according to claim 1 , wherein the first single sequence consists of the full-length sequence set forth in SEQ ID NO: 8.

20. A combination of primers comprising:

a 3′ primer comprising a mixture of oligonucleotides selected from the group consisting of:

(i) an equimolar mixture of each different oligonucleotide defined by a second single sequence consisting of at least 12 consecutive nucleotides of SEQ ID NO:4, where:

B represents C, G, or T,

R represents A or G, and

Y represents C or T; and

(ii) an equimolar mixture of the full-length complementary sequences of the different oligonucleotides set forth in (i).

21. A combination of primers comprising:

a 3′ primer consisting of:

at least 12 consecutive nucleotides of SEQ ID NO: 6; or

the full-length complementary sequence of at least 12 consecutive nucleotides of SEQ ID NO: 6.

22. A combination of primers comprising:

a 3′ primer comprising the mixture of oligonucleotides according to claim 1 , wherein the first single sequence consists of at least 18 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 8; and

a 5′ primer consisting of:

at least 12 consecutive nucleotides of SEQ ID NO: 7; or

the full-length complementary sequence of at least 12 consecutive nucleotides of SEQ ID NO:7.

Assignments (2)

MERGER Recorded Oct 18, 2012

From: UNIVERSITE DE LA MEDITERRANEE (AIX-MARSEILLE II)

To: UNIVERSITE D'AIX-MARSEILLE

Reel/Frame 029162/0518 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 18, 2007

From: LA SCOLA, BERNARD; RAOULT, DIDIER

To: UNIVERSITE DE LA MEDITERRANEE (AIX-MARSEILLEII); CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS)

Reel/Frame 019484/0415 →

Priority Claims (1)

FR 05 02630 · Mar 17, 2005 · national

Continuity (1)

Related Publication 20090123916A1 · May 14, 2009