Methods for identification of sepsis-causing bacteria

• US 8,546,082 B2
• Filed: 05/25/2007
• Issued: 10/01/2013
• Est. Priority Date: 09/11/2003
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Hall, Thomas A., Sampath, Rangarajan, Harpin, Vanessa, Hofstadler, Steven A., Jiang, Yun
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US11/754,182
• Publication Number:
  US 20080145847A1
• Time in Patent Office: 2,321 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides compositions, kits and methods for rapid identification and quantification of sepsis-causing bacteria by molecular mass and base composition analysis.

Claims

1. A method for identifying a sepsis-causing bacterium in a sample, comprising:

a) amplifying a nucleic acid from said sample using an oligonucleotide primer pair comprising a forward primer and a reverse primer, said forward primer and reverse primer respectively comprising sequences selected from the group consisting of SEQ ID NOs;

(forward;

reverse) 1448;

1461, 430;

1321, and 1454;

1468;

wherein said amplifying step generates an amplification product; and

b) determining the molecular mass of said amplification product by mass spectrometry without fragmentation of said amplification product prior to mass spectrometry analysis.

2. The method of claim 1, further comprising comparing said molecular mass to a database comprising a plurality of molecular masses of amplification product from known sepsis-causing bacteria, wherein a match between said determined molecular mass and a molecular mass in said database identifies said sepsis-causing bacterium in said sample.

3. The method of claim 1, further comprising calculating a base composition of said amplification product using said molecular mass.

4. The method of claim 3, further comprising comparing said calculated base composition to a database comprising a plurality of base compositions of amplification products from sepsis-causing bacteria, wherein a match between said calculated base composition and a base composition included in said database identifies said sepsis-causing bacterium in said sample.

5. The method of claim 1, wherein said forward primer consists of a sequence selected from the group consisting of SEQ ID NO:

1448, 430, and 1454.

6. The method of claim 1, wherein said reverse primer consists of a sequence selected from the group consisting of SEQ ID NO:

1461, 1321, and 1468.

7. The method of claim 1 further comprising repeating said amplifying and determining steps using at least one additional oligonucleotide primer pair wherein the primers of each of said at least one additional primer pair are designed to hybridize to conserved sequence regions within a bacterial gene selected from the group consisting of 16S rRNA, 23S rRNA, tufB, rpoB, valS, rplB, and gyrB.

8. The method of claim 1, wherein said molecular mass identifies the presence of said sepsis-causing bacterium in said sample.

9. The method of claim 8, further comprising determining either sensitivity or resistance of said sepsis-causing bacterium in said sample to one or more antibiotics.

10. The method of claim 1, wherein said molecular mass identifies a sub-species characteristic, strain, or genotype of said sepsis-causing bacterium in said sample.

11. A method for identification of a sepsis-causing bacterium in a sample comprising:

providing one or more primer pairs that hybridize to ribosomal RNA and one or more primer pairs that hybridize to one or more housekeeping genes, wherein said primer pairs that hybridize to one or more housekeeping genes are selected from the group consisting of SEQ ID NOs;

1448;

1461, 430;

1321, and 1454;

1468;

obtaining an amplification product from each of said one or more primer pairs that hybridize to ribosomal RNA and an amplification product from each of said one or more primer pairs that hybridize to one or more housekeeping genes;

measuring molecular masses of said amplification products without fragmentation of said amplification products prior to molecular mass analysis;

calculating base compositions of said amplification products from said molecular masses; and

comparing said base compositions to known base compositions of amplification products of known sepsis-causing bacteria produced with said one or more primer pairs, thereby identifying said sepsis-causing bacterium in said sample.

12. The method of claim 11, wherein said molecular masses are measured by mass spectrometry.

13. The method of claim 12, wherein said mass spectrometry is electrospray time-of-flight mass spectrometry.

14. The method of claim 11, further comprising obtaining one or more amplification products from one or more additional primer pairs, wherein said one or more additional primer pairs hybridize to one or more of rpoC, valS, rpoB, rplB, gyrA or tufB.

15. The method of claim 11, wherein said sepsis-causing bacterium is Bacteroides fragilis, Prevotella denticola, Porphyromonas gingivalis, Borrelia burgdorferi, Mycobacterium tuburculosis, Mycobacterium fortuitum, Corynebacterium jeikeium, Propionibacterium acnes, Mycoplasma pneumoniae, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus mitis, Streptococcus pyogenes, Listeria monocytogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus coagulase-negative, Staphylococcus epidermis, Staphylococcus hemolyticus, Campylobacter jejuni, Bordatella pertussis, Burkholderia cepacia, Legionella pneumophila, Acinetobacter baumannii, Acinetobacter calcoaceticus, Pseudomonas aeruginosa, Aeromonas hydrophile, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Moxarella catarrhalis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Pantoea agglomerans, Bartonella henselae, Stenotrophomonas maltophila, Actinobacillus actinomycetemcomitans, Haemophilus influenzae, Escherichia coli, Klebsiella oxytoca, Serratia marcescens, or Yersinia enterocolitica.

16. The method of claim 11, wherein said sample is a blood sample obtained from a human.

17. The method of claim 16, further comprising selecting an antibiotic known to kill said sepsis-causing bacterium and treating said human with said antibiotic.

18. An oligonucleotide primer pair comprising a forward primer having SEQ ID NO:

1448 and a reverse primer having SEQ ID NO;

1461.

19. The oligonucleotide primer pair of claim 18, wherein said forward primer is SEQ ID NO:

1448.

20. The oligonucleotide primer pair of claim 18, wherein said reverse primer is SEQ ID NO:

1461.

21. The oligonucleotide primer pair of claim 18, wherein at least one of said forward primer and said reverse primer comprises at least one modified nucleobase.

22. The oligonucleotide primer pair of claim 21, wherein at least one of said at least one modified nucleobases is a mass modified nucleobase.

23. The oligonucleotide primer pair of claim 22, wherein said mass modified nucleobase is 5-Iodo-C.

24. The oligonucleotide primer pair of claim 22, wherein said mass modified nucleobase comprises a molecular mass modifying tag.

25. The oligonucleotide primer pair of claim 21, wherein at least one of said at least one modified nucleobases is a universal nucleobase.

26. The oligonucleotide primer pair of claim 25, wherein said universal nucleobase is inosine.

27. The oligonucleotide primer pair of claim 18, wherein at least one of said forward primer and said reverse primer comprises a non-templated T residue at its 5′

end.

28. The oligonucleotide primer pair of claim 18, wherein said forward primer is SEQ ID NO:

1448 and said reverse primer is SEQ ID NO;

1461.

29. The oligonucleotide primer pair of claim 28, wherein at least one of said forward primer and said reverse primer comprises at least one modified nucleobase.

30. The oligonucleotide primer pair of claim 29, wherein at least one of said at least one modified nucleobases is a mass modified nucleobase.

31. The oligonucleotide primer pair of claim 30, wherein said mass modified nucleobase is 5-Iodo-C.

32. The oligonucleotide primer of claim 30, wherein said mass modified nucleobase comprises a molecular mass modifying tag.

33. The oligonucleotide primer pair of claim 29, wherein at least one of said at least one modified nucleobases is a universal nucleobase.

34. The oligonucleotide primer pair of claim 33, wherein said universal nucleobase is inosine.

35. A kit for identifying a sepsis-causing bacterium, comprising:

i) a first oligonucleotide primer pair comprising a forward primer having SEQ ID NO;

1448 and a reverse primer having SEQ ID NO;

1461; and

ii) at least one additional primer pair, wherein the primers of each of said at least one additional primer pair are configured to hybridize to conserved sequence regions within a bacterial gene selected from the group consisting of;

16S rRNA, 23S rRNA, tufB, rpoB, valS, rplB, and gyrB.

36. The kit of claim 35, wherein each of said at least one additional primer pairs is a primer pair comprising a forward primer and a reverse primer, said forward primer and said reverse primer each between 13 to 35 linked nucleotides in length and each having at least 100% sequence identity with the corresponding forward and reverse primers of primer pair numbers 346 (SEQ ID NOs:

202;

1110), 347 (SEQ ID NOs;

560;

1278), 348 (SEQ ID NOs;

706;

895), 349 (SEQ ID NOs;

401;

1156), 360 (SEQ ID NOs;

409;

1434), 361 (SEQ ID NOs;

697;

1398), 2249 (SEQ ID NOs;

430;

1321), 3361 (SEQ ID NOs;

1454;

1468), 354 (SEQ ID NOs;

405;

1072), 358 (SEQ ID NOs;

385;

1093), 359 (SEQ ID NOs;

659;

1250), or 449 (SEQ ID NOs;

309;

1336).

37. The kit of claim 35, wherein said forward primer of said first oligonucleotide primer pair is SEQ ID NO:

1448 and said reverse primer of said first oligonucleotide primer pair is SEQ ID NO;

1461; and

said at least one additional primer pair consists of at least three additional oligonucleotide primer pairs, each of said three oligonucleotide primer pairs comprising a forward primer and a reverse primer, said forward primer and said reverse primer each between 13 to 35 linked nucleotides in length and each having at least 100% sequence identity with the corresponding forward and reverse primers of primer pair numbers, 346 (SEQ ID NOs;

202;

1110), 348 (SEQ ID NOs;

706;

895), and 349 (SEQ ID NOs;

401;

1156).

38. The kit of claim 35, further comprising one or more additional primer pairs, said additional primer pairs comprising a forward primer and a reverse primer, said forward primer and said reverse primer each between 13 to 35 linked nucleotides in length and each having at least 100% sequence identity with corresponding forward and reverse primers selected from the group consisting of primer pair numbers:

3360 (SEQ ID NOs;

1444;

1457), 3350 (SEQ ID NO;

309;

1458), 3351 (SEQ ID NOs;

309;

1460), 3354 (SEQ ID NO;

309;

1459), 3355 (SEQ ID NOs;

1446;

1458), 3353 (SEQ ID NOs;

1447;

1460), 3352 (SEQ ID NOs;

1445;

1458), 3347 (SEQ ID NOs;

1448;

1464), 3348 (SEQ ID NOs;

1451;

1464), 3349 (SEQ ID NOs;

1450;

1463), 3359 (SEQ ID NOs;

1449;

1462), 3358 (SEQ ID NOs;

1453;

1466), 3356 (SEQ ID NOs;

1452;

1467), 3357 (SEQ ID NOs;

1452;

1465), 3361 (SEQ ID NOs;

1454;

1468), 3362 (SEQ ID NOs;

1455;

1469), and 3363 (SEQ ID NOs;

1456;

1470).