METHOD FOR DETECTING PNEUMONIA CAUSATIVE BACTERIA USING NUCLEIC ACID CHROMATOGRAPHY

• US 20130023443A1
• Filed: 03/30/2011
• Published: 01/24/2013
• Est. Priority Date: 03/31/2010
• Status: Active Grant

• Current Assignee: Yamaguchi Technology Licensing Organization, Ltd.
• Original Assignee: Yamaguchi Technology Licensing Organization, Ltd.
• Inventors: Ezaki, Takayuki, Shirai, Mutsunori, Hayashi, Tsukasa, Ujiiye, Takeshi, Ganaha, Makoto, Yamamoto, Shigekazu
• Granted Patent: US 9,347,944 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/137 Chromatographic separation
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: G01N 33/56916 Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
• CPC Class Codes: G01N 33/56933 Mycoplasma
• CPC Class Codes: G01N 33/56938 Staphylococcus
• CPC Class Codes: G01N 33/56944 Streptococcus
• 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

Provided are a method and a kit for accurately and rapidly detecting ten types of targeting pneumonia bacteria: Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus. A set of primer pairs directed to their respective target regions contained in the DnaJ gene, etc., of the ten types of pneumonia causative bacteria is designed for the ten bacterial strains and used to amplify gene products. A set of bacterial strain-specific probe pairs is further designed for the ten bacterial strains such that the probe pairs hybridize with the amplification products via sequences in the respective target regions differing from the sequences hybridized by the set of primer pairs. A first probe-bound labeled high molecular carrier in which plural types of first probes for the pneumonia bacteria are bound to a labeled high molecular carrier and a solid-phase second probe-carrying developing support are used as the set of probe pairs to perform nucleic acid chromatography.

Claims

1. A method for detecting pneumonia causative bacteria targeting at least three types of pneumonia bacteria selected from Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus, the method comprising the following steps (a) to (f):

1) step (a) of amplifying a pneumonia bacterium-specific target nucleic acid arbitrarily extracted from a sample as a single-stranded nucleic acid, using a set of at least three types of NASBA multiplex primer pairs differing by the pneumonia bacteria;

2) step (b) of preparing at least three types of probe pairs differing by the pneumonia bacteria, wherein the probe pairs are selected from a nucleotide sequence complementary to an amplification product;

3) step (c) of binding a first probe for the at least three types of pneumonia bacteria to a labeled high molecular carrier to prepare a first probe-bound labeled high molecular carrier;

4) step (d) of immobilizing a second probe for the at least three types of pneumonia bacteria paired with the first probe, to a predetermined position distinguishable for each of the pneumonia bacteria to prepare a second probe-carrying developing support;

5) step (e) of hybridizing the amplification product with the second probe carried by the developing support and the first probe bound to the labeled high molecular carrier, followed by a detection; and

6) step (f) of evaluating and assessing the detection image,

and wherein the set of at least three types of NASBA multiplex primer pairs differing by the pneumonia bacteria used in the above step (a) is selected from a primer pair represented by any one of the following (i) to (x), or a primer set capable of amplifying a pneumonia bacterium-specific target nucleic acid, constituted of a nucleotide sequence in which 1 to 3 bases are deleted, substituted or added in the nucleotide sequence shown by SEQ ID NOs;

21 to 40 constituting the primer pair;

(i) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

21 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

31 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(ii) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

22 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

32 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(iii) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

23 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

33 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(iv) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

24 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

34 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(v) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

25 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

35 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(vi) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

26 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

36 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(vii) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

27 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

37 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(viii) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

28 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

38 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(ix) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

29 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

39 and an RNA polymerase promoter sequence added to the 5′

end thereof; and

(x) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

30 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

40 and an RNA polymerase promoter sequence added to the 5′

end thereof.

3. The detection method according to claim 1, wherein the first probe for the at least three types of pneumonia bacteria consists of at least three types of DNAs selected from the nucleotide sequences represented by SEQ ID NOs:

1 to 10, and the second probe for the at least three types of pneumonia bacteria to be paired with the first probe is at least three types of DNAs selected from the nucleotide sequences represented by SEQ ID NOs;

11 to 20.

2. (canceled)

4. A kit for detecting pneumonia causative bacteria targeting at least three types of pneumonia bacteria selected from Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus, the kit comprisingat least three types of NASBA multiplex primer pairs differing by the pneumonia bacteria, selected from a primer pair represented by any one of the following (xi) to (xx) which are capable of amplifying a pneumonia bacterium-specific target nucleic acid arbitrarily extracted from a sample, or a primer pair capable of amplifying a pneumonia bacteria-specific target nucleic acid, constituted of a nucleotide sequence in which 1 to 3 bases are deleted, substituted or added in the nucleotide sequence shown by SEQ ID NOs:

21 to 40 constituting the primer pair,

(xi) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

21 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

31 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(xii) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

22 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

32 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(xiii) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

23 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

33 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(xiv) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

24 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

34 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(xv) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

25 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

35 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(xvi) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

26 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

36 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(xvii) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

27 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

37 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(xviii) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

28 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

38 and an RNA polymerase promoter sequence added to the 5′

end thereof;

(xix) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

29 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

39 and an RNA polymerase promoter sequence added to the 5′

end thereof; and

(xx) a forward primer consisting of the nucleotide sequence represented by SEQ ID NO;

30 and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO;

40 and an RNA polymerase promoter sequence added to the 5′

end thereof, further comprising

2) a first probe-bound labeled high molecular carrier in which a first probe for the at least three types of pneumonia bacteria differing by the pneumonia bacteria is bound to a labeled high molecular carrier, wherein the first probe is selected from a nucleotide sequence complementary to an amplification product; and

3) a second probe-carrying developing support in which a second probe for the at least three types of pneumonia bacteria to be paired with the first probe is immobilized at a predetermined positions distinguishable for each of the pneumonia bacteria.

6. The kit according to claim 4, wherein the first probe for the at least three types of pneumonia bacteria consists of at least three types of DNAs selected from the nucleotide sequences represented by SEQ ID NOs:

1 to 10, and the second probe for the at least three types of pneumonia bacteria to be paired with the first probe is at least three types of DNAs selected from the nucleotide sequences represented by SEQ ID NOs;

11 to 20.

5. (canceled)