Method for detecting pneumonia causative bacteria using nucleic acid chromatography

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 one 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 represented 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.

2. 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.

3. 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 comprising

1) at 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.

4. The kit according to claim 3 , 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.