JP2005211027A - Primer for amplification of target base sequence of legionella bacteria, method for amplifying target base sequence of legionella bacteria, primer for amplification of target base sequence of legionella pneumophila and method for amplifying target base sequence of legionella pneumophila - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a primer for the amplification of a target base sequence of Legionella bacteria enabling efficient detection of Legionella bacteria. <P>SOLUTION: General Legionella bacteria are efficiently amplified and detected by using a specific base sequence as the upstream and downstream primers. Concretely, the target base sequence of Legionella bacteria can be amplified by a step of hybridizing the 1st primer containing the base sequence of 5'-ccttacatcctcctcggctc-3' and the 2nd primer containing the base sequence of 5'-cgctcgtttccagctcccc-3' to a nucleic acid containing the target base sequence and a step of extending the hybridized 1st and 2nd primers on the nucleic acid. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a primer for amplification of a target base sequence of Legionella spp., A method of amplification of a target base sequence of Legionella spp., A primer for amplification of a target base sequence of Legionella pneumophila, and a method of amplification of a target base sequence of Legionella pneumophila .

Legionella species are known to cause legionellosis such as Legionella pneumonia, which exists in natural soil and fresh water.
A technique using nucleic acid amplification for rapidly detecting and identifying Legionella pneumophila has been disclosed (see Patent Document 1).
JP 2002-320486 A

However, it is difficult to detect Legionella in general with the technique disclosed in Patent Document 1. Moreover, it does not mean that the detection of Legionella spp.
In view of the above, the present invention is a primer for amplification of a target base sequence of Legionella genus that can efficiently detect Legionella, a method for amplification of a target base sequence of Legionella, a primer for amplification of a target base sequence of Legionella pneumophila, An object of the present invention is to provide a method for amplifying the target nucleotide sequence of Legionella pneumophila.

A. In order to achieve the above object, the target nucleic acid amplification primer for Legionella spp. According to the present invention is selected from 5'-ccttacatcctcctcggctc-3 '(SEQ ID NO: 1) and 5'-cgctcgtttccagctcccc-3' (SEQ ID NO: 2) The base sequence is provided.
By using the nucleotide sequences of SEQ ID NOs: 1 and 2 as upstream and downstream primers, Legionella in general can be efficiently amplified and detected.

  That is, the nucleic acid containing the target base sequence contains the first primer containing the base sequence of 5'-ccttacatcctcctcggctc-3 '(SEQ ID NO: 1) and the base sequence of 5'-cgctcgtttccagctcccccc-3' (SEQ ID NO: 2) The target base sequence of Legionella can be amplified by the step of hybridizing with the second primer and the step of extending the hybridized first and second primers on the nucleic acid.

  For this amplification, any of PCR method, SDA method, tSDA method, and real-time fluorescence tSDA method can be used.

B. The primer for amplification of the target nucleic acid of Legionella pneumophila according to the present invention is 5'-cccttgctgaattggagaac-3 '(SEQ ID NO: 3), 5'-gttgcattgacattgcctca-3' (SEQ ID NO: 4), 5'-tgctttaggacatcccagctat-3 ' (SEQ ID NO: 5), 5'-cactgttcttgtccgctaattg-3 '(SEQ ID NO: 6), 5'-tcgtcgtatggaacgagaagt-3' (SEQ ID NO: 7), 5'-gagggtcaaatgtctgtccaa-3 '(SEQ ID NO: 8) An array is provided.
A pair of base sequences of SEQ ID NOs: 3 and 4, a pair of base sequences of SEQ ID NOs: 5 and 6, and a pair of base sequences of SEQ ID NOs: 7 and 8 are used as upstream and downstream primers to efficiently amplify Legionella pneumophila Can be detected.

  That is, any one of 5′-cccttgctgaattggagaac-3 ′ (SEQ ID NO: 3), 5′-tgctttaggacatcccagctat-3 ′ (SEQ ID NO: 5), and 5′-tcgtcgtatggaacgagaagt-3 ′ (SEQ ID NO: 7) is added to the nucleic acid containing the target base sequence. A first primer containing a base sequence selected from 5′-gttgcattgacattgcctca-3 ′ (SEQ ID NO: 4), 5′-cactgttcttgtccgctaattg-3 ′ (SEQ ID NO: 6), 5′-gagggtcaaatgtctgtccaa-3 ′ (SEQ ID NO: 4) 8) Legionella pneumophila by a step of hybridizing a second primer containing a base sequence selected from any of the above and a step of extending the hybridized first and second primers on the nucleic acid. Can be amplified.

  For this amplification, any of PCR method, SDA method, tSDA method, and real-time fluorescence tSDA method can be used.

  The primer for amplification is a primer for amplifying the target sequence by extension of the primer after hybridizing to the target sequence. The primer for amplification is generally 10 to 75 nucleotides in length, and preferably 15 to 50 nucleotides in length.

  The total length of primers for SDA amplification is about 25-50 nucleotides. The 3 'end (target binding sequence) of the primer for SDA amplification hybridizes at the 3' end of the target sequence. The target binding sequence is approximately 10-25 nucleotides in length and provides hybridization properties to the primers for amplification. Primers for SDA amplification further include recognition sites for restriction endonucleases from 5 'to the target binding sequence. The recognition site nicks (cuts one strand) the DNA double helix when semi-modifying the recognition site. This is due to the restriction endonuclease. The nucleotide from the 5 'to the restriction endonuclease recognition site ("tail") functions as a polymerase repriming site when cleaving the remainder of the amplification primer and replacing it in SDA. The tail nucleotide repriming function continues the SDA reaction and allows the synthesis of multiple amplicons from a single target molecule. This tail is generally about 10-25 nucleotides in length. Its length and sequence are generally not critical and can be routinely selected and modified.

  In the case of amplification methods where the target binding sequence does not require a unique sequence at the end of the target, the primers for amplification generally consist essentially of the target binding sequence only. For example, for amplification of a target sequence using the polymerase chain reaction (PCR), an amplification primer consisting of a target binding sequence of the amplification primer is used. Amplification methods that require a SDA nickable restriction endonuclease recognition site and a unique sequence added to a target other than the tail (eg, Self-Sustained Sequence Replication: 3SR), nucleic acid sequence-based amplification ( In the case of Nucleic Acidsequence-Based Amplification (NASBA) or RNA polymerase promoters for transcription-based amplification systems (TAS), it is necessary to use a standard oligonucleotide preparation method without changing the hybridization specificity of the primers Unique sequence can be linked to the target binding sequence.

  The term target or target sequence refers to the nucleic acid sequence to be amplified. These include any strand of the original nucleic acid sequence to be amplified, a second strand complementary to the original nucleic acid sequence to be amplified, and a copy of the original sequence generated by the amplification reaction. It is. Since amplifiable targets include copies of sequences to which amplification primers are hybridized, these copies serve as amplifiable targets.

  The copy of the target sequence generated during the amplification reaction is called the amplification product, amplimer or amplicon.

  The term extension product refers to a copy produced by primer hybridization and extension of the primer by a polymerase using the target sequence as a template.

  The term species-specific refers to detection, amplification or oligonucleotide high to a species of organism or group of related species that does not substantially detect, amplify or hybridize oligonucleotides to species of other or different genera of the same genus. Refers to hybridization.

  The term assay probe is an oligonucleotide used to facilitate detection or identification of nucleic acids. The detector probes, detector primers, capture probes, signal primers and reporter probes described below are examples of assay probes.

  The signal primer contains a 3 'target binding sequence that hybridizes to a complementary sequence in the target and a 5' tail sequence (adapter sequence) that is not complementary to the target. This adapter sequence is an indirectly detectable marker selected such that its complementary sequence hybridizes to the 3 'end of the reporter probe described below. The signal primer hybridizes to the target sequence at least some downstream downstream of the hybridization site of the amplification primer. This signal primer is extended by the polymerase in a manner similar to the extension of the amplification primer. The extension part of the primer for amplification is replaced with the extension product of the signal primer depending on the target amplification. A 5 'adapter sequence, single stranded product, is generated that includes a downstream target binding sequence and a hybridization specific 3' binding sequence to a primer for flanking SDA amplification. Hybridization and extension of this flanking amplification primer, and subsequent nicking and extension, yields an amplification product that includes the complement of the adapter sequence, which can be detected as an indicator of target amplification.

  The reporter probe comprises a label that functions as a detector oligonucleotide and is preferably at least one donor / quencher dye pair (ie, a donor fluorescent dye and a quencher for the donor fluorophore). The label is linked to a sequence or structure (reporter moiety) in the report probe that does not hybridize directly to the target sequence. The sequence from the reporter probe 3 'to the reporter moiety is selected to hybridize to the complement of the signal primer adapter sequence. Generally, the 3 'end of the reporter probe does not contain sequences that have significant complementarity to the target sequence. If an amplification product is present that contains the complement of the adapter sequence described above, the amplification product can hybridize to the 3 'end of the reporter probe. Reporter partial complement can be formed by priming and extension from the 3 'end of the adapter complement sequence. This formation causes the reporter moiety to become double-stranded so that the reporter probe label can be detected and can indicate the presence or amplification of the target.

  The term amplicon refers to the product of an amplification reaction that results from the extension of either or both of a pair of amplification primers. If both primers used hybridize to the target sequence, the amplicon may comprise an exponentially amplified nucleic acid. Alternatively, amplicons may be generated by linear amplification if one of the primers used does not hybridize to the target sequence. Thus, this term does not imply the presence of exponentially amplified nucleic acid.

  As described above, according to the present invention, a primer for amplification of the target base sequence of Legionella, which can efficiently detect Legionella, a method for amplifying the target base sequence of Legionella, and a target base sequence of Legionella pneumophila Amplification primer, Legionella pneumophila target base sequence amplification method can be provided.

  The present invention provides oligonucleotide primers that can be used to amplify target sequences in Legionella spp. More specifically, this target sequence is included in the 16S-23S rRNA intergenic region with tRNA-Ala-like sequence (GeneBank: AF000655) (or 16S-23S ribosomal RNA intergenic spacer, 16S-23S ribosomal RNA gene spacer region, etc. Also called). The present invention also provides an oligonucleotide primer that can be used for amplification of a target sequence for specifically detecting Legionella pneumophila. Specifically, this target sequence is contained in the grpE gene (GeneBank: D89498), which encodes one of the heat shock proteins. Primers for amplification are useful for both high-efficiency high-specificity amplification (for example, tSDA and PCR) and low-temperature amplification reactions such as SDA, 3SR, TAS, and NASBA at high temperatures. Oligonucleotide reporter probes that hybridize to target-specific signal primers are used to indirectly detect amplification products.

  The oligonucleotide of the present invention can be used as a means for confirming the identity of a cultured microorganism after culturing. Alternatively, to detect and identify Legionella nucleic acids using known amplification methods, human clinical samples (respiratory samples), or environmental samples (eg, cooling tower water in a facility, Hot spring water, bathtub water, etc.). In any case, Legionella and other microorganisms can be quickly distinguished from each other by using the oligonucleotide and analysis method of the present invention.

(Base sequence)
The oligonucleotides of SEQ ID NOs: 1 to 8 are shown below.
Sequence number 1 (LP / F2-1): 5'-ccttacatcctcctcggctc-3 '
Sequence number 2 (LP-R4): 5'-cgctcgtttccagctcccc-3 '
Sequence number 3 (grpE1-f): 5'-cccttgctgaattggagaac-3 '
Sequence number 4 (grpE1-r): 5'-gttgcattgacattgcctca-3 '
Sequence number 5 (grpE2-f): 5'-tgctttaggacatcccagctat-3 '
Sequence number 6 (grpE2-r): 5'-cactgttcttgtccgctaattg-3 '
Sequence number 7 (grpE3-f): 5'-tcgtcgtatggaacgagaagt-3 '
Sequence number 8 (grpE3-r): 5'-gagggtcaaatgtctgtccaa-3 '

  SEQ ID NO: 1 is an oligonucleotide sequence that can be used as a forward primer for amplification of the target sequence of Legionella spp. SEQ ID NO: 2 is an oligonucleotide sequence that can be used as a downstream primer for amplification of the target sequence of Legionella spp. The sequences 1 and 2 are not limited to Legionella pneumophila but can be used for general detection of Legionella spp.

On the other hand, SEQ ID NOs: 3 to 8 are not general Legionella spp., But are oligonucleotide sequences that can be used particularly for detection of Legionella pneumophila.
SEQ ID NOs: 3 and 4 are oligonucleotide sequences that can be used as a pair of upstream and downstream primers for amplification of a target sequence of Legionella pneumophila, respectively.
SEQ ID NOs: 5 and 6 are oligonucleotide sequences that can be used as a pair of upstream and downstream primers for amplification of a target sequence of Legionella pneumophila, respectively.
SEQ ID NOs: 7 and 8 are oligonucleotide sequences that can be used as a pair of upstream and downstream primers for amplification of the target sequence of Legionella pneumophila, respectively.

The oligonucleotides of SEQ ID NOs: 3 to 8 can be used as primers specific to Legionella pneumophila. That is, it is possible to prevent detection of Legionella bacteria other than Legionella pneumophila. Further, the detection sensitivity is extremely high, and detection can be performed from three Legionella pneumophila. Moreover, it can be easily applied to quantitative detection using quantitative PCR or the like.
As described above, the set of oligonucleotides of SEQ ID NOs: 3 to 8 has 1) a sequence specific to Legionella pneumophila, 2) has good detection sensitivity as a primer, and 3) is suitable for quantitative PCR. Easy to apply.

  SEQ ID NOs: 3 to 8 are designed based on the grpE gene (GeneBank: D89498) encoding one kind of heat shock protein. Primers designed based on this grpE gene are included in the technical scope of the present invention.

  Since nucleic acids do not require perfect complementarity to hybridize, the oligonucleotide sequences disclosed herein do not lose their usefulness as primers for amplification of Legionella target sequences, It can be modified to some extent. For example, by adjusting hybridization conditions (ie, hybridization pH, temperature or buffer salt content) to enhance or reduce stringency, complementary and partially complementary nucleic acid sequences Can be obtained. Such minor modifications of the disclosed sequences and adjustment of the hybridization conditions necessary to maintain specificity for Legionella are within the ordinary skill in the art.

  Amplification products generated using the primers described herein can be detected by a characteristic size, for example, on polyacrylamide gels or agarose gels stained with ethidium bromide. Alternatively, assay probes (oligonucleotides labeled with a detectable label) can be used to detect the amplified target sequence.

  The detectable label of the assay probe can be detected either directly or indirectly as an indication of the presence of the target nucleic acid. In the case of direct detection of the label, the assay probe may be labeled with a radioisotope and detected by autoradiography, or labeled with a fluorescent moiety and detected with fluorescence. Alternatively, the assay probe can be detected indirectly by labeling with a label that requires additional reagents to be detectable. Labels that can be detected indirectly include, for example, chemiluminescent agents, enzymes that produce visible reaction products, and ligands that can be detected by binding to a labeled binding partner (eg, antibody or antigen / hapten) ( For example, hapten, antibody or antigen). The ligand is also useful for immobilizing a ligand-labeled oligonucleotide (capture probe) to a solid phase to facilitate its detection. Particularly useful labels include biotin (which can be detected by binding to labeled avidin or streptavidin) and enzymes such as horseradish peroxidase or alkaline phosphatase (by adding an enzyme substrate to produce a colored reactive organism). Can be detected).

  Primers for amplification for specific detection and identification of nucleic acids can be packaged in the form of a kit. In general, such kits include at least a pair of amplification primers. Along with primers for target-specific amplification, reagents for performing a nucleic acid amplification reaction, such as buffers, additional primers, nucleotide triphosphates, enzymes, etc. may also be included. Other optional components, such as oligonucleotides labeled with a label suitable for use as an assay probe, and / or reagents or means for detecting the label may also be included in the kit.

Examples of specific detection methods that can be used include a chemiluminescence method described in US Pat. No. 5,470,723 that uses a biotinylated capture probe and an enzyme complex detector probe to detect an amplification product. is there. After hybridizing these two assay probes to different sites in the target sequence assay region (between the binding sites of the two amplification primers), the capture probe was used to bind the complex to a streptavidin-coated trace amount. Capture on titration plate, generate chemiluminescent signal and read with luminometer.
As another alternative for detecting amplification products, signal primers as described in European Patent EP 0 678 582 may be included in the SDA reaction. In yet another alternative for detecting the amplification product, the signal primer may comprise a sequence that does not hybridize to the target sequence, ie an adapter sequence.

  The reporter probe bound to the label can hybridize to the complement of the adapter sequence. In both embodiments of the signal primer, it is possible to generate a secondary amplification product in the SDA in a target amplification dependent manner, which can be detected as an indicator of target amplification.

  If the target binding sequence of the present invention is modified for amplification methods other than SDA, routine methods for preparing primers for amplification (eg, chemical synthesis) and well-known necessary for primers of selected amplification reactions Use the structural requirements of Thus, the target binding sequences of the present invention are readily modified for Legionella specific target amplification and detection in a variety of amplification reactions using only routine methods for production, screening and optimization. .

  SDA is the excision of primers, semi-modified restriction endonuclease recognition / cleavage site nicking, displacement of single-stranded extension products, annealing of primers to extension products (or original target sequence) and subsequent primer Elongation is an isothermal process that occurs simultaneously in the reaction mixture. This is in contrast to PCR where the reaction steps are performed in a discontinuous phase or cycle as a result of the temperature cycling characteristics of the reaction. SDA has the following features: 1) restriction endonuclease can cleave the unmodified strand of its hemiphosphorothioate-type double strand recognition / cleavage site, and 2) a specific polymerase initiates replication at a nick and downstream non-template. Based on the ability to replace the chain. In order to anneal the primers, the double-stranded target sequence is first denatured by first incubating at an elevated temperature (about 95 ° C.) followed by amplification and replacement of the newly synthesized strand at a constant temperature. The creation of each new copy of the target sequence consists of 1) binding the amplification primer to the original target sequence or a prepolymerized and displaced single-stranded extension product, and 2) α-thiodeoxynucleoside triphosphate. Extending the primer with a 5′-3 ′ exonuclease deficient polymerase that incorporates 3), 3) cleaving the semi-modified double stranded restriction site, 4) separating the restriction enzyme from the nick site, 5 ) 5 steps consisting of 5′-3 ′ exonuclease deficient polymerase extending from the 3 ′ end of the nick and replacing the newly synthesized strand downstream. Since extension from the nick regenerates another cleavable restriction site, nicking, polymerization and displacement occur simultaneously and sequentially at a constant temperature. Amplification is exponential when a pair of amplification primers are used and each hybridizes to one of the two strands of a double stranded target sequence. This is because the sense and antisense strands serve as templates for the opposite primer in subsequent circular amplification. When using one amplification primer, amplification is linear because only one strand serves as a template for primer extension.

  In order to prevent cross-contamination of one SDA reaction by another amplification product, dUTP can be incorporated into SDA amplified DNA instead of dTTP without inhibiting the amplification reaction. Subsequently, by treating with uracil DNA glycosylase (UDG), it is possible to specifically recognize and inactivate the uracil-modified nucleic acid. Thus, if dUTP is incorporated into the SDA amplified DNA in a prior reaction, the subsequent SDA reaction can be treated with UDG before amplifying the double stranded target, and the DNA from the previously amplified reaction The dU containing is made unamplifiable. The target dNA to be amplified in subsequent reactions does not contain dU and is not affected by UDG treatment. UDG can then be inhibited by treatment with UGI prior to target amplification. Alternatively, UDG may be heat-inactivated. In the case of tSDA, the high temperature of the reaction itself (≧ 50 ° C.) can be used simultaneously to inactivate UDG and amplify the target.

  SDA requires a polymerase that lacks 5′-3 ′ exonuclease activity and initiates polymerization at a single nick in a double stranded nucleic acid, replacing the strand downstream of the nick while Use as a template to generate a new complementary strand. The polymerase must be extended by adding nucleotides to the free 3'-OH. In order to optimize the SDA reaction, it is also desirable that the polymerase be very advanced in maximizing the length of the target sequence that can be amplified. Extremely advanced polymerases can polymerize new chains of considerable length before separating and terminating extension products. The displacement activity creates a target that can be used for further copy synthesis, and in an exponential amplification reaction, produces a single-stranded extension product to which the second amplification primer can hybridize. Therefore, it is indispensable for the amplification reaction. This is also very important because the nicking activity of the restriction enzyme is a nick that perpetuates the reaction and allows the subsequent circulation of target amplification to begin.

  tSDA is performed essentially the same as conventional SDA, replacing the appropriate thermostable polymerase and thermostable restriction endonuclease. The reaction temperature is adjusted to a high temperature suitable for the replaced enzyme, and the HincII restriction endonuclease recognition / cleavage site is replaced with a restriction endonuclease recognition / cleavage site appropriate for the selected thermostable endonuclease. Also, if the enzyme is sufficiently stable at the denaturation temperature, the specialist will include the enzyme in the reaction mixture prior to the first denaturation step. Preferred restriction endonucleases for use in tSDA are BsrI, BstNI, BsmAI, BslI and BsoBI (New England BioLabs), and BstOI (Promega). Preferred thermophilic polymerases are Bca (Panvera) and Bst (New England BioLabs).

  Homogeneous real-time fluorescent tSDA is an improved version of tSDA. This results in low fluorescence quenching in a target dependent manner using detector oligonucleotides. This detector oligonucleotide comprises a donor / acceptor dye pair linked so that fluorescence quenching occurs in the absence of the target. In the presence of the target, unfolding and linearization of the intramolecular base-pairing secondary structure in the detector oligonucleotide increases the distance between the dyes and decreases fluorescence quenching. Base pairing secondary structure unfolding generally involves intermolecular base pairing between the secondary structure sequence and the complementary strand so that the secondary structure is at least partially disrupted. In the presence of a sufficiently long complementary strand, the secondary structure can be sufficiently linearized.

In a preferred embodiment, the restriction endonuclease recognition site (RERS) is 2 so that RERS becomes double stranded by intermolecular base pairing between the secondary structure and the complementary strand and can be cleaved by the restriction endonuclease. Exists between two pigments. By cleaving with a restriction endonuclease, the donor dye and the acceptor dye are separated on separate nucleic acid fragments, which further helps to reduce quenching.
In either embodiment, accompanying fluorescence parameter changes (eg, increased donor fluorescence intensity, decreased acceptor fluorescence intensity, or ratio of fluorescence before and after unfolding) are monitored as an indication that the target sequence is present. Since the change in donor fluorescence intensity is generally larger than the change in acceptor fluorescence intensity, it is preferable to monitor the change in donor fluorescence intensity. Other fluorescence parameters such as fluorescence lifetime may be monitored.
Oligonucleotide cleavage refers to breaking the phosphodiester bonds of both strands of the DNA double helix, or breaking the phosphodiester bonds of single stranded DNA. This is in contrast to nicking, which refers to breaking the phosphodiester bond of only one of the two strands in the DNA double helix.

  Detector oligonucleotides for allogeneic real-time fluorescent tSDA include single stranded 5 'or 3' sections (target binding sequences) that hybridize to the target sequence, as well as intramolecular base pairing secondary adjacent to the target binding sequence. It may be an oligonucleotide containing both of the structures. In a preferred embodiment, the detector oligonucleotide is a reporter probe comprising a single stranded 5 ′ section or 3 ′ section that does not hybridize to the surface telosequence. Rather, the single-stranded 5 ′ section or 3 ′ section hybridizes to the complement of the signal primer adapter sequence (adapter-complement binding sequence). A further feature of the reporter probe is that this hybridizing section is adjacent to an intramolecular base-pairing secondary structure.

The detector oligonucleotides of the present invention for allogeneic real-time fluorescent tSDA contain sequences that form an intramolecular base-pairing secondary conformation at the reaction conditions selected for primer extension or hybridization. In one embodiment, this secondary structure is placed close to the target binding sequence of the detector oligonucleotide such that at least a portion of the target binding sequence forms a single stranded 3 ′ tail or 5 ′ tail. May be.
In a preferred embodiment, the secondary structure is the adapter-complement of the reporter probe detector oligonucleotide so that at least a portion of the adapter-complement binding sequence forms a single stranded 3 'tail or 5' tail. Located near the binding sequence. As used herein, the terms “near a target binding sequence” or “near an adapter-complement binding sequence” refer to whether all or part of a target / adapter-complement binding sequence is a target / adapter-complement. It means that it remains single-stranded at the 5 ′ tail or 3 ′ tail that can be used for hybridization to the body. That is, the secondary structure does not include the entire target / adapter-complement binding sequence.

  The target / adapter-complement binding sequence may be partly involved in intramolecular base pairing in the secondary structure, and the first sequence involved in intramolecular base pairing in the secondary structure May be part or all of, but preferably does not reach within its complementary sequence. For example, if the secondary structure is a stem-loop structure (eg, “hairpin”) and the target / adapter-complement binding sequence of the detector oligonucleotide is present as a single-stranded 3 ′ tail, the target / Adapter-complement binding sequences may extend through all or part of the first arm of the stem and optionally all or part of the loop. However, it is preferred that the target / adapter-complement binding sequence does not reach within the second arm of the sequence involved in stem intramolecular base pairing. That is, it is desirable to avoid having both sequences involved in intramolecular base pairing in the secondary structure that can hybridize to the target / adapter-complement.

  Mismatches in the intramolecular base-pairing portion of the detector oligonucleotide secondary structure may reduce the magnitude of the change in fluorescence in the presence of the target, but can be tolerated if analytical sensitivity is not an issue. Mismatches in single-stranded tail target / adapter-complement binding sequences can be tolerated, but analytical sensitivity and / or specificity may be similarly reduced. However, it is a feature of the present invention that complete base pairing in both the secondary structure and the target / adapter-complement binding sequence is not compromised. Perfect matches in sequences involved in hybridization improve analytical specificity without negatively impacting reaction kinetics.

  When added to an amplification reaction, the detector oligonucleotide reporter probe of the present invention is converted to a double-stranded form by hybridization and extension. Strand displacement by polymerase also unwinds or linearizes the secondary structure and converts it to the double-stranded form by synthesis of the complementary strand. RERS, if present, is also double-stranded and can be cleaved by a restriction endonuclease. As the secondary structure is unwound or linearized by the strand displacement activity of the polymerase, the distance between the donor and acceptor dyes increases, resulting in a decrease in donor fluorescence quenching. Concomitant changes in fluorescence of either the donor or acceptor dyes can be monitored or detected as an indicator of target sequence amplification.

In general, when RERS is cleaved, the magnitude of the fluorescence change is further increased by generating two separate double-stranded secondary amplification product fragments, each with one of the two dyes. To do. These fragments diffuse freely in the reaction solution, further increasing the distance between the dyes of the donor / acceptor pair.
An increase in donor fluorescence intensity or a decrease in acceptor fluorescence intensity may be detected and / or monitored as an indication that target amplification has occurred or has occurred, but other fluorescence affected by the proximity of the donor / acceptor dye pair. Parameters may be monitored.
A change in donor or acceptor fluorescence intensity can also be detected as a change in the ratio of donor and / or acceptor fluorescence intensity. For example, the change in fluorescence intensity may be a) an increase in the ratio of donor fluorophore fluorescence after linearization or unfolding of secondary structure to fluorophore fluorescence in the detector oligonucleotide before linearization or unfolding, or b) It can be detected as a decrease in the ratio of acceptor dye fluorescence after linearization or unfolding of the secondary structure to acceptor dye fluorescence in the detector oligonucleotide before linearization or unfolding.

For PCR by using standard amplification DNA polymerases that lack PCR amplification primers and 5 '→ 3' exonuclease activity in PCR (eg Promega's Sequencing GradeTaq or New England BioLabs exo ^- Vent or exo ^- Deep Vent) The method may be modified. The signal primer hybridizes at least partially to the downstream target from the PCR amplification primer and is displaced and double-stranded after hybridization to the detector oligonucleotide reporter probe and subsequent extension.
Since PCR generally does not have modified deoxynucleotide triphosphates that can induce nicking rather than cleavage of RERS, RERS can be arbitrarily selected for detector oligonucleotides. Since thermocycling is a feature of amplification by PCR, restriction endonucleases are preferably added at low temperatures after the final period of primer annealing and extension for detection of amplification endpoints. However, thermophilic restriction endonucleases that remain active throughout the hot phase of the PCR reaction may also be present throughout amplification to provide a real-time assay. As in the case of the SDA system, the fluorescence quenching is reduced by the separation of the dye pair, and the change of the fluorescence parameter such as intensity serves as an indicator of target amplification.

  Changes in fluorescence due to unfolding or linearization of the detector oligonucleotide can be detected at selected endpoints of the reaction. However, since the linearized secondary structure occurs simultaneously with hybridization or primer extension, it is possible to monitor changes in fluorescence as the reaction takes place, ie, “real time”. This homogeneous real-time assay format can be used to provide semi-quantitative or quantitative information regarding the initial amount of target present. For example, the rate at which fluorescence intensity changes during an unfolding or linear reaction (as part of target amplification or in a non-amplified detection method) is an indication of the initial target level. As a result, when there are more initial copies of the target sequence, the donor fluorescence reaches the selected threshold more quickly (ie, it is ensured in a short time). The decrease in acceptor fluorescence intensity is likewise detected in a shorter time and is detected as the time required to reach the selected minimum value.

  Assays for the presence of target sequences selected according to the methods of the present invention can be performed in solution or in the solid phase. Real-time or endpoint homogeneous assays in which the detector oligonucleotide functions as a primer are generally performed in solution. Hybridization assays using the detector oligonucleotides of the invention can be performed in solution (eg, homogeneous real-time assays), but are particularly suitable for solid-phase assays for real-time detection of targets or endpoint detection. Yes. In solid phase assays, detector oligonucleotides can be immobilized on a solid phase (eg, beads, membranes or reaction vessels) by internal or end labeling using methods well known in the art. For example, a biotin-labeled detector oligonucleotide can be immobilized on an avidin-modified solid phase, where exposure to the target under appropriate hybridization conditions results in a change in fluorescence. Capturing the target in this manner facilitates the separation of the target from the sample and removes substances in the sample that may interfere with the detection of assay signals or other characteristics. An example of a solid phase system that can be used is an array format, such as those well known in the art.

Specific examples of the present invention will be described below.
A. Detection Results by Sequences 1 and 2 (1) Detection Sensitivity FIG. 1 is a diagram showing detection results of Legionella pneumophila when sequences 1 and 2 are used as primers.

This detection result was obtained as follows.
10, 10 ^double the bacterial solution which number of Legionella pneumophila contained in phosphate buffer solution (PBS) is known, 10 ³ fold, 10 ⁴ fold, diluted 10 ⁵ fold (stock solution 1mL extract Add 9 mL of phosphate buffer for dilution at each concentration).
Extract 2.5 μL from each dilution. This extract contains about 1400, 140, 14, 1.4, and 0.14 Legionella pneumophila N depending on the stage of dilution.
The target sequence is amplified by the PCR method using the sequences 1 and 2 as primers. At this time, the total amount of the solution used for PCR was 25 μL, and the PCR cycle was 40 cycles.
Furthermore, the amplified target sequence was detected using agarose gel electrophoresis.

In FIG. 1, the molecular weight marker (M) and the result of electrophoresis in each dilution are shown in comparison. “277 bp” is the size of the amplification product when sequences 1 and 2 are used as a primer pair.
As can be seen from FIG. 1, it can be seen that Legionella pneumophila can be detected from N = 14 in 2.5 μL.

(2) Detection in an actual sample FIG. 2 is a diagram illustrating a detection result of Legionella pneumophila in an actual sample.
This detection result was obtained as follows.
[Please enter the details of the filter in the underlined portion]
Centrifugal concentration using the water inside the filter installed in the circulation bath as a stock solution and adding a phosphate buffer (PBS) to obtain a 100-fold concentrated solution (sample water).
A target sequence is amplified by PCR using sequences 1 and 2 as primers for 2.5 μL of this concentrated solution. At this time, the total amount of the solution used for PCR was 25 μL, and the PCR cycle was 40 cycles.

Here, when bacteria in the phosphate buffer solution (sample water) were detected by other methods, the following results were obtained.
Legionella bacteria count: 9.9 × 10 ^4/100 mL
Number of general bacteria: 3.8 × 10 ⁵ / mL
Number of heterotrophic bacteria: 1.2 × 10 ⁶ / mL

  In addition to general bacteria, heterotrophic bacteria that can amplify and survive in low-temperature, low-temperature water environments are also found in the water inside the filter.

FIG. 2 includes a molecular weight marker (M), a solution containing only 10 ⁵ / mL Legionella bacteria (ST: reference water), sample water (Samp), and Pseudomonus aeruginosa. The results of electrophoresis in a solution (Pa: comparative water) are shown in comparison. In any of these solutions, the target sequence is amplified by the PCR method, and then the electrophoresis method is applied.
Pseudomonas aeruginosa is widely distributed in nature, including soil, water, and sewage, and is often present in the region where Legionella lives.

As shown in FIG. 2, there is a detection reaction from the reference water ST and the sample water Samp. a. Is not detected.
The result of FIG. 2 shows that by using the sequences 1 and 2 as a primer, Legionella pneumophila is amplified, and other common bacteria, heterotrophic bacteria, and Pseudomonas aeruginosa with similar gene sequences are not amplified. Indicates. That is, sequences 1 and 2 indicate that Legionella pneumophila can be specifically distinguished from other bacteria.

(3) Detection of Legionella genus FIG. 3 is a diagram showing a detection result of Legionella in general.
Here, a colony (colon colony) of a medium in which Legionella spp. Was cultured was used as a sample, amplified by a PCR method using sequences 1 and 2 as primers, and measured by electrophoresis.

In FIG. 3, the molecular weight marker (M) is compared with the result of electrophoresis in a solution containing Legionella spp.
Sequences 1 and 2 from FIG. 3 represent that not only Legionella pneumophila but also general Legionella spp. With many serogroups can be detected.
Here, PCR is carried out using a colony (bacteria colony) of the medium closer to the actual sample as compared with the purified pure fungal DNA.

B. FIG. 4 is a graph showing the results of quantitative detection of Legionella pneumophila using sequences 5 and 6 as primers.
Here, a phosphate buffer solution (PBS) containing 3, 30, 300, 3000 Legionella pneumophila N in 2.5 μL is used as a sample, and quantitatively by a quantitative PCR method using a Roche light cycler. Detected. The horizontal axis of this graph is the number of PCR cycles, and the vertical axis is the intensity of fluorescence. When the number N is 0.3, Legionella spp. Is not amplified, but when the number N is 3 or more, Legionella pneumophila is amplified. That is, the intensity of fluorescence (corresponding to the concentration of the target sequence) increases with the number of cycles.
As can be seen from the above, the number N per 2.5 μL is 3 and it can be seen that Legionella New can also detect the filler quantitatively.

  FIG. 5 is a standard curve prepared from the results of detection of Legionella pneumophila by real-time quantitative PCR using a light cycler manufactured by Roche, using the sequences 5 and 6 of FIG. 4 as primers. The vertical axis represents the cycle number, and the horizontal axis represents the logarithmic value. By using this standard curve, it is technically possible to perform quantitative PCR on a sample of unknown concentration.

FIG. 6 is an electrophoresis photograph showing the results showing the detection limit of Legionella pneumophila using sequences 5 and 6 as primers.
Here, a phosphate buffer solution (PBS) containing 3, 30, 300, 3000 Legionella pneumophila N in 2.5 μL was used as a sample and detected by PCR. In this photograph, the product amplified by the PCR method is visualized by an agarose gel electrophoresis method. As can be seen from this figure, it is understood that Legionella pneumophila is detected when the number N is 3 or more.
The embodiments of the present invention are not limited to the above-described embodiments, and can be expanded and modified. The expanded and modified embodiments are also included in the technical scope of the present invention.

It is a figure showing the result of having detected Legionella pneumophila using arrangement | sequence 1 and 2 as a primer. It is a figure showing the result of having detected Legionella pneumophila in the actual sample using arrangement | sequence 1 and 2 as a primer. It is a figure showing the result of having detected Legionella spp. It is a graph showing the result of having detected Legionella pneumophila by real-time quantitative PCR by the light cycler made from Roche, using arrangement | sequence 5 and 6 as a primer. It is a standard curve created from the results of detection of Legionella pneumophila by real-time quantitative PCR using a light cycler manufactured by Roche using sequences 5 and 6 as primers. It is the photograph which amplified Legionella pneumophila by PCR method using arrangement | sequences 5 and 6 as a primer, and showed the detection limit of Legionella pneumophila by agarose electrophoresis.

Claims (6)

  A primer for amplification of a target nucleic acid of Legionella, comprising a base sequence selected from 5'-ccttacatcctcctcggctc-3 '(SEQ ID NO: 1) and 5'-cgctcgtttccagctcccc-3-3 (SEQ ID NO: 2). A nucleic acid containing the target base sequence includes a first primer containing the base sequence of 5'-ccttacatcctcctcggctc-3 '(SEQ ID NO: 1) and a second primer containing the base sequence of 5'-cgctcgtttccagctcccc-3-3 (SEQ ID NO: 2) And hybridizing with a primer of:
Extending the hybridized first and second primers on the nucleic acid;
A method for amplifying a target base sequence of Legionella spp.   The method for amplifying a target base sequence of Legionella spp. According to claim 2, wherein the amplification method is any one of a PCR method, an SDA method, a tSDA method, and a real-time fluorescence tSDA method.   5'-cccttgctgaattggagaac-3 '(SEQ ID NO: 3), 5'-gttgcattgacattgcctca-3' (SEQ ID NO: 4), 5'-tgctttaggacatcccagctat-3 '(SEQ ID NO: 3), 5'-gttgcattgacattgcctca-3' (SEQ ID NO: 4) ), 5'-tgctttaggacatcccagctat-3 '(SEQ ID NO: 5), 5'-cactgttcttgtccgctaattg-3' (SEQ ID NO: 6), 5'-tcgtcgtatggaacgagaagt-3 '(SEQ ID NO: 7), 5'-gagggtcaaatgtctgtccaa-3' (sequence) No. 8), a primer for amplification of a target nucleic acid of Legionella pneumophila, comprising a base sequence selected from Select nucleic acid containing target base sequence from 5'-cccttgctgaattggagaac-3 '(SEQ ID NO: 3), 5'-tgctttaggacatcccagctat-3' (SEQ ID NO: 5), 5'-tcgtcgtatggaacgagaagt-3 '(SEQ ID NO: 7) A first primer containing the base sequence, 5'-gttgcattgacattgcctca-3 '(SEQ ID NO: 4), 5'-cactgttcttgtccgctaattg-3' (SEQ ID NO: 6), 5'-gagggtcaaatgtctgtccccaa-3 '(SEQ ID NO: 8) Hybridizing with a second primer comprising a base sequence selected from any one of the following:
Extending the hybridized first and second primers on the nucleic acid;
A method for amplifying a target base sequence of Legionella pneumophila comprising: The method for amplifying a target base sequence of Legionella pneumophila according to claim 5, wherein the amplification method is any one of a PCR method, an SDA method, a tSDA method, and a real-time fluorescence tSDA method.

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