JP2010284090A - Primer set for detecting streptococcus group b and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method, by which the presence or absence of Streptococcus group B in a test sample can more quickly, simply and highly sensitively be confirmed than by conventional methods, and to provide a material for performing the method. <P>SOLUTION: The production of a set of primers for LAMP, capable of amplifying the specific region of a CAMP factor gene characteristic to Streptococcus group B has been succeeded. The primer set could have been used to highly sensitively and specifically amplify the region by LAMP method, consequently having more quickly, simply and highly sensitively detected the Streptococcus group B than by the conventional methods. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a primer set for detecting group B streptococci. The present invention further relates to a method for detecting group B streptococci using the primer set and a kit for carrying out the detection method.

Group B Streptococcus ( Streptococcus agalactiae ) is the leading bacterial species of neonatal sepsis and meningitis. In order to prevent vertical transmission between mother and child during childbirth, it is recommended in the US CDC and Europe that penicillin be administered prophylactically at the time of delivery to pregnant women carrying group B streptococci. In Japan, such preventive medication has recently been widely used. Group B Streptococcus is known not only as a causative bacterium for neonatal sepsis and meningitis but also as a causative bacterium for pneumonia in adults.

Up to now, β-lactam drugs such as penicillin have been effective for group B streptococci, and the first-line drugs have been penicillin and cephalosporins. However, in recent years, some strains of group B streptococci have acquired mutations in penicillin-binding proteins and are resistant to β-lactam drugs. For example, the present inventors have reported a group B streptococci that has acquired resistance to penicillin (Non-patent Document 1).

Therefore, in the treatment of the above diseases in newborns and adults, after confirming the presence or absence of group B streptococci in clinical samples, whether or not the group B streptococci are resistant to β-lactam drugs There is a need to inspect. Confirmation of the presence of group B streptococci so far can be done by using an identification kit for group B streptococci (for example, an identification kit for Streptococcus agalactiae "Streptex") from a test sample derived from a living body such as a vaginal swab. Had been implemented.

K. Kimura et al., Antimorobial Agents and Chemothery, (2008), 52 (8), pp. 2890-2897.

However, the method for detecting group B streptococci using the above-described kit for identifying group B streptococcus requires a two-step operation of culturing and identification, and therefore requires a period of at least two days. Two different procedures are required for identification. Furthermore, the above method requires a certain number of group B streptococci having proliferative activity. Since such a method is difficult to cope with an examination at the time of emergency delivery, it is desired to develop a rapid, simple and highly sensitive detection method for group B streptococcus in view of clinical applicability. .

Accordingly, an object of the present invention is to provide a method capable of confirming the presence or absence of group B Streptococcus in a test sample, and a material for carrying out the method, more quickly, conveniently and with a higher sensitivity than the conventional method. Is to provide.

As a result of intensive studies to solve the above problems, the present inventors succeeded in producing a set of LAMP primers that can amplify a specific region of a CAMP factor gene unique to group B streptococci. did. In addition, by using this primer set, the specific region can be specifically amplified with high sensitivity by the LAMP method, and as a result, group B streptococci can be detected more quickly, simply and with higher sensitivity than the previous method. did it. The present invention has been completed based on these findings.

Therefore, this invention consists of the following structures.
[1]
A primer set for detecting group B streptococci ( Streptococcus agalactiae ) capable of amplifying a target region in a CAMP factor gene by the LAMP method.
[2]
Arbitrary sequences F3c, F2c, F1c, R1, R2 and R3 arranged from the 3 ′ end to the 5 ′ end in the target region in the CAMP factor gene,
Inner primer forward (IPF) containing the same sequence as F1c on the 5 ′ side of sequences F2 and F2 complementary to F2c,
Outer primer forward (OPF) comprising the sequence F3 complementary to F3c,
Inner primer reverse (IPR) containing the same sequence as R2 and a sequence R1c complementary to R1 on the 5 ′ side of the sequence, and outer primer reverse (OPR) containing the same sequence as R3
The primer set according to [1], comprising:
[3]
The primer set according to [1] or [2], wherein at least one of IPF and IPR has a total free energy of 6 bases of 3 ′ end and 5 ′ end of −4 kcal / mol or less.
[4]
The primer set according to any one of [1] to [3], wherein at least one of OPF and OPR has a Tm value of 59 to 61 ° C.
[5]
[1] to [4], further comprising a loop primer including a sequence of a single-stranded loop part of a dumbbell structure in a target region in the CAMP factor gene and / or a sequence complementary to the sequence of the single-stranded loop part. The primer set according to any one of the above.
[6]
The primer set according to any one of [1] to [5], wherein at least one of the loop primers has a Tm value of 64 to 66 ° C.
[7]
The primer set according to any one of [1] to [6], wherein the target region in the CAMP factor gene is between positions 130 to 540 of the base sequence described in SEQ ID NO: 1 in the Sequence Listing.
[8]
IPF includes the following sequence (a) or (a ′); OPF includes the following sequence (b) or (b ′); IPR includes the following sequence (c) or (c ′) And the primer set according to any one of [1] to [7], wherein the OPR includes the following sequence (d) or (d ′):
(A) the sequence described in SEQ ID NO: 2 in the sequence listing (b) the sequence described in SEQ ID NO: 3 in the sequence listing (c) the sequence described in SEQ ID NO: 4 in the sequence listing (d) described in SEQ ID NO: 5 in the sequence listing The sequence (a ′) in the sequence set forth in SEQ ID NO: 2 in the sequence listing has at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases, In addition, in the sequence described in SEQ ID NO: 3 in the sequence (b ′) sequence listing, wherein the total free energy of 6 bases at each of the 3 ′ end and the 5 ′ end is −4 kcal / mol or less, 1 to a plurality of bases Described in SEQ ID NO: 4 in the sequence listing (c ′) having at least one mutation selected from the group consisting of deletion, substitution, inversion, addition, and insertion, and having a Tm value of 59-61 ° C. From the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases The sequence (d ′) described in SEQ ID NO: 5 has at least one kind of mutation and the total free energy of 6 bases at the 3 ′ end and 5 ′ end is −4 kcal / mol or less. A sequence having at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases and having a Tm value of 59 to 61 ° C. [9] ]
The loop primer consists of forward and reverse, the forward of the loop primer includes the following sequence (e) or (e ′), and the reverse of the loop primer includes the following sequence (f) or (f ′): The primer set according to any one of [5] to [8].
(E) the sequence set forth in SEQ ID NO: 6 in the sequence listing (e ′) from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases in the sequence set forth in SEQ ID NO: 6 in the sequence listing In the sequence described in SEQ ID NO: 7 in the sequence (f ′) sequence listing of the sequence (f) sequence listing having at least one selected mutation and having a Tm value of 64 to 66 ° C. A sequence having at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases and having a Tm value of 64-66 ° C. [10]
The primer set according to any one of [1] to [9], wherein at least one of IPF, OPF, IPR, OPR, and loop primer has a GC content of 40 to 65%.
[11]
A method for detecting group B Streptococcus, comprising detecting the presence or absence of a CAMP factor gene in a test sample by performing a LAMP method using the primer set according to any one of [1] to [10].
[12]
The detection method according to [11], wherein the presence of the amplified DNA is confirmed by the turbidity of the solution after the amplification operation.
[13]
The detection method according to [11] or [12], wherein the test sample is a human-derived sample.
[14]
The detection method according to [13], wherein the human-derived sample is vaginal swab, sputum, pus or exudate.
[15]
A kit for carrying out the detection method according to any one of [11] to [14], comprising the primer set according to any one of [1] to [10].

According to the method for detecting group B streptococci using the primer set of the present invention, group B streptococci can be detected more quickly, simply and with higher sensitivity than the method for detecting group B streptococci using a conventional identification kit. be able to. The primer set of the present invention is used for the LAMP method. Since the LAMP method does not require a special device as required by PCR, the primer set of the present invention is used for general detection of group B streptococci in normal facilities, for example, clinical facilities such as hospitals. Can be used for

The primer set of the present invention detects only group B streptococci, does not detect group A streptococci, pneumococci, enterococci and the like belonging to the same gram-positive cocci, and has high specificity for group B streptococci. Show. Furthermore, the detection method using the primer set of the present invention can be expected as a highly sensitive test because it can theoretically detect about 4 group B streptococci per 1 μl.

It is a graph of the results of examining the detection sensitivity of the LAMP method using one embodiment of the primer set of the present invention.

The present invention will be described in detail.
[Primer set]
The primer set of the present invention is used to detect group B streptococci ( Streptococcus agalactiae ) that can amplify the target region in the CAMP factor gene by the LAMP method. The primer set of the present invention is designed using, for example, LAMP method primer design support software (PrimerExplorer Ver.4, Eiken Chemical), and a primer set that can be a candidate is synthesized from the designed primer set, For each primer set, the primer set of the present invention can be prepared based on detection sensitivity and specificity by trying the LAMP method using DNA purified from group B streptococci as a template.

The CAMP factor gene (Christie-Atkins-Munch-Peterson factor gene) is possessed by most group B streptococci and is specific to group B streptococci. This is reported by Menard et al. (Ke D, Menard C, Picard FJ, Boissinot M, Ouellette M, Roy PH, Bergeron MG., Clin Chem. 2000 Mar; 46 (3): 324-31). . As a result of intensive studies, the present inventors made this CAMP factor gene a detection target gene, and further used the LAMP method, for example, without using a special device such as a PCR device, for example, bacteria in hospitals. We succeeded in detecting group B streptococci easily and quickly in laboratories. A specific example of the CAMP factor gene is the base sequence set forth in SEQ ID NO: 1 in the sequence listing. The CAMP factor gene consists of a base sequence having one to a plurality of base deletions, substitutions, additions and / or insertions in the base sequence shown in SEQ ID NO: 1 in the sequence listing, and the base set forth in SEQ ID NO: 1 It may be a gene encoding a protein having a function equivalent to a CAMP factor consisting of a sequence.

LAMP is an abbreviation for Loop-mediated Isometric Amplification, and is also referred to as a cyclic isothermal amplification reaction or a loop-mediated isothermal amplification method, and details can be referred to International Publication No. 00/28082.

In the LAMP method, a nucleotide as a template (for example, a target region in a CAMP factor gene containing the base sequence described in SEQ ID NO: 1 in the sequence listing) is annealed at its 3 ′ end to serve as a starting point for complementary strand synthesis. This is a nucleic acid amplification method that enables isothermal complementary strand synthesis reaction by combining a primer that anneals with the loop formed at this time.

In the LAMP method, since the 3 ′ end of the primer always anneals to the region derived from the sample, the check mechanism based on complementary binding of the base sequence functions repeatedly, resulting in high sensitivity and specificity. High nucleic acid amplification reaction is enabled.

One of the specific embodiments of the primer set of the present invention is the arbitrary sequences F3c, F2c, F1c, R1, R2 and R3 arranged from the 3 ′ end to the 5 ′ end in the target region in the CAMP factor gene.
Inner primer forward (IPF) containing the same sequence as F1c on the 5 ′ side of sequences F2 and F2 complementary to F2c,
Outer primer forward (OPF) comprising the sequence F3 complementary to F3c,
Inner primer reverse (IPR) containing the same sequence as R2 and a sequence R1c complementary to R1 on the 5 ′ side of the sequence, and outer primer reverse (OPR) containing the same sequence as R3
including.

Recognize at least 4 base sequences of arbitrary 6 regions included in the target region in the CAMP factor gene, that is, the region of F3c, F2c, F1c from the 3 ′ end side and the region of R3, R2, R1 from the 5 ′ end side These types of primers are referred to as inner primer forward (IPF) and inner primer reverse (IPR), and outer primer forward (OPF) and outer primer reverse (OPR), respectively. The complementary base sequences of F1c, F2c, and F3c are called F1, F2, and F3, and the complementary base sequences of R1, R2, and R3 are called R1c, R2c, and R3c, respectively.

An inner primer is an arbitrary nucleic acid synthesis reaction product that recognizes a “specific nucleotide sequence region” on a target region and has a base sequence that gives a synthesis starting point at the 3 ′ end and at the same time starts from this primer. An oligonucleotide having a base sequence complementary to the region at the 5 ′ end. Here, a primer including “a base sequence selected from F2” and “a base sequence selected from F1c” is an inner primer forward (hereinafter referred to as IPF), and “a base sequence selected from R2” and “R1c are selected. A primer containing “a base sequence” is called an inner primer reverse (hereinafter referred to as IPR). The inner primer is between “a base sequence selected from F2” and “a base sequence selected from F1c” or “a base sequence selected from R2” and “a base sequence selected from R1c”. You may have arbitrary base sequences of 0-50 bases.

The outer primer is an oligonucleotide having a base sequence that recognizes “a specific nucleotide sequence region existing on the 3 ′ end side of“ a specific nucleotide sequence region ”” on the target region and provides a starting point for synthesis. Here, a primer including “a base sequence selected from F3” is referred to as an outer primer forward (hereinafter OPF), and a primer including “a base sequence selected from R3” is referred to as an outer primer reverse (hereinafter referred to as OPR).

Here, forward in each primer is a primer display that binds complementarily to the sense strand of the target region and provides a starting point for synthesis, while reverse is complementary to the antisense strand of the target region, Primer display that provides a starting point for synthesis. Here, the length of the oligonucleotide used as a primer is 10 bases or more, preferably 15 bases or more, and may be either chemically synthesized or natural. Each primer may be a single oligonucleotide, It may be a mixture of oligonucleotides.

By the LAMP method using the primer set of the present invention, a target region in the CAMP factor gene and a region complementary to the target region can be amplified.

In the primer set of the present invention, IPF, OPF, IPR and OPR are not particularly limited as long as they are primers containing the above sequences. Among these, preferable specific examples of IPF and IPR are such that the total free energy of 6 bases at the 3 'end and the 5' end is -4 kcal / mol or less.

Calculation of the free energy of 6 bases at the 3 ′ end and 5 ′ end of each primer can be calculated using the Nearest-Neigbor method. For example, the free energy of 6 bases at the 3 ′ end and 5 ′ end of IPF and IPR can be measured by Eiken Chemical HP (URL:
https://primerexplorer.jp/lamp4.0.0/index.html), and using the PrimerExplorer provided on the above-mentioned HP, specifically PrimerExplorer V4, the 5 ′ end of F1c and R1c It can be calculated as the free energy of each 6 bases at the 3 ′ end of F2 and R2. In addition, if PrimerExplorer V4 is used, by entering and operating information such as the base sequence described in SEQ ID NO: 1 in the sequence listing, the total free energy of 6 bases at each of the 3 ′ end and the 5 ′ end is − A series of primers that are less than 4 kcal / mol can be obtained. Among these, preferred IPF and IPR can be selected.

Preferable specific examples of OPF and OPR have a melting temperature value, that is, a Tm value of 59 to 61 ° C. The Tm value can be calculated, for example, using the Nearest-Neigbor method with an oligo concentration of 0.1 μM, a sodium ion concentration of 50 mM, and a magnesium ion concentration of 4 mM, and the above-described PrimerExplorer V4 can also be used. The Tm value is preferably 64 to 66 ° C. for F1c and R1c, and 59 to 61 ° C. for F2, R2, F3 and R3. Furthermore, the GC content of each primer of IPF, OPF, IPR and OPR is more preferably about 40 to 65%.

In the LAMP nucleic acid amplification, in addition to the inner primer and the outer primer, another primer, that is, a loop primer can be used. The loop primer (Loop Primer) is a primer having a base sequence complementary to the base sequence of the single-stranded portion of the loop structure having a dumbbell structure. When this primer is used, the starting point of nucleic acid synthesis is increased, and the reaction time can be shortened and the detection sensitivity can be increased (for details, see WO02 / 24902).

The base sequence of the loop primer is not particularly limited as long as it is complementary to the base sequence of the single-stranded part of the loop structure described above, and is preferably selected from the base sequence of the target region or its complementary strand. More preferably, it is on the 5 ′ end side of the dumbbell structure. The loop primer is more preferably composed of a forward having a sequence complementary to the base sequence of the single-stranded part of the loop structure and a reverse having a base sequence of the single-stranded part of the loop structure.

The loop primer preferably has a Tm of 64 to 66 ° C. The total free energy of the 6 bases at the 3 'end of the loop primer is more preferably -4 kcal / mol or less, but this condition does not necessarily have to be satisfied. The GC content of the loop primer is more preferably about 40 to 65%.

The target region in the CAMP factor gene is not particularly limited as long as it is a region where the above IPF, IPR, OPF and OPR, and a loop primer can be designed. For example, the 130th position of the nucleotide sequence described in SEQ ID NO: 1 in the sequence listing To 540th position, 200th to 540th position is more preferable, 250th to 540th position is more preferable, 300th to 540th position is still more preferable, and 320th to 530th position is particularly preferable.

Specific examples of IPF, IPR, OPF and OPR in the case where the target region in the CAMP factor gene is positions 320 to 530 of the base sequence described in SEQ ID NO: 1 of the sequence listing are shown in SEQ ID NO: 2, 3 of the sequence listing, respectively. The sequences described in 4 and 5 can be mentioned, but the IPF, IPR, OPF and OPR included in the primer set of the present invention are not limited to these. In addition, as a specific example of IPF and IPR, at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases in the sequences of SEQ ID NOs: 2 and 4 in the sequence listing And the total free energy of 6 bases at the 3 ′ end and the 5 ′ end is −4 kcal / mol or less. Furthermore, as a specific example of OPF and OPR, at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases in the sequences of SEQ ID NOs: 3 and 5 in the sequence listing And a sequence having a Tm value of 59 to 61 ° C.

Specific examples of the loop primer when the target region in the CAMP factor gene is from position 320 to position 530 of the base sequence described in SEQ ID NO: 1 of the Sequence Listing are the sequences described in SEQ ID NO: 6 and 7 of the Sequence Listing, and Examples of these sequences include sequences having at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases and having a Tm value of 64-66 ° C. However, the primer set included in the primer set of the present invention is not limited thereto.

In the present specification, the range of “1 to plural” in “at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases” is the kind of primer As long as it is a sequence in which the total free energy of 6 bases at the 3 ′ end and 5 ′ end is −4 kcal / mol or less, or a Tm value is 59 to 61 ° C. or 64 to 66 ° C. For example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, more preferably 1 to 5, still more preferably 1 to 4, particularly preferably 1 Means about three. Also, “base deletion” means the loss or disappearance of a base in the sequence, “base replacement” means that the base in the sequence is replaced with another base, “base inversion” Indicates that the positions of two or more adjacent bases are reversed, “adding a base” means adding a base, and “inserting a base” means that another base is inserted between the bases in the sequence. It means that it is inserted.

Each primer included in the primer set of the present invention includes a CAMP factor gene, in particular, based on the information of the CAMP factor gene consisting of the base sequence described in SEQ ID NO: 1 in the sequence listing, as long as the above-described sequence is included. It can be made by any method commonly known to those skilled in the art, such as engineering techniques or mutagenesis. For example, it can be synthesized by a commercially available DNA synthesizer.

[Detection method]
According to another aspect of the present invention, there is provided a method for detecting group B streptococcus comprising detecting the presence or absence of a CAMP factor gene in a test sample by performing a LAMP method using the primer set of the present invention. The One embodiment of the detection method of the present invention includes, for example, subjecting the primer set of the present invention and a test sample or a solution containing DNA obtained from the test sample to a nucleic acid amplification operation by the LAMP method, and amplification in the solution after the nucleic acid amplification operation Confirming the presence of the purified DNA. Furthermore, another aspect of the detection method of the present invention includes, for example, preparing the primer set of the present invention, and subjecting the primer set and a test sample or a solution containing DNA obtained from the test sample to a nucleic acid amplification operation by the LAMP method. Providing and confirming the presence of the amplified DNA in the solution after the nucleic acid amplification operation.

Regarding the primer set of the present invention, the description of the above [Primer set] can be referred to.

In the method of the present invention, the presence or absence of the CAMP factor gene in the test sample is detected by performing the LAMP method using the primer set of the present invention. Specifically, first, the primer set of the present invention and a test sample or a solution containing DNA obtained from the test sample is subjected to a nucleic acid amplification operation by the LAMP method.

Examples of the test sample include a growth colony separated and cultured, a culture solution or a patient catheter wiped specimen, a vaginal swab, sputum, pus juice, exudate, etc., and these are preferably human-derived, and human-derived vaginal swabs. More preferably, sputum, pus or exudate. The test sample may be, for example, the test sample itself, or may be a test sample diluted with an appropriate buffer, physiological saline, water, or the like.

As a method for obtaining DNA from a test sample, a growth colony obtained by separating and culturing the test sample, a culture solution or a patient catheter wiped specimen, a vaginal swab, sputum, pus juice, exudate, etc. After decomposing the derived protein, a known DNA extraction / purification method such as a method using phenol and chloroform, a method using a commercially available extraction kit, or the like can be used. In addition, an unpurified sample processing solution can be used for rapid detection.

Specifically, examples of the method for obtaining DNA from the separately cultured growth colonies include a method of treating the growth colonies with a DNA extraction reagent such as GeneReleaser (Funakoshi) according to the instructions. As a method for obtaining DNA from a liquid medium inoculated with a growing colony, for example, a method shown in Examples described later can be used.

The nucleic acid amplification operation by the LAMP method can be performed, for example, as follows. A reagent (for example, Bst DNA polymerase, reaction buffer, dNTPs, primer set, distilled water) is mixed, dispensed into a reaction tube, and then a test sample, DNA obtained from the test sample, or a solution containing DNA obtained from the test sample. Add. As the reagent, for example, a loopamp DNA amplification reagent kit manufactured by Eiken Chemical is preferably used. Then, it is incubated at a temperature suitable for enzyme activity, for example, 60 to 70 ° C, preferably 65 ° C. The incubation time can be set to a time sufficient for completion of DNA amplification by the LAMP method, for example, 30 to 120 minutes, preferably 60 to 90 minutes.

The presence of the amplified DNA is confirmed in the solution after the nucleic acid amplification operation. When group B streptococci are contained in the test sample, DNA corresponding to the CAMP factor gene is amplified by the nucleic acid amplification operation. Conversely, if the test sample does not contain group B streptococci, there is no DNA that can be amplified by the nucleic acid amplification operation.

The presence or absence of DNA amplified in the solution after the nucleic acid amplification operation, that is, the CAMP factor gene can be confirmed without limitation by a technique known so far. For example, measuring the turbidity of the solution or fluorescence In addition to confirmation by visual detection or the like, a method using a labeled oligonucleotide or a fluorescent intercalator (Japanese Patent Application Laid-Open No. 2001-242169) that specifically recognizes an amplified base sequence, a reaction after completion of the reaction The solution can be confirmed by a method of subjecting the solution to agarose gel electrophoresis as it is. In agarose gel electrophoresis, a large number of bands with different base lengths are detected in a ladder (ladder) form in the amplified DNA.

In the LAMP method, a large amount of substrate is consumed by DNA synthesis, and pyrophosphate as a by-product reacts with the coexisting magnesium to become magnesium pyrophosphate, and the reaction solution becomes cloudy enough to be confirmed with the naked eye. Therefore, it is also possible to confirm the white turbidity by using a measuring device capable of optically observing an increase in turbidity after the reaction or during the reaction, for example, a change in absorbance at 650 nm using a normal spectrophotometer ( (Patent Document WO 01/83817 pamphlet). The fluorescent visual detection can be performed by adding a fluorescent visual reagent for reaction, and visually observing the fluorescent color of the reaction solution by applying a UV lamp.

From the viewpoint of simplicity and versatility, the presence of DNA amplified in the solution after the nucleic acid amplification operation is preferably confirmed by turbidity measurement. The turbidity of the amplified DNA can be measured as follows, for example. The turbidity of the final amplification product is measured over time with a real-time turbidity measuring device or with a turbidity measuring device. The wavelength for detecting turbidity is preferably 650 nm.

Specifically, using the real-time turbidity measuring device LA-320C manufactured by Eiken Chemical, it is possible to determine that amplified DNA exists when the turbidity is 0.1 or more.

According to another aspect of the present invention, a kit for carrying out the detection method of the present invention comprising the primer set of the present invention is also included. The kit of the present invention is not particularly limited as long as it includes the primer set of the present invention. For example, the following can be included.
1. Template-dependent nucleic acid synthase with strand displacement activity
2. Reaction buffer
3.dNTPs
4.Primer set (primer of the present invention)
5. Control DNA (positive control)
6. Reaction tube
7. Manual

As described above, in the method of the present invention, the presence or absence of DNA by-products can be determined by turbidity measurement or visual fluorescence detection. In the case of detecting with fluorescent visual observation, a fluorescent visual inspection reagent can be included in the kit. When using a turbidity measuring device, the fluorescent visual detection reagent is not included in the kit.

The enzyme included in the kit of the present invention is not particularly limited as long as it is a template-dependent nucleic acid synthase having strand displacement activity. Examples of such an enzyme include Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase I, and preferably Bst DNA polymerase (large fragment).

The reaction buffer is not particularly limited as long as it mixes reagents suitable for the reaction of the LAMP method. For example, a buffer solution or salts that give suitable conditions for the enzyme reaction, a protective agent that stabilizes the enzyme or the template is used. A mixture can be used. The kit of the present invention can contain reagents necessary for confirmation of amplified DNA as necessary.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

1 Design of primer set for LAMP to detect group B streptococci Based on the full-length sequence of CAMP factor gene (cfb) of group B streptococcus ATCC12403 (NEM316) strain (SEQ ID NO: 1 in the sequence listing) Designed as shown in Table 1 using Explorer V4. The primers listed in Table 1 were synthesized and purified by Sigma-Aldrich Japan.

2 Sensitivity of LAMP method
Using chromosomal DNA purified from Group B Streptococcus ATCC BAA-611 (2603V / R) (purified with Promega Wizard genomic DNA purification kit and not heat-treated) as a template, using a loopamp DNA amplification reagent kit from Eiken Chemical, a template The LAMP method was carried out at 65 ° C. for 90 minutes using the reaction mix shown in Table 3 while changing the amount of the solution as shown in Table 2. Amplification was confirmed by measuring turbidity. The results are shown in FIG. As shown in FIG. 1, an increase in turbidity was observed up to 10 fg, and detection was possible.

3 Specificity of LAMP method Chromosomal DNA was purified from the following various bacteria (purified with Promega Wizard genomic DNA purification kit, without heat treatment), and 1 ng was used as a template for the LAMP method under the same conditions as in 2 above. The presence or absence of amplification was determined by turbidity measurement. The results are shown in Table 4 with “+” indicating amplification and “−” indicating no amplification. As shown in Table 4, amplification was observed only in Streptococcus agalactiae , and amplification was not observed in other bacterial species.

The sequence listing is based on the following description.
SEQ ID NO: 1 CAMP factor gene:

Sequence number 2 IPF (NEM316FIP): CAG CTT AGT TAT CCC AAA TCC CAT A GAA GCA ATC ACT TTT TCA ACT CA
Sequence number 3 OPF (NEM316F3): AGA AGC CTT AAC AGA TGT GA
Sequence number 4 IPR (NEM316BIP): ATT CGC ATT TTA GAT CCA TTT GCT T GCC TTT ACA TCG TTA ACT TGA G
Sequence number 5 OPR (NEM316B3): CAG GAT AAG TTA AAA CCT TTT GTT C
Sequence number 6 LPF (LF2): TGC TTG ACT AAC CTT ATT TGC
Sequence number 7 LPR (LB): CAG TTG ATT CAA TTA AAG

Claims (15)

A primer set for detecting group B streptococci ( Streptococcus agalactiae ) capable of amplifying a target region in a CAMP factor gene by the LAMP method. Arbitrary sequences F3c, F2c, F1c, R1, R2 and R3 arranged from the 3 ′ end to the 5 ′ end in the target region in the CAMP factor gene,
Inner primer forward (IPF) containing the same sequence as F1c on the 5 ′ side of sequences F2 and F2 complementary to F2c,
Outer primer forward (OPF) comprising the sequence F3 complementary to F3c,
Inner primer reverse (IPR) containing the same sequence as R2 and a sequence R1c complementary to R1 on the 5 ′ side of the sequence, and outer primer reverse (OPR) containing the same sequence as R3
The primer set according to claim 1, comprising: The primer set according to claim 1 or 2, wherein at least one of IPF and IPR has a total free energy of 6 bases of 3 'end and 5' end of -4 kcal / mol or less. The primer set according to any one of claims 1 to 3, wherein at least one of OPF and OPR has a Tm value of 59 to 61 ° C. The loop primer containing a sequence complementary to the sequence of the single-stranded loop part of the dumbbell structure in the target region in the CAMP factor gene and / or the sequence of the single-stranded loop part. The primer set according to claim 1. The primer set according to any one of claims 1 to 5, wherein at least one of the loop primers has a Tm value of 64 to 66 ° C. The primer set according to any one of claims 1 to 6, wherein the target region in the CAMP factor gene is between positions 130 to 540 of the base sequence described in SEQ ID NO: 1 in the sequence listing. IPF includes the following sequence (a) or (a ′); OPF includes the following sequence (b) or (b ′); IPR includes the following sequence (c) or (c ′) The primer set according to any one of claims 1 to 7, wherein the OPR comprises the following sequence (d) or (d '):
(A) the sequence described in SEQ ID NO: 2 in the sequence listing (b) the sequence described in SEQ ID NO: 3 in the sequence listing (c) the sequence described in SEQ ID NO: 4 in the sequence listing (d) described in SEQ ID NO: 5 in the sequence listing The sequence (a ′) in the sequence set forth in SEQ ID NO: 2 in the sequence listing has at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases, In addition, in the sequence described in SEQ ID NO: 3 in the sequence (b ′) sequence listing, wherein the total free energy of 6 bases at each of the 3 ′ end and the 5 ′ end is −4 kcal / mol or less, 1 to a plurality of bases Described in SEQ ID NO: 4 in the sequence listing (c ′) having at least one mutation selected from the group consisting of deletion, substitution, inversion, addition, and insertion, and having a Tm value of 59-61 ° C. From the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases The sequence (d ′) described in SEQ ID NO: 5 has at least one kind of mutation and the total free energy of 6 bases at the 3 ′ end and 5 ′ end is −4 kcal / mol or less. A sequence having at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases and having a Tm value of 59 to 61 ° C. The loop primer consists of forward and reverse, the forward of the loop primer includes the following sequence (e) or (e ′), and the reverse of the loop primer includes the following sequence (f) or (f ′): The primer set according to any one of claims 5 to 8.
(E) the sequence set forth in SEQ ID NO: 6 in the sequence listing (e ′) from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases in the sequence set forth in SEQ ID NO: 6 in the sequence listing In the sequence described in SEQ ID NO: 7 in the sequence (f ′) sequence listing of the sequence (f) sequence listing having at least one selected mutation and having a Tm value of 64 to 66 ° C. A sequence having at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of bases and having a Tm value of 64-66 ° C The primer set according to any one of claims 1 to 9, wherein at least one of IPF, OPF, IPR, OPR and loop primer has a GC content of 40 to 65%. A method for detecting group B streptococcus, comprising detecting the presence or absence of a CAMP factor gene in a test sample by performing a LAMP method using the primer set according to any one of claims 1 to 10. The detection method according to claim 11, wherein the presence of the amplified DNA is confirmed by the turbidity of the solution after the amplification operation. The detection method according to claim 11 or 12, wherein the test sample is a human-derived sample. The detection method according to claim 13, wherein the human-derived sample is vaginal swab, sputum, pus or exudate. The kit for implementing the detection method in any one of Claims 11-14 containing the primer set of any one of Claims 1-10.