CN1863925B - Oligonucleotide to be used in determining sporogenous aerobic bacterium and determination method and determination kit for sporogenous aerobic bacerium using the same - Google Patents
Oligonucleotide to be used in determining sporogenous aerobic bacterium and determination method and determination kit for sporogenous aerobic bacerium using the same Download PDFInfo
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Abstract
An oligonucleotide which contains a base sequence specific to 16S ribosomal RNA gene of a sporogenous aerobic bacterium or a sequence complementary to the above base sequence is provided. The specific base sequence as described above is a base sequence represented by any of SEQ ID NOS : 1 to 6. This oligonucleotide is employed as a primer in a PCR reaction wherein a nucleic acid prepared from a subject bacterium to be determined is used as a specimen and the specimen serves as a template. Thus, it can be determined whether a subject bacterium is a sporogenous aerobic bacterium or not based on the amplification of a specific DNA fragment as an indication.
Description
Technical field
The present invention relates to and to be used for identifying that whether the bacterium of being identified as identifying object has been oligonucleotide and the application thereof that the P C R of gas property spore-producing bacterium uses; Specifically; Relate to above-mentioned oligonucleotide, through being that primer advances performing PCR with this oligonucleotide; Detected the specificity D N A fragment of gas property spore-producing bacterium, thereby identified and whether bacterium has been the method for gas property spore-producing bacterium and the identification kit that is used to carry out this authentication method.
Background technology
In the past; The method of bacterium is identified in classification, and the method for known with good grounds Physiology and biochemistry proterties is according to the method for quinone composition, thalline lipid acid composition, cell wall constituent etc.; Method according to the G+C content of D N A; According to the method for D N A homology, use the method for D N A probe, and carry out method that the base sequence of 16S rrna R N A gene analyzes etc.
Particularly in recent years, be that phylogenetic systematics is carried out on the basis through base sequence with 16S rrna R N A gene, resulting flora (bunch) carry out the Classification and Identification of bacterium as index, become general method.
, be distributed widely in the occurring in nature of soil, water, air etc. with the good gas property spore-producing bacterium headed by the bacillus (Bacillus).In the bacillus, human body is shown pathogenic bacterium, known anthrax bacillus (Bacillus anthracis) arranged, cause the bacillus cereus (Bacilluscereus) of food poisoning etc.And; About the phylogenetic systematics and the taxonomic hierarchies of the good gas property spore-producing bacterium that contains bacillus, for example, non-patent literature 1 [control very much by letter; " classification of good gas property spore-producing bacterium and evaluation "; Japan's refreshment drink (Soft Drinks) technical information calendar year 2001 No. 3,221-227] described in, each beyond the bacillus belongs to also known.These good gas property spore-producing bacteriums have the characteristic that forms gemma, but because of thermotolerance and the resistance of its gemma are strong, so sterilization is difficult, and from the manufacturing processed of food factories fully removal be very difficult.So, in most cases, can in process inspection of food factories etc., isolate gas property spore-producing bacterium.
Therefore, for example, in the food mfg workshop, from product and manufacturing processed, during separation of bacterial,, it is important to identify whether separated bacterium forms gemma for verifying pollution cause.So particularly in food service industry, expectation can identify rapidly whether bacterial isolated forms gemma from product or manufacturing processed.
Usually, whether separated bacterium has been gas property spore-producing bacterium, and authentication method commonly used is to form to cultivate on the substratum as the quilt of identifying object at gemma to identify bacterium (supplying the examination bacterium), confirms with microscope whether this thalline forms gemma again.In this method, identified bacterium, so need the time of growing very much in a few days to 1 week till identify because of forming on the substratum to cultivate at gemma.In this general authentication method, detect qualification time (incubation time) through shortening, though can shorten the time in a way, might can not detect the slow bacterial strain of rate of propagation this moment, so risk is bigger.
For this reason, in above-mentioned general authentication method,, just need to cultivate for a long time for having identified gas property spore-producing bacterium reliably.So above-mentioned authentication method we can say that the inspection before dispatching from the factory is unaccommodated to the food service industry of the rapid property of special needs.
Beyond the above-mentioned general authentication method; Evaluation has belonged to the technology of the bacterium of gas property spore-producing bacterium; For example, patent documentation 1 [Japan's publication communique " japanese kokai publication hei 9-94100 communique " (open day: on April 8th, 1997)] and non-patent literature 2 [Shida, O et al.; Proposal forTwo New Genera; Brevibacillus gen.nov.and Aneurinibacillus gen.nov., International Journal of Systematic Bacteriology, 939-946 (1996)] in disclosed method.In this technology; Belong to bacillus brevis and belong to (Brevibacillus), VitB1 bacillus (Aneurinibacillus), series bacillus and belong to the specificity base sequence D N A that exists in the 16S rrna R N A genes good gas property spore-producing bacterium, bacterium such as (Paenibacillus) having as primer; With from being identified that the karyomit(e) D N A that obtains the bacterium is a template; Use P C R method; With the amplification of specific D N A as index, thereby carry out being identified the evaluation of the genus level of bacterium.
In addition; The known sorting technique that utilizes the bacillus of 16S rrna R N A gene is like non-patent literature 3 (Goto, K et al.; Application of the partial 16S rDNA sequence asan index for rapid identification of species in the genus Bacillus; The Journal ofGeneral and Applied Microbiology, 46,1-8 (2000)) middle disclosed method.The about 275bp of 5 ' side that is known as the hypervariable region [hypervariable region (HV region)] of 16S rrna R N A gene is the specific specificity zone; So; In this technology,, can carry out the classification that the genus bacillus bacterium belongs to bacterium through carrying out this regional base sequence analysis.
Other; Patent documentation 2 [Japan's patent gazette " Japan special fair 7-89956 communique " (day for announcing: October 4 nineteen ninety-five, corresponding Japan publication communique " japanese kokai publication hei 3-49696 communique " (open day: on March 4th, 1991))] in put down in writing and derive from primer bacillus cereus, that the β-Nei Xiananmei gene test is used through use and detect this gene, thereby detect the method for bacillus cereus.
But, whether be the effective ways that form the good gas property spore-producing bacterium of gemma because of also not identifying rapidly and at present with covering, so, the food service industry that the expectation exploitation can be used for the special rapid property of the needs preceding technology of checking etc. of dispatching from the factory.
Specifically, at first, the method described in above-mentioned patent documentation 1 and the non-patent literature 1 is not the technology of inspection etc. before being used for food service industry and dispatching from the factory from the beginning, but focuses on the technology of Bacteria Identification.Therefore, this method, though since P C R method detect and can assure rapid property owing to be to belong to specificity method, so need to adopt the primer that adapts to each genus, trivial operations not only, but also can produce the problem that can not be suitable for for the bacterium that detects the genus that does not come out.
In addition, the method described in the above-mentioned non-patent literature 3, effective when identifying the kind of bacillus, inapplicable to the bacterium of other genus, and the method described in the above-mentioned patent documentation 2 is only effective to the detection of bacillus cereus.
Therefore, up to now, though the existing technology that more promptly detects the good gas property spore-producing bacterium of (or evaluation) a certain specified genus, rapidly and identified that the technology of gas property spore-producing bacterium is also not known with covering.
Summary of the invention
The present invention is point in view of the above problems; Its purpose is; Provide that can preferably be applicable to can be rapidly and identified the oligonucleotide of the method for gas property spore-producing bacterium with covering, and, detect specificity D N A fragment for aerobic bacteria through being that primer carries out amplified reaction with this oligonucleotide; Thereby identify and whether bacterium has been the method for gas property spore-producing bacterium, and the test kit that carries out this authentication method.
Present inventors etc. are for solving above-mentioned problem, are conceived to more common base sequence zone in the 16S rrna R N A gene of good gas property spore-producing bacterium of bacillus and nearly edge thereof, have carried out studying with keen determination.Its result advances performing PCR amplification through the oligonucleotide relevant with the present invention being used for primer, has found that for the good gas property spore-producing bacterium headed by the bacillus, the specificity DN A fragment of about 100bp length has obtained amplification, and the present invention is accomplished.Be that the present invention comprises following invention.(1) oligonucleotide, it contains for the specificity base sequence of the 16S rrna R N A gene of good gas property spore-producing bacterium or the complementary sequence of this base sequence, and above-mentioned specificity base sequence be the base sequence shown in the sequence number 1～6 any.
(2) oligonucleotide described in (1); Wherein, above-mentioned good gas property spore-producing bacterium belongs at least a kind the bacterium of from bacillus, series bacillus genus, VitB1 bacillus, native bacillus, ring grease acid bacillus, bacillus brevis genus, diplobacillus genus, twig spore Bacillaceae, happiness salt bacillus, thin walled bar Pseudomonas, need salt bacillus, bacillus marinus genus and Sporolactobacillus bacterium, selecting.
(3) authentication method of good gas property spore-producing bacterium; It is used as seized sample by the nucleic acid of being identified the bacterium preparation of identifying object; The amplification that is template with this seized sample, carries out resulting specific DNA fragments of PCR when reaction as index; Identify above-mentionedly identified whether bacterium has been gas property spore-producing bacterium, and be used for the primer of PCR reaction, use at least a kind that contains the oligonucleotide of the complementary sequence of the special base sequence of the 16S ribosomal RNA gene of good gas property spore-producing bacterium or this base sequence; Simultaneously, above-mentioned specificity base sequence be the base sequence shown in the sequence number 1～6 any.
(4) authentication method of the good gas property spore-producing bacterium described in (3); Wherein, In the above-mentioned primer; The oligonucleotide that contains the complementary sequence of the base sequence described in sequence number 1 or 2 or its base sequence uses as sense primer, and simultaneously, the oligonucleotide that contains the complementary sequence of the base sequence shown in any in the sequence number 3～6 or its base sequence uses as antisense primer.
(5) authentication method of the good gas property spore-producing bacterium described in (3) or (4), at least one side of wherein above-mentioned sense primer and antisense primer uses 2 kinds or greater than 2 kinds contain above-mentioned specificity base sequence or the oligonucleotide of the complementary sequence of this base sequence.
(6) authentication method of each said good gas property spore-producing bacterium in (3)～(5), wherein, the above-mentioned specific dna fragmentation that is increased has through gel electrophoresis separation, near the length that can 0.1kb, be detected.
(7) authentication method of each said good gas property spore-producing bacterium in (3)～(6), wherein above-mentioned good gas property spore-producing bacterium belong at least a kind the bacterium of from bacillus, series bacillus genus, VitB1 bacillus, native bacillus, ring grease acid bacillus, bacillus brevis genus, diplobacillus genus, twig spore Bacillaceae, happiness salt bacillus, thin walled bar Pseudomonas, need salt bacillus, bacillus marinus genus and Sporolactobacillus bacterium, selecting.
(8) identification kit, it is used for carrying out the authentication method of each described good gas property spore-producing bacterium of (3)～(7).
(9) identification kit of good gas property spore-producing bacterium; It is used as seized sample by the nucleic acid of being identified the bacterium preparation of identifying object; Being template with this seized sample, advancing the segmental amplification of performing PCR resulting specific D N A of when reaction as index; Whether be used to identify above-mentioned has been gas property spore-producing bacterium by the evaluation bacterium; And the primer that is used for P C R reaction contains the base sequence of the 16S rrna R N A gene specific of good gas property spore-producing bacterium or the complementary sequence of this base sequence, and contains at least a kind of some oligonucleotide that this specificity base sequence is the base sequence shown in the sequence number 1～6.
(10) identification kit of the good gas property spore-producing bacterium described in (9); Wherein, Also contain as gel electrophoresis with index affinity tag, that comprise multiple different lengths with the segmental D N of D N A A fragment mixture; And in this D N A fragment mixture, contain have 0.1kb length index with DN A fragment.
(11) identification kit of good gas property spore-producing bacterium described in (9) or (10) wherein, also contains by the reagent set when identifying that bacterium prepares nucleic acid.
(12) identification kit of each described good gas property spore-producing bacterium in (9)～(11) wherein, also contains the reagent that is useful on P C R reaction.
Can make much of other purpose, characteristic and advantage of the present invention through explanation as follows.In addition, also can very clear and definite benefit of the present invention through following explanation with reference to accompanying drawing.
Description of drawings
Fig. 1 is expressed as to obtain the oligonucleotide relevant with the present invention, the result of the base sequence of the 16S rrna R N A gene of more various bacteriums and be used as the synoptic diagram of the specific D N A segment area of index.
Fig. 2 is illustrated among the embodiment 1, uses the oligonucleotide relevant with the present invention as primer, is the synoptic diagram of the electrophoresis result of template when advancing performing PCR, amplified production with the genome D N A of good gas property spore-producing bacterium and other bacterium thereof.
Fig. 3 is illustrated among the embodiment 2; Using the oligonucleotide relevant with the present invention as primer, is the synoptic diagram of the electrophoresis result of template when advancing performing PCR, amplified production with the isolating genome D N A that does not identify bacterium 2 strain No.1 and No.2 from the food mfg environment.
Embodiment
According to Fig. 1 one of embodiment of the present invention is carried out following detailed description, but the invention is not restricted to following content.
In the mode of the present invention; By the oligonucleotide relevant with the present invention, use this oligonucleotide with the present invention relevant good gas property spore-producing bacterium authentication method (below; Sometimes only abbreviate authentication method as) and the identification kit of the good gas property spore-producing bacterium relevant with the present invention (below; Sometimes only abbreviate identification kit as) order, specify the present invention.
(1) oligonucleotide relevant with the present invention
The oligonucleotide relevant with the present invention; Contain the base sequence of the 16S rrna R NA gene specific of good gas property spore-producing bacterium or the complementary sequence of this base sequence; Specifically; Above-mentioned specificity base sequence is the some oligonucleotide as above-mentioned specificity base sequence that contain the base sequence shown in the handlebar sequence number 1～6.
Above-mentioned oligonucleotide does not all have special the qualification so long as contain the base sequence shown in each in the sequence number 1～6 or the complementary sequence of this base sequence.More particularly, for example, can be following 6 kinds of oligonucleotide (a)～(f).
(a) have base sequence as follows (sequence number 1), contain 24 bases.
Oligonucleotide (a): (5 ') d-ACGATGAGTGCTAAGTGTTGGGGG (3 ')
(b) have base sequence as follows (sequence number 2), contain 24 bases.
Oligonucleotide (b): (5 ') d-ACGATGAGTGCTAGGTGTTGGGGG (3 ')
(c) have base sequence as follows (sequence number 3), contain 22 bases.
Oligonucleotide (c): (5 ') d-TTGAGTTTCAGTCTTGCGAGCG (3 ')
(d) have base sequence as follows (sequence number 4), contain 22 bases.
Oligonucleotide (d): (5 ') d-TTGAGTTTCAGTCTTGCGACCG (3 ')
(e) have base sequence as follows (sequence number 5), contain 22 bases.
Oligonucleotide (e): (5 ') d-TTGAGTTTCACTCTTGCGAGCG (3 ')
(f) have base sequence as follows (sequence number 6), contain 22 bases.
Oligonucleotide (f): (5 ') d-TTGAGTTTCACTCTTGCGACCG (3 ')
The oligonucleotide relevant with the present invention is not limited to above-mentioned (a)～(f), also can be the material with complementary sequence beyond the base sequence shown in each, that have its base sequence in the sequence number 1～6.And, in the oligonucleotide relevant, also can contain additional base sequence beyond the complementary sequence of base sequence shown in each in the sequence number 1～6 and base sequence thereof with the present invention.Particularly in the oligonucleotide relevant with the present invention, also can be in sequence number 1～6 change in the complementary sequence of the base sequence shown in each and base sequence thereof, make the base in 1～3 scope be substituted, lack, insert and/or add.
The preparation method of the oligonucleotide relevant with the present invention does not have special qualification.Specifically, any base sequence or complementary sequence in the available known method chemosynthesis sequence number 1～6.
The oligonucleotide relevant with the present invention preferably uses as the primer of P C R, but its base sequence and length (size), as after state shown in the embodiment, use B L A S T DB to measure.Specifically, at first, from DB (B L A S T), extract the 16S rrna R N A gene order that Bacillus belongs to and closely the gemma of edge forms bacterium and non-gemma formation bacterium.Use the AlignX genetic analysis software of VectorNTI to compare afterwards, and select and the more common zone of good gas property spore-producing bacterium.
In these multiple zones, make it to satisfy each condition that is suitable as primer as follows as far as possible, and confirm its base sequence and length.
1. suitable length (size) is 20～25 bases.
(2.G+C guanine+cytosine(Cyt)) content preferred about 50%.
3. consider the complementarity between primer, form, avoid 3 ' terminal complementary primer for preventing dimer.
4. in primer, form secondary structure for avoiding, can not contain from the body complementary sequence.
5. select the approaching primer of Tm value of 2 kinds of primers as far as possible.
Its result through comparison as shown in Figure 1, has designed above-mentioned oligonucleotide (a)～(f).Zone shown in the conduct " D N A amplification region " is equivalent to be used to judge " the specific D N A fragment " that be taken as index when whether the quilt evaluation bacterium of identifying object has been gas property spore-producing bacterium in same figure.And; Left side at same width of cloth figure; Paenib validus above (upside of figure center line); The formal name used at school that expression Bacillus belongs to and closely the gemma of edge forms bacterium, the formal name used at school of the non-gemma formation of (downside of figure center line) expression below Staphy aureus bacterium, netting twine partly representes to have the base of homology.In addition, in the arrow with the figure below, the arrow of representing with F is corresponding to oligonucleotide (a) zone (b), and the arrow of representing with R is the zone corresponding to oligonucleotide (c)～(f).
(2) authentication method of the good gas property spore-producing bacterium relevant with the present invention
The authentication method of the good gas property spore-producing bacterium relevant with the present invention is through advancing the performing PCR reaction to above-mentioned oligonucleotide (a)～(f) of explanation in (1) as primer, make specific D N A fragment amplification, thereby is the method that index is identified with it.
Specifically; In the authentication method relevant with the present invention; Preferably comprise at least above-mentioned oligonucleotide (a)～(f) as primer, to advance " the P C R process " of performing PCR reaction; With identify in the P C R product that obtains by PC R process whether contain the specific D N A that is increased segmental " D N A fragment amplification is confirmed step ", but also can comprise other the step of " seized sample preparation steps " etc.
< good gas property spore-producing bacterium >
Be used as the good gas property spore-producing bacterium of identifying object in the present invention; Specifically; So long as genus bacillus (Bacillus) belongs to and the bacterium of nearly edge; There is not special the qualification; But specifically; For example, can give an example belong to (Brevibacillus) into bacillus, series bacillus belong to (Paenibacillus), VitB1 bacillus (Aneurinibacillus), native bacillus (Geobacillus), ring grease acid bacillus (Alicyclobacillus), bacillus brevis, diplobacillus belongs to (Amphibacillus), twig spore Bacillaceae (Virgibacillus), happiness salt bacillus (Halobacillus), thin walled bar Pseudomonas (Gracillibacillus), needs salt bacillus (Salibacillus), bacillus marinus to belong to the bacterium of (Marinibacillus), Sporolactobacillus (Sporolactobacillus) etc.Above-mentioned good gas property spore-producing bacterium, its good gas property spore-producing bacterium have the characteristic that forms gemma.
Above-mentioned each good gas property spore-producing bacterium, according to its molecular system etc., can classify becomes a plurality of inferior floras.Specifically, for example, can give an example is the various inferior flora of bacillus; The inferior flora of ring grease acid bacillus; The inferior flora that series bacillus belongs to; The inferior flora that bacillus brevis belongs to; The inferior flora of VitB1 bacillus; The inferior flora of soil bacillus; Twig spore Bacillaceae, happiness salt bacillus, thin walled bar Pseudomonas (Gracillibacillus), and need the inferior floras of salt bacillus (Salibacillus); The inferior flora of diplobacillus genus and Sporolactobacillus etc.The present invention is all applicable for the purposes of the good gas property spore-producing bacterium of identifying above-mentioned any inferior flora.
In above-mentioned each good gas property spore-producing bacterium; Each inferior flora that various, the series bacillus genus of bacillus, VitB1 bacillus, native bacillus, ring grease acid bacillus and bacillus brevis belong to; Be to close at food especially to fasten often separated bacterium; To the evaluation of these good gas property spore-producing bacteriums, can especially preferably use the present invention.
In the authentication method relevant,, use the oligonucleotide that contains the complementary sequence of the special base sequence of the 16S ribosomal RNA gene of good gas property spore-producing bacterium or this base sequence as primer like above-mentioned oligonucleotide (a)～(f) etc. with the present invention.Therefore, can identify whether by the evaluation bacterium be gas property spore-producing bacterium with covering.
So; The authentication method relevant with the present invention; In the past technological different of cause and specific indivedual genus can promptly be identified and whether bacterium is the bacterium that forms gemma, especially in food service industry; Can be used as rapid evaluation detected bacterium from product or manufacturing processed and whether form the inspection method of gemma, the preferred use.
< P C R step >
P C R step is to be seized sample with the nucleic acid of being identified bacterium preparation by the quilt of identifying object, and is the step that template is advanced the performing PCR reaction with this seized sample, at this moment, uses the oligonucleotide relevant with the present invention, and for example above-mentioned oligonucleotide is as primer.
At this, in the above-mentioned primer, the oligonucleotide that contains the complementary sequence of the base sequence shown in sequence number 1 or 2 or its base sequence can be used as sense primer and uses.Specifically, in the above-mentioned oligonucleotide (a)～(f), oligonucleotide (a) and/or (b) use as sense primer.In addition, the oligonucleotide that contains the complementary sequence of the base sequence shown in any in the sequence number 3～6 or its base sequence can be used as antisense primer and uses.Specifically, in the above-mentioned oligonucleotide (a)～(f), at least one during oligonucleotide (c), (d), (e) reach (f) can be used as antisense primer and uses.
And, in above-mentioned sense primer or antisense primer or its any primer, can only use a kind of oligonucleotide, also can use 2 kinds or greater than 2 kinds oligonucleotide.Specifically, as sense primer, can only use oligonucleotide (a) or (b), but also both use all.Equally,, can use any in the oligonucleotide (c)～(f), also can use in 4 kinds 2 kinds or greater than 2 kinds as antisense primer.
In P C R step; The condition that is used for reagent, device or the P C R reaction of P C R does not have special qualification; Reagent can preferably use the P C R test kit or the device of market sale, and conditions such as temperature of reaction, reaction times, cycle index can the suitable setting corresponding to the kind of P C R test kit or device.
< D N A fragment amplification is confirmed step >
So long as in above-mentioned P C R step, in the resulting P C R product, identify the D N A fragment amplification affirmation the step whether segmental step of specific D N A that is increased is arranged, all there be not special the qualification.In the authentication method relevant with the present invention, be index with the segmental amplification of above-mentioned specific D N A, can identify that being used as the quilt of identifying object identifies whether bacterium has been gas property spore-producing bacterium.
Confirm the method for the segmental amplification of above-mentioned specific D N A, do not have qualification especially, but the method for general preferred use gel electrophoresis.Through the method for gel electrophoresis, be the technology that in the experiment of using nucleic acid, generally is widely used, can be accurately and when separating nucleic acid such as D N A, R N A effectively, also can confirm its size (length) through the dyeing of gel.Do not have special restriction for device that carries out gel electrophoresis or deposition condition, can preferably use the electrophoresis apparatus of market sale etc., and carry out electrophoresis with the condition that is adapted to this device and get final product.
The segmental length of in D N A fragment amplification affirmation step, confirming of above-mentioned specific D N A also can clearly be known from " D N A amplification region " shown in Figure 1, is about 100bp.In other words, the above-mentioned specific D N A fragment that is increased is separated through gel electrophoresis, has near the length that 0.1kb, is detected.For example, in the instance as shown in Figure 1,101 bases have been increased.Because the kind of good gas property spore-producing bacterium is different, and considers disappearance or the interpolation that base takes place in this zone, can be increased so have 90～110bp scope D N A fragment interior, that be preferably 100bp～110bp range size at least.In other words, as long as in above-mentioned scope, can regard size as with 0.1kb.Therefore, confirm in the step, only need to confirm whether can detect D N A fragment and get final product with near the length that can 0.1kb, be detected through the gel electrophoresis separation at D N A fragment amplification.
< other steps >
In the authentication method relevant,, but also can comprise other steps, for example seized sample preparation steps as long as comprise above-mentioned P C R step and D N A fragment amplification affirmation step with the present invention.
Seized sample preparation steps at the leading portion of above-mentioned P C R step, is got final product by a certain amount of nucleic acid of being identified that bacterium utilizes known method to prepare karyomit(e) D N A etc.The preparation method of the nucleic acid through thalline does not have special qualification, because of can preferably using known method, so preferably utilize the D N A of market sale to prepare test kit etc.
Other also can comprise, identified that isolating from the product of inspection object or the mechanism the manufacturing processed etc. bacterium carries out the quilt evaluation bacterium culturing process of a certain amount of cultivation etc.Substratum or the culture condition of this moment do not have special qualification yet, can suit to select known substratum or culture condition.
(3) identification kit of the good gas property spore-producing bacterium relevant with the present invention
The identification kit of the good gas property spore-producing bacterium relevant with the present invention all can so long as carry out the identification kit of the authentication method of the above-mentioned good gas property spore-producing bacterium of explanation in (2).Specifically; As the primer that is used for P C R reaction; Can give an example into; At least be contain comprise to the 16S rrna R N A gene specific of good gas property spore-producing bacterium, the composition of the oligonucleotide of the complementary sequence of any base sequence or this base sequence in the sequence number 1～6, be preferably the composition that comprises the reagent class of carrying out above-mentioned each step etc.
For example; In the identification kit relevant with the present invention; The oligonucleotide that contains the complementary sequence of any base sequence in the above-mentioned sequence number 1～6 or this base sequence; Preferably comprise above-mentioned oligonucleotide (a) at least one side (b) as sense primer, as antisense primer preferably comprise oligonucleotide (c)～(f) at least any.Above-mentioned oligonucleotide as long as can use as the primer of P C R reaction, can be any state, for example, also can be the solution state that is dissolved in damping fluid.
The identification kit relevant with the present invention; Can comprise and be used to reagent class of implementing above-mentioned each process etc.; This kind reagent class can give an example into, be used for P C R reaction reagent set, be used to confirm the reagent set of D N A fragment amplification, by the reagent set when identifying that bacterium prepares nucleic acid, be used to cultivate the reagent set of being identified bacterium etc.These reagent set contain a kind at least for well.And reagent set described herein preferably contains the reagent that is useful on said process and implements, experiment with material or 1 property experiment apparatus etc. 2 kinds or greater than 2 kinds.
The reagent set that is used for P C R specifically, can be given an example to d N T P, Taq polysaccharase (polymerase) and is used for damping fluid (Taq Buffer) of Taq polysaccharase (polymerase) etc.Other as required, also can add the test of miniature centrifuge tube etc. and use material.
Be used to confirm the reagent set of D N A fragment amplification, when confirming amplification through gel electrophoresis, can give an example for be used to process electrophoresis with the agarose of gel, damping fluid, gel electrophoresis with affinity tag etc.Wherein, affinity tag is used in gel electrophoresis, though preferably contain the segmental D N of the D N A A fragment mixture as index of multiple different lengths, in this D N A fragment mixture, need contain the D N A fragment as index with 0.1kb length.As stated, as the specific D N A fragment of identification of indicator, because of having the size of about 100bp, thus very preferably gel electrophoresis with affinity tag in, the index that contain on electrophoresis, can clearly distinguish this big or small band is with D N A fragment.And, be used for the DN A segmental source of this gel electrophoresis with affinity tag, there is not qualification especially.
Other, the reagent set when identifying that by quilt bacterium prepares nucleic acid can be given an example to known D N A prepares test kit, is used to cultivate the reagent set of being identified bacterium, and can give an example is substratum or its raw material.
(4) purposes of the present invention
The oligonucleotide relevant with the present invention and use its authentication method and the purposes of identification kit not to have special qualification can be used for whole purposes of having identified that gas property spore-producing bacterium is required.Specifically, for example, because in the manufacturing process of microbial contamination and various Industrial products that quality is made a big impact, identify, can preferably use from whether being the fashionable of gas property spore-producing bacterium with isolating bacterium Industrial products or its manufacturing environment etc.
Become the above-mentioned Industrial products of being identified the bacterium source of identifying object, giving an example of representative is food, beverage, pharmaceuticals reagent Medicines and Health Product, disposable medical appliance etc., but do not have special the qualification.Above-mentioned food; More particularly; Can give an example is processing of farm products article, dietary supplement (nutrient supplement food etc.), the food with long preservation period (can, frozen product, high-temperature cooking food etc.) of bread, Japanese Western-style pastry (comprising the cold spot heart), non-staple foodstuff food, milk-product, cereal breakfast food, bean curd fried bean-curd, wheaten food class, packed meal class, condiment, wheat-flour or meat etc., but does not have special the qualification.Beverage, more particularly can give an example is various refreshment drinks, mineral water, cow's milk milky-drinks, pure mellow wine, beer, sparkling wine etc., but does not have special the qualification.Pharmaceuticals reagent Medicines and Health Product more particularly, for example can be whole etc. with reagent class, other inspection medicines of the pharmaceuticals towards various medical institutions, various nonprescription drug, clinical examination medicine, test, but does not have special qualification.Disposable medical appliance more particularly for example, can be syringe etc., but does not have special the qualification.
In the producing apparatus of Industrial products, swim in the air bacterium might as well, the surface attachment bacterium might as well, other bacterium might as well, all can be used as and identified bacterium.Swim the in the air separation of bacterium can be adopted the method for plate sedimentation, aerial bacterioplankton sampling thief method etc., but not have special the qualification.In the separation of surface attachment bacterium, can use the method for smearing sheet test method(s), stampagar method etc., but not have special the qualification.
And the present invention is not limited to above-described each formation; In the scope shown in the patent claimed range, can carry out all changes; Resulting example after the various technique means that have been disclosed also is contained in the technical scope of the present invention in the different example of appropriate combination.
Below, be that more specifically explain on the basis with the present invention with embodiment, Fig. 2 and Fig. 3, but the invention is not restricted to this.
(embodiment 1: the good gas property spore-producing bacterium of primer detects the mensuration of power)
Use to differentiate that bacillus (Bacillus genuss) reaches designed primer after the gene order of the 16S rrna R N A gene specific of the good gas property spore-producing bacterium of its nearly edge, advances performing PCR, and estimated whether to have detected gas property spore-producing bacterium and bacterium in addition thereof.
< seized sample prepares process >
Use belongs to (Paenibacillus genus), VitB1 bacillus (Aneurinibacillus genus), ring grease acid bacillus (Alicyclobacillus genus) with bacillus (Bacillus genus), series bacillus, reaches the more similar non-gemma formation bacterium of gene order of bacillus (Bacillus genus) bacterium.The employed bacterium of seized sample is as shown in table 1.Use the bacterial strain that the righttest culture condition of above-mentioned each bacterial strain is cultivated, use genomic dna purification kit (Edge Biosystems corporate system),, prepared genome D N A according to the interpolation scheme.
(table 1)
< primer design is with synthetic >
The good gas property spore-producing bacterium of extraction bacillus (Bacillus genus) edge nearly with it [for example from DB (B L A S T); Series bacillus belongs to (Paenibacillus genuss), VitB1 bacillus (Aneurinibacillus genuss), native bacillus (Geobacillus genuss), ring grease acid bacillus (Alicyclobacillus genus), bacillus brevis genus (Brevibacillus genus) etc.], reach the 16S rrna R N A gene order of non-gemma formation bacterium; Use the genetic analysis software of the AlignX of VectorNTI to compare, select with good gas property spore-producing bacterium and compare the common zone.Wherein, select the sequence of sequence number 1～6, chemosynthesis has the oligonucleotide of same sequence with it, and uses as primer.Below, each primer is called oligonucleotide respectively
(a)～(f)。
< P C R step D N A fragment amplification is confirmed step >
With above-mentioned genome D N A is template, uses above-mentioned oligonucleotide (a)～(f), has prepared to carry out P C R reaction behind the sample shown in the table 2.And P C R reaction uses the TGRAGIENT of Biometra corporate system to carry out, and P C R condition is as shown in table 3.After P C R reaction finishes, analyze, confirmed the amplified band of D N A and the size of amplified band thereof according to additional scheme in the Agilent2100 biological analyser (Agilent Technologies corporate system).
< result >
It is as shown in Figure 2 to carry out electrophoretic result through biological analyser.Represented respectively among Fig. 2 that swimming lane 1 is molecular weight marker; Swimming lane 2 is subtilis (Bacillus subtilis) results (IFO13719); Swimming lane 3 is results of Bacillus sphaericus (B.sphaericus) (NBRC 15095); Swimming lane 4 is bacillus cereus (B.cereus) results (IAM12605); Swimming lane 5 is Bacillus coagulans (B.coagulans) results (IAM12463); Swimming lane 6 is bacillus megaterium (B.megaterium) results (JCM2506); Swimming lane 7 is results of Paenibacillus validus (BUB75), and swimming lane 8 is results of P.illinoisensis (IFO 15379), and swimming lane 9 is results of Aneurinibacillus aneurinolyticus (IAM 1077); Swimming lane 10 is sour soil ring grease genus bacillus (Alicyclobacillus acidoterrestris) results (ATCC49025); Swimming lane 11 is acid heat alicyclic acid genus bacillus (A.acidocaldarius) results (JCM5260), and swimming lane 12 is streptococcus aureus (Staphylococcus aureus) results (ATCC9144), and swimming lane 13 is head staphylococcus (Staphylococcus capitis) results (ATCC27840); Swimming lane 14 is streptococcus-salivarius (Streptococcus salivarius) results (SAM0180), and swimming lane 15 is lactobacillus delbruckii (Lactobacillus delbrueckii) results (IAM1928).
Have only the good gas property spore-producing bacterium shown in the swimming lane 2～11 near 100bp, to detect amplified band among Fig. 2, and shown in the bacterium result's beyond the good gas property spore-producing bacterium the swimming lane 12～15, do not detect near the amplified band of 100bp.Therefore, have the DN A fragment of about 100bp size, to good gas property spore-producing bacterium tool specificity, we can say, D N A is segmental has or not as index with this, and whether can judge at an easy rate has been gas property spore-producing bacterium.
(embodiment 2: the isolating detection evaluation of not identifying bacterial strain from the food mfg environment)
For not identifying bacterial strain; D N A fragment through having or not 100bp judges whether it has been the result of gas property spore-producing bacterium as index; Compare with the qualification result of this bacterium, inquired into the appropriate property of the detection authentication method of the good gas property spore-producing bacterium relevant with the present invention.
< seized sample preparation steps >
Do not identify that isolating from the food mfg environment bacterium 2 strains (No.1 and No.2) place pancreas casein peptone soy agar substratum (B B L corporate system), in 35 ℃, aerobic conditions is cultivated down.1 bacterium colony of thalline on the picking substratum uses Genomic DNA Purification Kit (Edge Biosystems corporate system), extracts D N A according to the scheme of adding, and with it as seized sample (seized sample preparation steps).
< P C R step D N A fragment amplification is confirmed step >
With above-mentioned analytic liquid is template, uses aforesaid oligonucleotide (a)～(f), and the sample shown in the preparation table 2 has carried out P C R.And P C R uses the T GRAGIENT of Biometra corporate system to carry out, and P C R condition is as shown in table 3.P C R according to additional scheme in the Agilent2100 biological analyser (Agilent Technologies corporate system), has confirmed the amplified band of D N A and the size of amplified band thereof after finishing.
(table 2)
(table 3)
< result >
Electrophoresis result is as shown in Figure 3.Shown respectively among Fig. 3 that swimming lane 1 is molecular weight marker, swimming lane 2 is results that do not identify bacterial strain of No.1, and swimming lane 3 is results that do not identify bacterial strain of No.2.
Among the No.1, near 100bp, detect amplified band, this bacterium of decidable has been a gas property spore-producing bacterium.And when using microscopic examination, also confirmed the formation of gemma.In addition, use Api50CHB (Japanese Biomerieux corporate system), according to additional scheme, when carrying out the evaluation of this bacterium, identifying has been the Bacillus licheniformis (Bacillus licheniformis) of gas property spore-producing bacterium.Therefore be that the result that judges of index is consistent with amplified band with 100bp.
In addition, in No.2, do not detect amplified band near the 100bp, it has not been gas property spore-producing bacterium for a decidable.Check order when identifying for the part base sequence of the 16S rrna R N A gene of this bacterium, distinguished that it has not been a gas property spore-producing bacterium, but methyl Bacillaceae (Methylobacterium) bacterium.Therefore, No.2 and amplified band with 100bp are that the result that judges of index is consistent, near the amplified band that will have or not the 100bp can be described as index, identify it whether has been that the method for gas property spore-producing bacterium is appropriate to detect.
By above result, be primer, when carrying out the amplified reaction of PC R amplification etc., whether increased with the oligonucleotide relevant (a)～(f) by specificity D N A fragment (about 100bp) with the present invention, can identify easily whether by the evaluation bacterium be gas property spore-producing bacterium.That is to say that the authentication method relevant with the present invention has been the effective detection and the identification of means of gas property spore-producing bacterium.Simultaneously, also we can say,, the identification kit of gas property spore-producing bacterium can be provided like the above-mentioned oligonucleotide test kitization that will contain.
And; The practical implementation form or the embodiment that in embodiment, enumerate; In any case the also content of clear and definite technology contents of the present invention only; And be not interpreted as with not answering narrow sense and be only limited to its specific examples, in the scope of the claim of spirit of the present invention and the following stated, can carry out various changes.
Through being primer with the oligonucleotide relevant with the present invention; With from being identified that karyomit(e) DN A of obtaining the bacterium etc. is a template; Advance the amplified reaction of performing PCR amplification etc., the specificity D N A fragment that exists in the 16S rrna R N A gene of the good gas property spore-producing bacterium of can increase bacillus and nearly edge thereof.Thus,, whether increased, can promptly identify and identified whether bacterium is the good gas property spore-producing bacterium of bacillus and nearly edge thereof by this specificity D N A fragment (about 0.1kb) through above-mentioned amplified reaction.In addition, through the above-mentioned oligonucleotide test kitization that will contain, the identification kit of gas property spore-producing bacterium can be provided.
Therefore, according to the present invention, can be rapidly and think the detection of good gas property spore-producing bacterium of difficulty up to now with covering, and effective.
Up to now, whether be when forming the bacterium (good gas property spore-producing bacterium) of gemma for identifying, must form to cultivate on the substratum at gemma and identified bacterium, confirm with microscope whether this bacterium has formed gemma again.So, till identifying, need the very long time in a few days～1 week.And whether according to the present invention, can identify about half day has been gas property spore-producing bacterium, and whether make easy and promptly identify has been that gas property spore-producing bacterium becomes possibility.
So the present invention is capable of using in hygiene control, process management, the quality control of foodstuff manufacturing, catering industry, medicine trade etc.And, just in case when having detected microbial contamination, can whether be gas property spore-producing bacterium through this bacterium of rapid evaluation also, and can take measures rapidly to situation.
Claims (6)
1. the identification kit of good gas property spore-producing bacterium is characterized in that, contains as PCR with sense primer or the PCR following oligonucleotide with antisense primer:
(a) oligonucleotide, the base sequence of being represented by sequence number 1 constitutes;
(b) oligonucleotide, the base sequence of being represented by sequence number 2 constitutes;
(c) oligonucleotide, the base sequence of being represented by sequence number 3 constitutes;
(d) oligonucleotide, the base sequence of being represented by sequence number 4 constitutes;
(e) oligonucleotide, the base sequence of being represented by sequence number 5 constitutes; And
(f) oligonucleotide, the base sequence of being represented by sequence number 6 constitutes.
2. the application of the described test kit of claim 1 in having identified gas property spore-producing bacterium; Wherein, Above-mentioned good gas property spore-producing bacterium belongs to from bacillus (Bacillus); Series bacillus belongs to (Paenibacillus); VitB1 bacillus (Aneurinibacillus); Soil bacillus (Geobacillus); Ring grease acid bacillus (Alicyclobacillus); Bacillus brevis belongs to (Brevibacillus); Diplobacillus belongs to (Amphibacillus); Twig spore Bacillaceae (Virgibacillus); Happiness salt bacillus (Halobacillus); thin walled bar Pseudomonas (Gracillibacillus); need salt bacillus (Salibacillus); bacillus marinus belongs at least a kind the bacterium of selecting in (Marinibacillus) and Sporolactobacillus (Sporolactobacillus) bacterium
And this application is used for the purpose of the diagnosis of non-disease.
3. the test kit described in claim 1 is characterized in that, also contain and use, and comprise the dna fragmentation mixture of the different dna fragmentation that is used as index of multiple length with affinity tag as gel electrophoresis,
And in this dna fragmentation mixture, contain dna fragmentation as index with 0.1kb length.
4. the test kit described in claim 1 is characterized in that, also contains by the reagent set when identifying that bacterium prepares nucleic acid.
5. the test kit described in claim 1 is characterized in that, also contains the reagent set that is useful on the PCR reaction.
6. the authentication method of good gas property spore-producing bacterium; It is used as seized sample by nucleic acid of being identified the bacterium preparation of identifying object, with this seized sample be template carry out PCR resulting specific dna fragmentation when reacting amplification as index, identify whether above-mentioned be gas property spore-producing bacterium by the evaluation bacterium; It is characterized in that; Use any described test kit in the claim 1,3～5, and this authentication method is used for the purpose of the diagnosis of non-disease.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003346167A JP4242244B2 (en) | 2003-10-03 | 2003-10-03 | Oligonucleotide used for determination of spore aerobic bacteria, determination method and determination kit of spore aerobic bacteria using the same |
| JP346167/2003 | 2003-10-03 | ||
| PCT/JP2004/014240 WO2005033337A1 (en) | 2003-10-03 | 2004-09-29 | Oligonucleotide to be used in determining sporogenous aerobic bacterium and determination method and determination kit for sporogenous aerobic bacerium using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1863925A CN1863925A (en) | 2006-11-15 |
| CN1863925B true CN1863925B (en) | 2012-09-05 |
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| CN2004800288051A Expired - Fee Related CN1863925B (en) | 2003-10-03 | 2004-09-29 | Oligonucleotide to be used in determining sporogenous aerobic bacterium and determination method and determination kit for sporogenous aerobic bacerium using the same |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JP4242244B2 (en) |
| KR (1) | KR101113974B1 (en) |
| CN (1) | CN1863925B (en) |
| TW (1) | TWI342338B (en) |
| WO (1) | WO2005033337A1 (en) |
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|---|---|---|---|---|
| ES2423921T3 (en) * | 2004-08-04 | 2013-09-25 | Suntory Holdings Limited | Bacteria detection instrument, bacteria detection method and bacteria detection kit |
| AU2008201391A1 (en) * | 2007-03-30 | 2008-10-16 | Gc Corporation | A method for measuring the number of oral lactobacillus, and a PCR primers-probe set used for the same |
| JP5279204B2 (en) * | 2007-06-04 | 2013-09-04 | サントリーホールディングス株式会社 | Bacteria detection instrument, bacteria detection method, and bacteria detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH0994100A (en) * | 1995-07-03 | 1997-04-08 | Higeta Shoyu Co Ltd | Bacteria identification and kit therefor |
| JP3423597B2 (en) * | 1997-11-05 | 2003-07-07 | 三井農林株式会社 | Bacterial identification method |
| JP2002051783A (en) * | 2000-08-09 | 2002-02-19 | Denso Corp | Method of detection/determination for eubacteria |
-
2003
- 2003-10-03 JP JP2003346167A patent/JP4242244B2/en not_active Expired - Fee Related
-
2004
- 2004-09-29 KR KR1020067007783A patent/KR101113974B1/en not_active IP Right Cessation
- 2004-09-29 WO PCT/JP2004/014240 patent/WO2005033337A1/en active Application Filing
- 2004-09-29 CN CN2004800288051A patent/CN1863925B/en not_active Expired - Fee Related
- 2004-10-01 TW TW093129860A patent/TWI342338B/en not_active IP Right Cessation
Non-Patent Citations (1)
| Title |
|---|
| Goto et al.Application of partial 16S rDNA sequences as an index forrapid identification of species in the genus Bacillus.The Journal of general and applied microbiology46.2000,461-8. * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2005110530A (en) | 2005-04-28 |
| TWI342338B (en) | 2011-05-21 |
| CN1863925A (en) | 2006-11-15 |
| KR20060076786A (en) | 2006-07-04 |
| JP4242244B2 (en) | 2009-03-25 |
| KR101113974B1 (en) | 2012-03-06 |
| TW200525039A (en) | 2005-08-01 |
| WO2005033337A1 (en) | 2005-04-14 |
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