CN1507496A - Detection of spore forming bacteria - Google Patents

Abstract

Disclosed are methods, probes, and nucleic acid sequences for the systematic identification of sporulation genes in spore forming bacteria. A probe comprises a nucleic acid sequence able to form a detectable hybrid with highly conserved regions of the spoOA gene of certain spore forming bacteria Bacillus and Clostridium bacteria species, said nucleic acid sequence being unable to form a detectable hybrid with genetic material of non-spore forming bacteria. Amplification of a portion of the spoOA gene from the cellular DNA of a spore forming bacteria by a polymerase chain reaction using such probe, primer or nucleic acid sequence as one member of a primer set results in the generation of a detectable 346-365 nucleotide long DNA product. One disclosed method comprises a) combining a tagged or labeled probe with a sample, b) hybridizing the probe to the target spore forming bacteria spoOA gene; and c) detecting the hybridized product.

Description

The detection of spore forming bacteria

Technical field

The present invention relates to be used to detect the method for spore forming bacteria (spore forming bacterium).The present invention is specially adapted to sheet paper products and papermaking flow process are carried out Bacteria Detection.According to the present invention spore forming bacteria detected and to have comprised the method that relates to polymerase chain reaction (PCR).The invention also discloses and be specially adapted to primer that spore forming bacteria is detected.

Background technology

The sheet paper products that is used for food product pack should not contain the various microorganisms that are unfavorable for food sanitation.The manufacturing processed of sheet paper products is the modal approach that this type of microbial contamination takes place, and microorganism can grow in sheet paper products and breed.For this pollution, normally take to use biocide (biocide) or handle by heating.But, in the paper mill and the danger that in final sheet paper products, may exist limited the use of biocide.In addition, some microorganisms are because it has the intrinsic protection mechanism, i.e. sporulation (sporulation) can be avoided being eliminated.

For paper-making industry, control spore forming bacteria (SFB) is to cost dearly the most and one of obstinate problem.Be different from most of bacteriums, SFB can be survived from this link of drying machine in paper mill, and causes the danger of pollution in the use (for example food product pack) of sheet paper products.Simultaneously, except the strongest biocide of toxicity, spore forming bacteria is usually to all biocide resistances.Identified some and caused the SFB of problem to paper-making industry, of (Journal of Applied Bacteriology 81,445-458 (1996)) such as Pirttij  rvi, its full content is incorporated herein by reference at this.

Some industrial trend make to be paid close attention to more to the microbiological quality of the paper that is used for food product pack.The regenerated fibre (Recycled fiber) that contains starch and coating material helps microbial growth.Because the ratio of the twice-laid stuff that uses constantly increases aborning, the chance that causes finished product to be polluted also will increase thereupon.The increase of regenerated fibre correspondingly requires to reduce the biocide that uses the controlling microbial growth.Quality product is carried out monitoring rapid, reliable, easy and that valency is cheap, can make that the bacterium that causes problem is controlled, and simultaneously can be where necessary and particular place use biocide, thereby improve the overall manufacturing benefit.

In the U.S., the current industrial standard of food product pack level material is 250 spores of every gram paper.This is measured by Dairyman ' s method, and this method is that technology (plate count enumeration technique) is enumerated in plate count, need carry out 48 hours culturing process.Diagnosis is faster made in pollution problem can make waste product significantly reduce and improve comprehensively the productivity in paper mill.

Being not only paper-making process need detect fast to spore forming bacteria.For example, by the heat resistanceheat resistant spore that the member produced of bacillus, series bacillus genus and fusobacterium, can in the processing of for example food, medicine and medical treatment product, (it is not suitable for carrying out the hot high pressure sterilization) cause variety of issue.In these processes, must take special measure to avoid polluting and when occurring polluting, source of pollution to be assessed.Starting materials with contaminative is made rapid evaluation can be prevented unnecessary production pause and can save substantial contribution.

In therapeutic treatment, also need spore forming bacteria is identified.Sometimes, for example when treatment infectation of bacteria (as bronchitis, upper respiratory tract infection, otalgia or the like), selected microbiotic can resist the microorganism that causes infection effectively, for example is the bacterial population of clostridium bacterial strain (a kind of spore forming bacteria) class but can't kill.Although fusobacterium can not cause problem usually, under situation about lacking from the competitions of other microorganisms (it is killed by the microbiotic of junior one wheel), fusobacterium is able to a large amount of breedings, causes the potential severe infections.Therefore, need in biological sample, detect whether there is this type of bacterial classification.

Summary of the invention

The present invention relates to the method for bacterial detection.

More specifically, the present invention relates to use nucleic acid primer and probe to come the method for bacterial detection.

The present invention be more particularly directed to detect spore forming bacteria with this type of primer.Detection method of the present invention comprises the polymerase chain reaction use that combines with electrophoresis or fluorescence technique.

The invention further relates to the nucleic acid primer that is used to detect spore forming bacteria, more specifically, relate to the test kit of the nucleic acid primer that is used to detect spore forming bacteria.

These and other aspect of the present invention is to carry out the method that systematicness identifies and realize by being provided for sporulation gene to spore forming bacteria, described method comprises by using a primer sets that the part from a kind of gene of the total cell dna of spore forming bacteria is increased, and described primer sets comprises 5-AGTATCATTCATGAAATTGG-3 (SEQ ID NO.1), 5-AAAAAAGCAGTTGACT-3 (SEQ ID NO.2), 5-CGGCTTGCCGTTGTATT-3 (SEQ ID NO.3), 5-GAAGATGTGACGAAAAAG-3 (SEQ ID NO.4), 5-CAAGAAGATGTGACGAAA-3 (SEQ ID NO.5), 5-GTTGTATTATATTTCTTTGC-3 (SEQ ID NO.6), and 5-GTTGTGTTAAATTTTTTGGC-3 (SEQ ID NO.7), and 5-AGTATCATTCATGAAATTGGCGTTCC-3 (SEQ ID NO.8); And the existence of detection amplified production.Spore forming bacteria includes but not limited to bacillus megaterium (Bacillus megaterium), Bacillus licheniformis (Bacillus lichenformis), bacillus cereus group (Bacillus cereus group), bacillus pumilus (Bacilluspumilus), and Paenibacillus macerans (Paenbacillus macerans), Paenibacillus polymyxa (Paenbacillus polymyxa), Paenbacillus pabuli, crooked genus bacillus (Bacillus flexus), subtilis (Bacillus subtilis), Bacillus anthracis (Bacillus anthracis), Bacillus sporothermodurans, Bacillus sphaericus (Bacillus sphaericus), clostridium perfringens (Clostridium perfringens), clostridium butylicum (Clostridium butyricum), Clostridium baratii (Clostridiumpasteurianum), clostridium cochlearium (Clostridium cochlearium), clostridium scatologenes (Clostridium scatologenes), Soxhlet clostridium (Clostridium sordellii), clostridium lituseburense (Clostridium lituseburense), arguement clostridium (Clostridiumparadoxum), thermal fiber clostridium (Clostridium thermocellum), the hot anaerobic bacillus(cillus anaerobicus) of Bu Shi (Thermoanaerobacter brockii), hot autotrophy Moore Salmonella (Moorellathermoautotrophica), avette mouse spore bacterium (Sporomusa ovata), Theraobrachiunacelere, bacillus acidocaldarius (Bacillus acidocaldarus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus brevis (Bacillus brevis), bacillus thuringiensis (Bacillus thuringiensis), bacstearothermophilus (Bacillusstearothermophilus), clostridium difficile (Clostridium dificile), separate fiber clostridium (Clostridium cellulolyticum), clostridium bifermentans (Clostridium biferfnentans), and clostridium acetobutylicum (Clostridium acetobutylicum).Amplification can comprise the use polymerase chain reaction, can comprise amplified production is carried out electrophoresis and the electrophoresis substrate staining is observed and detect.In some embodiments, the electrophoresis substrate comprises sepharose; In some embodiments, dyeing comprises uses the pyridine of bromination second.

Another aspect of the present invention comprises a pair of primer, it comprises a kind of SEQ of being selected from ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQID NO.6, sequence among SEQ ID NO.7 and the SEQ ID NO.8, and another aspect of the present invention comprises a nucleotide sequence, it can be a kind of primer or probe, and described nucleotide sequence comprises a kind of SEQ of being selected from ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQID NO.4, SEQ ID NO.5, SEQ ID NO.6, sequence among SEQ ID NO.7 and the SEQ ID NO.8.The present invention also comprises the primer that is selected among SEQ ID NO.1, SEQ ID NO.2, SEQ IDNO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and the SEQ ID NO.8.

The present invention also further relates to a kind of composition, it comprises the material and at least a primer of at least a cellulose, and described primer comprises the sequence of a kind of SEQ of being selected from ID NO.1, SEQ IDNO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.The material of described cellulose can comprise paper pulp.

Being used to measure the test kit that whether has spore forming bacteria also belongs within the scope of the present invention, wherein said test kit comprises the substratum of at least a primer and the growth of at least a support spore forming bacteria, and described primer comprises the sequence among a kind of SEQ of being selected from ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQID NO.7 and the SEQ ID NO.8.Described test kit can be designed as and is used to measure paper-making process, or is common to any material of mensuration.The using method of this type of test kit also belongs within the scope of the present invention.

The present invention also further relates to and is used for the method whether working sample exists spore forming bacteria.A kind of method comprises a) at least two kinds of nucleotide primers is combined with a kind of sample, wherein said nucleotide primer i) is complementary to from least a forward kernel acid sequence in the total cell dna of described bacterium (forward nucleic acid sequence) and at least a inverse kernel acid sequence (reverse nucleic acid sequence), ii) can with the spoOA gene conservative area hybridization of spore forming bacteria rather than non-spore forming bacteria (non-spore forming bacteria), and iii) it is such nucleotide primer, promptly can produce the DNA product of a length as 346-365 Nucleotide by using this type of primer that the part from the cell DNA spoOA gene of spore forming bacteria is increased; B) with primer the cell DNA in the sample is increased; And c) measures the DNA that whether has amplification.Described sample can be the sample of cellulose, and can be the sample of taking from the paper-making process.This type of sample includes but not limited to the sample from plain boiled water (white water), a box (head box), waste paper, additive storage tank and coating roller calender.Other samples comprise air, soil, water, blood, movement, starch, protein or a kind of Epicholorohydrin (epichlorohydrin) reaction product.It is right that the nucleotide sequence that any the application disclosed all can be used for described primer, and this type of sequence comprises SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQID NO.4, SEQ TD NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

The present invention also provides the sample that is used for measuring cellulose whether to have the method for spore forming bacteria, wherein said method comprises at least a primer is combined with a kind of sample of cellulose, and described primer comprises the sequence of a kind of SEQ of being selected from ID NO.1, SEQ ID NO.2, SEQID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.The invention still further relates to the sample that is used for measuring cellulose and whether have the method for spore forming bacteria.A kind of these class methods comprise a) at least two kinds of nucleic acid primers of the present invention are combined with a kind of sample of cellulose, and described primer is complementary to from least a forward kernel acid sequence of the total cell dna of bacterium and at least a inverse kernel acid sequence; And b) primer of hybridization is observed.Described at least two kinds of nucleic acid primers preferably comprise at least a among SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and the SEQ ID NO.8.

The present invention also provides the method for the spore forming bacteria colony that is used for controlling the industrial production flow process, described method comprises a) uses a kind of primer in described Production Flow Chart bacterium to be detected, and described primer comprises the sequence among a kind of SEQ of being selected from ID NO.1, SEQ ID NO.2, SEQID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and the SEQ ID NO.8; And b) concentration of the biocide in the described Production Flow Chart is adjusted to is enough to reduce number of bacteria.Described industrial production flow process can be, for example, and paper flow process or food-processing flow process.

Other aspects of the present invention comprise that the sporulation gene that is used for spore forming bacteria carries out the method that systematicness is identified, described method comprises: a) use at least a among SEQ ID NO.1, SEQID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and the SEQ ID NO.8, the part of a kind of gene of the total cell dna that comes from described spore forming bacteria is increased; And b) detects whether there is a kind of amplified production.

On the other hand, the invention provides and be used for the probe whether test sample exists spore forming bacteria, described probe comprises a kind of nucleotide sequence, described sequence can be with following (a) or (b) the high conservative zone of the spoOA gene of the bacterial classification of listed spore forming bacteria bacillus and fusobacterium form detectable crossbred (hybrid):

A) bacillus cereus, bacillus megaterium, Bacillus anthracis and Clostridium baratii;

B) bacillus cereus, bacillus megaterium, Bacillus sphaericus and Clostridium baratii,

Described nucleotide sequence can not form detectable crossbred with the genetic material of non-spore forming bacteria.(a) the high conservative zone of listed preferred spore forming bacteria bacterial classification is shown in the Table II and the Table III of following examples 2 and (b).This preferred probes on the one hand of the present invention is meant, use this probe as in one group of primer a kind of, by polymerase chain reaction the part from the spoOA gene of the cell DNA of spore forming bacteria is increased, can produce a length is the detected DNA product of 346-365 Nucleotide.For above (a), preferred bacillus in addition and the bacterial classification of fusobacterium are subtilis and hot vinegar clostridium.For above (b), preferred bacillus in addition and the bacterial classification of fusobacterium are bacstearothermophilus and hot vinegar clostridium.The preferred high conservative zone of these bacterial classifications also is shown in the Table II and the Table III of following examples 2.

On the other hand, the invention provides and be used for the probe whether test sample exists spore forming bacteria, described probe comprises a kind of nucleotide sequence, described sequence can be with following (a) or (b) the high conservative zone of the spoOA gene of the bacterial classification of listed spore forming bacteria bacillus and fusobacterium form detectable crossbred:

A) bacillus cereus, bacillus megaterium, subtilis and Clostridium baratii;

B) bacillus cereus, bacillus megaterium, Bacillus sphaericus and Clostridium baratii,

Described nucleotide sequence can not form detectable crossbred with the genetic material of non-spore forming bacteria.(a) the high conservative zone of listed preferred spore forming bacteria bacterial classification is shown in the Table VI and the Table VII of following examples 3 and (b).This preferred probes on the one hand of the present invention is meant, use this probe as in one group of primer a kind of, by polymerase chain reaction the part from the spoOA gene of the cell DNA of spore forming bacteria is increased, can produce a length is the detected DNA product of 346-365 Nucleotide.For above (a) and (b) both, preferred bacillus in addition and the bacterial classification of fusobacterium are bacillus thuringiensis and hot vinegar clostridium.The preferred high conservative zone of these bacterial classifications also is shown in the Table VI and the Table VII of following examples 3.

The present invention also provides and has been used for the probe whether test sample exists spore forming bacteria, described probe comprises a kind of nucleotide sequence, described sequence can form detectable crossbred with the spoOA gene of spore forming bacteria, but can not form detectable crossbred with the genetic material of non-spore forming bacteria, wherein said nucleotide sequence is made up of VITAMIN B4, guanine, cytosine(Cyt) and thymus pyrimidine basically.This type of nucleotide sequence can comprise SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ IDNO.6, SEQ ID NO.7 or SEQ ID NO.8.

Another embodiment of the invention is a kind of probe whether test sample exists spore forming bacteria that is used for, described probe comprises a kind of nucleotide sequence, described sequence can form detectable crossbred with the spoOA gene of spore forming bacteria, but can not form detectable crossbred with the genetic material of non-spore forming bacteria, wherein said nucleotide sequence can form detectable crossbred to 93 bit bases with the 76th of the spoOA gene (corresponding to GenBank registration number gbU09972) of bacillus cereus.This nucleotide sequence can comprise SEQ ID NO.4 and SEQ ID NO.5.

The present invention also provides a kind of probe whether test sample exists spore forming bacteria that is used for, described probe comprises a kind of nucleotide sequence, described sequence can form detectable crossbred with the spoOA gene of spore forming bacteria, but can not form detectable crossbred with the genetic material of non-spore forming bacteria, wherein said nucleotide sequence can form detectable crossbred to 422 bit bases with the 403rd of the spoOA gene (corresponding to GenBank registration number gb U09972) of bacillus cereus.This nucleotide sequence can comprise SEQ ID NO.3, SEQ ID NO.6 or SEQ ID NO.7.

The invention still further relates to the composition that comprises at least a primer, described primer comprises a kind of sequence that is selected among SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ IDNO.5, SEQ ID NO.6, SEQ ID NO.7 and the SEQ ID NO.8.Described composition can comprise a kind of material of cellulose, for example paper pulp.

The present invention also comprises and is used for the method whether test sample exists spore forming bacteria, and the probe that described method comprises a) a kind of mark combines with sample; B) the spoOA gene of described probe and purpose spore forming bacteria is hybridized; And c) the hybridization product is detected.SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8 can be used as the probe of mark.The sample that can determined sample includes but not limited to air, soil, water, blood, movement, starch, protein and/or a kind of Epicholorohydrin reaction product.

Another aspect of the present invention comprises and is used for the probe whether test sample exists spore forming bacteria, this type of probe comprises a kind of nucleotide sequence, described sequence can form detectable crossbred with the spoOA gene of spore forming bacteria, but can not form detectable crossbred with the genetic material of non-spore forming bacteria, wherein said nucleotide sequence can form detectable crossbred to 427 bit bases with the 70th of the spoOA gene of bacillus cereus, and this nucleotide sequence is made up of guanine, cytosine(Cyt), adenine and thymine basically.

Another aspect of the present invention comprises and is used for the probe whether test sample exists spore forming bacteria, this type of probe comprises a kind of nucleotide sequence, described sequence can form detectable crossbred with the spoOA gene of spore forming bacteria, but can not form detectable crossbred with the genetic material of non-spore forming bacteria, wherein said nucleotide sequence can form detectable crossbred to 427 bit bases or with the 570th of the spoOA gene (gb M10082) of subtilis to 930 bit bases with the 70th of the spoOA gene of bacillus cereus.This nucleotide sequence preferably comprises at least a among SEQ ID NO.1, SEQ ID NO.2, SEQID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and the SEQ ID NO.8.

The preferred probes of aforementioned aspects of the present invention is meant, use a kind of as in one group of primer of this probe, by polymerase chain reaction the part from the spoOA gene of the cell DNA of spore forming bacteria is increased, can produce a length is the detected DNA product of 346-365 Nucleotide.

Another aspect of the present invention comprises that manufacturing is used for detecting the method whether spore forming bacteria exists a kind of nucleotide sequence of conservative gene, and described method comprises the conservative region of a) determining from the conservative gene of at least two kinds of spore forming bacteria bacterial strains; And b) preparation can with the nucleotide sequence of described conservative region hybridization, wherein said nucleotide sequence is made up of VITAMIN B4, guanine, cytosine(Cyt) and thymus pyrimidine basically.Described conservative gene can comprise spoOA, ssp and/or dpaAIB, is preferably spoOA.

The invention still further relates to the system that is used to identify spore forming bacteria, described system comprises: a) be used to make the DNA of spore forming bacteria to be easy to the method with at least a nucleotide primer hybridization; B) at least a nucleotide primer; And c) is used to detect the method for the hybridisation events of the DNA of described spore forming bacteria and described at least a nucleotide primer.The DNA of described spore forming bacteria can comprise the spoOA gene, and described at least a nucleotide primer can be made up of VITAMIN B4, guanine, cytosine(Cyt) and thymus pyrimidine basically.Described at least a nucleotide primer can comprise a kind of sequence that is selected among SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and the SEQID NO.8.The method that the described DNA that is used to make spore forming bacteria is easy to hybridize can comprise a growth step, and wherein said bacterium is placed in the environment that can promote to grow, and what continue is a cleavage step, and wherein said bacterium is cleaved.Described cleavage step can comprise heating.The described method that is used to detect hybridisation events can comprise polymerase chain reaction.The described method that is used to detect hybridisation events can comprise detection technique of fluorescence.

Embodiment

The present invention relates to be used for the method for bacterial detection, particularly, is the method that detects spore forming bacteria (SFB).Spore forming bacteria is meant the bacterium that can form spore, and this bacterioid is known in the art.The example of this type of SFB includes but not limited to bacillus megaterium, Bacillus licheniformis, bacillus cereus family, bacillus pumilus, and Paenibacillus macerans, Paenibacillus polymyxa, Paenbacillus pabuli, crooked genus bacillus, subtilis, Bacillus anthracis, Bacillus sporothermodurans, Bacillus sphaericus, clostridium perfringens, clostridium butylicum, Clostridium baratii, clostridium cochlearium, clostridium scatologenes, the Soxhlet clostridium, clostridium lituseburense, the arguement clostridium, the thermal fiber clostridium, the hot anaerobic bacillus(cillus anaerobicus) of Bu Shi, hot autotrophy Moore Salmonella, avette mouse spore bacterium, Thermobrachium celere, bacillus acidocaldarius, bacillus amyloliquefaciens, bacillus brevis, bacillus thuringiensis, bacstearothermophilus, clostridium difficile, separate the fiber clostridium, clostridium bifermentans and clostridium acetobutylicum.

The present invention is applicable at paper-making process and detects SFB, but is not limited to this class process.(at this, term " paper (paper) " is a generalized.That is to say that " paper " in " paper-making process " means and comprise paper (paper), paperboard (paperboard), cardboard (cardboard), or the like.) when in paper-making process, measuring, can itself measure water treatment procedure (process water).Can measure it in any link of water treatment procedure, but preferably in a box or storage tank, measure.This type of storage tank can contain additive for paper making, can measure the latter and whether have SFB.Examples of such additives comprises starch, latex (latex), clay, protein and Epicholorohydrin reaction product, include but not limited to gather (resin acid-altogether-diethylenetriamine) (poly (adipic acid-codiethylenetriamine)) and Epicholorohydrin reaction product, commodity are called Kimene.Except in paper-making process, water treatment procedure being measured, also can measure papermaking equipment and whether have SFB.Usually, preferably measure the shower faucet settling and whether have SFB.

The present invention also can be used for detecting the SFB in air, soil, food and the water, and water comprises waste water, process water and tap water.The present invention can be used for detecting the SFB that contains in the protein sample.The present invention can be used in the medical diagnosis SFB detection for example is included in blood or the movement at least a SFB is measured.As said, the method for bacterial detection is similar to the detection method in paper-making process in these other media.

The focus that the present invention pays close attention to is to mediate the evolution conservative of the gene of sporulation process.A kind of multifarious bacterium hypotype (a subset of phylogeneticallydiverse bacteria) that is in phylogeny can form spore.Modal spore forming bacteria is the member of bacillus (aerophil) and fusobacterium (anerobe).Sporulation is the growth course of a complexity, is the reaction to the bad environment condition, and strictness is subjected to the cells physiological regulation and control.Heat, hungry and chemistry disorderly (chemical perturbation) comprise some but the not every factor that can induce the sporulation approach.The gene that sporulation relates in early days has high homology in each bacterial classification.SpoOA is one of this genoid, and it can be considered to " effector " in the sporulation process.

The kinases of a kind of responsible signal transduction of spoOA genes encoding by phosphorylation, makes other gene activations in this process.The kinase whose phosphorylation state of SpoOA is represented its activity in cell.Because its this central role in triggering sporulation, spoOA belongs to a kind of high conservative gene and therefore becomes the good goal gene of detection.

The present invention is based on a such discovery, promptly spore forming bacteria have some can be as the conservative genetic material of testing goal thing.According to the present invention, described can be spoOA gene or the gene that has homology with it as the conservative genetic material of testing goal thing.By with this gene (or its homologous gene) as object, the present invention can detect a large amount of bacteriums.Each can be detected according to the present invention bacterium all be considered to have spoOA gene or its homologous gene, it can participate in sporulation.Other genes that can be used as object according to the present invention can comprise ssp gene and dpaA/B gene, all are present in spore forming bacteria (sporogenic bacteria) and are not present in non-spore forming bacteria (asporogenicbacteria).

Design of the present invention is based on this discovery of genetic material that specific short chain Nucleotide can be incorporated into the purpose bacterium.By some different technology, even this combination can be observed by quantitative.Identify that with nucleic acid probe or primer the basic fundamental of purpose genetic material is known in the art and describes.About the argumentation of spore forming bacteria and with its method that detects of spoOA gene pairs, can be described referring to Brill and (Molecular Microbiology 14 (3) (1994) 411-426) such as Wiegel (Journal of MicrobiologicalMethods 31 (1997) 29-36) and Brown, be incorporated herein by reference at this.About using probe and primer to identify bacterium, can be referring to U.S. Patent No. 5,747,252,5,969,122,5,430,137,5,714,321 and 5,958,679, all be incorporated herein by reference at this.

Therefore, the present invention relates to the purposes of the specificity of spoOA gene part as the nucleotide sequence of object.These nucleotide sequences can combine with the purpose part of the genetic material of SFB or hybridize.The span of the purpose part of described spoOA gene is to start from about the 70th bit base of bacillus cereus and terminate in about 427 bit bases (GenBank registration number gb U09972).Nucleotide sequence of the present invention also can with from the homologous sequence of other SFB as object.

Obviously, bacterial strain is different with the base number order of bacterial strain.But, by using CLUSTAL sequence alignment program (Baylor College of Medicine NucleotideSearch Launcher) to seek homologous sequence in the GenBank database, those skilled in the art can determine other SFB and corresponding genetic material thereof easily.(certainly, also can use other sequence alignment program.) nonrestrictive example as, the 570th Nucleotide to 930 Nucleotide of subtilis (gb M10082) can be used as object.

This technology of polymerase chain reaction (PCR) can be used to observe the existence of sporulation gene.This method is based on the base complement of DNA.DNA is made of two antiparallel strands, and described chain is made of Nucleotide " base ".These bases, promptly VITAMIN B4, guanine, cytosine(Cyt) and thymus pyrimidine can form specific hydrogen bond each other.VITAMIN B4 and thymus pyrimidine pairing, guanine and cytosine(Cyt) pairing.Two chains of DNA can be through alkali or thermal treatments and sex change are taken place or change single stranded form into.Under appropriate condition, DNA can reassociate becomes its double-stranded conformation.

Polymerase chain reaction (Mullis, U.S. Patent No. 4,683,195,4,683,202 and 4,800,159, its full content is incorporated herein by reference at this) but be a kind of common method that target DNA fragment is expanded to detection level.Current its is widely used in detecting multiple malignant bacteria.In the method, use the dna primer of the specific sequence that is complementary to purpose scope two side areas, the enzyme that carries out DNA by means of archaeal dna polymerase is synthetic.Archaeal dna polymerase needs a primer to begin the synthetic of a complementary dna chain.

Device that carries out PCR and system that number of different types is arranged.Device commonly used comprises Mini Cycler (MJ Instruments), Delta CyclerI System (EriComp) and Smart Cycler (Cepheid).Other system also can be used for the present invention, and the some of them example can be referring to the U.S. Patent No. 5,882 of authorizing NORTHRUP etc., 496,5,674,742,5,646,039,5,589,136,5,639,423, the U.S. Patent No. 5 of authorizing the U.S. Patent No. 5,527,510 of ATWOOD etc. and authorizing PETERSEN etc., 958,349.Because of the argumentation of its relevant PCR system, with the U.S. Patent No. 5,882 of NORTHRUP etc., 496,5,674,742,5,646,039,5,589,136,5,639,423, the U.S. Patent No. 5 of the U.S. Patent No. 5,527,510 of ATWOOD etc. and PETERSEN etc., 958,349 are incorporated herein by reference at this.

Primer is one section short chain Nucleotide (being typically about 15-22 base).In the PCR process, initial by temperature control in the annealed step.The annealing conditions that is determined by experiment each group primer is to guarantee specificity.After the annealing, the synthetic complementary DNA chain of polysaccharase has promptly begun polymerization process.PCR reaction heated so that all double-stranded DNA generation sex change thereafter.Use a kind of thermostability archaeal dna polymerase that is located away from super thermophilic thermus aquaticus (Thermus aquaticus), can anneal repeatedly, the circulation of polymerization and sex change, and enzymic activity is descended.The pcr amplification method is a kind of laboratory method of routine, can use automatic thermal cycler to carry out.Its result makes the amplification of target DNA fragment being carried out exponential form, and the object after the amplification can be detected immediately.Preferred primer of the present invention, primer to and primer sets can make amplification can produce the detectable DNA product that length is 346-365 Nucleotide.Nucleotide sequence of the present invention comprises SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQID NO.7 and SEQ ID NO.8, can be used as primer.

Detecting a kind of method that whether has amplified production is that agarose gel electrophoresis continues with dyeing.Other detection method includes but not limited to detection technique of fluorescence.In a kind of technology based on fluorescence, a kind of insertion dyestuff, for example Syber Green or the pyridine of bromination second can be in conjunction with the DNA of two strands, and sends fluorescence.The result who mixes this type of dyestuff in the PCR reaction is that along with the carrying out of PCR reaction and synthesizing of double-stranded DNA, fluorescence intensity increases thereupon.The thermally denature of product can be used for finding out the size of PCR product and GC content (%) (%GC is that the base number of G or C is divided by the base sum).

TaqMan ^TMSystem is one of fluorescence detecting system of commercial PCR-based.Be applicable to this system reporting dyes for example for 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, FAM), four-6-Fluoresceincarboxylic acid (tetra-6-carboxyfluorescein, TET) and chlordene-6-Fluoresceincarboxylic acid (hexachloro-6-carboxyfluorescein, HEX), see P.M.Holland, R.D.Abramson, R.Watson, S.Will, R.K.Sakai and D.H.Gelfand (1992.Detection of specific PCR product by utilizing the 5 '-3 ' exonuclease activity of Thermus aquaticus DNA polymerase.Clin.Chem., 38:462-463) described.Another commercial detection system adopts molecular signal, sees that S.Tayagi and F.R.Kramer (1996.Molecular Beacons:Probes that fluoresceupon hybridization.Nature Biotechnology 14:303-308) are described.

Another kind of technology adopts the nucleotide sequence of mark that hybridization is detected.For example, can adopt fluorescently-labeled oligonucleotide sequence to come whether to exist in the test sample object from an interior region of spoOA PCR product.Along with the carrying out of PCR reaction, this fluorescent marker can be cut on probe, therefore can be observed fluorescence.There is direct dependency in the increase of the object in the increase of fluorescence intensity and this testing sample.Two examples of this type of sequence are 5-AGTATCATTCATGAAATTGG-3 (SEQ ID NO.1) and 5-AGTATCATTCATGAAATTGGCGTTCC-3 (SEQ ID NO.8).Sequence not be only used for illustrating, other conserved sequences in the spoOA also can be used as the testing goal thing.Other nucleotide sequences of the present invention also can be used for this respect, and described sequence comprises SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ IDNO.6 and SEQ ID NO.7.

Except the method for having mentioned, also can adopt some other method to detect hybridisation events between nucleotide sequence of the present invention and the object.Described additive method does not need polymerase chain reaction or primer to obtaining to detect the method for crossbred particularly including those.For example, can predict, nucleotide sequence of the present invention can be labeled and be used for by oligonucleotide probe testing goal sequence.These sequences can be carried out mark with fluorescence or Geigers.If use fluorescent mark, the nucleotide sequence that hybridization takes place can launch with non-hybridization form different energy spectrum, and can detect it by currently known methods.

For radiolabeled probe, the sequence that hybridization takes place can be by means of radioactive automatic developing, detect by being exposed to radioactivity susceptibility film, see Maniatis, T., E.F.Fritsch and J.Sambrook (1982 Molecular Cloning:A Laboratory Manual.ColdSpring Harbor Laboratories, Cold Spring Harbor, New York) described.Nucleotide sequence of the present invention comprises SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, can be used as probe and is used for this respect.

Nucleotide sequence of the present invention is to obtain by the spoOA gene from a large amount of spore forming bacterias being carried out sequence contrast (sequence comparison).This method relies on the high conservative zone that nucleotide sequence comparison software program is found gene.In these zones, select specific initiation site, and synthesize suitable primer.Need make repeated attempts and to determine to can be used for carrying out the optimal sequence of primer screening.Preferred primer satisfies following standard:

I. can in the spore forming bacteria of a stack features, detect spoOA;

Obtain negative findings when ii. measuring non-SFB; And

Iii. can from separate the non-distinctive spore forming bacteria from paper or sheet paper products sample, detect spoOA.

Should be noted in the discussion above that nucleotide sequence of the present invention in this announcement is based on it and can filters out with the ability of the goal gene hybridization of SFB.Be specially, described nucleotide sequence at be the conservative purpose genetic material of SFB.Consider that with SFB as object, described nucleotide sequence of the present invention should be considered to be highly preferred.But, will be appreciated that, do not need just can obtain satisfied result with the identical sequence of the present invention.That is to say, will be appreciated that the replacement that has one or more bases still can carry out the hybridization with the goal gene of SFB.Identical with sequence of the present invention is highly preferred, and homology or conservative replace poorer are slightly taken place, but still is acceptable.This compromise proposal might be a kind of " pardon (inclusiveness) " than low degree, that is to say, the sequence of having only less SFB can be taken place to replace is identified.In some cases, for example, only need to identify that this pardon is acceptable under the situation of a kind of SFB.

The detection that provides spore forming bacteria has been provided method of the present invention.According to the present invention, can hang down the detection level of spore and to reach 200 spores of every gram paper (even may be lower).The detection that following steps can obtain to optimize:

A. 1% pulp sample (containing 1g paper pulp in the 100ml sterilized water) with 10ml combines with the tryptic soy broth substratum (tryptic soy broth medium) (Difco Laboratories) of 40ml, and places 7 hours at 37 ℃.

B. with this sample centrifugation in Eppendorf tube of 4ml.

C. wash this precipitation, recentrifuge with 100 μ l sterilized waters (deionization).

D. precipitation is resuspended in the 30 μ l sterilized waters, and boiled 5 minutes.

E. the solution that boils of getting 5 μ l carries out PCR, and on sepharose observations.

Notice that under the concentration condition with higher of SFB, the incubation time in the step a) can shorten, and for some sample, the incubation time of step a) even can be longer.For example, for those doubt samples, can hatch 16 hours (but in step b) only the sample of centrifugal 1ml).For example, if think the SFB that has extremely low quantity, or under the situation that has the pcr amplification inhibitor, can carry out hatching of longer time.Therefore, adopt long incubation time, detection level can hang down and reach about 100 spores/gram paper.In addition, in step a), process water, additive or the alternative paper pulp of stock's sample are as initial sample.In step e), can adopt other observational technique, for example fluorescent method.

Can believe that according to the above, those skilled in the art need not to pay too much effort, can maximally utilise the present invention.Therefore, below preferred, specific embodiment should to be regarded as only be illustrative, and have no intention by any way to other guide of the present invention any restriction in addition.

More than and all patents quoted subsequently and the disclosure of publication, just as being incorporated herein by reference at this in full with it.

Following embodiment has further set forth implementation process of the present invention, and it should not be considered to restrictive.

Embodiment 1-primer sets numbering 1

Access one group of spoOA sequence and adopt CLUSTAL sequence alignment program to compare from GenBank.By sequence alignment, filter out the oligonucleotide initiation site and select a preliminary primer sets.Synthesized a forward primer, 5-AAAAAAGCAGTTGACT-3 (SEQID NO.2), and a reverse primer, 5-CGGCTTGCCGTTGTATT-3 (SEQID NO.3).Use this primer sets expection can obtain being approximately the PCR product of 300-400 base pair---use PCR pearl (the Pharmacia Biotech of " ready (Ready to Go) ", Piscataway N.J.) optimizes the reaction conditions of the PCR of thermal cycling program with different annealing temperatures.This step can be used any PCR instrument, and non-limitative example wherein is MiniCycler and Delta Cycler.Distinctive SFB and one group of non-distinctive SFB used in this test separate from a paper mill.

It is detected subsequently and confirm that there is the spoOA gene in it to detect not the non-distinctive sample of male (from the sample in paper mill).Distinctive SFB the results are shown in table 1.

Table 1

Bacterial strain	The PCR product?
		Bacillus cereus	????+
Subtilis	????+
		Bacillus megaterium	????+
Clostridium perfringens	????-

The result shows that except clostridium perfringens, all SFB all illustrate the band that a molecular weight is lower than about 369 base pairs just.In order to determine that spoOA is object, on nylon membrane, carry out Southern hybridization from the sepharose of PCR.Positive according to expectation, except clostridium perfringens, all SFB are all hybridized with the amplified production from the mark of bacillus cereus.But, although use this primer sets not detect clostridium perfringens, the positive findings that comes from non-distinctive paper mill sample points out this primer sets to be applicable to this purpose.

Embodiment 2-primer sets numbering 2

According to the result of above embodiment 1,, produced one second primer sets by comparing with a bigger data set.What table 2 showed is the data of the forward primer of improved primer sets, and table 3 then is the data of reverse primer.

Table 2

Bacillus cereus	????G	????A	????A	????G	????A	????T	????G	????T	????G	????A	????C	????G	????A	????A	????A	????A	????A	????A	????G
																				Bacillus megaterium	????G	????A	????A	????G	????A	????C	????G	????T	????A	????A	????C	????G	????A	????A	????A	????A	????A	????A	????G
Bacstearothermophilus	????G	????A	????A	????G	????A	????C	????G	????T	????G	????A	????C	????G	????A	????A	????A	????A	????A	????G	????G
																				Bacillus thuringiensis	????G	????A	????A	????G	????A	????T	????G	????T	????G	????A	????C	????G	????A	????A	????A	????A	????A	????A	????G
Bacillus sphaericus	????G	????A	????A	????G	????A	????T	????G	????T	????A	????A	????T	????G	????A	????A	????A	????C	????A	????G	????G
																				Bacillus anthracis	????G	????A	????A	????G	????A	????T	????G	????T	????G	????A	????C	????G	????A	????A	????A	????A	????A	????A	????G
Clostridium baratii	????G	????A	????C	????A	????A	????A	????A	????T	????T	????A	????C	????T	????C	????A	????A	????A	????G	????A	????G
																				Clostridium innocuum	????G	????A	????T	????C	????T	????C	????A	????T	????C	????G	????T	????G	????G	????C	????A	????G	????G	????T	????G
Hot vinegar clostridium	????G	????A	????G	????A	????G	????T	????A	????T	????G	????A	????C	????C	????A	????T	????G	????C	????G	????G	????T

Table 3

Bacillus cereus	A	?A	?T	?A	?C	?A	?A	?C	?A	?G	?C	?A	?A	?G	?C	?C	?G
																		Bacillus megaterium	A	?A	?T	?A	?C	?A	?A	?C	?G	?G	?C	?A	?A	?G	?C	?C	?G
Bacstearothermophilus	A	?A	?C	?A	?C	?A	?A	?C	?G	?G	?C	?A	?A	?G	?C	?C	?G
																		Bacillus thuringiensis	A	?A	?T	?A	?C	?A	?A	?C	?A	?G	?C	?A	?A	?G	?C	?C	?G
Bacillus sphaericus	A	?A	?T	?A	?C	?A	?A	?C	?A	?C	?C	?G	?T	?C	?A	?C	?G
																		Bacillus anthracis	A	?A	?T	?A	?C	?A	?A	?C	?A	?G	?C	?A	?A	?G	?C	?C	?G
Clostridium baratii	A	?A	?T	?A	?C	?T	?A	?C	?T	?G	?C	?A	?A	?G	?C	?C	?G
																		Clostridium innocuum	G	?C	?A	?A	?C	?C	?A	?C	?G	?G	?C	?A	?T	?C	?C	?C	?G
Hot vinegar clostridium	A	?T	?G	?A	?C	?T	?A	?C	?T	?C	?C	?C	?A	?G	?T	?C	?G

From table 2 and table 3 as can be seen, have mutually big sequence homogeny and homology between the genetic material of SFB.Use has been synthesized a kind of new forward primer from the data of sequence alignment, 5-GAAGATGTGACGAAAAAG-3 (SEQ ID NO.4).5-CGGCTTGCCGTTGTATT-3 described in this primer and the previous embodiment 1 (SEQ ID NO.3) has together constituted new primer sets.

(SEQ ID NO.4 and 3) tests known SFB and non-SFB respectively with this primer sets.This primer sets can produce the spoOA product in distinctive SFB, but does not produce product in non-SFB.After the PCR, detect whether have positive findings, the band that it is 346-365 base pair that positive findings is shown as a size on sepharose with agarose gel electrophoresis.Table 4 is depicted as the result who comes from the characteristic SFB that is tested.

Table 4

Bacterial strain	The PCR product?
		Bacillus cereus	????+
Subtilis	????+
		Bacillus megaterium	????+
Clostridium perfringens	????+
		Streptococcus aureus	????-
Staphylococcus epidermidis	????-
		Pseudomonas aeruginosa	????-
Klebsiella pneumonia	????-
		Bacstearothermophilus	????+
Bacillus licheniformis	????+

From table 4 as can be seen, adopt new primer sets that the clostridium perfringens except other bacterial strains is detected.As can be seen, this primer sets has required characteristics from the result: with SFB hybridization, and do not hybridize with non-SFB.Similarly, (from embodiment's 1) is similar with aforementioned primer sets, and resulting positive findings confirms that this primer sets can be used for its intended purposes in the sample of non-distinctive paper mill.

Have the function of expection in case determine this primer sets, promptly implement further test how with the susceptibility of determining this primer sets.Therefore, carry out following process to determine " sensing range " of this primer sets.Although this process adopts outturn as test material, this process is applicable to the various samples of test, comprises air, soil, food and water, and water includes but not limited to waste water, process water and tap water.Should be noted that this process is further improved and optimized among the embodiment 3 below.

Determine sensing range

1. the bacillus cereus culture with 100ml grows to the lag phase, places 80 ℃ to induce sporulation then.

With this culture with 10 times of phosphate buffered saline(PBS) (PBS) dilutions, get on the outturn of this diluent point of 0.1ml and grade dissimilar, comprise A to 0.5g) kraft paper, the B of recycle) alkaline kraft and C) acid meticulous paper.

3. outturn is placed 10ml PBS, and carried out vortex oscillation 2 minutes.

4. sample was placed 10 minutes at 80 ℃, then the 1ml sample is placed 9ml PBS to reach 10 times dilution once more.

5. the sample with 0.1ml joins (sample of adding 0.1ml and sample diluting liquid are so that the result of PCR is consistent with colony forming unit) in the aseptic Eppendorf tube that contains 0.1ml pancreatin soya broth.

6. sample was hatched 45 minutes so that its sprouting (germination) at 37 ℃.

[annotate: this step is the optimization step among the embodiment 3, under the lower situation of bacterial concentration, may need longer incubation time.

7. this Eppendorf tube was placed boiling water 5 minutes, get 5 μ l and be used for PCR, PCR adopts " ready " PCR pearl (Pharmacia).

8. the program setting of thermal cycler is as follows:

A.94 ℃, 5 minutes

B.30 a circulation: 94 ℃, 0.5 minute; 52 ℃, 0.5 minute; 72 ℃,

0.5 minute

C.72 ℃, 3 minutes

Table 5 is depicted as the sensing range of determined this primer sets.

Table 5

Sample	Spore/0.5g Paper #	SpoOA PCR product?
			No paper *	????171±6.0	????+
No paper	????22±1.0	????-
			????A	????114.5±1.5	????+
????A	????1.5±0.5	????-
			????B	????59±6.0	????+
????B	????7±7.0	????-
			????C	????149±1.0	????+
????C	????19.5±18.5	????-

^#Determine with plate count

^*The broth culture of bacillus cereus spore, no paper

In table 5, A) kraft paper of recycle, B) alkaline kraft and C) acid meticulous paper.

Embodiment 3-primer sets numbering 3

The primer sets of embodiment 2 may be inconsistent to the detection of Bacillus sphaericus.For addressing this problem, prepared new primer sets.Prepare data presentation that this improved primer sets considers in table 6 (forward primer) and table 7 (reverse primer).

Table 6

Bacillus cereus	????C	????A	????A	????G	????A	????A	????G	????A	????T	????G	????T	????G	????A	????C	????G	????A	????A	????A
																			Bacillus megaterium	????C	????A	????A	????G	????A	????A	????G	????A	????C	????G	????T	????A	????A	????C	????G	????A	????A	????A
Bacstearothermophilus	????C	????A	????G	????G	????A	????A	????G	????A	????C	????G	????T	????G	????A	????C	????G	????A	????A	????A
																			Bacillus thuringiensis	????C	????A	????A	????G	????A	????A	????G	????A	????T	????G	????T	????G	????A	????C	????G	????A	????A	????A
Bacillus sphaericus	????C	????A	????A	????G	????A	????A	????G	????A	????T	????G	????T	????A	????A	????T	????G	????A	????A	????A
																			Bacillus anthracis	????C	????A	????A	????G	????A	????A	????G	????A	????T	????G	????T	????G	????A	????C	????G	????A	????A	????A
Subtilis	????C	????A	????G	????G	????A	????A	????G	????A	????T	????G	????T	????C	????A	????C	????G	????A	????A	????A
																			Clostridium baratii	????C	????A	????A	????G	????A	????C	????A	????A	????A	????A	????T	????T	????A	????C	????C	????A	????A	????A
Clostridium innocuum	????A	????A	????C	????G	????A	????T	????C	????T	????C	????A	????T	????C	????G	????T	????G	????G	????C	????A
																			Hot vinegar clostridium	????C	????A	????G	????G	????A	????G	????A	????G	????T	????A	????T	????G	????A	????C	????C	????A	????T	????G
Clostridium perfringens	????C	????A	????A	????G	????A	????C	????A	????A	????A	????A	????T	????T	????A	????C	????T	????C	????A	????A

Table 7

Bacillus cereus	G	?C	?A	?A	?A	?G	?A	?A	?A	?T	?A	?T	?A	?A	?T	?A	?C	?A	?A	?C
																					Bacillus megaterium	G	?A	?A	?A	?A	?A	?A	?A	?A	?T	?A	?T	?A	?A	?T	?A	?C	?A	?A	?C
Bacstearothermophilus	G	?C	?C	?A	?A	?A	?A	?A	?A	?T	?A	?C	?A	?A	?C	?A	?C	?A	?A	?C
																					Bacillus thuringiensis	G	?C	?A	?A	?A	?G	?A	?A	?A	?T	?A	?T	?A	?A	?T	?A	?C	?A	?A	?C
Bacillus sphaericus	G	?C	?A	?A	?A	?G	?A	?A	?A	?T	?T	?C	?A	?A	?T	?A	?C	?A	?A	?C
																					Bacillus anthracis	G	?C	?G	?A	?A	?G	?A	?A	?A	?T	?A	?T	?A	?A	?T	?A	?C	?A	?A	?C
Subtilis	G	?C	?C	?A	?A	?A	?A	?A	?A	?T	?T	?T	?A	?A	?C	?A	?C	?A	?A	?C
																					Clostridium baratii	G	?C	?A	?A	?A	?A	?A	?A	?A	?T	?A	?T	?A	?A	?T	?A	?C	?T	?A	?C
Clostridium innocuum	G	?C	?C	?A	?A	?G	?A	?A	?A	?T	?A	?T	?G	?C	?A	?A	?C	?C	?A	?C
																					Hot vinegar clostridium	G	?C	?C	?C	?G	?C	?A	?A	?G	?T	?A	?T	?A	?T	?G	?A	?C	?T	?A	?C
Clostridium perfringens	G	?C			?A	?G	?G	?C	?A	?T	?G	?C	?A	?A	?G	?G	?C	?T	?T	?T

According to the resulting data of sequence alignment, prepared new primer sets.This new primer sets comprises a forward primer and two reverse primers.New primer sets is:

5-CAAGAAGATGTGACGAAA-3 (SEQ ID NO.5) (forward),

5-GTTGTATTATATTTCTTTGC-3 (SEQ ID NO.6) (oppositely) and

5-GTTGTGTTAAATTTTTTGGC-3 (SEQ ID NO.7) (oppositely).

This primer sets can produce the spoOA product in distinctive SFB, but does not produce product in non-SFB.After the PCR, detect whether have positive findings, the band that it is 347-356 base pair that positive findings is shown as a size on sepharose with agarose gel electrophoresis.Table 8 is depicted as the result who comes from the characteristic SFB that is tested.

Table 8

Bacterial strain	The PCR product?
		Bacillus cereus	????+
Subtilis (ATCC 6051)	????+
		Subtilis (ATCC 23059)	????+
Bacillus megaterium	????+
		Bacstearothermophilus	????+
Bacillus licheniformis	????+
		Bacillus sphaericus	????+
Clostridium perfringens	????+
		Streptococcus aureus *	????-
Staphylococcus epidermidis *	????-
		Suppurative staphylococcus *	????-
Pseudomonas aeruginosa *	????-
		Klebsiella pneumonia *	????-

^*Non-SFB

In case determine that this new primer sets has the function of expection, promptly test to determine its sensing range.The process that is used for determining sensing range is similar to previous embodiment 2, but different.

1. 1% pulp sample (food grade wrapping plate) of 10ml is mixed with 40ml tryptic soy broth substratum, and placed 7 hours at 37 ℃.

In Eppendorf tube with the centrifugation of 4ml sample.

3. will precipitate with the washing of 100 μ l sterilized waters, and recentrifuge.

4. precipitation is resuspended in the 30 μ l sterilized waters, and boiled 5 minutes.

5. get the solution that 5 μ l boiled and be used for polymerase chain reaction.

As mentioned above, some samples incubation time that may need to reach 16 hours detects to optimize.(in sample, contain under the situation of the clay of high density or other pollutents, should need the long time.) if incubation time is 16 hours, only has the sample of 1ml precipitated.The long time can be brought up to detection level and can detect low 100 spores/every gram paper that reaches.

The sensing range of the primer sets of embodiment 3 is shown in following table 9.

Table 9

Sample	Spore/0.5g paper #	SpoOA PCR product?
			????A	????605±43	????+
????B	????590+90	????+
			????C	????520±50	????+
????D	????340±0	????+
			????E	????255±55	????-
????F	????175±15	????-

^#Determine with plate count

In table 9, sample A-F is the food grade wrapping plate of same type, has the SFB of different levels.

Embodiment 4-detects spore forming bacteria in paper-making process

In the process water in a box zone in paper mill, get the sample of 10ml.Each sample is mixed with 40ml tryptic soy broth substratum respectively.Hatched 7 hours, sample is centrifugal so that the bacterium composition concentrates.Remove supernatant, will precipitate resuspended.

Resuspended sample is boiled with the cracking bacterium, and mix with primer cracked sample cooling back, then this testing mixture is placed the PCR thermal cycler.The operation thermal cycler carries out electrophoresis with the PCR product on sepharose, with bromination second pyridine dyeing, observe under the UV-light.

If show the excessive number of spore forming bacteria, then add biocide with kill bacteria.

Embodiment 5-detects spore forming bacteria in food processing process

Before and after pasteurization, respectively get the milk sample of processing of 10ml.Also regular draw samples is checked in packaged product.Every part of 10ml sample to be measured is mixed with 40ml tryptic soy broth substratum respectively.Hatched subsequently 7 hours, sample is centrifugal to concentrate the bacterium composition.Remove supernatant, will precipitate resuspended.

Resuspended sample is boiled with the cracking bacterium, and mix with primer cracked sample cooling back, then this testing mixture is placed the PCR thermal cycler.The operation thermal cycler carries out electrophoresis with the PCR product on sepharose, with bromination second pyridine dyeing, observe under the UV-light.

According to the result who measures, can in the suitable link of production process, take adequate measures to remove spore forming bacteria.

Embodiment 6-detects spore forming bacteria in biological sample

In movement to be measured, take out the 100mg sample.Every part of 100mg sample to be measured is mixed with 50ml tryptic soy broth substratum respectively.Hatched subsequently 7 hours, sample is centrifugal to concentrate the bacterium composition.Remove supernatant, will precipitate resuspended.

Resuspended sample is boiled with the cracking bacterium, and mix with primer cracked sample cooling back, then this testing mixture is placed the PCR thermal cycler.The operation thermal cycler carries out electrophoresis with the PCR product on sepharose, with bromination second pyridine dyeing, observe under the UV-light.

According to the result who measures, leave the effective antibiotic prescription that the treatment spore forming bacteria infects.

In embodiment 4,5 and 6 (and in the embodiment at other), pollutent can influence the detectivity of this measuring method to spore forming bacteria.For example, if having clay or some enzymes in the sample, may influence polymerase chain reaction.In this case, suggestion is diluted raw sample, can not exert an influence up to the concentration of pollutent again.

Equally,, it should be noted that,, also can adopt other method although it has taught the method as the hybridization between detection probes and the purpose sample with PCR for embodiment 4,5 and 6.For example, probe can be connected to a kind of fluorescence (other are detectable) molecule, again with sample mix.Through hybridization, and under appropriate condition, the molecule of this mark can discharge a kind of detectable energy, for example fluorescence.

According to the content of above specification sheets, those skilled in the art can determine essential feature of the present invention easily, and in the case of without departing from the spirit and scope, can make various changes and improvement to the present invention, so that it is suitable for various uses and condition.

For example, be to screen with the ability that the goal gene of SFB is hybridized at the nucleotide sequence of the present invention that this disclosed according to it.Be specially, the nucleotide sequence of this aspect at be conservative purpose genetic material among the SFB.Therefore, should think that other belong within the scope of the present invention with the nucleotide sequence that the purpose zone of SFB gene combines.

But, it should be understood that betide the base in the nucleotide sequence of the present invention replacement still can so that itself and goal gene hybridize.Therefore, this type of replacement still belongs within the scope of the present invention, does not have substantial difference and can not cause between itself and the present invention.

In addition, as implied above, nucleotide sequence of the present invention can be combined with other nucleotide sequence, and still can obtain same result.Embodiment 1 and 2 has confirmed this effect, wherein only one of two primers has been carried out improvement and can improve detection level.Therefore, will be understood that the applied in any combination between nucleotide sequence of the present invention and other nucleotide sequences also belongs to this

Within the scope of invention.

Sequence table

＜110〉Hercules Inc

＜120〉detection of spore forming bacteria

<130>B1113P-PCT

<160>8

<170>PatentIn?version?3.0

<210>1

<211>20

<212>DNA

＜213〉bacillus cereus

<400>1

agtatcattc?atgaaattgg

20

<210>2

<211>16

<212>DNA

＜213〉bacillus cereus

<400>2

aaaaaagcag?ttgact

16

<210>3

<211>17

<212>DNA

＜213〉bacillus cereus

<400>3

cggcttgccg?ttgtatt

17

<210>4

<211>18

<212>DNA

＜213〉bacillus cereus

<400>4

gaagatgtga?cgaaaaag

18

<210>5

<211>18

<212>DNA

＜213〉bacillus cereus

<400>5

caagaagatg?tgacgaaa

18

<210>6

<211>20

<212>DNA

＜213〉bacillus cereus

<400>6

gttgtatctat?atttctttgc

20

<210>7

<211>20

<212>DNA

＜213〉bacillus cereus

<400>7

gttgtgttaa?attttttggc

20

<210>8

<211>26

<212>DNA

＜213〉bacillus cereus

<400>8

agtatcattc?atgaaattgg?cgttcc

26

Claims (40)

1, a kind ofly is used for the probe whether test sample exists spore forming bacteria, described probe comprises a nucleotide sequence, described nucleotide sequence can be with following (a) or the high conservative of the spoOA gene of the bacterial classification of spore forming bacteria bacillus (b) and fusobacterium zone form detectable crossbred

A) bacillus cereus

Bacillus megaterium

Bacillus anthracis and

Clostridium baratii;

B) bacillus cereus

Bacillus megaterium

Bacillus sphaericus and

Clostridium baratii,

Described nucleotide sequence can not form detectable crossbred with the genetic material of non-spore forming bacteria.

2, the probe of claim 1, the high conservative zone of spore forming bacteria bacillus in wherein said (a) and the spoOA gene of fusobacterium is

Bacillus cereus G A A G A T G T G A C G A A A A A A G

Bacillus megaterium G A A G A C G T A A C G A A A A A A G

Bacillus anthracis G A A G A T G T G A C G A A A A A A G

The high conservative zone of the spoOA gene of Clostridium baratii G A C A A A A T T A C T C A A A G A G and spore forming bacteria bacillus (b) and fusobacterium is

Bacillus cereus A A T A C A A C A G C A A G C C G

Bacillus megaterium A A T A C A A C G G C A A G C C G

Bacillus sphaericus A A T A C A A C A C C G T C A C G

Clostridium baratii A A T A C T A C T G C A A G C C G

3, a kind ofly be used for the probe whether test sample exists spore forming bacteria, described probe comprises a nucleotide sequence, described nucleotide sequence can be with following (a) or the high conservative of the spoOA gene of the bacterial classification of spore forming bacteria bacillus (b) and fusobacterium zone form detectable crossbred

A) bacillus cereus

Bacillus megaterium

Subtilis and

Clostridium baratii;

B) bacillus cereus

Bacillus megaterium

Bacillus sphaericus and

Clostridium baratii,

Described nucleotide sequence can not form detectable crossbred with the genetic material of non-spore forming bacteria.

4, the probe of claim 3, the high conservative zone of spore forming bacteria bacillus in wherein said (a) and the spoOA gene of fusobacterium is

Bacillus cereus C A A G A A G A T G T G A C G A A A

Bacillus megaterium C A A G A A G A C G T A A C G A A A

Subtilis C A G G A A G A T G T C A C G A A A

The high conservative zone of the spoOA gene of Clostridium baratii C A A G A C A A A A T T A C C A A A and spore forming bacteria bacillus (b) and fusobacterium is

Bacillus cereus G C A A A G A A A T A T A A T A C A A C

Bacillus megaterium G A A A A A A A A T A T A A T A C A A C

Bacillus sphaericus G C A A A G A A A T T C A A T A C A A C

Clostridium baratii G C A A A A A A A T A T A A T A C T A C

5, each probe in the claim 1 to 4, wherein said nucleotide sequence comprise at least a sequence among SEQID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and the SEQ ID NO.8.

6, each probe in the claim 1 to 5, wherein said nucleotide sequence is as the primer of the polymerase chain reaction of the described detectable crossbred that is used for increasing.

7, each probe in the claim 1 to 5, wherein said nucleotide sequence with a kind of fluorescent derivative mark to be used to detect described detectable crossbred.

8, each probe in the claim 1 to 5, wherein said nucleic acid with a kind of labelled with radioisotope to be used to detect described detectable crossbred.

9, a kind ofly be used for the probe whether test sample exists spore forming bacteria, described probe comprises a nucleotide sequence, described nucleotide sequence can form detectable crossbred with the spoOA gene of spore forming bacteria and can not form detectable crossbred with the genetic material of non-spore forming bacteria, wherein said nucleotide sequence can form detectable crossbred with the 76th to the 93rd base of the spoOA gene of bacillus cereus, and the spoOA gene of described bacillus cereus is equivalent to GenBank registration number gb U09972.

10, the probe of claim 9, wherein said nucleotide sequence comprise at least a sequence among SEQ ID NO.4 and the SEQ ID NO.5.

11, a kind ofly be used for the probe whether test sample exists spore forming bacteria, described probe comprises a nucleotide sequence, described nucleotide sequence can form detectable crossbred with the spoOA gene of spore forming bacteria and can not form detectable crossbred with the genetic material of non-spore forming bacteria, wherein said nucleotide sequence can form detectable crossbred with the 403rd to the 422nd base of the spoOA gene of bacillus cereus, and the spoOA gene of described bacillus cereus is equivalent to GenBank registration number gb U09972.

12, the probe of claim 11, wherein said nucleotide sequence comprise at least a sequence among SEQ ID NO.3, SEQ ID NO.6 and the SEQ ID NO.7.

13, a kind ofly be used for the probe whether test sample exists spore forming bacteria, described probe comprises a nucleotide sequence, described nucleotide sequence can form detectable crossbred with the spoOA gene of spore forming bacteria and can not form detectable crossbred with the genetic material of non-spore forming bacteria, wherein said nucleotide sequence can form detectable crossbred with the 70th to the 427th base of the spoOA gene of bacillus cereus, and the spoOA gene of described bacillus cereus is equivalent to GenBank registration number gb U09972.

14, a kind ofly be used for the probe whether test sample exists spore forming bacteria, described probe comprises a nucleotide sequence, described nucleotide sequence can form detectable crossbred with the spoOA gene of spore forming bacteria and can not form detectable crossbred with the genetic material of non-spore forming bacteria, wherein said nucleotide sequence can form detectable crossbred with the 570th to the 930th base of the spoOA gene of subtilis, and the spoOA gene of described subtilis is equivalent to gb M10082.

15, each probe in the claim 1,3,9,11,13 or 14, the part of spoOA gene is one detectable to produce, length is the DNA product of 346 to 365 Nucleotide wherein to use this probe to increase in the cell DNA of spore forming bacteria as a member of a primer sets, by polymerase chain reaction.

16, primer sets, it comprises at least a sequence that is selected among SEQ ID NO.1, SEQ ID NO.2, SEQID NO.3, SEQ ID NO.4 and the SEQ ID NO.8.

17, the primer sets of claim 16, wherein second member is selected from SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7.

18, a kind of nucleotide sequence, it comprises a kind of sequence that is selected among SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and the SEQ ID NO.8.

19, a kind of primer, it is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.8.

20, a kind of composition, it comprises material and at least one primer of at least a cellulose, and described primer comprises a kind of sequence that is selected among SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO, SEQ ID NO.5, SEQ ID NO.6, SEQ IDNO.7 and the SEQ ID-NO.8.

21, the composition of claim 20, the material of wherein said at least a cellulose comprises paper pulp.

22, be used for whether existing in the paper-making process test test kit of spore forming bacteria, it comprises

A) primer, described primer comprise a kind of sequence that is selected among SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ IDNO.6, SEQ ID NO.7 and the SEQ ID NO.8, and

B) substratum of at least a support spore forming bacteria growth.

23, preparation is used for detecting the method whether spore forming bacteria exists the nucleotide sequence of conservative gene, it is characterized in that

A) determine to come from the high conservative zone of the spoOA gene of at least three kinds of spore forming bacteria genus bacillus bacterial classifications and a kind of spore forming bacteria clostridium species; And

B) at least a forward of preparation and at least a inverse kernel thuja acid primer sequence, described nucleotide primer sequence can with this spoOA gene conservative area hybridization of spore forming bacteria, but, wherein use these primer sequences, in the cell DNA of spore forming bacteria, increase the part of spoOA gene to produce the DNA product that a length is 346 to 365 Nucleotide by polymerase chain reaction with the hybridization of non-spore forming bacteria.

24, the method for claim 23, the spoOA gene region that wherein is used for preparing the described high conservative of three kinds of genus bacillus bacterial classifications of forward primer and a kind of clostridium species is

Bacillus cereus C A A G A A G A T G T G A C G A A A

Bacillus megaterium C A A G A A G A C G T A A C G A A A

Subtilis C A G G A A G A T G T C A C G A A A

The spoOA gene region that Clostridium baratii C A A G A C A A A A T T A C C A A A and being used for prepares the described high conservative of three kinds of genus bacillus bacterial classifications of reverse primer and a kind of clostridium species is

Bacillus cereus G C A A A G A A A T A T A A T A C A A C

Bacillus megaterium G A A A A A A A A T A T A A T A C A A C

Bacillus sphaericus G C A A A G A A A T T C A A T A C A A C

Clostridium baratii G C A A A A A A A T A T A A T A C T A C

25, a kind of system that is used to identify spore forming bacteria, it comprises:

A) make the DNA of spore forming bacteria be easy to the device with at least a nucleotide probe hybridization;

B) at least a being used for detected the nucleotide probe that whether has spore forming bacteria at sample, described probe comprises a nucleotide sequence, described nucleotide sequence can with following i) or ii) in the spore forming bacteria bacillus and the high conservative zone of the spoOA gene of the bacterial classification of fusobacterium form detectable crossbred

I) bacillus cereus

Bacillus megaterium

Subtilis and

Clostridium baratii;

Ii) bacillus cereus

Bacillus megaterium

Bacillus sphaericus and

Clostridium baratii,

Described nucleotide sequence can not form detectable crossbred with the genetic material of non-spore forming bacteria; And

C) be used to detect the device of the hybridisation events of the DNA of described spore forming bacteria and described at least a nucleotide probe.

26, the system of claim 25, the spoOA gene region that wherein is used for preparing the described high conservative of three kinds of genus bacillus bacterial classifications of forward primer and a kind of clostridium species is

Bacillus cereus C A A G A A G A T G T G A C G A A A

Bacillus megaterium C A A G A A G A C G T A A C G A A A

Subtilis C A G G A A G A T G T C A C G A A A

The spoOA gene region that Clostridium baratii C A A G A C A A A A T T A C C A A A and being used for prepares the described high conservative of three kinds of genus bacillus bacterial classifications of reverse primer and a kind of clostridium species is

Wax shape bud bag bacillus G C A A A G A A A T A T A A T A C A A C

Bacillus megaterium G A A A A A A A A T A T A A T A C A A C

Bacillus sphaericus G C A A A G A A A T T C A A T A C A A C

Clostridium baratii G C A A A A A A A T A T A A T A C T A C

27, the system of claim 25, wherein said probe can make and use this probe as a member of a primer sets, increase the part of spoOA gene to produce the DNA product that a length is 346 to 365 Nucleotide in the cell DNA of spore forming bacteria by polymerase chain reaction.

28, the system of claim 25, wherein said at least a nucleotide probe comprises a kind of sequence that is selected among SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQID NO.5, SEQ ID NO.6, SEQ ID NO.7 and the SEQ ID NO.8.

29, the system of claim 25, the wherein said device that is used to detect hybridisation events does not comprise the use polymerase chain reaction.

30, be used for the method whether working sample exists spore forming bacteria, it comprises:

A) with at least two nucleotide primers and sample mix,

Wherein said nucleotide primer

I) be complementary at least a forward and at least a inverse kernel acid sequence of the total cell dna that comes from described bacterium,

Ii) can hybridize, but not hybridize with non-spore forming bacteria with this type of spoOA gene conservative region of spore forming bacteria, and

Iii) can make and use this nucleotide primer, the amplification of the part of the spoOA gene of the cell DNA that comes from spore forming bacteria produced the DNA product that a length is 346 to 365 Nucleotide by polymerase chain reaction; And

B) cell DNA of the bacterium in the described sample of usefulness primer amplification; And

C) detect the DNA that whether has amplification.

31, the method for claim 29, wherein said at least two nucleic acid primers comprise at least a sequence among SEQ IDNO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and the SEQ ID NO.8.

32, the method for claim 29, wherein the another one primer is selected from SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7.

33, the method for claim 29, wherein said sample comprise at least a in air, soil or the water.

34, the method for claim 29, wherein said sample comprise at least a in blood and the movement.

35, the method for claim 29, wherein said sample comprise at least a in starch, protein and a kind of Epicholorohydrin reaction product.

36, a kind of method that is used for the sporulation gene of systematicness evaluation spore forming bacteria, it comprises

A) with the claim 1 of mark or 2 probe and sample mix;

B) with the spoOA gene recombination of described probe and purpose spore forming bacteria; And

C) the hybridization product is detected.

37, a kind of method that is used for the sporulation gene of systematicness evaluation spore forming bacteria, it comprises

A) with each probe and sample mix in the claim 9,11,13 and 14 of mark;

B) with the spoOA gene recombination of described probe and purpose spore forming bacteria; And

C) the hybridization product is detected.

38, claim 36 or 37 method, wherein said probe is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ IDNO.6, SEQ ID NO.7 and SEQ ID NO.8.

39, each probe in the claim 36 to 38, wherein said nucleotide sequence with a kind of fluorescent derivative mark to be used to detect detectable crossbred.

40, each probe in the claim 36 to 38, wherein said nucleic acid with a kind of labelled with radioisotope to be used to detect detectable crossbred.