CN103060926A - Enzyme digestion based preparation method of bacteria nucleic acid fingerprint characteristic spectrums and application thereof - Google Patents
Enzyme digestion based preparation method of bacteria nucleic acid fingerprint characteristic spectrums and application thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing bacteria nucleic acid fingerprint spectrums. The method comprises the steps of PCR (polymerase chain reaction) amplification, SAP (super absorbent polymer) enzymatic digestion, transcription and nuclease digestion, purification, mass spectrometer detection and the like. A nucleic acid fingerprint spectrum database of common bacteria is created based on the method. According to the mass spectrum peak chart generated through experiments, the bacteria to be detected can be classified and identified and the results can be widely applied to the fields of bacteria classification and identification, genetic evolution analysis, drug resistance screening application, import and export inspection and the like.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method of bacterial nucleic acid finger printing preparation, and use the method, bacterium is carried out the method for classification and identification.
Background technology
The classification of early stage bacteriology mainly is the traditional bacteriology sorting technique that adopts take form cultural characteristic and physiological and biochemical property as foundation.But because this surface characteristic of bacterium often is subjected to impact and the restriction of a lot of human factors, particularly the difference of people's subjective judgement is difficult to reflect the natural sibship of bacterium on the contrary, and this has just embodied the limitation that only is based upon the traditional taxonomy on morphological specificity and the physiological and biochemical property basis.
Therefore, Colweel has proposed a new division bacteria technics " polyphase sort (Polyphasic taxonomy) ", and it refers to fully utilize the multiple different information of microorganism, comprises the method that phenotype and genotypic information are classified.Molecular classification and phylogeny abundant information the content of polyphase sort, it is close towards natural classification system jointly to promote division bacteria.The molecular indexes that is applied to classify is mainly DNA and RNA, and the dna homology analysis is to determine correct classification position, sets up the most direct method of natural classification system.Method is simple for this type of, and resolving power is high, plays a part more and more important in bacterial systematics research.
The principle of using mass-spectrometric technique to carry out division bacteria and evaluation is, the elementary cell that forms hereditary material DNA---there is mass discrepancy between four kinds of Nucleotide, upper certain or certain the several fragments of bacterial genomes DNA are carried out after PCR and enzyme cut, molecular weight and the different fragment of abundance will be produced, use mass spectrometric detection can produce the nucleic acid finger printing, genomic dna there are differences between the different bacterium, different nucleic acid finger printings will be produced, behind the building database, can experimental result and database compare, can finish the evaluation to bacterium.
More existing open source literatures use mass-spectrometric techniques are classified to microorganism and are identified, for example, Chinese patent application CN102337223A, " penicillium chrysogenum antifungal protein Pc-Arctin and preparation method thereof ", a kind of MALDI-TOF authentication method that detects penicillium chrysogenum antifungal protein Pc-Arctin is disclosed, wherein picking Penicllium chrysogenum A096 spore inoculating is cultivated in the SGY liquid nutrient medium from the flat board, pre-treatment obtains the separation and purification on chromatographic column of crude protein solution, and separation and purification on carboxymethyl cation-exchange chromatography post, collect each elution fraction, each component centrifugal ultrafiltration is concentrated into volume required, take paecilomyces varioti as responsive tested indicator, follow the trail of the anti-mycotic activity component, definite activeconstituents is judged the purity that obtains albumen; Extract the single band on the SDS-PAGE electrophorogram, carry out MALDI-TOF and identify.The method is only applicable to specified microorganisms, and needs the multiplexed protein purge process, finally uses MALDI-TOF identification mark albumen Pc-Arctin, and its process is loaded down with trivial details, applicable surface is narrow, can not realize the purpose of mass spectral classification bacterium or microorganism.
Chinese patent application 200910157210, " analytical procedure of fatty acid component in a kind of listeria cell " discloses a kind of method of utilizing gaschromatographic mass spectrometry (GC-MS) analytical method directed toward bacteria lipid acid to classify, comprise: the listeria bacteria rejuvenation, separate respectively and the purifying listeria bacteria with Oxford agar plate and trypticase soy yeast extract agar flat board, cultivate the listeria bacteria of single colonies typical and make bacteria suspension, process with formalin-inactivated, bacteria suspension average mark after formaldehyde treated is contained in the centrifuge tube washing, with the mixed solution esterification of hydrochloric acid and methyl alcohol, the lipid acid that makes is carried out gaschromatographic mass spectrometry (GC-MS) analysis.The limitation of systematic bacteriology although the method breaks traditions, reduce the human factor error that classification brings to traditional form, identify the strong instrument that provides for the classification of new bacterial classification and seed culture of viruses simultaneously, utilize mass spectroscopy to carry out the CYTOCHEMICAL ANALYSIS classification but this method still belongs to, do not detect for nucleic acid.
International Patent Application WO 2010/021548, " Method for identifying biological material e.g. bacteria in sample of patient; involves separating stream of liquid containing sample into successive portions to form flying drops and ionizing flying drops to measure mass spectra ", the method and apparatus that a kind of use MALDI-MS (substance assistant laser desorpted and MALDI-MS) is used for the identification biomaterial is disclosed, comprise and prepare to comprise the liquid of sample and MALDI substrate material, and use it for the continuous a fluid stream that forms liquid.This a fluid stream is dispersed in succession part, is transmitted into aloft drop with formation, or a fluid stream is transmitted into in-flight, then be dispersed into drop.Can use drop formation technology known from ink-jet printer.Aloft drop is ionized out material.Measurement is from the mass spectrum of the ionization material of each drop.But the method purpose is how to improve the sensitivity of MALDI-MS detection of biological material, does not relate to Mass Spectrometric Identification and any microorganism of classification, therefore can not solve the problems of the technologies described above.
The people such as Zhu Jian (" high performance liquid chromatography-electrospray multi-stage mass method is to principium identification and the classification of each component in the geldanamycin crude product ", " Chinese microbiotic ", 03 phase in 2011) reported that application high performance liquid chromatography-electrospray multi-stage mass method (LC-ESI-MSn) carries out principium identification and the classification of the structural information aspect relevant with total mass number to each component in geldanamycin (GDM) crude product.The analysis and arrangement that the method is carried out for the multi-stage ms fragment of different components in the geldanamycin (GDM), and various compounds have been carried out accurate classification, thus but do not relate to the directed toward bacteria predetermined substance and detect the method that bacterium is classified.
Bang flood (" application of MALDI TOF MS in Bacteria Detection and evaluation ", " microbiology immunology progress ", 02 phase in 2003) reported " bacterial body contains chemical classification and the evaluation that a large amount of Biomarkers can be used for bacterium; for obtaining the finger printing detection such as the moiety according to bacterium and identifying bacterium ", and the method has been predicted its theoretic feasibility.Yet this research only is to have inquired in theory the direction of utilizing MALDI TOF MS that bacterium is classified, and its which kind of component that had not both indicated the institute directed toward bacteria detects, and concrete research method and process are not described yet.Because component (such as albumen, DNA, RNA, the polysaccharide etc.) kind that can be used for classifying in the bacterium is too many, and mass-spectrometric technique also exists combination and the selection of various experiment parameters for different determinands, does not therefore utilize MALDI TOF MS to carry out the new report of division bacteria in after this nearly 10 years.
As immediate prior art, Chinese patent application 201110154723, " MALDI TOF MS assistant identification singly increases the method for listeria spp " and 201110154469, " method of MALDI TOF MS assistant identification vibrio cholerae " discloses the method for a kind of MALDI of utilization TOF MS technology assistant identification bacterium, comprise: the pre-treatment bacterial cultures, gather the MALDI TOF MS collection of illustrative plates of all bacterial strain samples, prepare the bacterium standard diagram according to software, use identical method to detect and gather the collection of illustrative plates of tested bacteria, and compare the two collection of illustrative plates, judge according to the coupling mark.Because the method uses conventional processing (to pass through dehydrated alcohol, formic acid and acetonitrile treatment, and be aided with centrifugal, drawing at last supernatant liquor detects), although it can characterize the characteristic spectrum of this bacterium to a certain extent, but owing to contain protein in its determinand, lipid, lipopolysaccharides and fat oligosaccharides, DNA, polypeptide and the ionizable molecule of other energy, the collection of illustrative plates set that its collection of illustrative plates that obtains is in fact above-mentioned various molecules, therefore both needed the collection of illustrative plates quantity of information processing and compare excessive, and cause its collection of illustrative plates characteristic on the low side because molecule to be checked is too huge, be only applicable to certain concrete bacterium and can't be generalized in other a large amount of Bacteria Detection.
Therefore, need at present a kind of can directed toward bacteria in certain specific molecule to be checked carry out mass spectrometric detection, the method can generally be used for again mass spectral classification and the evaluation of most bacteriums simultaneously.
Summary of the invention
The principle of the invention is: the contriver is through groping in a large number and comparing, research for 841 kinds of bacteriums, after discovery selected universal primer that DNA of bacteria is carried out pcr amplification, the use specific enzymes was carried out mass spectrometric detection and can be obtained various bacterial nucleic acid fingerprint characteristic collection of illustrative plates after amplified production is processed.
Therefore, the present invention's the first purpose provides a kind of bacterial nucleic acid finger printing preparation method who cuts based on enzyme.It is characterized in that, which comprises at least following steps:
(1) PCR reaction: use the PCR universal primer of directed toward bacteria, the nucleic acid-templated of a plurality of bacteriums of increasing respectively obtains containing the PCR product of target area of increasing;
(2) SAP enzymic digestion: the PCR product that obtains with alkaline phosphatase (SAP enzyme) treatment step (1);
(3) transcribe with nuclease and cut: use and specifically transcribe and restriction endonuclease, respectively in a reaction system, the digestion product of each bacterium that step (2) is obtained is transcribed with nuclease and is cut, and obtains the nucleic acid fragment that a series of length differ, abundance differs;
(4) purifying: the enzyme that uses resin treatment step (3) to obtain is cut product;
(5) mass spectrograph detects: point is containing on the target sheet of matrix on the purified product that step (4) is obtained, and upper mass spectrograph detects, and obtains the feature nucleic acid fingerprint chromatogram of different bacterium.
In one embodiment, the bacterial nucleic acid sequence that the PCR reaction is increased is including but not limited to zone on the DNA of bacteria genome.In another embodiment, the zone on the described genome is selected from the zone shown in the SEQ ID NO:3, perhaps has the sequence of at least 60% homology with sequence shown in the SEQ ID NO:3.In the preferred embodiment therein, preferably this sequence has the sequence of 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.
In one embodiment, described universal primer includes but not limited to sequence shown in SEQ ID No:1 to the SEQ ID No:2.
In another embodiment, the described certain enzyme of step 3 includes but not limited to the RNaseA enzyme.In a specific embodiments, the purifying of step 5 is included in to transcribe to cut with enzyme and adds ultrapure water in the product, behind the mixing, adds resin again, and mixing 15 minutes turns upside down.In another embodiment, wherein said mass spectrograph is MALDI TOF MS mass spectrograph.
In above-mentioned arbitrary scheme, wherein said bacterium comprises the 836 kind bacteriums listed such as table 1.
Table 1
In above-mentioned arbitrary scheme, wherein said bacterium also comprises brucella (Brucella), mycobacterium tuberculosis (Mycobacterium tuberculosis), helicobacter pylori (Helicobacter pylori, Hp).Wherein, brucella comprises brucella melitensis (Brucella melitensis), B. abortus (Brucella abortus), pig kind brucella (Brucella suis).
The second purpose of the present invention provides the method for setting up bacterial nucleic acid fingerprint characteristic spectrum library, comprises at least: above-mentioned steps 1-5, and;
(6) the nucleic acid fingerprint characteristic collection of illustrative plates of the various bacteriums that step 5 obtained gathers and puts in order by computer software, obtains described bacterial nucleic acid fingerprint characteristic spectrum library.
In one embodiment, described software is the BioExplore software that the contriver researchs and develops voluntarily, and its copyright number is soft work step on word No. 136879, registration number 2009SR10700.
The 3rd purpose of the present invention provides a kind of method of utilizing above-mentioned characteristic spectrum storehouse to carry out Bacteria Identification, comprising:
(1) PCR reaction: use the PCR universal primer of directed toward bacteria, amplification tested bacteria nucleic acid-templated obtains containing the PCR product of target area of increasing;
(2) SAP enzymic digestion: the PCR product that obtains with alkaline phosphatase (SAP enzyme) treatment step (1);
(3) transcribe with nuclease and cut: use and specifically transcribe and restriction endonuclease, in a reaction system, the digestion product of the bacterium that step (2) is obtained is transcribed with nuclease and is cut, and obtains the nucleic acid fragment that a series of length differ, abundance differs;
(4) purifying: the enzyme that uses resin treatment step (3) to obtain is cut product;
(5) mass spectrograph detects: point is containing on the target sheet of matrix on the purified product that step (4) is obtained, and upper mass spectrograph detects, and obtains the nucleic acid fingerprint characteristic collection of illustrative plates of this bacterium;
(6) gained nucleic acid fingerprint characteristic collection of illustrative plates and bacterial nucleic acid fingerprint characteristic spectrum library are compared, thus classification or the kind of judgement tested bacteria.
In one embodiment, the bacterial nucleic acid sequence that the PCR reaction is increased is including but not limited to zone on the DNA of bacteria genome.In another embodiment, the zone on the described genome is selected from the zone shown in the SEQ ID NO:3, perhaps has the sequence of at least 60% homology with sequence shown in the SEQ ID NO:3.In the preferred embodiment therein, preferably this sequence has the sequence of 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.
In one embodiment, described universal primer includes but not limited to sequence shown in SEQ ID No:1 to the SEQ ID No:2.
In another embodiment, the described certain enzyme of step 3 includes but not limited to the RNaseA enzyme.In a specific embodiments, wherein said mass spectrograph is MALDI TOF MS mass spectrograph.In another embodiment, step 6 compares detection by BioExplore software.
The 4th purpose of the present invention provides and can screen the test kit of purposes for division bacteria and evaluation, resistance, comprising:
(1) universal primer that is used for the amplification DNA of bacteria to and damping fluid;
(2) SAP enzyme and damping fluid thereof;
(3) RNAase and damping fluid thereof;
(4) be used for the resin that purifying enzyme is cut product;
(5) for the analysis software of comparing nucleic acid fingerprint characteristic collection of illustrative plates.
In one embodiment, wherein said primer is SEQ ID NO:1-2.
In another embodiment, wherein said software is BioExplore software, and its copyright number is (soft work is stepped on word No. 136879, registration number 2009SR10700).
Definition
" division bacteria and evaluation " of the present invention, comprise to bacterium identify and identification, somatotype, minute kind, classification.For example in food safety detection, by division bacteria or evaluation, can accurately identify source of pollution and bacterium and form, ensure food safety in order to adopting rational approach.And for example, in the evolution of research bacterium, by identification and the classification/somatotype to bacterium, can determine the far and near relation of relationship between the various bacterium kinds.
Zone on the DNA of bacteria genome of the present invention preferably has the zone that high conservative has again certain polymorphism simultaneously.Because bacterium belongs to unicellular lower eukaryote, generally is rendered as the conservative property of certain homology in the genome of various bacteriums, so arbitrary relatively conservative zone in the directed toward bacteria genome, use universal primer can amplify the nucleic acid region that various bacteriums correspond to each other.The dna fragmentation enzyme of different microorganisms is cut, the collection of illustrative plates that takes on a different character, thereby the actual nucleic acid mass-spectrogram carried out bioinformatic analysis, can realize the classification and identification of bacterium.
Conservative region of the present invention or conservative fragments, preferably bacterium rRNA zone.RRNA is the important indicator of the evolution of research bacterium and sibship, its content reaches 80%, and be present in all bacteriums, the rRNA gene is comprised of conserved regions and variable region, conservative at the bacterium camber, have the title of " bacterial fossil ", is that bacterial systematics is learned useful and the most the most frequently used molecular clock in the research.Wherein, 16S rDNA sequence is because molecular size moderate (about 1.5kb), mutation rate is extremely low, become vital signs (the Olsen GJ that division bacteria and kind are identified, Pace NR, Nuell M, et al. Nucleic Acids Res, 1991,19 (supp l): 2017-2021.).The bacterium kind is identified and the standard method of classification although at present 16S rDNA sequential analysis has become, and the 16S rDNA complete sequence of about 2500 kinds is in the news, and according to their sequence homology, has made up the phylogenetic tree of each kind.But because the high conservative of 16S rDNA sequence in prokaryotic organism is relatively poor for the discriminating resolving power between the different strains in close kind or the same.
The method of in addition, at present carrying out division bacteria and evaluation with 16S rDNA mainly is to adopt PCR product direct Sequencing, the method that result and database are compared.Sequencing is applied to clinical detection, and there are the following problems at present: (1) cost is high; (2) consuming time; (3) for mixing sample, order-checking easily produces the cover peak, is difficult to effectively distinguish; (4) more than the 16S rDNA total length 1.5kb, generally need to splice through twice order-checking and with the result, in this process, easily introduce error.
As previously mentioned, 16S rDNA identifies division bacteria and has great importance, but high, the length consuming time of method testing cost such as tradition order-checking; Sample for polyinfection, the sequence peak figure that sequencing will obtain mixing, be difficult to effectively distinguish, and utilize mass spectrum to carry out analyte analysis, need to select suitable determinand and optimize the mass spectrum parameter, therefore need at present new division bacteria technology (such as mass spectroscopy) realize fast, accurately, cheapness, classification results easily.
" universal primer " of the present invention is the upstream and downstream that can be positioned at the zone to be amplified of various bacterial genomes, the primer of the respective segments that can increase in the different bacterium genome.Wherein, zone to be amplified on the genome of the present invention is selected from the sequence that has at least 60% homology with sequence shown in the SEQ ID NO:3, and preferably this sequence has the sequence of 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.Because for DNA of bacteria (for example 16S rDNA), its sequence is comprised of conserved regions and variable region usually, and the variable region mostly is discontinuous or short-movie section even SNP form are mixed in the middle of the conserved regions or two ends, therefore uses the universal primer respective segments that can increase in the different bacterium genome.Because this fragments of various bacteriums all has certain homology, thus have in the fragment of at least 60% homology with sequence shown in the SEQ ID NO:3, through enzyme cut with mass spectrum after can obtain the nucleic acid fingerprint characteristic collection of illustrative plates of tested bacteria.
In one embodiment, the specific region of bacterial 16 S rDNA is bacterial 16 S rDNA sequence C TGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCC TCTCAGGTCGGCTATGCATCGTTGCCTTGGTAGGCCATTACCCTACCAACTAGCTA ATGCACCGCGGGCCCATCTGTAAGCGATAGCCGAAACCATCTTTCAAAAGCGTGGC ATGCGCCACACTTTATCATTCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCCAAC TTACAGGCAGGTTGCCCACGTGTTACTCACCCGTCCGCCACTAACTTTGGAAGAGC AAGCTCTTCCTCCGTTCGTTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGT CCTGAGCCAGGATCAAACTCT, be SEQ ID NO:3(listeria bacteria, 358bp).In a specific embodiments, the primer of this specific region is SEQ ID No:1 and SEQ ID No:2.
In another embodiment, relate in the step 5 and use BioExplore software to carry out data analysis, and the database of Criterion bacterial strain.
In another embodiment, described certain enzyme of cutting for nuclease is the RNaseA enzyme.
Also in another embodiment, described mass spectrum is the MALDI-TOF mass spectrum.
Beneficial effect
1, the present invention's enzyme of repeatedly studying 836 kinds of bacteriums is cut and the mass spectrometric detection result, find and obtain various bacterial nucleic acid fingerprint characteristic collection of illustrative plates, can effectively identify the existence of various bacteriums, and not be subject to the interference of the non-bacterium microbe (such as fungi, virus etc.) similar to bacterial genomes.
2, owing to the high sensitivity of mass spectrometric detection, the detection lower limit that uses this programme that whether bacterium is existed can far exceed other technologies scheme (such as biochemistry detection, order-checking detection etc.);
3, for different samples, the present invention can compare the nucleic acid finger printing that they produce, and the nucleic acid finger printing of experiment generation and the collection of illustrative plates of database Plays bacterial strain is compared, through bioinformatic analysis, can judge to belong to which kind, or far and near with the sibship of a certain kind;
4, for the bacterium of all kinds of sudden changes of easy generation, the collection of illustrative plates of not mutated strain compares in the nucleic acid finger printing that experiment is produced and the database, through bioinformatic analysis, can judge whether to exist and suddenly change;
5, the sample that mixes for various bacteria, the comparing of the nucleic acid finger printing that experiment is produced and the collection of illustrative plates of database Plays bacterial strain through bioinformatic analysis, contains the bacterium of which kind in can judgement sample.
6, use this programme can carry out the classification and identification of bacterium, and be widely used in the many aspects such as division bacteria, food safety detection, disease prevention and control, disease propagation Study of way, epidemiology survey, clinical drug-resistant Mutation Screening, import and export inspection and quarantine.
7, with respect to the order-checking classification, whole process is only finished in a few hours, and is time saving and energy saving;
8, with respect to order-checking peak figure, the bacterial nucleic acid finger printing has better resolving power for biased sample.Utilize mass spectrometric detection to detect 16S rDNA zone through groping, designing first, obtain the flow process of bacterium finger printing;
9, in addition, the present invention proposes mass spectrometric detection is carried out as determinand in the 16S rDNA zone of bacterium first, to obtain the nucleic acid finger printing of different bacterium, is used for the phylogenetic analysis of bacterium and the research of classification and identification.
Description of drawings
Fig. 1 is listerial nucleic acid fingerprint characteristic collection of illustrative plates.
Fig. 2 is the nucleic acid fingerprint characteristic collection of illustrative plates of bacillus aceticus.
Fig. 3 is the nucleic acid fingerprint characteristic collection of illustrative plates of nitrogen-fixing rhizobia.
Fig. 4-Figure 100 is the nucleic acid fingerprint characteristic collection of illustrative plates of 97 kinds of bacteriums shown in the table 2, wherein, Fig. 4: No. 1 bacterial classification (Acaryochloris marina MBIC11017 chromosome) in the table 2, Figure 26: No. 23 bacterial strain in the table 2 (Capnocytophaga ochracea DSM 7271 chromosome), Figure 49: No. 46 bacterial classification (Megasphaera elsdenii DSM 20460), Figure 72: No. 69 bacterial classification (Rhodobacter sphaeroides 2.4.1 chromosome chromosome 1), Figure 95: No. 92 bacterial classification (Thiomicrospira crunogena XCL-2 chromosome), Figure 100: No. 97 bacterial classification (Xanthobacter autotrophicus Py2 chromosome).
Figure 101-103 is the nucleic acid fingerprint characteristic collection of illustrative plates of sheep kind (Brucella melitensis), ox kind (Brucella abortus), pig kind brucella (Brucella suis).
Figure 104 is the nucleic acid fingerprint characteristic collection of illustrative plates of mycobacterium tuberculosis.
Figure 105 is the nucleic acid fingerprint characteristic collection of illustrative plates of helicobacter pylori.
Figure 106 is the nucleic acid fingerprint characteristic collection of illustrative plates of the autotrophy Flavobacterium to be measured (Xanthobacter autotrophicus Py2 chromosome) of embodiment four.
Figure 107 is the bacterial nucleic acid fingerprint characteristic collection of illustrative plates of milk preparation to be measured among the embodiment five.
Figure 108 is the microbial culture figure of control experiment among the embodiment five.
Figure 109 is Ke's Albert'stain Albert figure of embodiment five control experiments.
Figure 110 is the nucleic acid fingerprint characteristic collection of illustrative plates of the meat product Bacteria Detection of embodiment six.
Figure 111 is Ke's Albert'stain Albert figure of embodiment six control experiments.
Figure 112 is gel electrophoresis figure behind the pcr amplification of embodiment six control experiments.
Figure 113 is the bacterial nucleic acid fingerprint characteristic collection of illustrative plates of the source of pollution to be checked of embodiment seven.
Figure 114 is the cultivation figure of Luo Shi solid medium of the control experiment of embodiment seven.
Figure 115 is the luxuriant sodium resistance to acid smear method colored graph of the control experiment of embodiment seven.
Figure 116 is the bacterial nucleic acid fingerprint characteristic collection of illustrative plates of the sputum sample of embodiment eight.
Figure 117 is the fluorescence quantitative PCR detection figure of embodiment eight.
Figure 118 is the bacterial nucleic acid fingerprint characteristic collection of illustrative plates of the source of pollution to be checked of embodiment nine.
Figure 119 is the magenta method figure as a result of the microbial culture of embodiment nine control experiments.
Figure 120 is the bacterial nucleic acid fingerprint characteristic collection of illustrative plates of the fresh milk to be checked of embodiment ten.
Figure 121 is the rear gel electrophoresis figure of bacteria PCR amplification of embodiment ten control experiments.
Figure 122 is the constructed bacterium Phylogenetic tree of embodiment 11.
Figure 123 is the nucleic acid fingerprint characteristic collection of illustrative plates of embodiment 11 bacterium to be measured.
Figure 124 is that the genetic evolution according to bacterium to be measured that evolutionary tree is determined concerns among the embodiment 11.
Embodiment
Only further describe the present invention with the mode with reference to following nonrestrictive embodiment now.But should be appreciated that the following examples only as illustration, should be by any way when doing the restriction overall to the invention described above.Unless other explanation is arranged, and embodiments of the invention use traditional molecular biology, cytobiology, pcr amplification and mutating technology in this area etc.These technology are known by the technical staff, and detailed explanation is arranged in the literature.Referring to, for example,
Sambrook and Russell″Molecular Cloning：A Laboratory Manual″(2001)；
Cloning：A Practical Approach，″Volumes I and II(D.N.Glover， ed.，1985)″；
T.A Brown “Genome” BIOS Scientific Publishers Limited;
PY Kwok Annu. Rev. Genomics Hum. Genet.(2001), 235–58.
The foundation of embodiment one, single bacterial nucleic acid fingerprint characteristic collection of illustrative plates
One, design and selection universal primer
According to the listeria bacteria (Listeria monocytogenes EGD-e, the NCBI accession number: in the conservative region of 16S rDNA NC_003210.1) (SEQ ID No:3), design universal PC R primer is respectively:
5-aggaagagagAGAGTTTGATCCTGGCTCAG-3（SEQ ID No：1）
5-cagtaatacgactcactatagggagaaggctCTGCTGCGTCCCGTAG-3（SEQ ID No：2）
Wherein sequence A GAGTTTGATCCTGGCTCAG and CTGCTGCGTCCCGTAG respectively with 16S rDNA Region Matching to be amplified, aggaagagag and cagtaatacgactcactatagggagaaggct are the additional sequences of adding at upstream and downstream PCR primer, guarantee for the PCR product is transcribed, the 5' end of SEQ ID No:1 primer contains the tag(aggaagagag of 10bp), the 5' end of SEQ ID No:2 primer contains the tag(cagtaatacgactcactatagggagaaggct of 31bp).
Relevant primer synthesizes in Sangon Biotech (Shanghai) Co., Ltd..
Two, universal primer amplification
To the DNA of the bacteriums such as listeria bacteria, use ABI 9700 type PCR instrument, carry out the biological experiment operation.
1、PCR
(1) reaction system of PCR is:
PCR reaction buffer (10x PCR Buffer with 20mM MgCl
2): 0.5ul;
dNTP mix（25mM each）：0.04ul；
Taq enzyme (PCR Enzyme, 5U/ul): 0.04ul;
SEQ ID No：1（1μM）：1ul
SEQ ID No：2（1μM）：1ul；
DNA of bacteria: 1ul;
Ultrapure water: 1.42ul.
Above reagent except DNA of bacteria, ultrapure water, primer, all comes from U.S. Sequenom company.
(2) loop parameter of PCR is:
95 ℃, 4 minutes, 1 circulation,
95 ℃, 20 seconds, 56 ℃, 30 seconds, 72 ℃, 60 seconds, 45 circulations,
72 ℃, 3 minutes, 1 circulation,
4 ℃, preserve.
2, SAP enzymic digestion
(1) reaction system of SAP enzymic digestion is:
In the PCR of 5ul product, add RNase-free ddH
2O:1.7ul, SAP enzyme (SAP enzyme, 1.7U/ul): 0.3ul.
Above reagent all comes from U.S. Sequenom company.
(2) reaction parameter of SAP enzymic digestion is:
37 ℃, 20 minutes, 1 circulation,
85 ℃, 5 minutes, 1 circulation,
4 ℃, preserve.
3, transcribe with nuclease and cut
(1) transcribing the reaction system of cutting with nuclease is:
Get the digestion product of 2ul, add:
RNase-free ddH2O：3.21ul，
5xT7 Polymerase Buffer：0.89μl
T Cleavage Mix：0.22μl
DTT（100mM）：0.22μl
T7 RNA & DNA Polymerase：0.4μl
RNaseA：0.06μl
Above reagent all comes from U.S. Sequenom company.
(2) transcribing the reaction parameter of cutting with enzyme is:
37℃，3h。
4, purifying
Cut in the product with enzyme transcribing of 7ul and to add the 20ul ultrapure water, behind the mixing, add 6mg resin (resin, U.S. Sequenom company) again, mixing 15 minutes turns upside down.
Three, set up the nucleic acid fingerprint characteristic collection of illustrative plates of single bacterium listeria bacteria (Listeria monocytogenes EGD-e)
Product behind the purifying uses to receive and rises point sample instrument (Nanodispenser, U.S. Sequenom company), put to the chip that contains matrix (SpectroCHIP, U.S. Sequenom company), and use time-of-flight mass spectrometer (U.S. Sequenom company) to detect with the result and judge.
The result can find out by experiment, the conservative region of the selected listerial 16S rDNA of this programme, through the biological experiment operation, produced the nucleic acid fragment combination of different lengths and different abundance this bacterium, through mass spectrometric detection, form special nucleic acid finger printing, detected result can be carried out the classification and identification of Related Bacteria through bioinformatic analysis.
Repeat twice, all obtain identical nucleic acid fingerprint characteristic collection of illustrative plates (Fig. 1).
Embodiment two, the preparation method of one nucleic acid fingerprint characteristic collection of illustrative plates is implemented in checking among a small circle
Use embodiment one described method and primer, Dichlorodiphenyl Acetate bacillus (Acetobacter pasteurianus IFO 3283-01), nitrogen-fixing rhizobia (Azorhizobium caulinodans ORS 571 chromosome) carry out that enzyme is cut and Mass Spectrometric Identification.
Repeat twice, all obtain identical nucleic acid fingerprint characteristic collection of illustrative plates, wherein Fig. 2, Fig. 3 show respectively the nucleic acid fingerprint characteristic collection of illustrative plates of bacillus aceticus and nitrogen-fixing rhizobia.
The foundation of the nucleic acid fingerprint characteristic spectrum library of embodiment three, bacterium
According to embodiment one described method and primer, listed bacterium carries out that enzyme is cut and Mass Spectrometric Identification in the his-and-hers watches 1, obtains germy nucleic acid fingerprint characteristic collection of illustrative plates.
With these characteristic spectrums, carry out confluence analysis by Bioexplore software, thereby obtain comprising the nucleic acid fingerprint characteristic spectrum library of most bacteriums.
As shown in table 2, Fig. 4-Figure 100 shows respectively the nucleic acid fingerprint characteristic collection of illustrative plates of 97 kinds of bacteriums.
Table 2(annotates: table 2 is the italic content in the table 1)
Figure 101-103 is sheep kind, ox kind, the brucellar nucleic acid fingerprint characteristic of pig kind collection of illustrative plates, and Figure 104 is the nucleic acid fingerprint characteristic collection of illustrative plates of mycobacterium tuberculosis, and Figure 105 is the nucleic acid fingerprint characteristic collection of illustrative plates of helicobacter pylori.
The nucleic acid fingerprint characteristic spectrum library of the bacterium that embodiment four, utilization are set up is identified the environmental safety of hospital
Locate at the generate heat water tap, ditch etc. of certain hospital of disease of doubtful outburst infant, collect the source of pollution sample, be divided into two after the dilution of sample to be tested appropriateness, wherein sample to be tested 1 is carried out after pcr amplification, enzyme cut according to the method for embodiment 1, carry out mass spectrometric detection, whole process 1-2 consuming time hour.
Use BioExplore software (its copyright number step on word for soft work No. 136879, registration number 2009SR10700), the mass spectral characteristic figure (Figure 106) of gained is compared with the bacterial nucleic acid fingerprint characteristic spectrum library of implementing to obtain in three, and the judging criterion of employing is:
When 2.300≤coupling mark≤3.000, the confidence level of expression strain identification is very high;
When 2.000≤coupling mark＜2.300, the identification of bacteria that expression is conservative or possible strain identification;
Between 1.700≤coupling mark＜2.000, the identification of bacteria that expresses possibility;
Between 0.000≤coupling mark＜1.700, represent incredible evaluation.
The comparative analysis result is as follows:
Testing sample collection of illustrative plates (Figure 106) is compared with Figure 100 in the spectrum library that embodiment three sets up, the coupling mark is 2.412, greater than 2.300, meet fully with desired value, show that this testing sample is autotrophy Flavobacterium (Xanthobacter autotrophicus Py2 chromosome), the confidence level of its qualification result is very high.
Control experiment: the detection of the biochemical method of sample to be tested 2
1, sample to be tested is carried out microscopy, present Gram-negative, thalline is shaft-like or the club shape, unpowered gemma and pod membrane.
2, the sample to be tested 2 of separation and Culture increases the bacterium cultivation with meat soup first, then directly is inoculated in blood agar or chocolate dull and stereotyped, cultivates the further biochemical test of faint yellow bacterium colony and microscopy that picking is suspicious 24 hours for 35 ℃.
3, biochemical reaction shows as: oxydase, catalase are positive, can decompose seminose, the ability of sugar fermentation slowly and slightly a little less than, aerobic growth can be hydrolyzed Vitamin C2 and liquefy gelatin.Single bacterium colony after the propagation is faint yellow, circular, diameter 1-1.5mm, and is smooth, transparent or semitransparent, slightly protruding, neat in edge.Above-mentioned reaction all meets the biochemical characteristic that singly increases the autotrophy Flavobacterium, shows consistent with MALDI TOF MS qualification result.
Show that by implementing the result that four nucleic acid fingerprint characteristic Atlas Method identifies there is the pollution of Flavobacterium in this bulletin place, need to the corresponding establishment disinfection, break out sporadic infection to prevent the public place.
The nucleic acid fingerprint characteristic spectrum library of the bacterium that embodiment five, utilization are set up detects milk preparation to be checked
To be divided into two after the milk preparation appropriateness to be checked dilution, wherein use following mode that testing sample 1 is detected:
One, extract the bacterial chromosomal dna of milk sample with the CTAB-NaCl method of optimizing, concrete steps are as follows:
(1) get the 1.5mL milk preparation, the centrifugal 10min of 10,000r/min abandons supernatant;
(2) with NET solution (50mM NaCl, 125mM EDTA, 50mM Tris-HCl, pH7.6) suspension is precipitated to 400 μ L, add 200 μ L 10% (w/v) SDS solution to final concentration 3.4% (w/v), put 80 ℃ of water-bath 10min, cooled on ice 3min;
(3) add respectively 3 μ L Proteinase Ks (200 mg/mL) and RNaseA (200mg/mL) to final concentration 1mg/mL, 50 ℃ of water-bath 2h;
(4) add 100 μ L 5M NaCl solution, adding 300 μ L behind the mixing is preheated to 65 ℃ the CTAB-NaCl solution that contains 10% (w/v) CTAB and 0.7M NaCl (taking by weighing 4.1g NaCl is dissolved in the 80mL water, slowly add 10g CTAB, heating for dissolving, be settled to 100mL), CTAB and the NaCl final concentration in extracting solution is respectively 3.4% (w/v) and 0.7M, 65 ℃ of water-bath 10min;
(5) add 600 μ L chloroforms: primary isoamyl alcohol (24: 1), room temperature concussion 2min, the centrifugal 5min of 10,000r/min; Carefully supernatant is transferred in the clean centrifuge tube, added 600 μ L chloroforms: primary isoamyl alcohol (24: 1) repeats extracting 1 time;
(6) in supernatant, add 2/3 volume Virahol, mixing, room temperature is placed 5min, and the centrifugal 10min of 10,000r/min abandons supernatant; Precipitation is washed 2 times 37 ℃ of dry 10min with 70% ethanol;
(7) with 5 μ L ultrapure water dissolving DNAs precipitation, be used for pcr amplification.
Two, carry out carrying out mass spectrometric detection, whole process 1-2 consuming time hour after pcr amplification, enzyme cut according to the method for embodiment 1.
Use BioExplore software (its copyright number step on word for soft work No. 136879, registration number 2009SR10700), mass spectral characteristic Figure 107 and the bacterial nucleic acid fingerprint characteristic spectrum library of implementing to obtain in three of gained are compared, the comparative analysis result is as follows:
The testing sample collection of illustrative plates is compared with Brucella abortus (Brucella abortus) standard diagram (Figure 102) in the spectrum library that the present invention sets up, the coupling mark is 2.308, greater than 2.300, be reported as Brucella abortus, meet fully with desired value, the confidence level of qualification result is very high.
The control experiment of embodiment five: the detection of the biochemical method of sample to be tested 1
According to standard method preparation serum glucose nutrient agar SDA, and add penicillin or cephamycin (100 μ g/100ml).
Choose fresh milk sample to be measured, draw the 0.5ml sample, be coated under gnotobasis in the previously prepared microbiotic SDA culture dish, 37 ℃ of lower cultivations 3 days, observe the colony growth situation, the result as shown in Figure 4.
Figure 108 shows, generates the small colonies of moistening, flash of light, water white transparency, circle, surface elevation, neat in edge in the culture dish.As parallel test, choose the fresh milk sample of 0.5ml process Bath sterilization sterilization in contrast, after cultivating 3 days, the SDA substratum generates without any bacterium colony.Therefore, the bacterium colony in the microbiotic SDA substratum is doubtful is Brucella abortus.
Be further this doubtful bacterium colony of identifying, use Ke's Albert'stain Albert method (husky Huang and Victoria Green WPB are to dying) staining to detect fresh milk sample to be measured.
Under gnotobasis, with the doubtful bacterium colony of aseptic liquid-transfering gun suction nozzle picking, place 10ml liquid SDA substratum, the vibration mixing.
Draw the 1ml sample, be coated under gnotobasis in previously prepared Ke's Albert'stain Albert culture dish, 37 ℃ of lower cultivations 3 days, observe the colony growth situation, the result as shown in Figure 7.
Figure 109 shows that doubtful bacterium colony presents red bead bacilliform at Ke's Albert'stain Albert culture dish, and two ends are the blunt round shape, meets brucellar biochemical characteristic.
Above-mentioned test all meets brucellar biochemical characteristic, shows consistent with MALDI TOF MS qualification result.
The result of embodiment five shows, the pollution of the existing Brucella abortus of these fresh milk goods to be checked needs to destroy immediately, and to the processing of quarantining of relevant milk cow.
The nucleic acid fingerprint characteristic spectrum library of the bacterium that embodiment six, utilization are set up detects barbecue's meat product
In the mutton sample of roadside barbecue, with aseptic technique, take by weighing lean meat sample 25 g after shredding, place in the sterilization homogeneous cup, add 25 mL, buffering peptone water enrichment liquid (penicillin 100 μ g/100ml), with 8000～10000r/min homogeneous 1min, immigration fills in the 500mL wide-necked bottle of 200 mL buffering peptone water enrichment liquid, mixes, and is lower than 6.6 such as pH, with sterilization 1mol/L sodium hydroxide solution, transfer pH to 6.8 ± 0.2, cultivate 4h (counting when reaching 37 ℃ with enrichment liquid) in 37 ℃ of water-baths, increase bacterium before carrying out; , pipette 10mL transferred species in the 250mL vial that fill 100mLGN enrichment liquid in, shake up, in 42 ± 1 ℃ of cultivation 20 ± 2h, carry out selective enrichment (penicillin 100 μ g/100ml) thereafter.In case of necessity, take by weighing in addition the lean meat sample that 25g shreds simultaneously, add the 25mLGN enrichment liquid, carry out equally homogeneous., move into the 500mL wide-necked bottle that fill 200mLGN enrichment liquid in (penicillin 100 μ g/100mls), mix, be lower than 6.6 such as pH, transfer pH to 6.8 ± 0.2 with sterilization 1mol/L sodium hydroxide solution, cultivate 24 ± 2 h in 37 ℃ thereafter.Then sample 2 to be checked is divided into two, wherein sample to be tested 2 is carried out carrying out mass spectrometric detection, whole process 1-2 consuming time hour after pcr amplification, enzyme cut according to the method for embodiment 1.
Use BioExplore software (its copyright number step on word for soft work No. 136879, registration number 2009SR10700), the mass spectral characteristic figure (Figure 110) of gained is compared with the bacterial nucleic acid fingerprint characteristic spectrum library of implementing to obtain in three, and the comparative analysis result is as follows:
The testing sample collection of illustrative plates is compared with Brucella melitensis (Brucella melitensis) standard diagram (Figure 101) in the spectrum library that the present invention sets up, the coupling mark is 2.420, greater than 2.300, is reported as Brucella melitensis, meet fully with given value, the confidence level of qualification result is very high.
The control experiment one of embodiment six: the biochemical method of sample to be tested 2 detects
According to the method for embodiment five control experiments, choose 1ml sample 2 to be checked, under gnotobasis, be coated in previously prepared Ke's Albert'stain Albert culture dish, 37 ℃ of lower cultivations 3 days, observe the colony growth situation, the result is shown in Figure 111.
Be not to select single bacterium colony (such as embodiment five) in the Yin Ben experiment, and contain other miscellaneous bacterias in the sample 2 to be checked, therefore Figure 111 is presented at around the green miscellaneous bacteria, is distributing and is justifying shape or the single bacterium colony of spherical redness, and Ke's Albert'stain Albert method result of this and Brucella melitensis is in full accord.
The control experiment two of embodiment six: the molecular Biological Detection of sample to be tested 2
One, design of primers
For Brucella melitensis (Brucella abortus)) specific PCR of outer membrane protein (OMP) 25kd gene design amplification Oligonucleolide primers:
BP1：5’-CGT GCC GCA ATT ACC CTC-3’
BP2：5’-CCG TCA GCT TGG CTT CGA-3
Two, PCR
Choose single red bacterium colony in Ke's Albert'stain Albert culture dish, ordinary method multiplication culture bacterium colony, and extract bacteria total DNA.
Carry out PCR according to following parameter:
Reaction conditions is 94 ℃ of 10min, 94 ℃ of 1min, and 50 ℃ of 1min, 72 ℃ of 2min circulate 35 times, and 72 ℃ are extended 10min.
After reaction finished, negate answered product 5 μ l to be added on 1.0% sepharose (the containing 0.5 μ g/ml EB) plate electrophoresis observation result in the TAE electrophoresis liquid.
Three, result and judgement
Set up simultaneously positive control and blank in the PCR test, after all setting up the PCR detected result is judged: 419bp amplified band person occurs in the test sample road positive as Infected with Brucella; 419bp amplified band person do not occur in the test sample road negative for Infected with Brucella, and the result is shown in Figure 112: swimming lane 1 is DL 2000DNA Marker; Swimming lane 2 and 3 is OMP 25 amplified fragments; Swimming lane 4 is negative controls.
According to the result of embodiment six, can determine that the mutton fresh meat that this barbecue sells is infected by Brucella melitensis, need to destroy at once, and the related personnel is isolated for treatment.
The concretion mycobacterium nucleic acid fingerprint characteristic spectrum library that embodiment seven, utilization are set up is identified the environmental safety of kindergarten
Locate in the water tap of certain public kindergarten of doubtful Child Pulmonary Tuberculosis contagium, ditch etc., collect the source of pollution sample, be divided into two after the dilution of sample to be tested appropriateness, wherein sample to be tested 1 is carried out after pcr amplification, enzyme cut according to the method for embodiment 1, carry out mass spectrometric detection, whole process 1-2 consuming time hour.
Use BioExplore software (its copyright number step on word for soft work No. 136879, registration number 2009SR10700), the mass spectral characteristic (Figure 113) of gained is compared with the bacterial nucleic acid fingerprint characteristic spectrum library of implementing to obtain in three, and the comparative analysis result is as follows:
The standard diagram 104 of the mycobacterium tuberculosis in testing sample collection of illustrative plates (Figure 113) and the spectrum library that the present invention sets up is compared, the coupling mark is 2.470, greater than 2.300, is reported as mycobacterium tuberculosis, meet fully with given value, the confidence level of qualification result is very high.
The control experiment of embodiment seven: the biochemical analysis control experiment of sample to be tested 1
One, the initial analysis of sample to be tested 1
1, the sample to be tested 1 of separation and Culture cultivated for 2 weeks upper 35 ℃ of Luo Shi solid medium (comprising potato, Victoria Green WPB etc.), produced the oyster white bacterium colony (seeing Figure 114) of particle or cauliflower form, the further biochemical test of bacterium colony and microscopy that picking is suspicious.
2, sample to be tested is carried out microscopy, luxuriant sodium resistance to acid smear method presents redness, and it is elongated that thalline is, and holds extremely blunt circle, unpowered gemma and pod membrane (Figure 115).
Thus, can determine tentatively that sample to be tested is mycobacterium tuberculosis.
Two, the biochemical analysis of sample to be tested 1
1, by following formulated mycobacterium tuberculosis substratum:
Wherein, the substratum making method: first with distilled water with after glycerine mixes, add successively again each composition, after dissolving, transfer ph value to 6.6～6.8 with 10% HCl, high pressure 0.7 * 105pa, 20min sterilizes.To be cooled to 56 ℃., add degerming calf serum, be mixed, be divided in the culture dish, behind 37 ℃ of aseptic mensuration 24h, it is for subsequent use to store 4 ℃ of refrigerators.
In addition, under aseptic condition, use the asepsis injector of hyperfiltration Rifampin (100mg/L) to be added in the dull and stereotyped culture dish of having made, after the coating evenly, again behind 37 ℃ of aseptic mensuration 24h.
2, experiment test
Sample to be tested 1 among the embodiment two and blank (sterilized water) are made bacteria suspension, get 0.1ml, slowly coat equably on the culture dish of blank substratum and Rifampin substratum, put in 37 ℃ of thermostat containers, report the result after 2 weeks.
Test-results criterion is as follows:
The flat board of (-) substratum is without colony growth;
(+) colony number accounts for the platen area 1/4 of substratum;
(++) colony number accounts for the platen area 1/2 of substratum;
(+++) colony number accounts for the platen area 3/4 of substratum;
(++ ++) bacterium colony is the growth of lawn sample.
The report colony number: when pastille substratum colony number at 20 when following, the number of report bacterium colony.
Above-mentioned test all meets the biochemical characteristic of mycobacterium tuberculosis, shows consistent with MALDI TOF MS qualification result.
Result by embodiment seven shows that there is the pollution of mycobacterium tuberculosis in this bulletin place, need to seal immediately the garden, and to the corresponding establishment disinfection, breaks out sporadic infection to prevent the kindergarten.
The nucleic acid fingerprint characteristic spectrum library of the bacterium that embodiment eight, utilization are set up is to the rapid detection of frontier port suspected patient
In certain port of entry, airport, to heating appears, acutely certain foreign nationality passenger of doubtful caking appears in cough and X-ray lung, after isolating, choose its sputum sample 5ml, add in the aseptic centrifuge tube of 50 ml, add equivalent NACL-NaOH solution, the vibration of vortex oscillator, abundant liquefy sputum sample, room temperature leaves standstill 15min, add 0.067 mol/L PBS solution, fully mixing with in and the Digestive system effect, centrifugal 15 min of 3 000 g carefully abandon supernatant, add again mixing of the about 2ml of 0.067mol/LPBS in the throw out, be postdigestive phlegm sample to be tested 2.
Sample to be checked is divided into two, wherein sample to be tested 2 is carried out carrying out mass spectrometric detection, whole process 1-2 consuming time hour after pcr amplification, enzyme cut according to the method for embodiment 1.
Use BioExplore software (its copyright number step on word for soft work No. 136879, registration number 2009SR10700), the standard diagram 104 of the mycobacterium tuberculosis in the mass spectral characteristic figure (Figure 116) of gained and the spectrum library that the present invention sets up is compared, it is 2.380 that the comparative analysis result is mated mark, greater than 2.300, be reported as mycobacterium tuberculosis, meet fully with given value, the confidence level of qualification result is very high.Judge that thus this passenger is the mycobacterium tuberculosis positive patient.
The control experiment of embodiment eight: the fluorescence quantitative PCR detection of sample to be tested 2
With the 4%NaOH of 4 times of volumes of the adding of the sputum sample after the digestion process, about liquefaction 30min, get 0.5ml to centrifuge tube, add 0.5ml 4%NaOH, centrifugal 5 min of 10000 r/min behind the 10min.Remove supernatant, precipitation adds stroke-physiological saline solution 1ml mixing, the centrifugal 5min of 10000r/min, repeated washing 1 time.Precipitation adds 50 μ lDNA extracting solution mixings, and boiling water bath 10min goes to 4 ℃ and leaves standstill 6-8h to guarantee abundant cracking.The centrifugal 5min of 10000r/min gets supernatant liquor 2 μ l and does amplification, 93 ℃ of 2min denaturations, 93 ℃ of 45s, 10 circulations of 55 ℃ of 60s amplifications, 93 ℃ of 30s, 55 ℃ of 45s, 30 circulations of increasing.
Other gets negative quality control product, each 40 μ l of positive quality control product add equivalent DNA extraction liquid mixing, and boiling water bath 10min processes.As a result judging criterion according to the detection reagent specification sheets is carried out interpretation of result, and the result is shown in Figure 117.
According to the result of embodiment eight, can make a definite diagnosis this passenger is the lunger, need to isolate for treatment to it.
The concretion mycobacterium nucleic acid fingerprint characteristic spectrum library that embodiment nine, utilization are set up is identified the safety of waterhead area
Waterhead area in certain waterworks of doubtful helicobacter pylori contagium, collect the source of pollution sample, be divided into two after the dilution of sample to be tested appropriateness, wherein sample to be tested 1 is carried out after pcr amplification, enzyme cut according to the method for embodiment 1, carry out mass spectrometric detection, whole process 1-2 consuming time hour.
Use BioExplore software (its copyright number step on word for soft work No. 136879, registration number 2009SR10700), the mass spectral characteristic (Figure 118) of gained is compared with the bacterial nucleic acid fingerprint characteristic spectrum library of implementing to obtain in three, the comparative analysis result is as follows: the standard diagram 105 of the helicobacter pylori in the spectrum library that the testing sample collection of illustrative plates divides with the present invention sets up (Helicobacter pylori) is compared, the coupling mark is 2.340, greater than 2.300, be reported as helicobacter pylori, meet fully with given value, the confidence level of qualification result is very high.
The control experiment of embodiment nine: the biochemical analysis control experiment of sample to be tested 1
One, the biochemical analysis of sample to be tested 1
1, by following formulated helicobacter pylori isolation medium:
Under aseptic condition, in prefabricated brain heart leach liquor agar (available from Difco) liquid nutrient medium 150ml, add defiber sheep blood 8ml, and adding mixes microbiotic (vancomycin 10mg/L, Trimethoprim lactic acid salt 0.005mg/L, amphotericin B 10mg/L, PXB 0.005mg/L), regulating pH is 7.5, then pour in a plurality of culture dish preparation helicobacter pylori isolation medium into.
Control medium (being that microbiotic replaces with amoxycilline Trihydrate bp 10mg/L, levofloxacin 2mg/L) is set simultaneously
3, experiment test
Sample to be tested 1 among the embodiment two and blank (sterilized water) are made bacteria suspension, get 0.1ml, slowly coat equably on the culture dish of isolation medium and control medium, put in 37 ℃ of thermostat containers, report the result after 3 days.
Test-results criterion is as follows:
The flat board of (-) substratum is without colony growth;
(+) colony number accounts for the platen area 1/4 of substratum;
(++) colony number accounts for the platen area 1/2 of substratum;
(+++) colony number accounts for the platen area 3/4 of substratum;
(++ ++) bacterium colony is the growth of lawn sample.
(+-) the suspicious growth of bacterium
The report colony number: when pastille substratum colony number at 20 when following, the number of report bacterium colony.
4, use the magenta method, the single bacterium colony of picking from the isolation medium, dyeing is carried out microscopy after processing.
Microscopically shows that this bacterium is red crooked shaft-like or spirrillum, and background be white, contrast gradient clear (referring to Figure 119).
Above-mentioned test all meets the biochemical characteristic of helicobacter pylori, shows consistent with MALDI TOF MS qualification result.
The result of embodiment nine shows that the waterhead area of this waterworks need to carry out disinfecting to this waterhead area by the pollution of helicobacter pylori, removes peripheral ight soil, livestock, excremental source of pollution.
The nucleic acid fingerprint characteristic spectrum library of the bacterium that embodiment ten, utilization are set up detects fresh milk to be checked
According to the report of the people such as Tian Yu (" dairy industry science and technology ", 2006), use to optimize the CTAB-NaCl method and extract DNA of bacteria in the fresh milk, and carry out carrying out mass spectrometric detection, whole process 1-2 consuming time hour after pcr amplification, enzyme cut according to the method for embodiment 1.
Use BioExplore software (its copyright number step on word for soft work No. 136879, registration number 2009SR10700), the mass spectral characteristic (Figure 120) of gained is compared with the bacterial nucleic acid fingerprint characteristic spectrum library of implementing to obtain in three, and the comparative analysis result is as follows:
The standard diagram 105 of helicobacter pylori (Helicobacter pylori) in testing sample collection of illustrative plates (Figure 120) and the spectrum library that the present invention sets up is compared, the coupling mark is 2.470, greater than 2.300, be reported as helicobacter pylori, meet fully with given value, the confidence level of qualification result is very high.
The control experiment of embodiment ten: the molecular Biological Detection of fresh milk
According to the helicobacter Pylori urease gene, in Genebank, retrieve 1 gene order (Accession M60398).Design following primer according to gene order, with the fragment of about 250bp that increases:
Forward primer: 5 ＇-AAGCTTTTAGGGGTGTTAGGGGTTT-3 ＇;
Reverse primer: 5 ＇-CAAGCCATCGCCGG TTTTAGC-3 ＇
Helicobacter pylori reference culture liquid and aseptic fresh milk standard control are set, carry out pcr amplification.
The PCR reaction system:
Reaction conditions:
5 ℃ of denaturation 2mi n; 94 ℃ of sex change 1min, 61 ℃ of annealing 20s, 72 ℃ are extended 30s, 35 circulations;
72 ℃ are extended 5mi n.
Amplified production is carried out gel electrophoresis, and the result is shown in following table and Figure 121
| Swimming lane | Specimen | The |
| 1 | 10 ml standard bacterium liquid | ++ |
| 2 | 10ml fresh milk to be measured | + |
| 3 | 10ml fresh milk to be measured | + |
| 4 | 9ml fresh milk+1ml standard bacterium liquid | + |
| 5 | The aseptic fresh milk contrast of 10ml | - |
Identify and the PCR detection method by implementing ten nucleic acid fingerprint characteristic Atlas Method, can determine that this fresh milk by helicobacter pylori, need to destroy at once, and the relevant pathogeny of making a thorough investigation.
The bacterial nucleic acid fingerprint characteristic collection of illustrative plates that embodiment 11, utilization are set up is used for foundation and the classification of the Phylogenetic tree of different bacterium strain isolated
Utilize existing bacterial nucleic acid fingerprint characteristic spectrum library, extract wherein nuclease and cut information, utilize Phylogenetic tree or the phylogenetic tree (Figure 122) of Clustalx2.0 and the described 841 kinds of bacterium kinds of MEGA4.1 software building.On evolutionary tree, can observe intuitively the evolutionary relationship of the not of the same race and different isolates of various Pseudomonas, can clearly judge bacterial strain to be measured simultaneously and this belongs to the evolutionary relationship of each bacterium.In addition, the foundation of phylogenetic tree also can be verified the analytical results of BE software, further proves the accuracy of Bacteria Identification.
For example, in the oral cavity, separate and obtain a strain suis, method according to previous embodiment, its collection of illustrative plates (Figure 123) is compared respectively with in the spectrum library that the present invention sets up, the result shows the standard diagram comparison of itself and Streptococcus sanguis (Streptococcus parasanguinis ATCC 15912), and the coupling mark is reported as Streptococcus sanguis greater than 2.300, meet fully with given value, the confidence level of qualification result is very high.
Also verified Pseudomonas to be measured in streptococcus according to phylogenetic tree, simultaneously nearest with the sibship of Streptococcus sanguis (Streptococcus parasanguinis ATCC 15912).
SEQUENCE LISTING
＜110〉to China
＜120〉the bacterial nucleic acid fingerprint characteristic of cutting based on enzyme is composed Preparation Method and uses thereof
<130> 2012
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
＜213〉artificial sequence
<400> 1
aggaagagag agagtttgat cctggctcag 30
<210> 2
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＜213〉artificial sequence
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cagtaatacg actcactata gggagaaggc tctgctgcgt cccgtag 47
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<211> 358
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＜213〉artificial sequence
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ctgctgcctc ccgtaggagt ctgggccgtg tctcagtccc agtgtggccg atcaccctct 60
caggtcggct atgcatcgtt gccttggtag gccattaccc taccaactag ctaatgcacc 120
gcgggcccat ctgtaagcga tagccgaaac catctttcaa aagcgtggca tgcgccacac 180
tttatcattc ggtattagcc ccggtttccc ggagttatcc ccaacttaca ggcaggttgc 240
ccacgtgtta ctcacccgtc cgccactaac tttggaagag caagctcttc ctccgttcgt 300
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cgtgccgcaa ttaccctc 18
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aagcttttag gggtgttagg ggttt 25
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Claims (11)
1. a bacterial nucleic acid finger printing preparation method who cuts based on enzyme is characterized in that, which comprises at least following steps:
(1) PCR reaction: use the PCR universal primer of directed toward bacteria, the nucleic acid-templated of a plurality of bacteriums of increasing respectively obtains containing the PCR product of target area of increasing;
(2) SAP enzymic digestion: the PCR product that obtains with alkaline phosphatase (SAP enzyme) treatment step (1);
(3) transcribe with nuclease and cut: use and specifically transcribe and restriction endonuclease, respectively in a reaction system, the digestion product of each bacterium that step (2) is obtained is transcribed with nuclease and is cut, and obtains the nucleic acid fragment that a series of length differ, abundance differs;
(4) purifying: the enzyme that uses resin treatment step (3) to obtain is cut product;
(5) mass spectrograph detects: point is containing on the target sheet of matrix on the purified product that step (4) is obtained, and upper mass spectrograph detects, and obtains the feature nucleic acid fingerprint chromatogram of different bacterium.
2. according to claim 1 method, wherein universal primer includes but not limited to sequence shown in SEQ ID No:1 and the SEQ ID No:2; And be used for the certain enzyme that nuclease is cut, include but not limited to the RNaseA enzyme.
3. claim 1 or 2 method, wherein the purifying of step 5 is included in to transcribe to cut with enzyme and adds ultrapure water in the product, behind the mixing, adds resin again, and mixing 15 minutes turns upside down; Described mass spectrograph is MALDI TOF MS mass spectrograph.
4. each method among the claim 1-3, wherein said bacterium comprises the 836 kind bacteriums listed such as table 1.
5. in above-mentioned arbitrary scheme, wherein said bacterium also comprise brucella (
Brucella), mycobacterium tuberculosis (
Mycobacterium tuberculosis), and helicobacter pylori (
Helicobacter pylori, Hp).
6. utilize the method for claim 1 for the method for setting up bacterial nucleic acid fingerprint characteristic spectrum library, comprise at least: above-mentioned steps 1-5, and;
(6) the nucleic acid fingerprint characteristic collection of illustrative plates of the various bacteriums that step 5 obtained gathers and puts in order by computer software, obtains described bacterial nucleic acid fingerprint characteristic spectrum library.
7. the method for claim 7, wherein said software is BioExplore software, No. 136879, registration number are 2009SR10700 for soft work is stepped on word for its copyright number.
8. the bacterial nucleic acid fingerprint characteristic collection of illustrative plates that utilizes the method for claim 5 or 6 to set up is used for the method for Bacteria Identification or classification, comprising:
(1) PCR reaction: use the PCR universal primer of directed toward bacteria, amplification tested bacteria nucleic acid-templated obtains containing the PCR product of target area of increasing;
(2) SAP enzymic digestion: the PCR product that obtains with alkaline phosphatase (SAP enzyme) treatment step (1);
(3) transcribe with nuclease and cut: use and specifically transcribe and restriction endonuclease, in a reaction system, the digestion product of the bacterium that step (2) is obtained is transcribed with nuclease and is cut, and obtains the nucleic acid fragment that a series of length differ, abundance differs;
(4) purifying: the enzyme that uses resin treatment step (3) to obtain is cut product;
(5) mass spectrograph detects: point is containing on the target sheet of matrix on the purified product that step (4) is obtained, and upper mass spectrograph detects, and obtains the nucleic acid fingerprint characteristic collection of illustrative plates of this bacterium;
(6) gained nucleic acid fingerprint characteristic collection of illustrative plates and bacterial nucleic acid fingerprint characteristic spectrum library are compared, thus classification or the kind of judgement tested bacteria.
9. the method for claim 7, wherein step 6 compares detection by BioExplore software.
10. provide and can screen the test kit of purposes for division bacteria and evaluation, resistance, comprising:
(1) universal primer that is used for the amplification DNA of bacteria to and damping fluid;
(2) SAP enzyme and damping fluid thereof;
(3) RNAase and damping fluid thereof;
(4) be used for the resin that purifying enzyme is cut product;
(5) for the analysis software of comparing nucleic acid fingerprint characteristic collection of illustrative plates.
11. the test kit of claim 9, wherein said primer are SEQ ID NO:1-2, described software is BioExplore software, and it is that No. 136879, registration number are 2009SR10700 that its soft work is stepped on word.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103352257A (en) * | 2012-05-08 | 2013-10-16 | 向华 | Method for preparing bacterial nucleic acid fingerprint characteristic spectrum library |
| CN104862217A (en) * | 2015-06-15 | 2015-08-26 | 杨亮 | Bacterial drug resistance quick projection system and method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103352257A (en) * | 2012-05-08 | 2013-10-16 | 向华 | Method for preparing bacterial nucleic acid fingerprint characteristic spectrum library |
| CN104862217A (en) * | 2015-06-15 | 2015-08-26 | 杨亮 | Bacterial drug resistance quick projection system and method |
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