CN102808223B - Nucleic acid fingerprint feature spectrum database of mycobacterium tuberculosis and usage of nucleic acid fingerprint feature spectrum database - Google Patents
Nucleic acid fingerprint feature spectrum database of mycobacterium tuberculosis and usage of nucleic acid fingerprint feature spectrum database Download PDFInfo
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Abstract
The invention discloses a method for rapidly classifying and identifying mycobacterium tuberculosis by the aid of the mass-spectrometric technique. The method includes the steps of PCR (polymerase chain reaction) amplification, SAP (severe acute pancreatitis) enzymatic digestion, transcription and nuclease digestion, purification, mass spectrometer detection and the like. A nucleic acid fingerprint spectrum database of the mycobacterium tuberculosis is established based on the method. The mycobacterium tuberculosis in samples to be detected can be rapidly identified according to mass spectrum peak diagrams generated by experiments, and results can be widely applied to typing and classification of the mycobacterium tuberculosis and the fields of environmental sanitation, public security quarantine and the like.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method setting up concretion mycobacterium nucleic acid finger printing, and use the method, mycobacterium tuberculosis is carried out to the method for Rapid identification.
Background technology
Tuberculosis is a kind of chronic infectious disease of serious harm people ' s health, and the current whole world 2,000,000,000 people that have an appointment are infected, newly occur that tuberculosis patient is about 800-1000 ten thousand every year, every year because tuberculosis death toll is about 200-300 ten thousand.Current China tuberculosis year number of the infected is about 1,300,000, because tuberculosis death toll reaches 130,000 every year, exceedes the summation of other Death of Infectious Diseases number.China is one of serious country of 22 TB endemic in the whole world, is also one of 27 popular countries seriously of multi-drug resistance tuberculosis in the whole world simultaneously.China's tuberculosis number of patients occupies second place of the world, is only second to India.Tuberculosis is one of major disease of China's priority control.
Mycobacterium tuberculosis (Mycobacterium tuberculosis), referred to as tubercule bacillus (tubercle bacilli), belongs to Mycobacterium, has multiple strain isolated.Test in laboratory mycobacterium tuberculosis is the Main Basis of clinical diagnosis of tuberculosis.At present, the main method of traditional detection mycobacterium tuberculosis is method of direct smear (luxuriant sodium method and fluorescence colour) and culture method, though luxuriant sodium smear method is simple and easy to do, recall rate is lower; Though fluorescence colour recall rate is higher, to equipment requirements also high (needing fluorescent microscope); Though culture method is gold standard, the cycle is oversize; All be difficult to meet clinical needs.In recent years, Real-time and Dynamic quantitative fluorescent PCR (fluorescent quantitative PCR, FQ-PCR) is also applied to clinical gradually, but this technology is owing to existing the problems such as false positive, false negative and expense are high.
In recent years, occur that mass-spectrometric technique is to detect nucleic acid and protein field, the theoretical basis that wherein mass-spectrometric technique is applied to field of nucleic acid detection is, the elementary cell of composition hereditary material DNA---there is mass discrepancy between four kinds of Nucleotide, molecular weight as ddAMP, ddCMP, ddGMP, ddTMP be followed successively by 271.2Da, 247.2Da, 287.2Da, 327.1Da(wherein ddTMP be through modification), minimum molecular weight difference between them, at 16Da, can be differentiated by mass spectrum completely.Use mass spectrum can detect multiple DNA change types such as base mutation or polymorphic site (SNP), insertion/deletion (InDel), methylation sites, gene quantification, copy numbers change (copy number variation, CNV).
More existing open source literature uses mass-spectrometric technique classify to microorganism and identify, such as, Chinese patent application CN102337223A, " penicillium chrysogenum antifungal protein Pc-Arctin and preparation method thereof ", disclose a kind of MALDI-TOF authentication method detecting penicillium chrysogenum antifungal protein Pc-Arctin, wherein cultivate from picking Penicllium chrysogenum A096 spore inoculating flat board in SGY liquid nutrient medium, pre-treatment obtains the separation and purification on a 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 the tested indicator of sensitivity, follow the trail of anti-mycotic activity component, the activeconstituents determined judges the purity obtaining albumen, extract the single band on SDS-PAGE electrophorogram, carry out MALDI-TOF qualification.The method is only applicable to specified microorganisms, and needs multiplexed protein purge process, and final with MALDI-TOF identification mark albumen Pc-Arctin, its process is loaded down with trivial details, applicable surface is narrow, can not realize the object of mass spectral classification bacterium or microorganism.
Chinese patent application 200910157210, the analytical procedure of fatty acid component " in a kind of listeria cell " discloses one and utilizes gaschromatographic mass spectrometry (GC-MS) analytical method directed toward bacteria lipid acid to carry out the method for classifying, comprise: listeria bacteria rejuvenation, with Oxford agar plate and trypticase soy yeast extract agar flat board abstraction and purification listeria bacteria respectively, cultivate the listeria bacteria of single colonies typical and make bacteria suspension, use formalin-inactivated process, bacteria suspension average mark after formaldehyde treated is contained in centrifuge tube washing, with the mixed solution esterification of hydrochloric acid and methyl alcohol, obtained lipid acid is carried out gaschromatographic mass spectrometry (GC-MS) analysis.The limitation of systematic bacteriology although the method breaks traditions, reduce human factor to classify the error brought to traditional form, simultaneously for the taxonomic identification of new bacterial classification and seed culture of viruses provides strong instrument, but this method still belongs to and utilizes mass spectroscopy to carry out CYTOCHEMICAL ANALYSIS classification, do not detect for nucleic acid.
International patent application WO2010/021548, " 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 ", disclose a kind of MALDI-MS (substance assistant laser desorpted and MALDI-MS) that uses for identifying the method and apparatus of biomaterial, comprise the liquid preparing to comprise sample and MALDI substrate material, and use it for the continuous a fluid stream forming liquid.This fluid stream is dispersed into part in succession, is transmitted into aloft drop to be formed, or a fluid stream is transmitted into in-flight, be then dispersed into drop.Drop formation technology known from ink-jet printer can be used.Material is ionized out to aloft drop.Measure the mass spectrum from the ionization material of each drop.But the method object is how to improve the sensitivity that MALDI-MS detects biological substance, does not relate to Mass Spectrometric Identification and any microorganism of classification, therefore can not solve the problems of the technologies described above.
Because mass-spectrometric technique bacterial detection also exists the problem being difficult to confirmed standard mass spectral characteristic collection of illustrative plates.That is, although the genomic dna between different bacterium there are differences, different nucleic acid fingerprint spectrum will be produced, if but there is no the mass-spectrogram database of Criterion, then because occur that the result of each mass spectrometric detection bacterium lacks repeatability because genome to be checked is too huge, accuracy is caused to decline.
Such as, the people such as Zhu Jian (" high performance liquid chromatography-electrospray multi-stage mass method is to the principium identification of component each in geldanamycin crude product and classification ", " Chinese microbiotic ", 03 phase in 2011) report application high performance liquid chromatography-electrospray multi-stage mass method (LC-ESI-MSn) carries out the structural information aspect relevant to total mass number principium identification and classification 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 geldanamycin (GDM), and Accurate classification has been carried out to various compound, but do not relate to directed toward bacteria predetermined substance (as nucleic acid) and carry out detecting thus the method for classifying to bacterium.
Bang flood (" application of MALDI TOF MS in Bacteria Detection and qualification ", " microbiology immunology progress ", 02 phase in 2003) report " chemical classification and the qualification of bacterium can be used in bacterial body containing a large amount of Biomarkers; obtain finger printing for the moiety such as according to bacterium and detect and qualification bacterium ", and its theoretic feasibility is predicted to the method.But this research is only inquired into the direction utilizing MALDI TOF MS to classify to bacterium in theory, its which kind of component both not indicated institute directed toward bacteria detects, and also concrete research method and process is not described.Because component (as albumen, DNA, RNA, the polysaccharide etc.) kind that can be used in bacterium classifying is too many, and mass-spectrometric technique also exists combination and the selection of various experiment parameter for different determinand, therefore MALDI TOF MS is not utilized to carry out the new report of Bacteria Identification in after this nearly 10 years.
Chinese patent application 201110154723, " MALDI TOF MS assistant identification list increases the method for listeria spp " and 201110154469, " method of MALDI TOF MS assistant identification vibrio cholerae " disclose a kind of method utilizing MALDI TOF MS technology assistant identification bacterium, comprise: pre-treatment bacterial cultures, gather the MALDI TOF MS collection of illustrative plates of all bacterial strain samples, bacterium standard diagram is prepared 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 coupling mark.Because the method uses conventional process (to pass through dehydrated alcohol, formic acid and acetonitrile treatment, and be aided with centrifugal, last Aspirate supernatant detects), although it can characterize the characteristic spectrum of this bacterium to a certain extent, but owing to containing protein in its determinand, lipid, lipopolysaccharides and fat oligosaccharides, DNA, polypeptide and the ionizable molecule of other energy, its collection of illustrative plates obtained is in fact the collection of illustrative plates set of above-mentioned various molecule, therefore both needed the profile information amount of process and comparison excessive, and cause its TuPu method on the low side because molecule to be checked is too huge, be only applicable to certain concrete bacterium and cannot be generalized in other a large amount of Bacteria Detection.
Due to the above-mentioned defect that mass-spectrometric technique bacterial detection exists, cause using mass-spectrometric technique the bacterial detection even drug resistant mutant genes of tubercule bacillus never progress at present.As immediate prior art, Chinese patent application 201110178412, " screening method of Mycobacterium tuberculosis drug-resistant protein " report a kind of in conjunction with sds gel electrophoresis and mass-spectrometric technique to screen the method for Mycobacterium tuberculosis drug-resistant protein, the method is mainly through after gel purified albumen, liquid chromatography mass spectrometric is used to carry out separation andpreconcentration drug-resistant protein, do not relate to and how to set up tubercule bacillus associated mass spectrometry feature database and utilize this storehouse to carry out the purposes of Bacteria Identification, somatotype, therefore cannot solve the problem yet.
Therefore need at present the qualification of new tubercule bacillus and analytical procedure (as mass spectroscopy) realizes fast, accurately, cheapness, easily classification results.
Summary of the invention
The principle of the invention is: the DNA for mycobacterium tuberculosis specific region designs suitable primer and carries out pcr amplification, then PCR primer is digested by specific restriction endonuclease, produce a series of length to differ the nucleic acid fragment that abundance differs, and carry out foranalysis of nucleic acids detection by MALDI-TOF MS mass spectrum, and form spectrogram.After increasing to mycobacterium tuberculosis target gene sequence, enzyme is cut, and obtains the collection of illustrative plates of mycobacterium tuberculosis.Mycobacterium tuberculosis standard diagram information can compare in experimental result and database, the classification to mycobacterium tuberculosis and qualification (qualification, somatotype, classification etc.) can be completed.The advantages such as this method has high specificity, highly sensitive, and cost is low, easy and simple to handle, and the used time is few.
Therefore, the present invention first object nucleic acid fingerprint characteristic spectrum library being to provide a kind of mycobacterium tuberculosis and uses thereof.It is characterized in that, which comprises at least following steps:
(1) PCR reaction: the PCR universal primer using directed toward bacteria, the nucleic acid-templated of multiple bacterium of increasing respectively, obtains the PCR primer containing amplification target area;
(2) SAP enzymic digestion: the PCR primer obtained with alkaline phosphatase (SAP enzyme) treatment step (1);
(3) transcribe and cut with nuclease: use and specifically transcribe and restriction endonuclease, respectively in a reaction system, the digestion product of each bacterium that step (2) obtains is transcribed and cuts with nuclease, obtain the nucleic acid fragment that a series of length differs, abundance differs;
(4) purifying: use the digestion products that resin treatment step (3) obtains;
(5) mass spectrograph detects: purified product step (4) obtained put on the target sheet containing matrix, and upper mass spectrograph detects, and obtains the nucleic acids characteristic fingerprint chromatogram of mycobacterium tuberculosis.
In one embodiment, described universal primer includes but not limited to sequence shown in SEQ ID No:1 to SEQ ID No:2.
In another embodiment, the certain enzyme described in step 3, includes but not limited to RNaseA enzyme.In a specific embodiment, the purifying of step 5 be included in transcribe with digestion products in add ultrapure water, after mixing, then add resin, mixing 15 minutes of turning upside down.In another embodiment, wherein said mass spectrograph is MALDI TOF MS mass spectrograph.
The second object of the present invention sets up the standard diagram of mycobacterium tuberculosis, at least comprises: above-mentioned steps 1-5, and;
(6) the nucleic acid fingerprint characteristic collection of illustrative plates of different isolates step 5 obtained, is undertaken gathering and arranging by computer software, obtains the standard nucleic acid fingerprint characteristic collection of illustrative plates of mycobacterium tuberculosis.
In one embodiment, described software is the BioExplore software that contriver researchs and develops voluntarily, and its copyright number steps on No. 136879th, word, registration number 2009SR10700 for soft work.
The third object of the present invention is to provide a kind of method utilizing above-mentioned characteristic spectrum storehouse Rapid identification mycobacterium tuberculosis, comprising:
(1) PCR reaction: use the PCR universal primer for mycobacterium tuberculosis, the nucleic acid-templated of bacterium to be measured of increasing, obtains the PCR primer containing amplification target area;
(2) SAP enzymic digestion: the PCR primer obtained with alkaline phosphatase (SAP enzyme) treatment step (1);
(3) transcribe and cut with nuclease: use and specifically transcribe and restriction endonuclease, in a reaction system, the digestion product of the bacterium that step (2) obtains is transcribed and cuts with nuclease, obtain the nucleic acid fragment that a series of length differs, abundance differs;
(4) purifying: use the digestion products that resin treatment step (3) obtains;
(5) mass spectrograph detects: purified product step (4) obtained put on the target sheet containing matrix, and upper mass spectrograph detects, and obtains the nucleic acid fingerprint characteristic collection of illustrative plates of this bacterium;
(6) concretion mycobacterium nucleic acid fingerprint characteristic collection of illustrative plates in gained nucleic acid fingerprint characteristic collection of illustrative plates and storehouse is compared, thus judge whether tested bacteria is mycobacterium tuberculosis.
The fourth object of the present invention be to provide can be used for mycobacterium tuberculosis classification and qualification, Clinical Laboratory test kit, comprising:
(1) for the DNA of bacteria that increases universal primer to and damping fluid;
(2) SAP enzyme and damping fluid thereof;
(3) RNAase and damping fluid thereof;
(4) for the resin of purifying digestion products;
(5) for the analysis software of comparison 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 steps on No. 136879th, word, registration number 2009SR10700).
Definition
" bacterium or mycobacterium tuberculosis classification and qualification " of the present invention, comprise bacterium identified and identifies, somatotype, point kind, classification.Such as in food safety detection, classified by mycobacterium tuberculosis or identify, accurately identifying source of pollution and bacterium composition, carrying out ensure food safety (as meat product, milk preparation safety) in order to adopting rational approach.And for example, in the evolution of research bacterium, by the identification of mycobacterium tuberculosis and classification/somatotype, the relationship distant relationships between various mycobacterium tuberculosis kind can be determined.A region on Mycobacterium tuberculosis DNA genome of the present invention, preferably has the region that high conservative has again certain polymorphism simultaneously.Conservative region of the present invention or conservative fragments, preferably mycobacterium tuberculosis rRNA region.RRNA is the important indicator of the evolution of research bacterium and sibship, its content reaches 80%, and be present in all bacteriums, rRNA gene is made up of conserved regions and variable region, guard at bacterium camber, have the title of " bacterial fossil ", is molecular clock useful and the most the most frequently used in bacterial systematics research.
In addition, at present with 16S rDNA carry out bacterium and mycobacterium tuberculosis classification and qualification method mainly adopt PCR primer 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 cover peak, is difficult to effectively distinguish; (4) 16S more than rDNA total length 1.5kb, generally needs through twice order-checking and result is spliced, easily introducing error in this process.
As previously mentioned, 16S rDNA has great importance to bacterium and mycobacterium tuberculosis taxonomic identification, but high, the consuming time length of method testing cost such as tradition order-checking; For the sample of polyinfection, sequencing will obtain the sequence peak figure mixed, be difficult to effectively distinguish, and utilize mass spectrum to carry out analyte analysis, need select suitable determinand and optimize mass spectrometry parameters, therefore need new division bacteria technology (as mass spectroscopy) to realize fast at present, accurately, cheapness, easily classification results.
" universal primer " of the present invention is the upstream and downstream in the region to be amplified that can be positioned at various bacterial genomes, the primer of the respective segments that can increase in different bacterium genome.Wherein, region to be amplified on genome of the present invention is selected from the sequence with sequence shown in SEQ ID NO:3 with at least 60% homology, 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.Due to for DNA of bacteria (such as 16S rDNA), its sequence is made up of conserved regions and variable region usually, and variable region mostly is discontinuous or short-movie section even SNP form be mixed in the middle of conserved regions or two ends, therefore use universal primer can to increase in different bacterium genome respective segments.Because this fragment of various bacterium all has certain homology, therefore have in the fragment of at least 60% homology with sequence shown in 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 mycobacterium tuberculosis 16S rDNA is 16S rDNA sequence C TGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAGTGTGGCCGGACACCC TCTCAGGCCGGCTACCCGTCGTCGCCTTGGTAGGCCGTCACCCCACCAACAAGCTG ATAGGCCGCGGGCTCATCCCACACCGCTAAAGCGCTTTCCACCACAAGACATGCAT CCCGTGGTCCTATCCGGTATTAGACCCAGTTTCCCAGGCTTATCCCGAAGTGCAGG GCAGATCACCCACGTGTTACTCACCCGTTCGCCACTCGAGTATCTCCGAAGAGACC TTTCCGTTCGACTTGCATGTGTTAAGCACGCCGCCAGCGTTCGTCCTGAGCCAGGA TCAAACTCT, i.e. SEQ ID NO:3.In a specific embodiment, the primer of this specific region is SEQ ID No:1 and SEQ ID No:2.
Beneficial effect
1, the present invention is based on mass spectrum detection, due to the high sensitivity of mass spectrometric detection, use this programme can far exceed other technologies scheme to the Monitoring lower-cut whether mycobacterium tuberculosis exists;
2, for different samples, the present invention can comparison their produce nucleic acid fingerprint spectrum, the collection of illustrative plates of mycobacterium tuberculosis reference culture in the nucleic acid fingerprint spectrum of experiment generation and database is contrasted, through bioinformatic analysis, can judge whether this bacterium is mycobacterium tuberculosis strain isolated;
3, use this programme can carry out the classification and identification of mycobacterium tuberculosis, and can be used for the aspects such as Clinical Laboratory.
4, relative to prior art, whole process only completes in a few hours, time saving and energy saving;
5, database of the present invention is open, constantly can supplement new strain isolated, constantly improves and expands database, to complete the qualification of mycobacterium tuberculosis more accurately;
6, in addition, the present invention proposes the 16S rDNA region of mycobacterium tuberculosis to carry out mass spectrometric detection as determinand, to obtain the nucleic acid fingerprint spectrum of the different isolates of mycobacterium tuberculosis, for the classification and identification of this kind first.
Accompanying drawing explanation
Fig. 1,2: the reproducible results of the nucleic acid fingerprint characteristic collection of illustrative plates of mycobacterium tuberculosis.
Fig. 3: to the mass spectrometric detection result of the mycobacterium tuberculosis of kindergarten's source of sewage.
Fig. 4: sample to be tested 1 is in the cultivation results of Roche solid medium.
Fig. 5: the coloration result of the luxuriant sodium resistance to acid smear method of sample to be tested 1.
Fig. 6: to the mass spectrometric detection result of the mycobacterium tuberculosis of inward passenger's (sample to be tested 2)
Fig. 7: to the fluorescence quantitative PCR detection result of sample to be tested 2
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment one: the foundation of concretion mycobacterium nucleic acid finger printing
One, design and select appropriate primer
According to the 16S gene order (SEQ ID NO:3) of mycobacterium tuberculosis (M. tuberculosis H37Rv), design PCR primer, is respectively:
| SEQ ID No：1 | 5-cagtaatacgactcactatagggagaaggctAGAGTTTGATCCTGGCTCAG-3（SEQ ID No：1） |
| SEQ ID No：2 | 5-aggaagagagCTGCTGCGTCCCGTAG-3（SEQ ID No：2） |
Wherein sequence A GAGTTTGATCCTGGCTCAG and CTGCTGCGTCCCGTAG mates with target area respectively, cagtaatacgactcactatagggagaaggct and aggaagagag is the additional sequences of adding in upstream and downstream PCR primer, guarantee to transcribe PCR primer, the tag(cagtaatacgactcactatagggagaaggct of 5' end containing 31bp of SEQ ID No:1 primer), the tag(aggaagagag of 5' end containing 10bp of SEQ ID No:2 primer).
Relevant primer synthesizes in Sangon Biotech (Shanghai) Co., Ltd..
Two, universal primer amplification
To the DNA of mycobacterium tuberculosis, use ABI 9700 type PCR instrument, carry out biological experiment operation.
1.PCR increases
(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 Sequenom company of the U.S..
(2) loop parameter of PCR is:
95 DEG C, 4 minutes, 1 circulation,
95 DEG C, 20 seconds, 56 DEG C, 30 seconds, 72 DEG C, 60 seconds, 45 circulations,
72 DEG C, 3 minutes, 1 circulation,
4 DEG C, preserve.
2.SAP enzymic digestion
(1) reaction system of SAP enzymic digestion is:
In the PCR primer of 5ul, add RNase-free ddH
2o:1.7ul, SAP enzyme (SAP enzyme, 1.7U/ul): 0.3ul.
Above reagent all comes from Sequenom company of the U.S..
(2) reaction parameter of SAP enzymic digestion is:
37 DEG C, 20 minutes, 1 circulation,
85 DEG C, 5 minutes, 1 circulation,
4 DEG C, preserve.
3. transcribe and cut with nuclease
(1) transcribing the reaction system of cutting with nuclease is:
Get the digestion product of 2ul, add:
| RNase-free ddH2O | 0.5ul |
| 5xT7 Polymerase Buffer | 0.04ul |
| T Cleavage Mix | 0.04ul |
| DTT（100mM） | 1ul |
| T7 RNA & DNA Polymerase | 1ul |
| RNaseA | 1ul |
Above reagent, all comes from Sequenom company of the U.S..
(2) transcribing the reaction parameter of cutting with enzyme is:
37℃，3h。
4. purifying
7ul transcribe with digestion products in add 20ul ultrapure water, after mixing, then add 6mg resin (resin, Sequenom company of the U.S.), mixing 15 minutes of turning upside down.
Three, concretion mycobacterium nucleic acid finger printing is set up
Product after purifying uses to receive and rises point sample instrument (Nanodispenser, Sequenom company of the U.S.), point extremely containing on the chip of matrix (SpectroCHIP, Sequenom company of the U.S.), and uses time-of-flight mass spectrometer (Sequenom company of the U.S.) to carry out detection and result judgement.
As can be seen from experimental result, the target area of the mycobacterium tuberculosis 16S gene order selected by the present embodiment, operate through biological experiment, create the nucleic acid fragment combination of different lengths and different abundance, through mass spectrometric detection, form special nucleic acid fingerprint spectrum, detected result, through bioinformatic analysis, can be used for detecting mycobacterium tuberculosis.
Fig. 1 with Fig. 2 repeats to test the identical nucleic acid fingerprint spectrum obtained.
The concretion mycobacterium nucleic acid fingerprint characteristic spectrum library that embodiment two, utilization are set up, identifies the environmental safety of public place
At the place such as water tap, ditch of certain public kindergarten of doubtful Child Pulmonary Tuberculosis contagium, collect source of pollution sample, be divided into two after the dilution of sample to be tested appropriateness, wherein according to the method for embodiment 1, pcr amplification is carried out to sample to be tested 1, after enzyme cuts, carry out mass spectrometric detection, whole process 1-2 hour consuming time.
Mass spectral characteristic Fig. 3 of gained and the bacteria nucleic acid fingerprint characteristic spectrum library implementing to obtain in three are compared, the judging criterion of employing is:
When 2.300≤coupling mark≤3.000, represent that the confidence level of strain identification is very high;
As 2.000≤coupling mark < 2.300, represent conservative identification of bacteria or possible strain identification;
Between 1.700≤coupling mark < 2.000, the identification of bacteria expressed possibility;
Between 0.000≤coupling mark < 1.700, represent incredible qualification.
Comparative analysis result is as follows:
The standard diagram comparison of the mycobacterium tuberculosis in the spectrum library that testing sample collection of illustrative plates is set up with the present invention respectively, coupling mark is 2.470, and be greater than 2.300, be reported as mycobacterium tuberculosis, meet completely with given value, the confidence level of qualification result is very high.
Embodiment three: 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 is cultivated 2 weeks upper 35 DEG C of Roche solid medium (comprising potato, Victoria Green WPB etc.), produces oyster white bacterium colony (see figure 4), the further biochemical test of bacterium colony that picking is suspicious and the microscopy of particle or cauliflower form.
2, sample to be tested is carried out microscopy, luxuriant sodium resistance to acid smear method presents redness, and thalline is elongated, holds extremely blunt circle, unpowered gemma and pod membrane (Fig. 5).
Thus, tentatively can determine 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, substratum making method: after first distilled water being mixed with glycerine, then add each composition successively, adjust ph value to 6.6 ~ 6.8 with 10% HCl after to be dissolved, high pressure 0.7 × 105pa, 20min sterilizing.To be cooled to 56 DEG C., add degerming calf serum, be mixed, be divided in culture dish, after 37 DEG C of aseptic mensuration 24h, store 4 DEG C of refrigerators for subsequent use.
In addition, aseptically, the asepsis injector of hyperfiltration is used to be added by Rifampin (100mg/L) in the slat chain conveyor ware made, after coating evenly, then after 37 DEG C of aseptic mensuration 24h.
2, experiment test
Sample to be tested 1 in embodiment two and blank (sterilized water) are made bacteria suspension, gets 0.1ml, slowly coat equably on the culture dish of blank cultures and Rifampin substratum, put in 37 DEG C 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.
Report colony number: when pastille substratum colony number is below 20, 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.
Being shown by the result implementing two and embodiment three, there is the pollution of mycobacterium tuberculosis in this kindergarten, needs to seal garden immediately, and to corresponding establishment disinfection, breaks out sporadic infection to prevent kindergarten.
The nucleic acid fingerprint characteristic spectrum library of the bacterium that embodiment four, utilization are set up, to the rapid detection of frontier port suspected patient
In certain port of entry, airport, to certain foreigner occurring heating, doubtful caking appears in violent cough and X-ray lung, after isolating, choose its sputum sample 5ml, add in 50 ml sterile centrifugation tube, add equivalent NACL-NaOH solution, vortex oscillator vibrates, abundant liquefy sputum sample, and room temperature leaves standstill 15min, add 0.067 mol/L PBS solution, abundant mixing with in and Digestive system effect, centrifugal 15 min of 3 000 g, carefully abandon supernatant, add 0.067mol/LPBS in throw out to be about 2ml and again to mix, be postdigestive phlegm sample to be tested 2.
Sample to be checked is divided into two, wherein according to the method for embodiment 1, pcr amplification carried out to sample to be tested 2, after enzyme cuts, carry out mass spectrometric detection, whole process 1-2 hour consuming time.
The mass spectral characteristic figure (Fig. 6) of gained and the bacteria nucleic acid fingerprint characteristic spectrum library implementing to obtain in are compared, comparative analysis result coupling mark is 2.380, is greater than 2.300, is reported as mycobacterium tuberculosis, meet completely with given value, the confidence level of qualification result is very high.Judge that this passenger is as mycobacterium tuberculosis positive patient thus.
The fluorescence quantitative PCR detection of embodiment five, sample to be tested 2
Sputum sample after digestion process is added the 4%NaOH of 4 times of volumes, liquefaction about 30min, gets 0.5ml in centrifuge tube, adds 0.5ml 4%NaOH, centrifugal 5 min of 10000 r/min after 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 DEG C of standing 6-8h to ensure abundant cracking.The centrifugal 5min of 10000r/min, gets supernatant liquor 2 μ l and increases, 93 DEG C of 2min denaturations, 93 DEG C of 45s, 55 DEG C of 60s increase 10 circulate, 93 DEG C of 30s, 55 DEG C of 45s, amplification 30 circulations.
Separately get negative quality control product, each 40 μ l of positive quality control product add equivalent DNA extraction liquid mixing, boiling water bath 10min process.Result judging criterion according to detection reagent specification sheets carries out interpretation of result, and result as shown in Figure 7.
By the result that the nucleic acid fingerprint characteristic Atlas Method implementing four and five is identified, can make a definite diagnosis this passenger is lunger, needs to isolate for treatment to it.
SEQUENCE LISTING
<110> is to China
Nucleic acid fingerprint characteristic spectrum library of <120> mycobacterium tuberculosis and uses thereof
<130> 2012
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> artificial sequence
<400> 1
aggaagagag agagtttgat cctggctcag 30
<210> 2
<211> 47
<212> DNA
<213> artificial sequence
<400> 2
cagtaatacg actcactata gggagaaggc tctgctgcgt cccgtag 47
<210> 3
<211> 358
<212> DNA
<213> known array
<400> 3
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
tcgacttgca tgtattaggc acgccgccag cgttcgtcct gagccaggat caaactct 358
Claims (5)
1. utilize mycobacterium tuberculosis fingerprint characteristic spectrum library for a mass spectrometry kit of classifying and identify, comprising:
(1) universal primer to and damping fluid;
(2) SAP enzyme and damping fluid thereof;
(3) RNAase and damping fluid thereof;
(4) for the resin of purifying digestion products;
(5) for the analysis software of comparison nucleic acid fingerprint characteristic collection of illustrative plates; Wherein
Described universal primer is to being specific primer pair for target area in the mycobacterium tuberculosis 16S rDNA that increases, and sequence is selected from
SEQ ID NO:15-cagtaatacgactcactatagggagaaggctAGAGTTTGATCCTGGCTCAG-3，
SEQ ID NO:25-aggaagagagCTGCTGCGTCCCGTAG-3；
Described AGAGTTTGATCCTGGCTCAG and CTGCTGCGTCCCGTAG mates with target area respectively, and cagtaatacgactcactatagggagaaggct and aggaagagag is the additional sequences of adding in upstream and downstream PCR primer, to guarantee to transcribe PCR primer;
In described 16S rDNA, target area is selected from the region shown in SEQ ID NO:3.
2. the test kit of claim 1, wherein said software is BioExplore software, and its soft work steps on that word is No. 136879, registration number is 2009SR10700.
3. utilize the test kit of claim 1 or 2 to prepare a preparation method for concretion mycobacterium nucleic acid fingerprint databases, comprise step:
(1) PCR reaction: use the described PCR universal primer pair for the target area in mycobacterium tuberculosis 16S rDNA, the mycobacterium tuberculosis of multiple strain isolated that increases respectively nucleic acid-templated, obtains the PCR primer containing amplification target area;
(2) SAP enzymic digestion: the PCR primer obtained with alkaline phosphatase (SAP enzyme) treatment step (1);
(3) transcribe and cut with nuclease: use and specifically transcribe and restriction endonuclease, respectively in a reaction system, the digestion product of each bacterium that step (2) obtains is transcribed and cuts with nuclease, obtain a series of length not
One, the nucleic acid fragment that differs of abundance;
(4) purifying: use the digestion products that resin treatment step (3) obtains;
(5) mass spectrograph detects: purified product step (4) obtained put on the target sheet containing matrix, and upper mass spectrograph detects, and obtains the nucleic acids characteristic fingerprint chromatogram of mycobacterium tuberculosis;
(6) the concretion mycobacterium nucleic acid fingerprint characteristic collection of illustrative plates of different isolates step (5) obtained, is undertaken gathering and arranging by computer software, obtains the nucleic acid fingerprint characteristic spectrum library of described mycobacterium tuberculosis.
4. utilize the test kit of claim 1 or 2, for the method that mycobacterium tuberculosis is identified or classified, comprising:
(1) PCR reaction: use described universal primer pair, amplification tested bacteria nucleic acid-templated, obtain containing amplification target area PCR primer;
(2) SAP enzymic digestion: the PCR primer obtained by alkaline phosphatase treatment step (1);
(3) transcribe and cut with nuclease: use and specifically transcribe and restriction endonuclease, in a reaction system, the digestion product of the bacterium that step (2) obtains is transcribed and cuts with nuclease, obtain the nucleic acid fragment that a series of length differs, abundance differs;
(4) purifying: use the digestion products that resin treatment step (3) obtains;
(5) mass spectrograph detects: purified product step (4) obtained put on the target sheet containing matrix, and upper mass spectrograph detects, and obtains the nucleic acid fingerprint characteristic collection of illustrative plates of this bacterium;
(6) by software, gained nucleic acid fingerprint characteristic collection of illustrative plates and concretion mycobacterium nucleic acid fingerprint characteristic spectrum library according to claim 4 are compared, thus judge whether tested bacteria is mycobacterium tuberculosis.
5. the method for claim 4, wherein universal primer is to being selected from sequence shown in SEQ ID No:1 to SEQ ID No:2, described mass spectrograph is MALDI TOF MS mass spectrograph, and described software is BioExplore software, and its soft work steps on that word is No. 136879, registration number is 2009SR10700.
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