CN102758012B - Nucleic acid fingerprint feature atlas database of mycobacterium tuberculosis rpoB gene and use of nucleic acid fingerprint feature atlas database - Google Patents
Nucleic acid fingerprint feature atlas database of mycobacterium tuberculosis rpoB gene and use of nucleic acid fingerprint feature atlas database Download PDFInfo
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Abstract
The invention discloses a method for detecting a mutant of a rifampicin resistance determining region (RRDR) for a mycobacterium tuberculosis rpoB gene. The method comprises polymerase chain reaction (PCR) amplification, shrimp alkaline phosphatase (SAP) digestion, transcription, restriction endonuclease, purification, mass spectrometer detection and other steps. Based on the method, a feature enzymatic mass spectrogram database for a wild RRDR or a mutant RRDR for the mycobacterium tuberculosis can be created. A mass spectrometer generated by an experiment is compared with the database to detect whether the RRDR of the mycobacterium tuberculosis rpoB gene mutates and the mutant, so as to determine the medicament resistance of the mycobacterium tuberculosis to rifampicin. The detection result is widely applied to clinical examination, disease prevention, epidemiological investigation, import and export commodity inspection and other fields.
Description
Technical field
The invention belongs to biological technical field, relate to the method for the nucleic acid finger printing preparation of a kind of mycobacterium tuberculosis rpoB gene RRDR, and use the method, the method that the mutant of mycobacterium tuberculosis rpoB gene RRDR to be checked is detected.
Background technology
In recent years, the drug resistance of tuberculosis epidemic situation is on the rise, and shows about investigating, and surpasses 50 countries and has the extensive tuberculosis patient of anti-the multiple medicines, and China ranks the second in the anti-multiple medicines tuberculosis height in 27, the whole world is born country, be only second to India.The breakthrough of early stage resistance diagnostic techniques is current control key lungy, therefore, sets up quick, sensitive drug resistant tuberculosis detection technique extremely urgent.Bacteriological resistance detection method remains the gold standard that drug resistant tuberculosis detects, but is limited by the poky characteristic of mycobacterium tuberculosis, and all tuberculosis bacteriology resistance detection methods all do not reach purpose fast at present.The advantage such as have fast, accurate, flux is high take the drug resistant gene detection as main various molecule susceptibility detection techniques, more and more become the focus of research.Rifampin RFP is a kind of broad spectrum antibiotic, can act on the RNA polymerase subunit β subunit (RNA polymerase B subunit, rpoB) that mycobacterium tuberculosis DNA relies on, thereby suppresses transcribing of mRNA.Mycobacterium tuberculosis is the major cause of tuberculosis chemotherapy failure to rifampin-resistance, and the detection of detection of rifampin resistant is the sign of judgement multiple-drug resistance tuberculosis sick (MDR-TB).
The rpoB gene is a single copy gene, sequence high conservative, 3543 bases of total length.Studies confirm that, when undergoing mutation, Rifampin can not be combined with RNA polymerase β subunit, suppresses to transcribe when high conservative nucleus (RRDR).The transgenation of the RFP Resistant strain of 95% left and right all concentrates on the zone of 27 amino acid (81bp) composition of 507～533, wherein, with 531 Ser → Leu and Trp conversion, the sudden change of 526 His → Tyr, Asp, Asn and Pro is the most common, and the sudden change in above two sites is the major causes that cause high-level resistance.Except above-mentioned two sites, also there is sudden change in 511,516,518,522 sites, and relative 531 and 526 is less, and are the reasons that causes low-level resistance.
more existing open source literatures are identified the rifampin-resistance site determining area (RRDR) of tubercule bacillus, for example, Chinese patent application 201110271351, " multiplex PCR-reverse dot blot hybridization technique detects the method for Drug Resistance of Mycobacterium Tuberculosis ", a kind of method that detects Drug Resistance of Mycobacterium Tuberculosis based on the reverse dot blot hybridization technique of multiplex PCR is disclosed, comprised the design of primer, the design of probe, cross experiment, result is judged, can detect simultaneously mycobacterium tuberculosis to RIF, the rpoB that the resistance of INH and EMB is relevant, katG, the base mutation of inhA generegulation district and embB transgenation hot zone.Yet, the method need to design multi-primers, and overcomes the defective of primer phase mutual interference in multiplex PCR, and the multiplex PCR process is loaded down with trivial details simultaneously, and reverse dot blot hybridization technique needs expensive immune biochemical reagent and plenty of time, therefore is difficult to realize testing goal fast and accurately.
Chinese patent application 200910114023, " detecting the Mycobacterium tuberculosis drug-resistant gene film chip " disclose a kind of detection Mycobacterium tuberculosis drug-resistant gene film chip, are also to hybridize for the mutational site designing probe of tubercule bacillus rpoB, kagG, embB, inhA, ahpC, gyrA, rrs, rpsL, pncA gene to detect on nylon membrane.Yet the method need to design nearly 54 kinds of probes, and testing process middle probe point sample and film chip all need accurate quantitative analysis and the sensitive operations such as elution of bound, therefore also is difficult to realize above-mentioned purpose.
Chen Shuanhong etc. (progress of tubercule bacillus laboratory diagnosis, Annali di Medicina Navale the 23rd the 2nd phase of volume in the 2002nd) roundup utilize bacillary detection, immunology detection, PCR diagnosis, DNA fingerprint technology, gene chip detecting technique and mass spectroscopy to come the somatotype tubercule bacillus.Wherein reported the good and bad point of various detection methods, and pointed out at present still to concentrate on round pcr for the detection of rpoB gene mutation site, this article is for mass spectrometric detection and analyze the mutational site and do not do further analysis.The theoretical basis that mass-spectrometric technique is applied to the detection of nucleic acids field is, the elementary cell that forms hereditary material DNA---there is mass discrepancy between four kinds of Nucleotide, as the molecular weight of ddAMP, ddCMP, ddGMP, ddTMP be followed successively by 271.2Da, 247.2Da, 287.2Da, 327.1Da(wherein ddTMP through modifying), minimum molecular weight difference between them can be differentiated by mass spectrum fully at 16Da.Use mass spectrum to change multiple DNA change types such as (copy number variation, CNV) and to detect base mutation or polymorphic site (SNP), insertion/deletion (InDel), the site that methylates, gene quantification, copy number.
Yet, exist the problem of the mass spectral characteristic collection of illustrative plates that is difficult to settle the standard due to the mass-spectrometric technique bacterial detection.That is to say, although the genomic dna between different bacterium there are differences, different nucleic acid finger printings will be produced, if but there is no the mass-spectrogram database of Criterion, lack repeatability because genome to be checked is too huge because the result of each mass spectrometric detection bacterium occurs, cause accuracy to descend.
For example, the people such as Zhu Jian (" principium identification and the classification of high performance liquid chromatography-electrospray multi-stage mass method to 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 relevant to total mass number structural information aspect 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 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 as the moiety according to bacterium and identifying bacterium ", and the method has been predicted its theoretic feasibility.Yet this research is only 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 the component that can be used in bacterium classifying (as albumen, DNA, RNA, polysaccharide etc.) kind is too many, therefore and mass-spectrometric technique also exists combination and the selection of various experiment parameters for different determinands, does not utilize MALDI TOF MS to carry out the new report of Bacteria Identification in after this nearly 10 years.
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 " disclose a kind of method of the 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 collection of illustrative plates more both, judge according to the coupling mark.because the conventional processing of the method use (is passed 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 containing 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.
Due to the defects that the mass-spectrometric technique bacterial detection exists, cause using at present the even drug resistant mutant genes progress never of tubercule bacillus of mass-spectrometric technique bacterial detection.As immediate prior art, Chinese patent application 201110178412, " screening method of Mycobacterium tuberculosis drug-resistant protein " have been reported a kind of method of screening Mycobacterium tuberculosis drug-resistant protein in conjunction with sds gel electrophoresis and mass-spectrometric technique, the method is mainly by after the gel electrophoresis purifying protein, separate and identify drug-resistant protein with the liquid phase mass spectrum, do not relate to and identify drug resistant gene and relevant mass spectral characteristic storehouse, therefore can't address the above problem yet.Therefore at present need the detection method (as mass spectroscopy) of the drug resistant mutant genes of new tubercule bacillus rpoB gene to realize fast, accurately, cheapness, classification results easily.
Summary of the invention
The principle of the invention is: the suitable primer of DNA design for mycobacterium tuberculosis rpoB gene high conservative nucleus (RRDR) carries out pcr amplification, then the PCR product is digested by specific restriction endonuclease, produce a series of length nucleic acid fragment that abundance differs that differs, and carry out foranalysis of nucleic acids and form spectrogram by MALDI-TOF MS mass spectrum.Owing to cutting for enzyme after the amplification of the rpoB gene RRDR of mycobacterium tuberculosis wild-type and mutant, dwindled significantly the genome area of mass spectrometric detection, and the mass spectrum after cutting by enzyme has stable characteristic spectrum, thereby realizes the detection to detection of rifampin resistant.
Therefore, the contriver is through groping in a large number and comparing, research for 71 kinds of mutation types of mycobacterium tuberculosis wild-type and rifampin-resistance zone, discovery selects specific primer to carry out pcr amplification to mycobacterium tuberculosis DNA, after using specific enzymes that amplified production is processed, carry out mass spectrometric detection and can obtain various mutant nucleic acid fingerprint characteristic collection of illustrative plates.
Therefore, first purpose of the present invention is to provide the test kit that can be used for mycobacterium tuberculosis detection of rifampin resistant detection applications, comprises for the RRDR zone specific primer of amplification mycobacterium tuberculosis DNA pair, and described primer is as shown in the table:
| SEQ ID No：1 | cagtaatacgactcactatagggagaaggctGAGCGGATGACCACCCAGG |
| SEQ ID No：2 | aggaagagagCACGCTCACGTGACAGACC |
Wherein, the sudden change in described genome area has caused the resistance of mycobacterium tuberculosis to Rifampin.
In one embodiment, described test kit also comprises:
(1) the right damping fluid of specific primer;
(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) be used 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 step on word No. 136879, registration number 2009SR10700.
Second purpose of the present invention is to provide the method for utilizing the mentioned reagent box to prepare the nucleic acid finger printing of mycobacterium tuberculosis rpoB gene RRDR, and it comprises the steps:
(1) PCR reaction: use the specific PCR primer of mycobacterium tuberculosis, amplification concretion mycobacterium nucleic acid template obtains containing the PCR product of RRDR;
(2) SAP enzymic digestion: the PCR product that obtains with alkaline phosphatase treatment step (1);
(3) endonuclease reaction: use specific restriction endonuclease, in a reaction system, the amplified production that step (2) is obtained carries out endonuclease reaction, 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, to avoid salt ion on the impact of subsequent detection;
(5) mass spectrograph detects: on the purified product that step (4) is obtained, point is containing on the target sheet of matrix, and upper mass spectrograph detects, and obtains the feature nucleic acid fingerprint chromatogram of different mycobacterium tuberculosis strain isolateds;
In one embodiment, the described certain enzyme of step 3 includes but not limited to the RNaseA enzyme.
In another embodiment, the purifying of step 5 is included in to transcribe to cut with enzyme and adds ultrapure water in product, after mixing, then adds resin, 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 mycobacterium tuberculosis comprises wild-type as listed in table 1 and 69 kinds of common mutation types.Mutation type described in table all occurs in and comprises the RRDR zone on the gene order of interior 200bp.
| The mutational site | Wild-type | Mutant |
| 489 | CCG | CCC |
| 490 | CAG | CAC |
| 491 | ACG | CAG |
| 501 | GCG | GCA |
| 507 | GGC | GAC/GGT |
| 508 | ACC | CCC |
| 510 | CAG | CAC |
| 511 | CTG | TTG/ATG/CCG/CGG/ |
| 512 | AGC | GGC/AGG |
| 513 | CAA | CGA/AAA/CCA/GAA |
| 515 | ATG | ATT/GTG |
| 516 | GAC | GCC/GTC/GAA/GGC/AAA/AAC/TAC |
| 517 | CAG | CTG/CGG |
| 518 | AAC | GAC/AGC/TAC |
| 519 | AAC | AAT |
| 521 | CTG | TTG |
| 522 | TCG | GCG/TTG/CCG/TCC/ACG/TGG |
| 523 | GGG | GCG/GGC |
| 525 | ACC | ACT |
| 526 | CAC | GCC/GAC/CTC/AAC/CCC/CAG/CGA/CGC/GTC/TAC |
| 529 | CGA | AAA/CAA/CGG |
| 530 | CTG | CGG |
| 531 | TCG | GCG/TTC/TTG/CCG/CAG/TGG/TAC |
| 532 | GCG | GAG/GTG |
| 533 | CTG | TTG/ATG/CCG/ |
Table 1
The 3rd purpose of the present invention is to provide the method for the nucleic acid fingerprint characteristic spectrum library of setting up mycobacterium tuberculosis rpoB gene RRDR, comprises at least:
Above-mentioned steps 1-5, and:
The nucleic acid fingerprint characteristic collection of illustrative plates of the mycobacterium tuberculosis wild-type that (6) step 5 is obtained and mutant gathers and arranges by computer software, obtains the nucleic acid fingerprint characteristic spectrum library of described mycobacterium tuberculosis rpoB gene RRDR.
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 4th purpose of the present invention is to provide the nucleic acid fingerprint characteristic spectrum library of the RRDR saltation zone of the prepared mycobacterium tuberculosis rpoB gene of aforesaid method.
The present invention's the 5th purpose is to provide a kind of above-mentioned characteristic spectrum storehouse of utilizing and carries out the method that the mycobacterium tuberculosis detection of rifampin resistant detects, and comprising:
(1) PCR reaction: uses specific PCR primer, the nucleic acid-templated of mycobacterium tuberculosis to be measured of increasing 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: on the purified product that step (4) is obtained, point is containing on the target sheet of matrix, and upper mass spectrograph detects, and obtains the nucleic acid fingerprint characteristic collection of illustrative plates of this bacterial strain rifampin-resistance determining area;
(6) gained nucleic acid fingerprint characteristic collection of illustrative plates and nucleic acid fingerprint characteristic spectrum library are compared, thereby whether the rpoB gene RRDR that judges mycobacterium tuberculosis to be measured undergos mutation and mutation type.
In one 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.
Definition
" detection of mycobacterium tuberculosis rpoB gene RRDR mutant " of the present invention comprises the judgement to mycobacterium tuberculosis mutation type and detection of rifampin resistant.For example, blood sample or tissue juice sample to individuality are analyzed, judge whether its mycobacterium tuberculosis rpoB gene RRDR undergos mutation and mutation type, thereby judge whether mycobacterium tuberculosis resistance occurs to Rifampin, thereby be conducive to the screening of related drugs.
" nucleic acid finger printing " of the present invention refers to comprise that a segment length of mycobacterium tuberculosis rifampin-resistance determining area is at the DNA sequence dna of 200bp left and right, the spectrogram that mass spectrometric detection obtains after enzyme is cut.Rifampin-resistance determining area (rifampicin resistance determing region, RRDR), refer to be positioned at that on mycobacterium tuberculosis rpoB gene, length is the conserved sequence of 81bp, the transgenation of RFP Resistant strain all concentrates on the zone of 27 amino acid (81bp) composition of 507～533, and 97% rifampin-resistant is by due to this sequence (511,513,515,516,518,519,522,526,531 and 533 site) sudden change.
The present invention utilizes the conservative section design universal primer (SEQ ID No:1 to SEQ ID No:2) of the 81bp of mycobacterium tuberculosis, the amplification of this primer comprises mycobacterium tuberculosis rifampin-resistance determining area (RRDR, 81bp) the sequence about 200 interior bp, mutant database of the present invention comprises that RRDR upward also comprises the part mutational site that this zone upstream and downstream has been found in common mutational site.
Be applied at present technological line that detection of rifampin resistant detects and be basically with PCR method to amplification basis, rpoB transgenation concentrated area on, to the amplified production evaluation that suddenlys change, existing method has direct sequencing, polymerase chain reaction-single-strand conformation polymorphism analysis method (PCR-SSCP), dideoxy fingerprinting method, heteroduplex forming method (HDF), reverse serial probe hybridization method (LiPA) etc.It is a kind of new technique means that mass-spectrometric technique is used for that detection of rifampin resistant detects, this technology of more existing molecular biology for detection, the advantage such as it is quick, accurate, cheap that the method has concurrently.
Beneficial effect
1. the present invention's rifampin-resistance determining area enzyme of repeatedly studying mycobacterium tuberculosis wild-type and 69 kinds of mutants is cut and the mass spectrometric detection result, find and obtain various concretion mycobacterium nucleic acid fingerprint characteristic collection of illustrative plates, building database can effectively detect the detection of rifampin resistant of mycobacterium tuberculosis;
2. the present invention proposes the rifampin-resistance determining area of mycobacterium tuberculosis is carried out mass spectrometric detection as determinand first, to obtain the different nucleic acid finger printing of mycobacterium tuberculosis, is used for the detection of detection of rifampin resistant;
3. through repeatedly testing and groping, design first and utilize mass spectrometric detection rifampin-resistance determining area, obtain the flow process of concretion mycobacterium nucleic acid finger printing;
4. due to the high sensitivity of mass spectrometric detection, use this programme to detect lower limit to bacterium amount in sample and can far exceed other technologies scheme (as biochemistry detection, order-checking detection etc.);
5. database of the present invention is open, can constantly replenish newfound mutation type, constantly improves and enlarges database, better serves the tuberculosis rifampin-resistance and detects and study;
6. with respect to traditional bacteriological resistance detection method, this testing process is only completed in a few hours, and is time saving and energy saving;
7. with respect to the direct Sequencing method, the bacterial nucleic acid finger printing has better resolving power for biased sample, and more saves time and save cost;
8. use this programme can judge resistance and the mutant thereof of mycobacterium tuberculosis Rifampin fast, and this technology can be widely used in clinical treatment lungy, and epiphytotics investigation.
Description of drawings
Fig. 1,2 is mycobacterium tuberculosis reference culture nucleic acid fingerprint characteristic collection of illustrative plates.
Fig. 3,4 is the nucleic acid fingerprint characteristic collection of illustrative plates of mycobacterium tuberculosis strain isolated 1.
Fig. 5,6 is the nucleic acid fingerprint characteristic collection of illustrative plates of mycobacterium tuberculosis strain isolated 2.
Fig. 7-Figure 76 is that the analogue enztme of 69 kinds of mycobacterium tuberculosis mutants shown in wild-type and table 2 is cut nucleic acid fingerprint characteristic collection of illustrative plates.
No. 1 mutant in Fig. 8 table 2 (489 CCG → CCC).
No. 20 mutant in Figure 27 table 2 (518 AAC → AGC).
No. 40 mutant in Figure 47 table 2 (513 CAA → CGA).
No. 60 mutant in Figure 67 table 2 (532 GCG → GTG).
Figure 77-78 are sputum sample 1 nucleic acid finger printing and sequencing result collection of illustrative plates in embodiment 3.
Figure 77 embodiment 3 sputum sample 1 mass spectrums.
Figure 78 embodiment 3 sputum sample 1 order-checking spectrograms.
Figure 79-80 are sputum sample 2 nucleic acid finger printings and sequencing result collection of illustrative plates in embodiment 4.
Figure 79 embodiment 4 sputum sample 2 mass spectrums.
Figure 80 embodiment 4 sputum sample 2 order-checking spectrograms.
Figure 81-82 are sputum sample 3 nucleic acid finger printings and sequencing result collection of illustrative plates in embodiment 4.
Figure 81 embodiment 4 sputum sample 3 mass spectrums.
Figure 82 embodiment 4 sputum sample 3 order-checking spectrograms.
Figure 83-84 are sputum sample 4 nucleic acid finger printings and sequencing result collection of illustrative plates in embodiment 4.
Figure 83 embodiment 3 sputum sample 4 mass spectrums.
Figure 84 embodiment 4 sputum sample 4 order-checking spectrograms.
Figure 85-86 are sputum sample 5 nucleic acid finger printings and sequencing result collection of illustrative plates in embodiment 4.
Figure 85 embodiment 4 sputum sample 5 mass spectrums.
Figure 86 embodiment 4 sputum sample 5 order-checking spectrograms.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1: the foundation of single mycobacterium tuberculosis rifampin-resistance determining area nucleic acid finger printing
One, design and select suitable primer
According to the 81bp sequence of the rpoB gene RRDR of mycobacterium tuberculosis (Mycobacterium tuberculosis H37Rv), design universal PC R primer is respectively:
| SEQ ID No：1 | cagtaatacgactcactatagggagaaggctGAGCGGATGACCACCCAGG |
| SEQ ID No：2 | aggaagagagCACGCTCACGTGACAGACC |
Wherein sequence GAGCGGATGACCACCCAGG and CACGCTCACGTGACAGACC respectively with rpoB Region Matching to be amplified, cagtaatacgactcactatagggagaaggct and aggaagagag are the additional sequences of adding on upstream and downstream PCR primer, guarantee for the PCR product is transcribed, the 5' end of SEQ ID No:1 primer contains the tag(cagtaatacgactcactatagggagaaggct of 31bp), the 5' end of SEQ ID No:2 primer contains the tag(aggaagagag of 10bp).
Relevant primer synthesizes in Sangon Biotech (Shanghai) Co., Ltd..
Two, universal primer amplification
According to the method for the extraction DNA of bacteria of " molecular cloning ", extract respectively tubercule bacillus standard bacterial classification 1 and 2 and the DNA of the mycobacterium tuberculosis of strain isolated 1 to be measured and 2, use ABI 9700 type PCR instrument, carry out the biological experiment operation.
1.PCR amplification
(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 | 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 U.S. Sequenom company.
(2) transcribing the reaction parameter of cutting with enzyme is:
37℃，3h。
4. purifying
Add the 20ul ultrapure water in transcribing of 7ul cut product with enzyme, after mixing, then add 6mg resin (resin, U.S. Sequenom company), mixing 15 minutes turns upside down.
Three, set up single mycobacterium tuberculosis rifampin-resistance determining area nucleic acid finger printing
Product after purifying uses to receive and rises point sample instrument (Nanodispenser, U.S. Sequenom company), point and uses time-of-flight mass spectrometer (U.S. Sequenom company) to detect with result to judge to the chip that contains matrix (SpectroCHIP, U.S. Sequenom company).
Use such scheme, carry out confirmatory experiment, mycobacterium tuberculosis strain isolated 1 and 2 is carried out enzyme is cut and Mass Spectrometric Identification.Two bacterial strains respectively repeat twice, all obtain identical nucleic acid fingerprint characteristic collection of illustrative plates, and wherein Fig. 3 and Fig. 4, Fig. 5 and Fig. 6 show respectively the nucleic acid fingerprint characteristic collection of illustrative plates of the rifampin-resistance determining area of mycobacterium tuberculosis strain isolated 1 and 2.
With the mass spectrum of strain isolated to be measured 1 and 2 as a result figure compare with contrast Fig. 1-2, can find out, the selected mycobacterium tuberculosis rifampin-resistance of the present embodiment determining area, through the biological experiment operation, produced the nucleic acid fragment combination of different lengths and different abundance, through mass spectrometric detection, formation is different from the specific nucleic acid finger printing (as 2000-3000m/z, 3400-4000 m/z zone) that contrasts collection of illustrative plates, and the collection of illustrative plates of strain isolated 1 and 2 also there are differences, this shows strain isolated 1 and 2 with respect to wild strain, has different sudden change resistance sites.Through biological analysis, can judge that the sudden change of 516 GAC → GGC has occured strain isolated 1, the sudden change of 526 CAC → AAC has occured in strain isolated 2.Thus, can be used for detection of rifampin resistant is judged.
Embodiment 2: the foundation of the nucleic acid fingerprint characteristic spectrum library of mycobacterium tuberculosis rpoB gene RRDR
Method described according to embodiment one and primer, in his-and-hers watches 1, listed mycobacterium tuberculosis mutant carries out that enzyme is cut and Mass Spectrometric Identification, obtains the nucleic acid fingerprint characteristic collection of illustrative plates of all common mutations types.
With these characteristic spectrums, carry out confluence analysis by Bioexplore software, thereby obtain comprising the nucleic acid fingerprint characteristic spectrum library of the rpoB gene RRDR of mycobacterium tuberculosis wild-type and common mutations type.
As shown in table 2, Fig. 7 is the nucleic acid fingerprint characteristic collection of illustrative plates of mycobacterium tuberculosis wild-type, and Fig. 8-Figure 76 shows respectively the nucleic acid fingerprint characteristic collection of illustrative plates of 69 kinds of mycobacterium tuberculosis mutants.
| The 1-23 mutation type | The 24-46 mutation type | The 47-69 mutation type |
| 489 CCG → CCC | 516 GAC → GGC | 526 CAC → AAC |
| 490 CAG → CAC | 516 GAC → AAA | 526 CAC → CCC |
| 491 ACG → CAG | 516 GAC → AAC | 526 CAC → CAG |
| 501 GCG → GCA | 516 GAC → TAC | 526 CAC → CGA |
| 507 GGC → GGT | 517 CAG → CTG | 526 CAC → CGC |
| 507 GGC → GAC | 517 CAG → CGG | 526 CAC → GTC |
| 508 ACC → CCC | 518 AAC → GAC | 526 CAC → TAC |
| 510 CAG → CAC | 518 AAC → AGC | 529 CGA → AAA |
| 511 CTG → TTG | 518 AAC → TAC | 529 CGA → CAA |
| 511 CTG → ATG | 519 AAC → AAT | 529 CGA → CGG |
| 511 CTG → CCG | 521 CTG → TTG | 530 CTG → CGG |
| 511 CTG → CGG | 522 TCG → GCG | 531 TCG → GCG |
| 512 AGC → GGC | 522 TCG → TTG | 531 TCG → TTC |
| 512 AGC → AGG | 522 TCG → CCG | 531 TCG → TTG |
| 513 CAA → CGA | 522 TCG → TCC | 531 TCG → CCG |
| 513 CAA → AAA | 522 TCG → ACG | 531 TCG → CAG |
| 513 CAA → CCA | 522 TCG → TGG | 531 TCG → TGG |
| 513 CAA → GAA | 523 GGG → GCG | 531 TCG → TAC |
| 515 ATG → ATT | 523 GGG → GGC | 532 GCG → GAG |
| 515 ATG → GTG | 525 ACC → ACT | 532 GCG → GTG |
| 516 GAC → GCC | 526 CAC → GCC | 533 CTG → TTG |
| 516 GAC → GTC | 526 CAC → GAC | 533 CTG → ATG |
| 516 GAC → GAA | 526 CAC → CTC | 533 CTG → CCG |
[0116]Table 2
Embodiment 3: utilize the nucleic acid fingerprint characteristic spectrum library of the mycobacterium tuberculosis rifampin-resistance determining area of setting up, clinical sample is detected
Get tuberculosis patient sputum 2ml.Wherein sample to be tested 1 is carried out following processing, add 2.5 times of volume 4% NaOH, 37 ℃ of incubation 30min.Sputum after liquefaction is centrifugal, abandon supernatant, the gained precipitation is used for the extraction of sample DNA.Carry out the extraction of sample DNA with commercial genome DNA extracting reagent kit.The genomic dna that obtains is divided into two, and sample 1 carries 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.Result as shown in Figure 7.
The nucleic acid fingerprint characteristic spectrum library of the mycobacterium tuberculosis rpoB gene RRDR that obtains in the mass spectral characteristic figure of gained and embodiment 2 is compared, and comparison process is completed by BioExplore software, mainly is divided into two steps, and is as follows:
1. in the collection of illustrative plates of experiment acquisition and storehouse, wild-type is compared, and the judging criterion of employing is:
When 2.300≤coupling mark≤3.000, the expression Sequence Identification is that the confidence level of wild-type is very high;
When 0.000≤coupling mark＜2.300, express possibility and undergo mutation.
May undergo mutation if be judged as, carry out the judgement of step 2.
2. mutant comparing in the collection of illustrative plates that obtains of experiment and storehouse, the judging criterion of employing is:
When 2.300≤coupling mark≤3.000, represent that the confidence level of this mutant is very high;
When 2.000≤coupling mark＜2.300, expression is undergone mutation but this mutant can not be determined;
When 1.700≤coupling mark＜2.000 between, express possibility and undergo mutation;
0.000≤coupling mark＜1.700 between, represent incredible evaluation.
The comparative analysis result is as follows:
Spectrogram contrast in the spectrum library that the testing sample collection of illustrative plates is set up with the present invention respectively is reported as this mycobacterium tuberculosis rifampin-resistance determining area and undergos mutation, and this mutation type is 511 CTG → CCG, and this bacterium has resistance to Rifampin.
Comparative examples 1:
Use the method for aforementioned extraction DNA of bacteria, sample 1 is carried out direct Sequencing detect, result as shown in Figure 8.
By analysis, sequencing result is consistent with the mass spectrometric detection result, and this bacterial strain is 511 sudden changes that CTG → CCG occurs.
The result that the nucleic acid fingerprint characteristic Atlas Method of embodiment 3 is identified shows, this sufferer resistance occurs to rifampicin medicine, needs in time to change treatment plan.
Embodiment 4 utilizes the nucleic acid fingerprint characteristic spectrum library of the mycobacterium tuberculosis rifampin-resistance determining area of setting up, and a plurality of clinical samples are detected
According to the method for embodiment 3, the sputum sample of a plurality of sufferers is carried out mass spectrometric detection, the detection of checking order simultaneously, result is as shown in table 3.
| Sample to be tested | Mass spectrum is figure as a result | Mutation type | Sequencing result figure | Mutation type |
| 2 | Figure 79 | CAC→TAC | Figure 80 | CAC→TAC |
| 3 | Figure 81 | CAC→GAC | Figure 82 | CAC→GAC |
| 4 | Figure 83 | TCG→TTG | Figure 84 | TCG→TTG |
| 5 | Figure 85 | CAC→CGC | Figure 86 | CAC→CGC |
SEQUENCE LISTING
＜110〉to China
＜120〉nucleic acid fingerprint characteristic spectrum library of mycobacterium tuberculosis rpoB gene and uses thereof
<130> 2012
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> DNA
＜213〉artificial sequence
<400> 1
cagtaatacg actcactata gggagaaggc tgagcggatg accacccagg 50
<210> 2
<211> 29
<212> DNA
＜213〉artificial sequence
<400> 2
aggaagagag cacgctcacg tgacagacc 29
Claims (1)
1. test kit comprises following primer:
SEQ ID No：1 cagtaatacgactcactatagggagaaggctGAGCGGATGACCACCCAGG
SEQ ID No：2 aggaagagagCACGCTCACGTGACAGACC
Wherein this primer is for the RRDR zone specific primer of amplification mycobacterium tuberculosis DNA pair, and this test kit can be used for detecting nucleic acid finger printing and the spectrum library of mycobacterium tuberculosis detection of rifampin resistant and preparation RRDR.
2
.Test kit claimed in claim 1 wherein also comprises:
The damping fluid that specific primer is right;
SAP enzyme and damping fluid thereof;
RNAase and damping fluid thereof;
Be used for purifying enzyme and cut the resin of product;
The analysis software that is used for comparison nucleic acid fingerprint characteristic collection of illustrative plates.
3
.Test kit claimed in claim 2, wherein said software are BioExplore software, and its copyright number is soft work step on word No. 136879, registration number 2009SR10700.
4
.Utilize arbitrary described test kit in claim 1 to 3 to prepare the method for the nucleic acid finger printing of mycobacterium tuberculosis rpoB gene RRDR, comprise step:
(1) PCR reaction: uses the PCR primer of specific resistance determining area, the concretion mycobacterium nucleic acid template that increases obtains containing the PCR product of rifampin-resistance determining area;
(2) SAP enzymic digestion: the PCR product that obtains with SAP enzyme treatment step (1);
(3) endonuclease reaction: use specific restriction endonuclease, in a reaction system, the amplified production that step (2) is obtained carries out endonuclease reaction, 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, to avoid salt ion on the impact of subsequent detection;
(5) mass spectrograph detects: on the purified product that step (4) is obtained, point is containing on the target sheet of matrix, and upper mass spectrograph detects, and obtains the feature nucleic acid fingerprint chromatogram of drug resistance of Mycobacterium tuberculosis determining area.
5
.Method according to claim 4, wherein the specific restriction endonuclease of step (3) includes but not limited to the RNaseA enzyme; The purifying of step (4) is included in enzyme and cuts and add ultrapure water in product, after mixing, then adds resin, and mixing 15 minutes turns upside down.
6
.Method claimed in claim 5, wherein said mass spectrograph are MALDI TOF MS mass spectrographs.
7
.Method claimed in claim 4, wherein said mycobacterium tuberculosis comprise wild-type as listed in table 1 and 69 kinds of mutation types.
8
.The nucleic acid fingerprint databases of a kind of mycobacterium tuberculosis rpoB gene RRDR, its preparation method comprises:
Step claimed in claim 4 (1)-(5), and;
The nucleic acid fingerprint characteristic collection of illustrative plates of the various mycobacterium tuberculosis that (6) step (5) obtained gathers and arranges by computer software, obtains the nucleic acid fingerprint characteristic spectrum library of described mycobacterium tuberculosis rpoB gene RRDR.
9
.The spectrum library of claim 8, wherein said software are BioExplore software, and its copyright number is soft work step on word No. 136879, registration number 2009SR10700.
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| US20110105531A1 (en) * | 2007-06-22 | 2011-05-05 | Ibis Biosciences, Inc. | Compositions and methods for identification of subspecies characteristics of mycobacterium tuberculosis |
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| CN101838683A (en) * | 2008-12-12 | 2010-09-22 | 深圳华大基因科技有限公司 | Detection method of nucleotide mutation points of KRAS gene and/or BRAF gene |
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