CN1635158A - Preparation of mycobacterium molecular bacteria identification reagent box and use thereof - Google Patents

Preparation of mycobacterium molecular bacteria identification reagent box and use thereof Download PDF

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CN1635158A
CN1635158A CN 200410083825 CN200410083825A CN1635158A CN 1635158 A CN1635158 A CN 1635158A CN 200410083825 CN200410083825 CN 200410083825 CN 200410083825 A CN200410083825 A CN 200410083825A CN 1635158 A CN1635158 A CN 1635158A
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mycobacterium
primer
identification
sequence
preparation
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CN1311085C (en
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吴雪琼
梁建琴
张俊仙
李洪敏
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BEIJING CONTROLS & STANDARDS BIOTECHNOLOGY CO., LTD.
309th Hospital of PLA
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309th Hospital of PLA
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Abstract

The invention relates to a mycobacterium molecule strain identification kit and its preparation method. The kit is prepared by the following steps: designing and synthesizing corresponding oligonucleotide probes according to different mycobacterium 16S rRNA gene sequences, dotting the oligonucleotide probes according to certain sequence on the pretreated nitrate fiber film by dot plot method, then obtaining detection identification film sheet, and hybridizing with the PCR amplification products having biotin marks. According to the bluish purple signal strength of the probes hybridization to interpret the result with naked eyes, the invented identification kit can quickly, precisely detect the mycobacterium, identify the mycobacterium and the non-mycobacterium, the tuberculosis mycobacterium complex and the non-tuberculosis mycobacterium, and the mycobacterium strain, and the identification results can be reported in 24 hours to assist clinical diagnose and guide early chemotherapy.

Description

The preparation of mycobacterium molecular bacteria identification reagent box and application thereof
Technical field
The present invention relates to the preparation and the application thereof of molecular bacteria identification reagent box, it belongs to Medical Molecular Biology diagnostic techniques field, specifically is about the preparation of mycobacterium molecular bacteria identification reagent box and the application in diagnosis of tuberculosis thereof.
Background technology
Still one of main transmissible disease of harm humans health in whole world tuberculosis, the immigrant of popular, the tuberculosis infection of acquired immune deficiency syndrome (AIDS) and the groups of people all living creatures reason such as poverty of living makes American-European developed country incidence of tuberculosis such as U.S. be rise trend since 1985, and especially the sickness rate of tubercule bacillus resistance problem and non-tuberculous mycobacteria disease rises year by year tuberculotherapy is made the matter worse especially.
At present the whole world has tuberculosis patient about 2,000 ten thousand, annual newly-increased tuberculosis patient 800～1,000 ten thousand, annual death toll about 3,000,000.The World Health Organization (WHO) announced " the global tuberculosis emergency state " unprecedentedly in 1993, reaffirmed again to contain that action lungy was very urgent in 1998.
Tuberculosis epidemic situation of China and resistance situation are all quite serious, are one of the high burden of 22 tuberculosis in whole world countries, and the tuberculosis patient numerical digit occupies the second in the world, is only second to India.2000 the 4th time national tuberculosis epidemiological random sampling survey PRELIMINARY RESULTS shows that China has 5.5 hundred million people to infect tubercule bacillus; Existing pulmonary tuberculosis patient 4,510,000, wherein the infectivity pulmonary tuberculosis patient 1,960,000; Annual death toll is about 130,000, and tuberculosis death occupies the 9th in the various causes of death of China, and the residence is first in transmissible disease; In the isolated mycobacterium, mycobacterium tuberculosis accounts for 86.4%, and Mycobacterium bovis accounts for 2.5%, and non-tuberculous mycobacteria accounts for 11.1%, the more preceding obvious increase of the ratio of non-tuberculous mycobacteria.
Tracing it to its cause is that the non-tuberculous mycobacteria patient has natural drug-fastness to most of line antitubercular agents, adopts the chemotherapy regimen of present standard to fail to respond to any medical treatment.At present, the sick evaluation according to traditional mycobacteria strain usually of non-tuberculous mycobacteria made a definite diagnosis, yet traditional mycobacteria strain is identified loaded down with trivial details, time-consuming, needs 1～2 month, can not satisfy the clinical early stage effectively needs of chemotherapy of carrying out, make the non-tuberculous mycobacteria patient be used as tuberculosis usually and treat routinely, patient is through the long-term rule chemotherapy and unsatisfactory curative effect, and prolong the course of treatment, become refractory, the people that cures the disease again, bacterium is sent out in the part.Therefore, the Rapid identification of mycobacteria strain is propagated early diagnosis, differential diagnosis, effective chemotherapy and the control of tuberculosis and non-tuberculous mycobacteria disease all extremely important meaning.
Traditional method for identification of mycobacterium species needs at modified Russell medium, after tentatively differentiating mycobacterium tuberculosis and non-tuberculous mycobacteria on p-nitrobenzoic acid and the thiophene-2-carboxylic acid hydrazine differential medium, observe the microbial culture growing state again and (comprise colonial morphology, the speed of growth, growth temperature and pigment produce), do a series of biochemical tests and (comprise heat-resisting catalase test, nitrate reduction test, the tween-80 hydrolysis experiment, urease test, the test of aromatic sulfuric acid esterase, niacin test, iron ion absorption test and tellurous acid reduction test) carry out the evaluation of system, operate loaded down with trivial details, time-consuming, need 4～8 time-of-weeks, influence factor is many, poor repeatability, now clinical labororatory is not conventional carries out.
BACTAC TB460 came out in 1977, was characterized in easy, quick, can tentatively differentiate mycobacterium tuberculosis mixture and non-tuberculous mycobacteria, had become one of standard reference system of diagnosis of tuberculosis at home and abroad.Came out in 1977, introduce the BACTAC TB460 mycobacterium fast culture instrument of China the beginning of the nineties, utilize radioactivity 14When the C palmitinic acid is the 7H 12B liquid nutrient medium cultivation mycobacterium tuberculosis of substrate, since bacteria containing amount difference in the sample, the radioactivity CO that mycobacterium growth produces 2Amount is also different, and the detected value required time that reaches instrument is also different, generally needs 4-25 days.In addition, because P-nitro-alpha-acetamido--beta-hydroxyphenyl acetone (P-Nitro-α-Acetylamino-β-hydroxypropi-phenone, being called for short NAP) growth has restraining effect to mycobacterium tuberculosis complex, and to non-tuberculous mycobacteria growth unrestraint effect, in BACTACTB460, add NAP and continue to cultivate after 2-6 days, can tentatively differentiate mycobacterium tuberculosis complex and non-tuberculous mycobacteria according to growth index.But BACTAC TB460 can only carry out preliminary bacterial classification to be differentiated, and compare with traditional identification method, its specificity has only about 95%, the non-tuberculous mycobacteria that the distinguishes further strain identification of still needing, in addition, BACTAC TB460 instrument costs an arm and a leg, need the patent liquid nutrient medium of special import producer (U.S. BectonDickinson company), expense is higher, and radioactivity is arranged, and is difficult to extensively carry out in common lab.
PCR strain identification method is with various mycobacteria specific primers specimen dna to be carried out a series of pcr amplifications or multiplex PCR amplification, identify according to the specific fragment that amplified production produced, or earlier with the special outside primer amplification of Mycobacterium, and then carry out the Chao Shi amplification with the special inside primer of bacterial classification and identify, this method sensitivity, fast, but at present PCR the mycobacteria strain that can identify still few, mainly contain mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium tuberculosis complex, mycobacterium avium, mycobacterium paratuberculosis and Mycobacterium leprae, and identify unknown infectious bacteria, need to adopt a series of species-specific primers to increase; The PCR-identification method that directly checks order is with the special primer of Mycobacterium the 16SrRNA of sample or 16S-23SrRNA transcribed spacer sequence or 65kD or 32kD antigen gene to be carried out pcr amplification, directly measure the nucleotide sequence of amplified production then, relatively the difference of its Nucleotide is identified bacterial classification, though this method is sensitive, quick, has become " gold standard " that mycobacterium molecular bacteria is identified, the technical requirements height, need special technician, required order-checking instrument costliness, the cost height, and be difficult to promote; PCR-restriction fragment length polymorphism (RestrictionFragment Length Polymorphism, abbreviation RFLP) the strain identification method is with the Mycobacterium Auele Specific Primer mycobacterium 65KD protein coding gene, 16SrDNA or 16S-23SrDNA in the sample to be increased, use different digestion with restriction enzyme amplified fragments then, identify bacterial classification by the electrophoretic analysis restriction enzyme mapping, can only identify the part bacterial classification at present, repeatability is desirable not enough; PCR-gene chip identification method is meant the mode of a large amount of nucleic acid molecule with design in advance is fixed on the carrier, detect the testing sample DNA of tape label, it is a kind of novel method of large scale analysis hereditary difference, some laboratory utilizes mycobacterium 16SrRNA or some locational Mycobacterium of rpoB gene order or species specific Nucleotide to change both at home and abroad at present, research applying gene chip technology carries out sequencing by hybridization, the identification of mycobacterium bacterial classification, but this technical requirements height, required point sample system and system of fluorescence analysis costliness, and be difficult to apply.
Summary of the invention
One of purpose of the present invention provides a kind of quick, responsive, efficient, inexpensive mycobacterium molecular bacteria identification reagent box;
Another object of the present invention provides the preparation method of mentioned reagent box.
The objective of the invention is to reach by the following technical programs:
A kind of mycobacterium molecular bacteria identification reagent box, contain mycobacterium molecular bacteria and identify diaphragm and mycobacterium pcr amplification reagent, it is characterized in that: described mycobacterium molecular bacteria identifies that diaphragm is to be fixed with a series ofly at Mycobacterium and various mycobacterium 16S rRNA (being called for short rRNA) and the oligonucleotide probe of design on nitrocellulose membrane, described oligonucleotide probe is added with 50-110 thymus nucleic acid (abbreviation dT) at 3 ' end; Described mycobacterium pcr amplification reagent comprises increase required 5-10 times of PCR damping fluid (10mMTris.HCl, the 1.5mM MgCl that comprise 50mM KCl, pH8.3 at Mycobacterium 16SrRNA 2With 0.01% gelatin), concentration respectively is dGTP, dCTP, dATP and the dTTP of 2.5mmol/L, concentration respectively for the 5 ' end of 2.5-12.5 μ mol/L with biotin labeled upstream primer and the not downstream primer and the 1-10U/ μ l Taq archaeal dna polymerase of tape label; The final concentration of described each component of mycobacterium pcr amplification reagent is as follows: 1 times of PCR damping fluid, the final concentration of dGTP, dCTP, dATP and dTTP respectively is 0.2mmol/L, 5 ' end with biotin labeled upstream primer and not the final concentration of the downstream primer of tape label respectively be 0.2-1.0umol/L, Taq archaeal dna polymerase 2-5U/100 μ l.
A kind of optimal technical scheme is characterized in that: the specificity nucleotides sequence of described oligonucleotide probe is classified 16-18 base as.
A kind of optimal technical scheme is characterized in that: described oligonucleotide probe sequence is as shown in the table.
The probe title Probe sequence
????M ?5’-TATTAGACCCAGTTTCCC-3’
????a ?5’-ACAAGACATGCATCCCGT-3’
????b ?5’-GACATGCGTCTTGAGGTC-3’
????c ?5’-TAAAGACATGCGCCTAAA-3’
????d ?5’-CGCCAAGTGGTCCTATCC-3’
????h ?5’-CACACCATGCAGCATG-3’
????i ?5’-CAGAACATGCATCCCA-3’
????j ?5’-CGCAGAATGGTCCTATCC-3’
????k ?5’-CATGTGTCCTGTGGTCCT-3’
????l ?5’-CATGCGCCTCGGGGTCCT-3’
????m ?5’-AGGACATGAATCCCGT-3’
????n ?5’-ATGCGACCAATAGGGAAT-3’
????o ?5’-CACACCAGGAAGTGCG-3’
????p ?5’-ACTCACCATGAAGTGTGT-3’
????q ?5’-CGACCAGCAGGGTGTATT-3’
????r ?5’-ACACACCATGAAGCGCGT-3’
????s ?5’-TCCCAGCCATGCAACCAG-3’
????t ?5’-CACACACCATGAAGCAT-3’
????u ?5’-GACATGCATCGCGTAG-3’
????w ?5’-CACCATGCGACATGTG-3’
????y ?5’-AGACAATGCAGCCAGA-3’
????z ?5’-CACCCCATGAAGAGCG-3’
????aa ?5’-CCACCAGGCCATGCGA-3’
????ab ?5’-CACATGCATGCCGTGG-3’
????ac ?5’-CAACCCATGAAGGCCA-3’
A kind of optimal technical scheme is characterized in that: described 5 ' end is with biotin labeled upstream primer and the downstream primer sequence of tape label is not as follows:
Target gene Primer sequence (5 ' → 3 ')
? 16S rRNA Upstream primer 5 '-bio-CGA GTG GCG AAC GGG TGA G--3 ' downstream primer 5 '-TTG TGC AAT ATT CCC CAC TGC TG--3 '
A kind of preparation method of mycobacterium molecular bacteria identification reagent box may further comprise the steps:
1. mycobacterium molecular bacteria identifies that the diaphragm-operated preparation method is as follows:
(1) according to the synthetic above-mentioned oligonucleotide probe of prior art;
(2) with 10mM Tris.HCl-1mM edta buffer liquid dilution synthetic oligonucleotide probe, making its concentration is 1.5-6pmol/ μ l;
(3) nitrocellulose membrane is immersed pre-treatment 10-30min in 2 * SSC solution, dry or oven dry below 42 ℃;
(4) get 0.6-1 μ l oligonucleotide probe point on pretreated nitrocellulose membrane, irradiation 10-20 minute down of 60-80 ℃ of baking 1-2 hour or ultraviolet lamp, the simple rinsing of water is dried standby;
2. the compound method of mycobacterium pcr amplification reagent is as follows:
(1) according to the synthetic above-mentioned 5 ' end of prior art with the biotin labeled upstream primer and the downstream primer of tape label not, with the biotin labeled upstream primer and the downstream primer of tape label not, making its concentration is 2.5-12.5 μ mol/L with 1mM Tris.HCl-0.1mM edta buffer liquid dilution synthetic 5 ' end;
(2) get 5-10 times of PCR damping fluid, concentration respectively for the dGTP of 2.5mmol/L, dCTP, dATP and dTTP, concentration respectively for the 5 ' end of 2.5-12.5 μ mol/L with biotin labeled upstream primer with the downstream primer of tape label and 1-10U/ μ l Taq archaeal dna polymerase are not respectively packed into standby in the aseptic plastics tubing;
3. the assembling of mycobacterium molecular bacteria identification reagent box: identify mycobacterium molecular bacteria after the diaphragm seal and the mycobacterium pcr amplification reagent container of packing into.
A kind of optimal technical scheme is characterized in that: the specificity nucleotides sequence of described oligonucleotide probe is classified 16-18 base as.
A kind of optimal technical scheme is characterized in that: described oligonucleotide probe sequence is as shown in the table.
The probe title Probe sequence
????M ?5’-TATTAGACCCAGTTTCCC-3’
????a ?5’-ACAAGACATGCATCCCGT-3’
????b ?5’-GACATGCGTCTTGAGGTC-3’
????c ?5’-TAAAGACATGCGCCTAAA-3’
????d ?5’-CGCCAAGTGGTCCTATCC-3’
????h ?5’-CACACCATGCAGCATG-3’
????i ?5’-CAGAACATGCATCCCA-3’
????j ?5’-CGCAGAATGGTCCTATCC-3’
????k ?5’-CATGTGTCCTGTGGTCCT-3’
????l ?5’-CATGCGCCTCGGGGTCCT-3’
????m ?5’-AGGACATGAATCCCGT-3’
????n ?5’-ATGCGACCAATAGGGAAT-3’
????o ?5’-CACACCAGGAAGTGCG-3’
????p ?5’-ACTCACCATGAAGTGTGT-3’
????q ?5’-CGACCAGCAGGGTGTATT-3’
????r ?5’-ACACACCATGAAGCGCGT-3’
????s ?5’-TCCCAGCCATGCAACCAG-3’
????t ?5’-CACACACCATGAAGCAT-3’
????u ?5’-GACATGCATCGCGTAG-3’
????w ?5’-CACCATGCGACATGTG-3’
????y ?5’-AGACAATGCAGCCAGA-3’
????z ?5’-CACCCCATGAAGAGCG-3’
????aa ?5’-CCACCAGGCCATGCGA-3’
????ab ?5’-CACATGCATGCCGTGG-3’
????ac ?5’-CAACCCATGAAGGCCA-3’
A kind of optimal technical scheme is characterized in that: described 5 ' end is with biotin labeled upstream primer and the downstream primer sequence of tape label is not as follows:
Target gene Primer sequence (5 ' → 3 ')
? 16S rRNA Upstream primer 5 '-bio-CGA GTG GCG AAC GGG TGA G--3 ' downstream primer 5 '-TTG TGC AAT ATT CCC CAC TGC TG--3 '
Mycobacterium molecular bacteria identification reagent box of the present invention and preparation method thereof, can be according to mycobacterium 16S rRNA gene order not of the same race, design, synthetic corresponding oligonucleotide probe, by the point sample method oligonucleotide probe is put in sequence on pretreated nitrocellulose membrane, make to detect and identify diaphragm, with be with the hybridization of biotin labeled pcr amplification product, according to the bluish voilet signal power of probe hybridization sentence read result with the naked eye, can be simultaneously fast, detect mycobacterium exactly, differentiate mycobacterium and non-branch bacillus and mycobacterium tuberculosis complex and non-tuberculous mycobacteria, the identification of mycobacterium bacterial classification, can report the result in 24 hours, thus auxiliary clinical diagnosis, instruct the clinical early stage effectively chemotherapy of carrying out.
The present invention will be further described below by the drawings and specific embodiments, but and do not mean that limiting the scope of the invention.
Description of drawings
Fig. 1 identifies the result of mycobacterium tuberculosis type strain H37Rv for using diaphragm of the present invention;
Fig. 2 identifies the result of mycobacterium avium type strain for using diaphragm of the present invention;
Fig. 3 is the result of direct sequencing with the mycobacterium clinical separation strain No.12027 of bioanalysis software BLAST demonstration;
Fig. 4 is the result with diaphragm identification of mycobacterium clinical separation strain No.12027 of the present invention;
Embodiment
Embodiment 1
A kind of preparation method of mycobacterium molecular bacteria identification reagent box may further comprise the steps:
1. mycobacterium molecular bacteria identifies that the diaphragm-operated preparation method is as follows:
(1) according to the synthetic following oligonucleotide probe of prior art:
The probe title Probe sequence The mycobacteria strain of being identified
????M ?5’-TATTAGACCCAGTTTCCC-3’ Mycobacterium
????a ?5’-ACAAGACATGCATCCCGT-3’ Mycobacterium tuberculosis
????b ?5’-GACATGCGTCTTGAGGTC-3’ Mycobacterium avium
????c ?5’-TAAAGACATGCGCCTAAA-3’ Mycobacterium intracellulare
????d ?5’-CGCCAAGTGGTCCTATCC-3’ Kansas, scrofula, stomach, mycobacterium habana
????h ?5’-CACACCATGCAGCATG-3’ Achromatic mycobacterium
????i ?5’-CAGAACATGCATCCCA-3’ Mycobacterium terrae
????j ?5’-CGCAGAATGGTCCTATCC-3’ Mycobacterium littorale
????k ?5’-CATGTGTCCTGTGGTCCT-3’ Mycobacterium gordonae
????l ?5’-CATGCGCCTCGGGGTCCT-3’ Suhl adds the branch bacillus
????m ?5’-AGGACATGAATCCCGT-3’ Mycobacterium marinum
????n ?5’-ATGCGACCAATAGGGAAT-3’ Little yellow mycobacterium
????o ?5’-CACACCAGGAAGTGCG-3’ Mycobacterium chelonei tortoise subspecies
????p ?5’-ACTCACCATGAAGTGTGT-3’ Mycobacterium chelonei abscess subspecies
????q ?5’-CGACCAGCAGGGTGTATT-3’ M. smegmatics
????r ?5’-ACACACCATGAAGCGCGT ?-3’ Mycobacterium fortuitum
????s ?5’-TCCCAGCCATGCAACCAG ?-3’ Mycobacterium phlei
????t ?5’-CACACACCATGAAGCAT-3’ Pale yellow mycobacterium
????u ?5’-GACATGCATCGCGTAG-3’ Golden mycobacterium
????w ?5’-CACCATGCGACATGTG-3’ Mycobacterium triviale
????y ?5’-AGACAATGCAGCCAGA-3’ The cow mycobacterium
????z ?5’-CACCCCATGAAGAGCG-3’ The Di Shi mycobacterium
????aa ?5’-CCACCAGGCCATGCGA-3’ The heat resistanceheat resistant mycobacterium
????ab ?5’-CACATGCATGCCGTGG-3’ Chu cloth mycobacterium
????ac ?5’-CAACCCATGAAGGCCA-3’ Like to know mycobacterium
All oligonucleotide probes are added with 100 dT at 3 ' end.
(2) with 10mM Tris.HCl-1mM edta buffer liquid dilution synthetic oligonucleotide probe, making its concentration is 6pmol/ μ l;
The preparation of (3) 20 * SSC solution: sodium-chlor 175.3g, Trisodium Citrate 88.2g is dissolved in the 800ml distilled water, transfers pH to 7.0 with 10M sodium hydroxide, is settled to 1000ml then, autoclaving.Get 20 * SSC solution and become 2 * SSC solution for 10 times, nitrocellulose membrane is immersed pre-treatment 10min in 2 * SSC solution, put 37 ℃ of incubator oven dry with distilled water diluting;
(4) get on the pretreated nitrocellulose membrane of 0.8 μ l oligonucleotide probe idea, irradiation is 10 minutes under ultraviolet lamp, and the simple rinsing of water is dried standby;
2. the compound method of mycobacterium pcr amplification reagent is as follows:
(1) according to the synthetic following 5 ' end of prior art with the biotin labeled upstream primer and the downstream primer of tape label not:
Target gene Primer sequence (5 ' → 3 ')
16S rRNA Upstream primer 5 '-bio-CGA GTG GCG AAC GGG TGA G--3 ' downstream primer 5 '-TTG TGC AAT ATT CCC CAC TGC TG--3 '
With the dilution of 1mM Tris.HCl-0.1mM edta buffer liquid, making its concentration is 3.75 μ mol/L;
(2) getting 10 times of PCR damping fluids, concentration respectively is that the 5 ' end of 3.75 μ mol/L is with biotin labeled upstream primer with the downstream primer of tape label and 1U/ μ l Taq archaeal dna polymerase are not respectively packed into standby in the aseptic plastics tubing for the dGTP of 2.5mmol/L, dCTP, dATP and dTTP, concentration respectively;
3. the assembling of mycobacterium molecular bacteria identification reagent box and preservation: identify mycobacterium molecular bacteria after the diaphragm seal and the mycobacterium pcr amplification reagent container of packing into.Mycobacterium molecular bacteria is identified diaphragm room temperature kept dry; Mycobacterium pcr amplification reagent is stored in-20 ℃ of refrigerators, is not kept at the refrigeration chamber of frost-free type refrigerator, if preservation is proper, can keep active 1 year.
The application of mycobacterium molecular bacteria identification reagent box:
(1) sample preparation and DNA extraction: after will coming from the mycobacterium tuberculosis type strain H37Rv deactivation of Nat'l Pharmaceutical ﹠ Biological Products Control Institute, extract DNA;
(2) pcr amplification:
1. reaction system cumulative volume 25 μ l.
2. in the 0.5ml plastic centrifuge tube, add following reagent:
Reagent application of sample order volume (μ l)
Water 1 12.5
10 * PCR damping fluid 2 2.5
3.75 μ mol/L 5 ' end band vitamin H marks 3 2.0
The upstream primer of note
3.75 μ mol/L is the downstream 4 2.0 of tape label not
Primer
2.5mmol/L dGTP, dCTP, 5 2.0
DATP and dTTP
DNA sample 6 2.0
Amount to 23.0
If it is more to detect sample, can prepare main suspension packing.Different DNA amount of samples may be different, make cumulative volume the same by the volume of adjusting water in the reaction system.
3. behind the mixing, in 95 ℃ of sex change 8min.
4. slightly after the cooling, add Taq archaeal dna polymerase 2 μ l (containing 2U), mixing adds whiteruss 40 μ l, in case water evaporates in the amplification procedure, it is good to make it layering in centrifugal 5 seconds.
5. put in the pcr amplification instrument of Perkinelmer Inc.'s production, in 94 ℃ of sex change 1 minute, annealed 1 minute for 58 ℃, 72 ℃ were extended 1 minute, and circulated 35 times.Last 72 ℃ were extended 5 minutes.
Get 5 μ l pcr amplification products electrophoresis detection in 2% sepharose, the fragment of the visible 280bp of result.
(3) prehybridization solution preparation: 6 * SSC solution, 5 * Denharts solution, 0.1mg/ml calf thymus DNA, 0.5 sodium lauryl sulphate (being called for short SDS) solution.
50 * Denharts solution 10ml
20 * SSC solution 30ml
10SDS solution 5ml
10mg/ml calf thymus DNA 1ml
Add water and be settled to 100ml
(4) prehybridization: mycobacterium molecular bacteria is identified that diaphragm and 2ml prehybridization solution pack in the hybridization bag, hatch 30min in 37 ℃ of water-baths;
(5) hybridization: will be with biotin labeled pcr amplification product 20 μ l to put 98 ℃ of sex change 10min, put in the ice bath cooling 5min after, add in the hybridization bag, hatch 30min in 37 ℃ of water-baths;
(6) wash film: wash film 10min with washing lotion I2 * SSC-0.1%SDS in room temperature, use washing lotion II0.2 * SSC-0.1%SDS to wash film 10min again in room temperature;
(7) enzyme-added: mycobacterium molecular bacteria is identified that diaphragm changes in the new hybridization bag, with streptavidin-alkaline phosphatase (SA-AP) of 1: 1000 in 37 ℃ of water-bath 30min;
(8) wash film: with washing lotion III (100M Tris-HCl-150mM NaCl-2mMMgCl 2-0.05%TritonX-100 (pH7.5)) washes film 10min in room temperature, with washing lotion IV (100MTris-HCl-150mM NaCl-1mM MgCl 2(pH9.5)) wash film 10min in room temperature;
(9) colour developing: mycobacterium molecular bacteria is identified that diaphragm changes in the new hybridization bag, black out colour developing 10min in 5-bromo-4-chloro-3-indoles phosphoric acid salt (being called for short BCIP) and nitroblue tetrazolium (NBT) (being called for short NBT) substrate.The results are shown in Figure 1.
(10) result judges: as shown in Figure 1, according to the bluish voilet signal power of mycobacterium probe hybridization, the visible M probe and a probe hybridization positive, all the other probe hybridization feminine genders, thus this bacterium of decidable belongs to Mycobacterium, and can be accredited as mycobacterium tuberculosis.
Embodiment 2
A kind of preparation method of mycobacterium molecular bacteria identification reagent box may further comprise the steps:
1. mycobacterium molecular bacteria identifies that the diaphragm-operated preparation method is as follows:
(1) according to the synthetic following oligonucleotide probe of prior art:
The probe title Probe sequence The mycobacteria strain of being identified
??M 5 '-TATTAGACCCAGTTTCCC-3 ' adds 102 dT 3 ' Mycobacterium
??a 5 '-ACAAGACATGCATCCCGT-3 ' adds 102 dT 3 ' Mycobacterium tuberculosis
??b 5 '-GACATGCGTCTTGAGGTC-3 ' adds 102 dT 3 ' Mycobacterium avium
??c 5 '-TAAAGACATGCGCCTAAA-3 ' adds 102 dT 3 ' Mycobacterium intracellulare
??d 5 '-CGCCAAGTGGTCCTATCC-3 ' adds 102 dT 3 ' Kansas, scrofula, stomach, mycobacterium habana
??h 5 '-CACACCATGCAGCATG-3 ' adds 104 dT 3 ' Achromatic mycobacterium
??i 5 '-CAGAACATGCATCCCA-3 ' adds 104 dT 3 ' Mycobacterium terrae
??j 5 '-CGCAGAATGGTCCTATCC-3 ' adds 102 dT 3 ' Mycobacterium littorale
??k 5 '-CATGTGTCCTGTGGTCCT-3 ' adds 102 dT 3 ' Mycobacterium gordonae
??l 5 '-CATGCGCCTCGGGGTCCT-3 ' adds 102 dT 3 ' Suhl adds the branch bacillus
??m 5 '-AGGACATGAATCCCGT-3 ' adds 104 dT 3 ' Mycobacterium marinum
??n 5 '-ATGCGACCAATAGGGAAT-3 ' adds 102 dT 3 ' Little yellow mycobacterium
??o 5 '-CACACCAGGAAGTGCG-3 ' adds 104 dT 3 ' Mycobacterium chelonei tortoise subspecies
??p 5 '-ACTCACCATGAAGTGTGT-3 ' adds 102 dT 3 ' Mycobacterium chelonei abscess subspecies mycobacterium
??q 5 '-CGACCAGCAGGGTGTATT-3 ' adds 102 dT 3 ' M. smegmatics
??r 5 '-ACACACCATGAAGCGCGT-3 ' adds 102 dT 3 ' Mycobacterium fortuitum
??s 5 '-TCCCAGCCATGCAACCAG-3 ' adds 102 dT 3 ' Mycobacterium phlei
??t 5 '-CACACACCATGAAGCAT-3 ' adds 103 dT 3 ' Pale yellow mycobacterium
??u 5 '-GACATGCATCGCGTAG-3 ' adds 104 dT 3 ' Golden mycobacterium
??w 5 '-CACCATGCGACATGTG-3 ' adds 104 dT 3 ' Mycobacterium triviale
??y 5 '-AGACAATGCAGCCAGA-3 ' adds 104 dT 3 ' The cow mycobacterium
??z 5 '-CACCCCATGAAGAGCG-3 ' adds 104 dT 3 ' The Di Shi mycobacterium
??aa 5 '-CCACCAGGCCATGCGA-3 ' adds 104 dT 3 ' The heat resistanceheat resistant mycobacterium
??ab 5 '-CACATGCATGCCGTGG-3 ' adds 104 dT 3 ' Chu cloth mycobacterium
??ac 5 '-CAACCCATGAAGGCCA-3 ' adds 104 dT 3 ' Like to know mycobacterium
(2) with 10mM Tris.HCl-1mM edta buffer liquid dilution synthetic oligonucleotide probe, making its concentration is 4pmol/ μ l;
The preparation of (3) 20 * SSC solution: sodium-chlor 175.3g, Trisodium Citrate 88.2g is dissolved in the 800ml distilled water, transfers pH to 7.0 with 10M sodium hydroxide, is settled to 1000ml then, autoclaving.Get 20 * SSC solution and become 2 * SSC solution for 10 times, nitrocellulose membrane is immersed pre-treatment 10min in 2 * SSC solution, put room temperature and dry with distilled water diluting;
(4) get 0.6 μ l oligonucleotide probe point on pretreated nitrocellulose membrane, put in the baking box in 60 ℃ of bakings 1 hour, the simple rinsing of water is dried standby;
2. the compound method of mycobacterium pcr amplification reagent is as follows:
(1) according to the synthetic following 5 ' end of prior art with the biotin labeled upstream primer and the downstream primer of tape label not:
Target gene Primer sequence (5 ' → 3 ')
16S rRNA Upstream primer 5 '-bio-CGA GTG GCG AAC GGG TGA G--3 ' downstream primer 5 '-TTG TGC AAT ATT CCC CAC TGC TG--3 '
With the dilution of 1mM Tris.HCl-0.1mM edta buffer liquid, making its concentration is 12.5 μ mol/L;
(2) getting 5 times of PCR damping fluids, concentration respectively is that the 5 ' end of 12.5 μ mol/L is with biotin labeled upstream primer with the downstream primer of tape label and 5U/ μ l Taq archaeal dna polymerase are not respectively packed into standby in the aseptic plastics tubing for the dGTP of 2.5mmol/L, dCTP, dATP and dTTP, concentration respectively;
3. the assembling of mycobacterium molecular bacteria identification reagent box and preservation: mycobacterium molecular bacteria is identified diaphragm and the mycobacterium pcr amplification reagent carton of packing into.Mycobacterium molecular bacteria is identified diaphragm room temperature kept dry; Mycobacterium pcr amplification reagent is stored in-20 ℃ of refrigerators, is not kept at the refrigeration chamber of frost-free type refrigerator, if preservation is proper, can keep active 1 year.
The application of mycobacterium molecular bacteria identification reagent box:
(1) sample preparation and DNA extraction: after will coming from the mycobacterium avium type strain deactivation of Nat'l Pharmaceutical ﹠ Biological Products Control Institute, extract DNA;
(2) pcr amplification:
1. reaction system cumulative volume 50 μ l.
2. in the 0.5ml plastic centrifuge tube, add following reagent:
Reagent application of sample order volume (μ l)
Water 1 22.0
5 * PCR damping fluid 2 10.0
12.5 μ mol/L 5 ' end band vitamin H marks 3 4.0
The upstream primer of note
12.5 μ mol/L is the downstream 4 4.0 of tape label not
Primer
2.5mmol/L dGTP, dCTP, 5 4.0
DATP and dTTP
DNA sample 6 5.0
Amount to 49.0
If it is more to detect sample, can prepare main suspension packing.Different DNA amount of samples may be different, make cumulative volume the same by the volume of adjusting water in the reaction system.
3. behind the mixing, in 95 ℃ of sex change 8min.
4. slightly after the cooling, add Taq archaeal dna polymerase 1 μ l (containing 5U), mixing adds whiteruss 40 μ l, in case water evaporates in the amplification procedure, it is good to make it layering in centrifugal 10 seconds.
5. put in the pcr amplification instrument of Perkinelmer Inc.'s production, in 94 ℃ of sex change 1 minute, annealed 1 minute for 58 ℃, 72 ℃ were extended 1 minute, and circulated 35 times.Last 72 ℃ were extended 5 minutes.
Get 8 μ l pcr amplification products electrophoresis detection in 2% sepharose, the fragment of the visible 278bp of result.
(3) prehybridization solution preparation: 6 * SSC solution, 5 * Denharts solution, 0.1mg/ml calf thymus DNA, 0.5% sodium lauryl sulphate (being called for short SDS) solution.
50 * Denharts solution 10ml
20 * SSC solution 30ml
10%SDS solution 5ml
10mg/ml calf thymus DNA 1ml
Add water and be settled to 100ml
(4) prehybridization: mycobacterium molecular bacteria is identified that diaphragm and 3ml prehybridization solution pack in the hybridization bag, hatch 30min in 37 ℃ of water-baths;
(5) hybridization: will be with biotin labeled pcr amplification product 30 μ l to put 98 ℃ of sex change 10min, put in the ice bath cooling 5min after, add in the hybridization bag, hatch 30min in 37 ℃ of water-baths;
(6) wash film: wash film 10min with washing lotion I2 * SSC-0.1%SDS in room temperature, use washing lotion II0.2 * SSC-0.1%SDS to wash film 10min again in room temperature;
(7) enzyme-added: mycobacterium molecular bacteria is identified that diaphragm changes in the new hybridization bag, with streptavidin-alkaline phosphatase (SA-AP) of 1: 1000 in 37 ℃ of water-bath 30min;
(8) wash film: with washing lotion III (100M Tris-HCl-150mM NaCl-2mMMgCl 2-0.05%TritonX-100 (pH7.5)) washes film 10min in room temperature, with washing lotion IV (100MTris-HCl-150mM NaCl-1mM MgCl 2(pH9.5)) wash film 10min in room temperature;
(9) colour developing: mycobacterium molecular bacteria is identified that diaphragm changes in the new hybridization bag, black out colour developing 10min in 5-bromo-4-chloro-3-indoles phosphoric acid salt (being called for short BCIP) and nitroblue tetrazolium (NBT) (being called for short NBT) substrate.The results are shown in Figure 2.
(10) result judges: as shown in Figure 2, according to the bluish voilet signal power of mycobacterium probe hybridization, the visible M probe and the b probe hybridization positive, all the other probe hybridization feminine genders, thus this bacterium of decidable belongs to Mycobacterium, and can be accredited as mycobacterium avium.
Comparative Examples
Adopt the PCR-identification method that directly checks order to identify the mycobacterium clinical separation strain No.12027 that comes from P.L.A. 309 Hospital.
1. sample preparation and DNA extraction: after will coming from the mycobacterium clinical separation strain No.12027 deactivation of P.L.A. 309 Hospital, extract DNA;
2. the compound method of mycobacterium pcr amplification reagent is as follows:
(1) according to synthetic following upstream primer of prior art and downstream primer:
Target gene Primer sequence (5 ' → 3 ')
16S rRNA Upstream primer 5 '-CGA GTG GCG AAC GGG TGA G--3 ' downstream primer 5 '-TTG TGC AAT ATT CCC CAC TGC TG--3 '
With the dilution of 1mM Tris.HCl-0.1mM edta buffer liquid, making its concentration is 3.75 μ mol/L;
(2) respectively respectively to be that the upstream primer of 3.75 μ mol/L and downstream primer and 5U/ μ l Taq archaeal dna polymerase are respectively packed into standby in the aseptic plastics tubing for the dGTP of 2.5mmol/L, dCTP, dATP and dTTP, concentration to get 10 times of PCR damping fluids, concentration;
3. the preservation of mycobacterium pcr amplification reagent: mycobacterium pcr amplification reagent is stored in-20 ℃ of refrigerators, is not kept at the refrigeration chamber of frost-free type refrigerator,, can keep active a year if preservation is proper.
4.PCR amplification:
1. reaction system cumulative volume 100 μ l.
2. in the 0.5ml plastic centrifuge tube, add following reagent:
Reagent application of sample order volume (μ l)
Water 1 57.0
10 * PCR damping fluid 2 10.0
3.75 μ mol/L 5 ' end band vitamin H marks 3 8.0
The upstream primer of note
3.75 μ mol/L is the downstream 4 8.0 of tape label not
Primer
2.5mmol/L dGTP, dCTP, 5 8.0
DATP and dTTP
DNA sample 6 8.0
Amount to 99.0
3. behind the mixing, in 95 ℃ of sex change 8min.
4. slightly after the cooling, add Taq archaeal dna polymerase 1 μ l (containing 5U), mixing adds whiteruss 40 μ l, in case water evaporates in the amplification procedure, it is good to make it layering in centrifugal 5 seconds.
5. put in the pcr amplification instrument of Perkinelmer Inc.'s production, in 94 ℃ of sex change 1 minute, annealed 1 minute for 58 ℃, 72 ℃ were extended 1 minute, and circulated 35 times.Last 72 ℃ were extended 5 minutes.
Get 5 μ l pcr amplification products electrophoresis detection in 2% sepharose, the fragment of the visible 278bp of result.
5. identification method directly checks order: remaining 95 μ l pcr amplification products and 20 μ l downstream primers are served the sea together give birth to worker Bioisystech Co., Ltd and carry out dna sequencing.
6. the result judges: sequencing result as shown in Figure 3, and is identical with the mycobacterium gordonae sequence with bioanalysis software BLAST analysis demonstration, illustrates that this strain isolated is a mycobacterium gordonae.
Embodiment 3
Adopt diaphragm of the present invention to identify the mycobacterium clinical separation strain No.12027 that comes from P.L.A. 309 Hospital.
A kind of preparation method of mycobacterium molecular bacteria identification reagent box may further comprise the steps:
1. mycobacterium molecular bacteria identifies that the diaphragm-operated preparation method is as follows:
(1) according to the synthetic following oligonucleotide probe of prior art:
The probe title Probe sequence The mycobacteria strain of being identified
????M ?5’-TATTAGACCCAGTTTCCC-3’ Mycobacterium
????a ?5’-ACAAGACATGCATCCCGT-3’ Mycobacterium tuberculosis
????b ?5’-GACATGCGTCTTGAGGTC-3’ Mycobacterium avium
????c ?5’-TAAAGACATGCGCCTAAA-3’ Mycobacterium intracellulare
????d ?5’-CGCCAAGTGGTCCTATCC-3’ Kansas, scrofula, stomach, mycobacterium habana
????h ?5’-CACACCATGCAGCATG-3’ Achromatic mycobacterium
????i ?5’-CAGAACATGCATCCCA-3’ Mycobacterium terrae
????j ?5’-CGCAGAATGGTCCTATCC-3’ Mycobacterium littorale
????k ?5’-CATGTGTCCTGTGGTCCT-3’ Mycobacterium gordonae
????l ?5’-CATGCGCCTCGGGGTCCT-3’ Suhl adds the branch bacillus
????m ?5’-AGGACATGAATCCCGT-3’ Mycobacterium marinum
????n ?5’-ATGCGACCAATAGGGAAT-3’ Little yellow mycobacterium
????o ?5’-CACACCAGGAAGTGCG-3’ Mycobacterium chelonei tortoise subspecies
????p ?5’-ACTCACCATGAAGTGTGT-3’ Mycobacterium chelonei abscess subspecies mycobacterium
????q ?5’-CGACCAGCAGGGTGTATT-3’ M. smegmatics
????r ?5’-ACACACCATGAAGCGCGT ?-3’ Mycobacterium fortuitum
????s ?5’-TCCCAGCCATGCAACCAG ?-3’ Mycobacterium phlei
????t ?5’-CACACACCATGAAGCAT-3’ Pale yellow mycobacterium
????u ?5’-GACATGCATCGCGTAG-3’ Golden mycobacterium
????w ?5’-CACCATGCGACATGTG-3’ Mycobacterium triviale
????y ?5’-AGACAATGCAGCCAGA-3’ The cow mycobacterium
????z ?5’-CACCCCATGAAGAGCG-3’ The Di Shi mycobacterium
????aa ?5’-CCACCAGGCCATGCGA-3’ The heat resistanceheat resistant mycobacterium
????ab ?5’-CACATGCATGCCGTGG-3’ Chu cloth mycobacterium
????ac ?5’-CAACCCATGAAGGCCA-3’ Like to know mycobacterium
All oligonucleotide probes are added with 90 dT at 3 ' end.
(2) with 10mM Tris.HCl-1mM edta buffer liquid dilution synthetic oligonucleotide probe, making its concentration is 1.5pmol/ μ l;
The preparation of (3) 20 * SSC solution: sodium-chlor 175.3g, Trisodium Citrate 88.2g is dissolved in the 800ml distilled water, transfers pH to 7.0 with 10M sodium hydroxide, is settled to 1000ml then, autoclaving.Get 20 * SSC solution and become 2 * SSC solution for 10 times, nitrocellulose membrane is immersed pre-treatment 10min in 2 * SSC solution, put 37 ℃ of incubator oven dry with distilled water diluting;
(4) get 1 μ l oligonucleotide probe point on pretreated nitrocellulose membrane, irradiation is 20 minutes under ultraviolet lamp, and the simple rinsing of water is dried standby;
2. the compound method of mycobacterium pcr amplification reagent is as follows:
(1) according to the synthetic following 5 ' end of prior art with the biotin labeled upstream primer and the downstream primer of tape label not:
Target gene Primer sequence (5 ' → 3 ')
16S rRNA Upstream primer 5 '-bio-CGA GTG GCG AAC GGG TGA G--3 ' downstream primer 5 '-TTG TGC AAT ATT CCC CAC TGC TG--3 '
With the dilution of 1mM Tris.HCl-0.1mM edta buffer liquid, making its concentration is 2.5 μ mol/L;
(2) getting 10 times of PCR damping fluids, concentration respectively is that the 5 ' end of 2.5 μ mol/L is with biotin labeled upstream primer with the downstream primer of tape label and 10U/ μ l Taq archaeal dna polymerase are not respectively packed into standby in the aseptic plastics tubing for the dGTP of 2.5mmol/L, dCTP, dATP and dTTP, concentration respectively;
3. the assembling of mycobacterium molecular bacteria identification reagent box and preservation: mycobacterium molecular bacteria is identified diaphragm and the mycobacterium pcr amplification reagent carton of packing into.Mycobacterium molecular bacteria is identified diaphragm room temperature kept dry; Mycobacterium pcr amplification reagent is stored in-20 ℃ of refrigerators, is not kept at the refrigeration chamber of frost-free type refrigerator, if preservation is proper, can keep active 1 year.
The application of mycobacterium molecular bacteria identification reagent box:
(1) sample preparation and DNA extraction: after will coming from the mycobacterium clinical separation strain No.12027 deactivation of 309 hospitals of PLA, extract DNA;
(2) pcr amplification:
1. reaction system cumulative volume 100 μ l.
2. in the 0.5ml plastic centrifuge tube, add following reagent:
Reagent application of sample order volume (μ l)
Water 1 57.0
10 * PCR damping fluid 2 10.0
3.75 μ mol/L 5 ' end band vitamin H marks 3 8.0
The upstream primer of note
3.75 μ mol/L is the downstream 4 8.0 of tape label not
Primer
2.5mmol/L dGTP, dCTP, 5 8.0
DATP and dTTP
DNA sample 6 8.0
Amount to 99.0
3. behind the mixing, in 95 ℃ of sex change 8min.
4. slightly after the cooling, add Taq archaeal dna polymerase 1 μ l (containing 5U), mixing adds whiteruss 40 μ l, in case water evaporates in the amplification procedure, it is good to make it layering in centrifugal 5 seconds.
5. put in the pcr amplification instrument of Perkinelmer Inc.'s production, in 94 ℃ of sex change 1 minute, annealed 1 minute for 58 ℃, 72 ℃ were extended 1 minute, circulated 35 times, and last 72 ℃ were extended 5 minutes.
6. get 5 μ l pcr amplification products electrophoresis detection in 2% sepharose, the fragment of the visible 278bp of result.
(3) prehybridization solution preparation: 6 * SSC solution, 5 * Denharts solution, 0.1mg/ml calf thymus DNA, 0.5 sodium lauryl sulphate (being called for short SDS) solution.
50 * Denharts solution 10ml
20 * SSC solution 30ml
10%SDS solution 5ml
10mg/ml calf thymus DNA 1ml
Add water and be settled to 100ml
(4) prehybridization: mycobacterium molecular bacteria is identified that diaphragm and 4ml prehybridization solution pack in the hybridization bag, hatch 30min in 37 ℃ of water-baths;
(5) hybridization: will be with biotin labeled pcr amplification product 40 μ l to put 98 ℃ of sex change 10min, put in the ice bath cooling 5min after, add in the hybridization bag, hatch 30min in 37 ℃ of water-baths;
(6) wash film: wash film 10min with washing lotion I2 * SSC-0.1%SDS in room temperature, use washing lotion II0.2 * SSC-0.1%SDS to wash film 10min again in room temperature;
(7) enzyme-added: mycobacterium molecular bacteria is identified that diaphragm changes in the new hybridization bag, with streptavidin-alkaline phosphatase (SA-AP) of 1: 1000 in 37 ℃ of water-bath 30min;
(8) wash film: with washing lotion III (100M Tris-HCl-150mM NaCl-2mMMgCl 2-0.05%TritonX-100 (pH7.5)) washes film 10min in room temperature, with washing lotion IV (100MTris-HCl-150mM NaCl-1mM MgCl 2(pH9.5)) wash film 10min in room temperature;
(9) colour developing: mycobacterium molecular bacteria is identified that diaphragm changes in the new hybridization bag, black out colour developing 10min in 5-bromo-4-chloro-3-indoles phosphoric acid salt (being called for short BCIP) and nitroblue tetrazolium (NBT) (being called for short NBT) substrate.The results are shown in Figure 4.
(10) result judges: as shown in Figure 4, according to the bluish voilet signal power of mycobacterium probe hybridization, the visible M probe and the k probe hybridization positive, all the other probe hybridization feminine genders, thus this bacterium of decidable belongs to Mycobacterium, and can be accredited as mycobacterium gordonae.

Claims (9)

1, a kind of mycobacterium molecular bacteria identification reagent box, contain mycobacterium molecular bacteria and identify diaphragm and mycobacterium pcr amplification reagent, it is characterized in that: described mycobacterium molecular bacteria identifies that diaphragm is to fix a series of oligonucleotide probes that design at Mycobacterium and various mycobacterium 16S rRNA on nitrocellulose membrane, and described oligonucleotide probe is added with 50-110 thymus nucleic acid at 3 ' end; Described mycobacterium pcr amplification reagent comprises the required 5-10 times of PCR damping fluid that increase at Mycobacterium 16S rRNA, concentration respectively is dGTP, dCTP, dATP and the dTTP of 2.5mmol/L, concentration respectively for the 5 ' end of 2.5-12.5 μ mol/L with biotin labeled upstream primer and the not downstream primer and the 1-10U/ μ l Taq archaeal dna polymerase of tape label; The final concentration of described each component of mycobacterium pcr amplification reagent is as follows: 1 times of PCR damping fluid, the final concentration of dGTP, dCTP, dATP and dTTP respectively is 0.2mmol/L, 5 ' end with biotin labeled upstream primer and not the final concentration of the downstream primer of tape label respectively be 0.2-1.0umol/L, Taq archaeal dna polymerase 2-5U/100 μ l.
2, identification kit according to claim 1 is characterized in that: described PCR damping fluid contains 10mM Tris.HCl, the 1.5mM MgCl of 50mM KCl, pH8.3 2With 0.01% gelatin.
3, identification kit according to claim 1 is characterized in that: the specificity nucleotides sequence of described oligonucleotide probe is classified 16-18 base as.
4, identification kit according to claim 1 is characterized in that: described oligonucleotide probe sequence is as shown in the table.
The probe title Probe sequence ????M ?5’-TATTAGACCCAGTTTCCC-3’ ????a ?5’-ACAAGACATGCATCCCGT-3’ ????b ?5’-GACATGCGTCTTGAGGTC-3’ ????c ?5’-TAAAGACATGCGCCTAAA-3’ ????d ?5’-CGCCAAGTGGTCCTATCC-3’ ????h ?5’-CACACCATGCAGCATG-3’ ????i ?5’-CAGAACATGCATCCCA-3’ ????j ?5’-CGCAGAATGGTCCTATCC-3’ ????k ?5’-CATGTGTCCTGTGGTCCT-3’ ????l ?5’-CATGCGCCTCGGGGTCCT-3’ ????m ?5’-AGGACATGAATCCCGT-3’ ????n ?5’-ATGCGACCAATAGGGAAT-3’ ????o ?5’-CACACCAGGAAGTGCG-3’ ????p ?5’-ACTCACCATGAAGTGTGT-3’ ????q ?5’-CGACCAGCAGGGTGTATT-3’ ????r ?5’-ACACACCATGAAGCGCGT-3’ ????s ?5’-TCCCAGCCATGCAACCAG-3’ ????t ?5’-CACACACCATGAAGCAT-3’ ????u ?5’-GACATGCATCGCGTAG-3’ ????w ?5’-CACCATGCGACATGTG-3’ ????y ?5’-AGACAATGCAGCCAGA-3’ ????z ?5’-CACCCCATGAAGAGCG-3’ ????aa ?5’-CCACCAGGCCATGCGA-3’ ????ab ?5’-CACATGCATGCCGTGG-3’ ????ac ?5’-CAACCCATGAAGGCCA-3’
5, identification kit according to claim 1 is characterized in that: described 5 ' end is with biotin labeled upstream primer and the downstream primer sequence of tape label is not as shown in the table. Target gene Primer sequence (5 ' → 3 ') ??16S ??rRNA Upstream primer 5 '-bio-CGA GTG GCG AAC GGG TGA G--3 ' downstream primer 5 '-TTG TGC AAT ATT CCC CAC TGC TG--3 '
6, a kind of preparation method of mycobacterium molecular bacteria identification reagent box may further comprise the steps:
[1]. mycobacterium molecular bacteria identifies that the diaphragm-operated preparation method is as follows:
(1) according to the synthetic described oligonucleotide probe of prior art;
(2) with 10mM Tris.HCl-1mM edta buffer liquid dilution synthetic oligonucleotide probe, making its concentration is 1.5-6pmol/ μ l;
(3) nitrocellulose membrane is immersed pre-treatment 10-30min in 2 * SSC solution, dry or oven dry below 42 ℃;
(4) get 0.6-1 μ l oligonucleotide probe point on pretreated nitrocellulose membrane, irradiation 10-20 minute down of 60-80 ℃ of baking 1-2 hour or ultraviolet lamp, the simple rinsing of water is dried standby;
[2]. the compound method of mycobacterium pcr amplification reagent is as follows:
(1) according to the synthetic described 5 ' end of prior art with the biotin labeled upstream primer and the downstream primer of tape label not, with the biotin labeled upstream primer and the downstream primer of tape label not, making its concentration is 2.5-12.5 μ mol/L with 1mM Tris.HCl-0.1mM edta buffer liquid dilution synthetic 5 ' end;
(2) get 5-10 times of PCR damping fluid, concentration respectively for the dGTP of 2.5mmol/L, dCTP, dATP and dTTP, concentration respectively for the 5 ' end of 2.5-12.5 μ mol/L with biotin labeled upstream primer with the downstream primer of tape label and 1-10U/ μ l Taq archaeal dna polymerase are not respectively packed into standby in the aseptic plastics tubing;
[3]. the assembling of mycobacterium molecular bacteria identification reagent box: identify mycobacterium molecular bacteria after the diaphragm seal and the mycobacterium pcr amplification reagent container of packing into.
7, the preparation method of identification kit according to claim 6 is characterized in that: the specificity nucleotides sequence of described oligonucleotide probe is classified 16-18 base as.
8, the preparation method of identification kit according to claim 6 is characterized in that: described oligonucleotide probe sequence is as shown in the table.
The probe title Probe sequence ????M ?5’-TATTAGACCCAGTTTCCC-3’ ????a ?5’-ACAAGACATGCATCCCGT-3’ ????b ?5’-GACATGCGTCTTGAGGTC-3’ ????c ?5’-TAAAGACATGCGCCTAAA-3’ ????d ?5’-CGCCAAGTGGTCCTATCC-3’ ????h ?5’-CACACCATGCAGCATG-3’ ????i ?5’-CAGAACATGCATCCCA-3’ ????j ?5’-CGCAGAATGGTCCTATCC-3’ ????k ?5’-CATGTGTCCTGTGGTCCT-3’ ????l ?5’-CATGCGCCTCGGGGTCCT-3’ ????m ?5’-AGGACATGAATCCCGT-3’ ????n ?5’-ATGCGACCAATAGGGAAT-3’ ????o ?5’-CACACCAGGAAGTGCG-3’ ????p ?5’-ACTCACCATGAAGTGTGT-3’ ????q ?5’-CGACCAGCAGGGTGTATT-3’ ????r ?5’-ACACACCATGAAGCGCGT-3’ ????s ?5’-TCCCAGCCATGCAACCAG-3’ ????t ?5’-CACACACCATGAAGCAT-3’ ????u ?5’-GACATGCATCGCGTAG-3’ ????w ?5’-CACCATGCGACATGTG-3’ ????y ?5’-AGACAATGCAGCCAGA-3’ ????z ?5’-CACCCCATGAAGAGCG-3’ ????aa ?5’-CCACCAGGCCATGCGA-3’ ????ab ?5’-CACATGCATGCCGTGG-3’ ????ac ?5’-CAACCCATGAAGGCCA-3’
9, the preparation method of identification kit according to claim 6 is characterized in that: described 5 ' end is with biotin labeled upstream primer and the downstream primer sequence of tape label is not as shown in the table. Target gene Primer sequence (5 ' → 3 ') ??16S ??rRNA Upstream primer 5 '-bio-CGA GTG GCG AAC GGG TGA G--3 ' downstream primer 5 '-TTG TGC AAT ATT CCC CAC TGC TG--3 '
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CN101948922A (en) * 2010-09-21 2011-01-19 天津生物芯片技术有限责任公司 Solid phase DNA chip hybridization solution and hybridization method
CN101560568B (en) * 2008-04-16 2012-08-01 亚洲基因科技股份有限公司 Method and kit for detecting mycobacterium tuberculosis and nontuberculous mycobacterium
CN103014135A (en) * 2011-09-22 2013-04-03 上海市肺科医院 Method for identifying mycobacterium tuberculosis
CN113528682A (en) * 2021-06-29 2021-10-22 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Multiplex TaqMan fluorescent quantitative PCR kit for simultaneously detecting three mycobacteria and application thereof

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CN101948922A (en) * 2010-09-21 2011-01-19 天津生物芯片技术有限责任公司 Solid phase DNA chip hybridization solution and hybridization method
CN103014135A (en) * 2011-09-22 2013-04-03 上海市肺科医院 Method for identifying mycobacterium tuberculosis
CN103014135B (en) * 2011-09-22 2016-02-10 上海市肺科医院 A kind of method identifying mycobacterium tuberculosis
CN113528682A (en) * 2021-06-29 2021-10-22 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Multiplex TaqMan fluorescent quantitative PCR kit for simultaneously detecting three mycobacteria and application thereof

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Patentee after: Chinese People's Liberation Army No.309 Hospital

Patentee after: BEIJING CONTROLS & STANDARDS BIOTECHNOLOGY CO., LTD.

Address before: 100091 Beijing city Haidian District Montenegro Hu No. seventeen

Patentee before: Chinese People's Liberation Army No.309 Hospital