CN101886124A - Kit for detecting nucleic acid of M.tuberculosis (TB) by RNA isothermal amplification - Google Patents

Abstract

The invention relates to a kit for detecting M.tuberculosis (TB) by performing ultrasonication on bacteria to release a target RNA and using a constant temperature nucleic acid synchronous amplification and test technique (SAT). The kit comprises TB diluent, TB reaction liquid, TB detection liquid, SAT enzyme solution, TB positive control and TB negative control. The kit has high specificity and sensitivity; and the amplified product RNA is easy to degrade in the natural environment but the pollution is less.

Description

A kind of concretion mycobacterium nucleic acid detection kit of utilizing the RNA constant-temperature amplification

Technical field

The present invention relates to examine the external molecular biosciences method of mycobacterium TB detection range, particularly relate to a kind of concretion mycobacterium nucleic acid detection kit of the RNA of utilization constant-temperature amplification.

Background technology

(M.Tuberculosis is the pathogenic bacteria of people and animal tuberculosis (tuberculosis) TB) to mycobacterium tuberculosis, belongs to radiation Zoopagales mycobacteriaceae Mycobacterium.The African type that can be divided into human-like, ox type, bird type, mouse type, cold blooded animal type and discovered in recent years.Human-like to being mainly of human disease, ox type bacillus is more rare, and the birds bacillus is more rare, and anonymous mycobacteria also can cause similar tuberculosis sample pathology, but rare.First three type is called as classical tubercule bacillus because obtain full confirmation later on the international tuberculosis meeting in London in 1901 morning of finding.People and animals are threatened maximum formal classical tubercule bacillus.Tubercule bacillus is the bacillus of elongated or microbend, long 1～4 μ m, and wide 0.2～0.5 μ m is single or divides dendritic arrangement, no pod membrane, atrichia, no brood cell.In outmoded focus and culture, form often is not true to type, and can be particulate state, spherical, the corynebacterium of string, long filament type.Ox type bacterium is shorter and thick than human-like bacterium.Fowl type bacterium has polytypism, be shaft-like sometimes or chain spherical etc.Tubercule bacillus is an obligate aerobe.The nutritional requirement height could be grown on the solid medium that contains yolk, potato, glycerine and asparagine etc.Growth conditions is 6.5～6.8 the most suitable with 37～37.5 degree, pH, and poor growth needed for 2～8 weeks the bacterium colony that naked eyes can be seen just occurred on solid medium.

Mainly rely on laboratory diagnosis to combine to diagnosis of tuberculosis at present with clinical Signs.Laboratory diagnosis mainly comprises plate coating checking, culture method, antigen-antibody detection, PCR and TMA etc.

1. bacteriological method

Smear for microscopic examination method: to be detected or pretreated sample is applied on the slide, dyes after fixing, carry out microscopy after waiting to do.

Acid-fast bacilli feminine gender (one): observe 300 different visuals field continuously, do not find acid-fast bacilli.

The acid-fast bacilli positive:

---report acid-fast bacilli bacterium number: 1～8/300 visuals field

---the acid-fast bacilli positive (+): 3～9/100 visuals field

---the acid-fast bacilli positive (++): 1～9/10 visuals field

---the acid-fast bacilli positive (+++): 1～9/every visual field

---the acid-fast bacilli positive (++ ++): 〉=10/every visual field

The direct microscopy of phlegm smear is to detect the most general laboratory diagnostic method of acid-fast bacilli (AFB), specificity is higher, but susceptibility is lower, mycobacterium will reach 103～104CFU/ml and just can detect positive findings in the smear, and only can detect the active tuberculosis patient of 1/2-3/4, loss is quite high.

Culture method: get postdigestive sputum mixing, be seeded in medium slant with aseptic straw, the inoculation back bevel is upwards in 37 ℃ of constant incubators, observe the bacterial growth situation weekly, positive give birth to the elder through smear luxuriant-report the result at any time after the checking of Nissl's staining method, be cultured to the person that 8 weeks saw the bacterial growth not yet, report mycobacterium feminine gender.

Luo Shi (

Jensen) culture method is " gold standard " that detects MTB at present, but its sense cycle long (8 week) causes in time making a definite diagnosis the tubercular, and can't in time treat.

2. immunological method

Tuberculin skin test (tuberculin skin test, TST): tuberculin typically refers to ot (OT) and tubercule bacillus purified protein derivative (PPD), is usually used in tuberculosis epidemiology investigation, tubercle bacillus affection condition monitoring, the preceding test of BCG (Bacille Calmette-Guerin) vaccination, tuberculosis auxiliary diagnosis.MTB infects in vivo and mainly causes cell-mediated immune response, it is the important method of screening mycobacterium tuberculosis latent infection always, be employed for many years, and admitted widely, though it is very extensive that this method is worldwide used, but from sensitivity and specificity, it is not an ideal detection method.Be the crowd of prevention tuberculosis bcg vaccination, will produce false sun reaction PPD.

Amynologic mechanism lungy mainly is that body is to the caused cell immune response of mycobacterium tuberculosis.Make T lymphocyte sensitization be converted into the memory T lymphocyte behind the human body primary infection mycobacterium tuberculosis, after human body contacts mycobacterium tuberculosis once more, body can produce effector T cell rapidly and produce various kinds of cell factor performance immunological effect, and wherein Interferon, rabbit (IFNl) is the most key cytokine.Therefore, the level of the effector T cell of detection IFN-y level or secretion IFN-1 just can judge whether to exist tuberculosis infection.

The ELISA method: serological method detects extremely Chinese scholars concern of tuberculosis antibody in recent years.Tuberculosis diagnoses the negative diagnosis of tuberculosis of bacterium especially to be still to demand urgently in the tuberculosis prevention and treatment problem of studying and solving.Tuberculosis antibody is still active tuberculosis at present, particularly the important auxiliary diagnosis means in negative tuberculosis of bacterium and the extrapulmonary tuberculosis diagnosis.Cause there are phasic specificity in the difference and the tubercular of MTB antigen recognition to antigenic identification owing to the treatment of tubercular's immunologic function, genetic background and acceptance is different, any one single antigen or commercial combined antigen detection kit all can't detect the tuberculosis antibody in all tubercular's serum at present, screening MTB specific antigens, forming associating antigen is present serodiagnostic research direction.

The most of ELISA method that adopts of the detection of mycobacterium tuberculosis antibody at present, the ELISA method has simple, cheap characteristics, but present many methods are also not overripened, having much room for improvement aspect specificity and the sensitivity.Along with the discovery and the purifying of several species-specific antigens of mycobacterium tuberculosis, the updating of experimental technique, ELISA detects serum tuberculosis antibody application of clinic experimentation at home, detects tuberculosis antibody and helps diagnosis and differential diagnosis lungy.It is simple to operate, economical to detect mycobacterium tuberculosis antibody with ELISA, is the efficient diagnosis method of active tuberculosis.

3. molecular biology method

At aspects such as mycobacteria strain evaluation, epidemiological study, genome comparative analysis, resistance monitorings, molecular biology method is to diagnosis of tuberculosis and treated important booster action, and major technique comprises PCR, order-checking, finger printing, biochip, TMA etc.

PCR: be a kind of amplification in vitro technology that adopts nucleic acid reproduction process in the molecular engineering analog cell according to the nucleic acid replication principle, developed multiple round pcrs such as nido or sleeve type PCR, multiplex PCR, reverse transcription PCR, chimeric fluorescent PCR at present, the round pcr emerging technology that produces that combines with other molecular engineering emerges in an endless stream especially.1989, Hance etc. report the detection that round pcr is applied to TB at first, its outstanding feature is highly sensitive, can detect the mycobacterium DNA of 1～100fg purifying, be equivalent to 1～20 MTB, compare with cultural method with traditional phlegm smear acid-fast stain, its susceptibility has obtained improving greatly.In addition, compare with traditional detection method, the reporting period that PCR detects is short, can send report in 1～2 day, therefore the early diagnosis for TB has original advantage, but the problems such as false negative that the PCR inhibitory substance causes in the false positive that exists of this method, the sample simultaneously are still waiting further improvement.

TMA: adopt the amplification technique of transcriptive intermediate that target nucleic acid is increased; adopt the dna probe of chemiluminescent substance bifurcation pyridine ester mark and the target gene of amplification to hybridize; probe and amplified production hybridization back utilize the hybridization protection test to remove the not label probe of hybridization, utilize Chemiluminescence Apparatus to detect chemiluminescence signal at last.Owing to adopted the molecular engineering of multiple sensitivities such as transcriptive intermediate amplification technique, hybridization protection detection technique and chemoluminescence, had the susceptibility and the specificity of height.Studies show that TMA reaches 92～100% to the susceptibility that is coated with positive TB diagnosis, also can reach 40～93% to the susceptibility that is coated with cloudy TB diagnosis, therefore the specificity that all clinical samples are detected has great application potential all more than 95%.The approval that the TMA detection kit of Gen-Probe company has obtained U.S. FDA is applied to the diagnosis of clinical TB, weak point is that operation is comparatively complicated, technology is difficult for grasping, and needs supporting chemiluminescence detector, thereby in the clinical comparatively difficulty of applying.

Our company has applied for patent constant temperature synchronous amplification detecting process for nucleic acid (Simultaneous Amplification andTestiTB, SAT) (application number: 200810111479.0); On the basis of this technology, what mycobacterium tuberculosis of the present invention (TB) kit for detecting nucleic acid adopted is the SAT technology, nucleic acid amplification uses M-MLV ThermoScript II and T7 RNA polymerase to realize simultaneously, ThermoScript II is used to produce a DNA copy of target nucleic acids (RNA), T7 RNA polymerase produces a plurality of RNA copies from the DNA copy, have the RNA copy specific combination that fluorescently-labeled optimization probe and amplification back produce, thereby produce fluorescence, this fluorescent signal can be caught by detecting instrument.This test kit can detect the TBrRNA in the sputum, has specificity height, highly sensitive and pollute the characteristics of low (amplified production RNA be easy to degraded) under physical environment.

Summary of the invention

Technical problem to be solved by this invention is that to overcome prior art specificity, susceptibility not high, the uppity shortcoming of experimental pollution provides a kind of ultrasonication bacterium thalline to discharge the test kit that thalline nucleic acid RNA and the synchronous amplification detection technology of constant temperature nucleic acid (SAT) detect mycobacterium tuberculosis (TB).

A kind of concretion mycobacterium nucleic acid detection kit of utilizing the RNA constant-temperature amplification of the present invention comprises:

(1) TB diluent: the sterilization DEPC aqueous solution that contains 0.1～1%RNAse inhibitor;

(2) TB reaction solution:, mainly comprise Tris 10～50mM, MgCl for dNTPs and the NTPs required component that increases ₂10～40mM, dNTP 0.5～5mM, NTP 1～10mM, PVP40 1～10%, KCl 5～40mM;

(3) TB detects liquid: primer and fluorescent probe essential during for constant-temperature amplification are dissolved in the TE solution (mixed solution of 10mM Tris and 1mM EDTA) formulated, the primer probe sequence all is selected from long 516bp TB 16SrRNA gene conserved regions, and sequence is seen sequence table SEQ .ID.NO14;

(4) SAT enzyme liquid: essential multienzyme components system during for constant-temperature amplification contains t7 rna polymerase 200～2000U/ reaction, M-MLV ThermoScript II 400～4000U/ reaction, 2～10mM HEPES pH7.5,10～100mMN-acetyl-L-cysteine, 0.04～0.4mM zinc acetate, 10～100mM trehalose, 40～200mM Tris-HCl pH 8.0,40～200mM KCl, 1～10mM EDTA, 2～20% (v/v) TritonX-100,10～40% (v/v) glycerol;

(5) TB positive control; Be 10 ²～10 ⁵The bacterium liquid of CFU/ml mycobacterium tuberculosis;

(6) TB negative control: for do not contain the SEQ.ID.NO14 nucleotide sequence or do not contain mycobacterium tuberculosis solution.

It is 0.1～1% that diluent mainly contains the RNase inhibitor concentration, the RNase inhibitor can make the rapid inactivation of RNase, effectively preserve RNA, the ultrasonication thalline makes target RNA obtain release, sample is placed in this diluent, prolonged the shelf time of sample, the diluent component is sterilization DEPC water and 0.1～1%RNase inhibitor.

Contained t7 rna polymerase, M-MLV ThermoScript II in the SAT enzyme liquid, its stability has directly determined the use validity period of the amplification efficiency and the test kit of amplified reaction.Be added with stablizer in this SAT enzyme liquid, through long-term stable experiment, stability can reach 10 months, has guaranteed that the test kit validity period reaches 6 months.

The TB fluorescent probe is a molecular beacon in the TB detection liquid, it is the molecular probe of a class high specific, hypersensitivity, form by the single stranded nucleic acid molecule of fluorescence dye and quencher by two ends difference covalent labeling, be hair clip type or loop-stem structure, the loop section of molecular beacon and target complementation, two becomes stem owing to complementary, and molecular beacon probe is compared with the TaqMan probe of linearity, because of opening of its hairpin structure needs certain power, thereby specificity is better than linear probe.TB detects TB primer in the liquid, according to the principle design of SAT amplification 5 ' end have the downstream primer of T7 promoter sequence tail and special upstream primer, downstream primer distinguished sequence and target are complementary fully, upstream primer and target are in full accord, have guaranteed that this test kit can accurately carry out the SAT reaction that TB detects.

The primer that TB detects in the liquid includes following a pair of or several to sequence:

TB T7: sequence is seen sequence table SEQ .ID.NO1-5;

TB nT7: sequence is seen sequence table SEQ .ID.NO6-10.

The probe that TB detects in the liquid includes following one or more sequences:

TB probe: sequence is seen sequence table SEQ .ID.NO11-13.

Owing to being subject to multiple factor affecting, the SAT amplification makes the amplification failure, test kit user of service error in judgement is got the wrong sow by the ear, TB positive control in the test kit of the present invention and TB negative control, all contain this test kit and addressed the TB diluent, wherein the TB positive control also contains certain density TB bacterium, and concentration is about 10 ²～10 ⁵CFU/ml.When using this test kit, should do the quality control of parallel control simultaneously, and the result should satisfy positive control dt≤35 simultaneously, negative control dt does not have numerical value or is 40 at every turn, otherwise that experiment this time is considered as is invalid.(dt represents the X-coordinate reading of sample curve and threshold line intersection point, and is similar with the ct value of general real-time fluorescence PCR experimental result)

TB bacterium liquid in the critical weak positive control of TB positive control and TB can prepare gained by several different methods, and wherein a kind of preparation method is as follows:

(1) H37Ra (ATCC25177) the reference culture enlarged culturing that will buy from Chinese medicine bacterium preservation center;

(2) adopt the bacterial count method that the bacterium liquid of cultivating is counted;

(3) culture with counting becomes positive control with the TB diluted, and contained bacterial concentration is 10 ²～10 ⁵CFU/ml.

(4) positive control is become critical weak positive control with 100 times of TB diluted, contained bacterial concentration is 1～10 ³CFU/ml.

The sensitivity for analysis that detection kit of the present invention detects TB bacterium liquid is 10CFU/ml, the Clinical Laboratory requirement is satisfied in this sensitivity fully, so detection kit specificity height of the present invention, highly sensitive, and amplified production RNA is easy to degraded and pollutes low under physical environment.Test kit of the present invention can be used for the detection of sputum sample.

Description of drawings

Fig. 1 is the amplification figure that embodiment 4 test kits of the present invention detect patient's sputum sample.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Embodiment 1

The extraction of TB nucleic acid RNA, concrete steps are:

1. get sputum 1.5ml, the 4%NaOH that looks 1～2 times of volume of sputum specimen proterties adding is in the sterile sampling pipe, and vortex vibration 1min makes the abundant homogenize of sputum, and room temperature is placed 15min～20min.

Sample processing tube (the 1.5ml centrifuge tube, quantity is: number of awaiting test sample+2), the respectively pending sample number into spectrum of mark and the positive, negative control;

3. get the good sputum sample 1ml of liquefaction in sample processing tube, the centrifugal 5min of 13000rpm abandons supernatant; Add the resuspended washing of 1ml physiological saline, the centrifugal 5min of 13000rpm abandons supernatant, and it is resuspended to add 50 μ L TB diluents.

4. the 50 μ l samples that will handle well and 50 μ l positive controls, 50 μ l negative controls are put into ultrasonic processor and are carried out supersound process 15min, and ultrasonic power is 300W.

5. after the ultrasonic end, get the adding of 2 μ l handled things and contain in the clean micro-reaction pipe of augmentation detection liquid, this augmentation detection liquid comprises 30 μ lTB reaction solutions and 2 μ l TB detect liquid, detects the primer and the probe sequence that comprise in the liquid and be respectively:

TB T7: sequence is seen sequence table SEQ .ID.NO1-5;

TB nT7: sequence is seen sequence table SEQ .ID.NO6-10;

TB Probe: sequence is seen sequence table SEQ .ID.NO11-13.

Embodiment 2

The SAT nucleic acid amplification detects

1. the clean micro-reaction pipe that will contain augmentation detection liquid is put into the constant temperature primary heater unit, and 60 ℃ are incubated 10 minutes, and 42 ℃ are incubated 5 minutes; Simultaneously SAT enzyme liquid also is preheating to 42 ℃;

2. in the micro-reaction pipe, add the 10 μ l enzyme liquid of preheating (application of sample tip head does not contact the micro-reaction pipe, do change the tip head if any contact), enzyme-added bonnet upper tube cap 1200rpm shake 15 second mixing;

3. the micro-reaction pipe is gone to fast suitable constant-temperature fluorescence detector device, 42 ℃ were reacted 40 minutes, set per 1 minute and detected first order fluorescence, detected altogether 40 times;

4. after reaction finishes, directly the taking-up of micro-reaction pipe is soaked in 10%84 thimerosals, gets the operation of micro-reaction pipe care should be used to, forbid to open reaction tubes (preventing to pollute reaction zone)! Experiment finishes the back with 10%84 thimerosal clean work area and apparatus, uses the clear water wiped clean at last.

5. reference value:

1. threshold setting: with the vertex of threshold line just above normal negative control amplification curve.

2.dt≤35 sample is positive;

3.35 the suggestion of the sample of＜dt＜40 detects again, the sample of detected result dt＜40 is positive;

4.dt do not have numerical value or be that 40 sample is negative.

Annotate: dt represents the X-coordinate reading (similar with the ct value of general real-time fluorescence PCR experimental result) of sample curve and threshold line intersection point

6. quality control: each detection all is provided with positive control and negative control, and the result should satisfy positive control dt≤35 simultaneously, and negative control dt does not have numerical value or is 40, otherwise that experiment this time is considered as is invalid.

Embodiment 3

The preparation and the assembling of TB test kit each component

Reagent constituents:

TB diluent: the sterilization DEPC aqueous solution that contains 0.1～1%RNAse inhibitor;

TB reaction solution: contain dNTPs and NTPs, mainly comprise Tris 10～50mM, MgCl ₂10～40mM, dNTP 0.5～5mM, NTP 1～10mM, PVP40 1～10%, KCl 5～40mM;

SAT enzyme liquid: contain t7 rna polymerase 200～2000U/ reaction, M-MLV ThermoScript II 400～4000U/ reaction, 2～10mMHEPES pH7.5,10～100mM N-acetyl-L-cysteine, 0.04～0.4mM zinc acetate, 10～100mM trehalose, 40～200mM Tris-HCl pH 8.0,40～200mM KCl, 1～10mM EDTA, 2～20% (v/v) Triton X-100,10～40% (v/v) glycerol;

TB detects liquid: contain TB special primer, specific fluorescence probe, primer and probe all are dissolved in TE solution (mixed solution of 10mM Tris and 1mM EDTA);

TB positive control: contain 10 ²CFU/ml～10 ⁵The solution of CFU/ml TB bacterium;

The TB negative control: for do not contain the SEQ.ID.NO14 nucleotide sequence or do not contain mycobacterium tuberculosis solution, be used for the validity of checked operation process and reagent.

The detection of the clinical sample sputum of embodiment 4 application models

This detecting pattern is best embodiment the of the present invention: the preparation of reagent is with embodiment 3, the production of reagent is carried out in the GMP workshop, the clinical experiment sample that mycobacterium tuberculosis sputum clinical sample provides for Changzhou third party people hospital, be numbered TB sputum sample 1～8, other establishes each one of negative control, positive control.The nucleic acid extraction of sample is with embodiment 1, and SAT augmentation detection and judgement are with embodiment 2, and employed fluorescent PCR instrument is Mx-3005P.The results are shown in accompanying drawing 1, identical with gold standard culture method detected result.

Sequence table

SEQUENCE?LISTING

＜110〉applicant: Shanghai Rendu Biotechnology Co., Ltd.

 

＜120〉a kind of concretion mycobacterium nucleic acid detection kit of utilizing the RNA constant-temperature amplification

 

<160>14

 

<170>PatentIn?version?3.3

 

<210>1

<211>51

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(51)

＜223〉according to the TB T7 primer of TB 16S rRNA gene conserved regions design

 

<400>1

aatttaatac?gactcactat?agggagacac?caacaagctg?ataggccgcg?g 51

 

<210>2

<211>53

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(53)

＜223〉according to the TB T7 primer of TB 16S rRNA gene conserved regions design

<400>2

aatttaatac?gactcactat?agggagagtc?accccaccaa?caagctgata?ggc 53

 

<210>3

<211>52

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(52)

＜223〉according to the TB T7 primer of TB 16S rRNA gene conserved regions design

 

<400>3

aatttaatac?gactcactat?agggagagta?ggccgtcacc?ccaccaacaa?gc 52

 

<210>4

<211>52

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(52)

＜223〉according to the TB T7 primer of TB 16S rRNA gene conserved regions design

 

<400>4

aatttaatac?gactcactat?agggagatca?ccccaccaac?aagctgatag?gc 52

<210>5

<211>56

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(56)

＜223〉according to the TB T7 primer of TB 16S rRNA gene conserved regions design

 

<400>5

aatttaatac?gactcactat?agggagagta?ggccgtcacc?ccaccaacaa?gctgat 56

 

<210>6

<211>22

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(22)

＜223〉according to the TB nT7 primer of TB 16S rRNA gene conserved regions design

 

<400>6

gagtggcgaa?cgggtgagta?ac 22

 

<210>7

<211>22

<212>RNA

＜213〉artificial sequence

<220>

<221>prim_bind

<222>(1)..(22)

＜223〉according to the TB nT7 primer of TB 16S rRNA gene conserved regions design

 

<400>7

gcaagtcgaa?cggaaaggtc?tc 22

 

<210>8

<211>27

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(27)

＜223〉according to the TB nT7 primer of TB 16S rRNA gene conserved regions design

 

<400>8

cgggtgagta?acacgtgggt?gatctgc 27

 

<210>9

<211>21

<212>RNA

＜213〉artificial sequence

<220>

<221>prim_bind

<222>(1)..(21)

＜223〉according to the TB nT7 primer of TB 16S rRNA gene conserved regions design

 

<400>9

ctgggaaact?gggtctaata?c 21

 

<210>10

<211>20

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(20)

＜223〉according to the TB nT7 primer of TB 16S rRNA gene conserved regions design

 

<400>10

gtggcgaacg?ggtgagtaac 20

 

<210>11

<211>23

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(23)

＜223〉according to the TB Probe probe of TB 16S rRNA gene conserved regions design

 

<400>11

cguccggata?ggaccacggg?acg 23

 

<210>12

<211>27

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(27)

＜223〉according to the TB Probe probe of TB 16S rRNA gene conserved regions design

 

<400>12

ccacggatag?gaccacggga?tgcgugg 27

 

<210>13

<211>25

<212>RNA

＜213〉artificial sequence

 

<220>

<221>prim_bind

<222>(1)..(25)

＜223〉according to the TB Probe probe of TB 16S rRNA gene conserved regions design

<400>13

ccaggatgca?tgtcttgtgg?ccugg 25

 

<210>14

<211>516

<212>RNA

＜213〉TB 16S rRNA gene conserved regions (M.Tuberculosis, TB)

 

<400>14

gcggcgtgct?taacacatgc?aagtcgaacg?gaaaggtctc?ttcggagata?ctcgagtggc 60

gaacgggtga?gtaacacgtg?ggtgatctgc?cctgcacttc?gggataagcc?tgggaaactg 120

ggtctaatac?cggataggac?cacgggatgc?atgtcttgtg?gtggaaagcg?ctttagcggt 180

gtgggatgag?cccgcggcct?atcagcttgt?tggtggggtg?acggcctacc?aaggcgacga 240

cgggtagccg?gcctgagagg?gtgtccggcc?acactgggac?tgagatacgg?cccagactcc 300

tacgggaggc?agcagtgggg?aatattgcac?aatgggcgca?agcctgatgc?agcgacgccg 360

cgtgggggat?gacggccttc?gggttgtaaa?cctctttcac?catcgacgaa?ggtccgggtt 420

ctctcggatt?gacggtaggt?ggaga?agaag?caccggccaa?ctacgtgcca?gcagccgcgg 480

taatacgtag?ggtgcgagcg?ttgtccggaa?ttactg 516

Claims (7)

1. concretion mycobacterium nucleic acid detection kit of utilizing the RNA constant-temperature amplification comprises:

(1) TB diluent: for containing the sterilization DEPC aqueous solution of RNase inhibitor;

(2) TB reaction solution:, specifically comprise Tris 10～50mM, MgCl for containing dNTPs and the NTPs required component that increases ₂10～40mM, dNTP 0.5～5mM, NTP 1～10mM, PVP40 1～10%, KCl 5～40mM;

(3) TB detects liquid: essential TB primer and TB fluorescent probe are dissolved in the TE solution formulatedly during for constant-temperature amplification, and the primer probe sequence all is selected from long 516bp TB 16S rRNA gene conserved regions, and sequence is seen sequence table SEQ .ID.NO14;

(4) SAT enzyme liquid: essential multienzyme components system during for constant-temperature amplification contains t7 rna polymerase 200～2000U/ reaction, M-MLV ThermoScript II 400～4000U/ reaction, 2～10mM HEPES pH7.5,10～100mMN-acetyl-L-cysteine, 0.04～0.4mM zinc acetate, 10～100mM trehalose, 40～200mM Tris-HCl pH8.0,40～200mM KCl, 1～10mM EDTA, 2～20% (v/v) TritonX-100,10～40% (v/v) glycerol;

(5) TB positive control; For containing 10 ²～10 ⁵The bacterium liquid of CFU/ml mycobacterium tuberculosis;

(6) TB negative control: for do not contain the SEQ.ID.NO14 nucleotide sequence or do not contain mycobacterium tuberculosis solution.

2. concretion mycobacterium nucleic acid detection kit according to claim 1 is characterized in that: the concentration of RNase inhibitor is 0.1～1% in the diluent liquid in described (1), and all the other compositions are sterilization DEPC water.

3. concretion mycobacterium nucleic acid detection kit according to claim 1 is characterized in that: the TB fluorescent probe is a molecular beacon in the TB detection liquid in described (3).

4. concretion mycobacterium nucleic acid detection kit according to claim 3 is characterized in that: the probe that described TB detects in the liquid includes following one or more sequences: TB Probe: sequence is seen sequence table SEQ .ID.NO11-13.

5. concretion mycobacterium nucleic acid detection kit according to claim 1 is characterized in that: described TB detects in the liquid, and 5 ' end of at least one TB primer has the T7 promoter sequence.

6. concretion mycobacterium nucleic acid detection kit according to claim 5 is characterized in that: described primer sequence contains following a pair of or several to sequence:

TB T7: sequence is seen sequence table SEQ .ID.NO1-5;

TB nT7: sequence is seen sequence table SEQ .ID.NO6-10.

7. concretion mycobacterium nucleic acid detection kit according to claim 1 is characterized in that: mycobacterium tuberculosis bacterium liquid in the critical weak positive control of described TB positive control and TB, the preparation method comprises the following steps:

(1) with H37Ra reference culture enlarged culturing;

(2) adopt the bacterial count method that the bacterium liquid of cultivating is counted;

(3) culture with counting becomes positive control with the TB diluted, and contained bacterial concentration is 10 ²～10 ⁵CFU/ml;

(4) the TB positive control is the critical weak positive control of TB with 100 times of TB diluted, and contained bacterial concentration is 1～10 ³CFU/ml.

Images