CN109880922A - A kind of primer, kit and detection method detecting mycobacterium tuberculosis TB - Google Patents
A kind of primer, kit and detection method detecting mycobacterium tuberculosis TB Download PDFInfo
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- CN109880922A CN109880922A CN201910155026.6A CN201910155026A CN109880922A CN 109880922 A CN109880922 A CN 109880922A CN 201910155026 A CN201910155026 A CN 201910155026A CN 109880922 A CN109880922 A CN 109880922A
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- mycobacterium tuberculosis
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Abstract
The invention discloses primer, kit and the detection methods of a kind of detection mycobacterium tuberculosis (Mycobacterium Tuberculosis, TB), belong to molecular biology field；By nucleotide sequence forward primer as shown in SEQ ID NO:1 and nucleotide sequence, the reverse primer as shown in SED ID NO:2 forms the primer；The forward primer is marked with biotin；The reverse primer is marked with FITC or Dig；The present invention is using recombinase-polymerase nucleic acid amplification technologies and solid phase developing technology, it is only necessary to by not needing PCR instrument and carrying out thermal cycle reaction, testing result naked eyes can directly be read from solid phase colour developing test strips to isothermal duplication after clinical sample progress DNA extraction；Primer detection mycobacterium tuberculosis TB high specificity of the invention, amplification efficiency are high；Kit and detection method response procedures of the invention is simple, can be effective, rapidly detects to mycobacterium tuberculosis TB, is applicable in simplicity on site, quickly, accurately carries out the diagnosis of human tuberculosis.
Description
Technical field
The present invention relates to molecular biology fields, and in particular to a kind of primer, kit for detecting mycobacterium tuberculosis TB
And detection method.
Background technique
Tubercle bacillus, i.e. mycobacterium tuberculosis (Mycobacterium Tuberculosis, TB) are obligate aerobic micro- lifes
Object, it is the pathogen of human tuberculosis.Tuberculosis is by airborne infectious disease, and main infection pathway is droplet and air
It infects, only sick (pulmonary tuberculosis) patient of pulmonary Mycobacterium has infectiousness, when infectiousness patient coughs, sneezes, speaks or spits,
It generates the droplet with tubercle bacillus and tubercle bacillus is discharged into air, if the droplet that accidentally sucking patient generates, just having can
Tuberculosis can be infected.The people in the whole world 1/3 is mycobacterium tuberculosis infection person, but the people for wherein only having 5%-10% can send out in life
Disease.Immune system meeting " barrier " tubercle bacillus, and tubercle bacillus it is thick because appearance has cere protection, can in human body suspend mode it is more
Year.When the immunity of people weakens, illness probability increases.If do not treated, every active tuberculosis patient can infect every year on average
10-15 people.Due to immune system reduced capability, it is easier to that tuberculosis occurs after People living with HIV infection.According to the World Health Organization
WHO report, every year there are about 100 Wan Xinfa tuberculosis patients, as many as number is only second to India for China.Therefore prevention and control pair lungy
The public health security of country plays particularly important effect, and controls propagation key lungy and be that getting up early diagnoses.
Clinically the common symptom of patient has cough, pectoralgia, weight loss, burnout, loss of appetite, fever, hemoptysis etc..It removes
These symptoms, other chronic chest diseases also will appear, therefore these clinical symptoms can only be as the reference in diagnosis.Therefore, it
The necessary complex clinical performance of diagnosis of tuberculosis is just calculated complete in addition the variation and laboratory inspection of radiology are confirmed.Mesh
The culture of preceding mycobacterium tuberculosis at home is still the goldstandard of tulase detection and drug sensitive test (DST).But traditional sieve
Family name's cultivation takes a long time, and about 4~6 weeks, is not able to satisfy the needs of quick diagnosis.Liquid culture system such as BACTEC and MGIT
(BD Diagnostics, Sparks, Maryland, USA) provides method more sensitive and quick compared with conventional solid culture,
The growth of mycobacteria can be detected within 1~3 week.And it is directed to the Molecular Detection of mycobacterium tuberculosis TB nucleic acid, predominantly PCR method is examined
The mycobacterium tuberculosis TB gene in sputum, this method specificity and high sensitivity are surveyed, but the time needs at least 60-90 minutes, and
Complex instrument is needed, point-of-care detection (point-of-care test is not particularly suited for；POCT it) uses.Therefore, it is necessary to one kind
Can quick and precisely, simply, without expensive instrument, can be used for the detection kit and detection of on-site test mycobacterium tuberculosis TB
Method.
Summary of the invention
There is provided it is an object of the invention to overcome the deficiencies in the prior art it is a kind of detect mycobacterium tuberculosis TB primer,
Kit and the method for inspection solve mycobacterium tuberculosis TB existing in the prior art and detect cumbersome to be difficult to asking for meet demand
Topic.
Based on recombinase-polymerase nucleic acid amplification technologies (Recombinase-polymerase amplification,
RPA intracellular nucleic acid synthesis mechanism) is simulated, within the scope of steady temperature, by recombinase, polymerase and single strand binding protein
So that DNA double chain is untwisted, and DNA fragment specific is promoted to expand, to achieve the effect that nucleic acid in vitro expands.Whole process is rapid
It (10-20 minutes) and is carried out in (37-42 DEG C) of low constant temperature.At present still in its infancy for the research of RPA technology, of the invention
Using recombinase-polymerase nucleic acid amplification technologies and combine solid phase developing technology, provide one it is simple, quickly, be not necessary to valuableness
Equipment can scene carry out the screening mode of mycobacterium tuberculosis TB, have the characteristics that highly sensitive, specificity, it is applicable on site
Rapid screening.
To achieve the above object, the technical solution adopted by the present invention is as follows:
A kind of primer detecting mycobacterium tuberculosis TB, the primer is as nucleotide sequence as shown in SEQ ID NO:1
Forward primer and the nucleotide sequence reverse primer as shown in SED ID NO:2 composition；The forward primer is marked with biotin
(Biotin)；The reverse primer is marked with FITC or Dig.
Primer of the invention is the pathogenic genes sequence and TwistDx that inventor refers to NCBI gene database
Instruction manual and Primer-BLAST design of primers principle, a plurality of primer of design and synthesis.In design process
In, consider not only the formation that avoid primer dimer, hairpin structure, it is also contemplated that different target gene is anti-at one
With answering in system coamplification problem.Primer screening experiment shows that primer specificity of the invention is strong, amplification efficiency is high, can effectively,
Rapidly detect mycobacterium tuberculosis TB.
The present invention also provides a kind of kits for detecting mycobacterium tuberculosis TB comprising above-mentioned primer.
The kit and detection method response procedures of above-mentioned technical proposal are simple, can be effective, rapidly to tuberculosis branch bar
Bacterium TB is detected, and is applicable in simplicity on site, quickly, is accurately carried out the diagnosis of the disease.
To be of the present invention for detecting the preferred embodiment of the kit of mycobacterium tuberculosis TB, the kit
Further include recombinase, polymerase, DNA binding protein, buffer solution, the positive in Quality Control, ddH2O and solid phase chromogenic detection reagents.
For it is of the present invention for detect mycobacterium tuberculosis TB kit preferred embodiment, it is described the positive in
Quality Control contains the primer pair being made of the nucleotide sequence as shown in SEQ ID NO:3 and SED ID NO:4.
To be of the present invention for detecting the preferred embodiment of the kit of mycobacterium tuberculosis TB, the recombinase
For T4 uvsX/uvsY；The polymerase is Bsu archaeal dna polymerase；The DNA binding protein is T4 gp32；The buffering is molten
Liquid contains following components: Tris-HCl buffer, potassium acetate, magnesium acetate, dithiothreitol (DTT), dNTPs, ATP, phosphocreatine, flesh
Acid kinase and Carbowax20M.
To be of the present invention for detecting the preferred embodiment of the kit of mycobacterium tuberculosis TB, the buffer
Component containing following concentration: Tris-HCl buffer 50mM, potassium acetate 100mM, magnesium acetate 14mM, dithiothreitol (DTT) 2mM,
DNTPs200 μM, ATP3mM, phosphocreatine 50mM, creatine kinase 100ng/ μ L, Carbowax20M5wt%；The Tris-HCl
The pH value of buffer is 7.9.
To be of the present invention for detecting the preferred embodiment of the kit of mycobacterium tuberculosis TB, the solid phase is aobvious
Color detection reagent includes solid phase colour developing test strips and color developing detection buffer.
To be of the present invention for detecting the preferred embodiment of the kit of mycobacterium tuberculosis TB, the colour developing
Detection buffer is made of the component of following concentration: BSA 5wt% and PBS buffer solution 10mM.
As of the present invention for detecting the preferred embodiment of the kit of mycobacterium tuberculosis TB, the sun
Quality Control is the recombinant plasmid for inserting people's GAPDH gene in property.
It is described the positive in Quality Control the preparation method comprises the following steps: respectively with nucleotide sequence such as SEQ ID No.3 and SEQ ID No.4
Shown in primer be upstream and downstream primer its PCR product genetic fragment is recombinated to plastid (pMD- with people's GAPDH gene cDNA
In 20T), to filter out single bacterium colony with antibiotic (ampicillin), then with Plasmid DNA Preparation Mini
Kit (QIAGEN, Valencia, CA) extracts Plasmid DNA, and correct recombinant plasmid is sequenced as Quality Control in the positive.
It is including following the present invention also provides the method for detection mycobacterium tuberculosis TB using above-mentioned kit a kind of
Step:
(1) DNA of sample to be tested is extracted as detection template；
(2) detection template being expanded, obtains DNA amplification sequence: preparing reaction system, concussion mixes reaction system,
Sample reacts 20 minutes in 37~42 DEG C, is expanded, obtains DNA amplification sequence；
(3) amplified production is detected using solid phase colour developing test strips: takes a certain number of solid phase colour developing test strips, needle
Different samples to be tested are marked, each sample takes 50 μ L color developing detection buffers, is added in reaction tube；20 μ L amplification is taken to produce
Object mixes in centrifuge tube, amplified production and color developing detection buffer totally 70 μ L；Test strips sample application zone is added in above-mentioned reaction solution,
It is incubated for 5 minutes；
(4) point is presented according to test strips reaction zone and judges testing result: after incubation, naked eyes are observed immediately；If examination
Paper slip reaction zone shows test paper quality control point (Marker) and Quality Control point (Internal Control), then this reaction is effectively anti-
It answers；If test strips reaction zone shows mycobacterium tuberculosis TB reflecting point, test sample is the positive；If test strips reaction zone
Mycobacterium tuberculosis TB reflecting point is not shown, then detects sample as feminine gender.
Above-mentioned detection method is combined using recombinase-polymerase nucleic acid amplification technologies with solid phase developing technology, simple,
Quickly, has the characteristics that highly sensitive, specificity.
Sample to be tested derives from human respiratory secretion, such as sputum.
Compared with prior art, the beneficial effects of the present invention are:
The present invention combines recombinase-polymerase nucleic acid amplification technologies with solid phase developing technology, provide one it is simple,
Quickly, being not necessary to expensive device can the live screening mode for carrying out mycobacterium tuberculosis TB.Without thermal cycle reaction and special
Instrument, testing result can directly be observed by vision, have the characteristics that highly sensitive, specificity, suitable for quickly examining immediately
It is disconnected, screening can be carried out in a non-laboratory environment, it is possible to provide the quick detection of live First Line.
Detailed description of the invention
Fig. 1 is the test paper that recombinase polymeric enzymatic amplification detection mycobacterium tuberculosis TB is carried out using kit of the present invention
Item diagram；
Fig. 2 is to carry out recombinase polymeric enzymatic amplification using kit of the present invention to detect mycobacterium tuberculosis TB, and needle
The result figure that different detection samples are marked.
Appended drawing reference: A. siphon area, B. reaction zone, the sample application zone C..
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention further illustrates.It will be appreciated by those skilled in the art that specific embodiment described herein is only to explain this hair
It is bright, it is not intended to limit the present invention.
Test method as used in the following examples is conventional method unless otherwise specified；Institute in following embodiments
Material, reagent for using etc., are commercially available unless otherwise specified.
Embodiment 1: primer and kit
1, design of primers
According to the respiratory tract infection pathogenic genes sequence of NCBI gene database, with reference to TwistDx instruction
Manual and Primer-BLAST design of primers principle, design primer.Primer length is about 30-35nt, due to currently without needle
To the primer-design software of RPA, is designed during previous work of the present invention and synthesized a large amount of primers, be screened out from it high sensitivity
And specific good primer is used for (table 1) of the invention.
2, primer synthesizes
According to primer sequence shown in table 1, sequent synthesis is carried out.It is characterized in that, its mycobacterium tuberculosis TB gene is just
To primer sequence as shown in SEQ ID NO:1, and modifies and Biotin is added；Reverse primer sequences as shown in SEQ ID NO:2, and
FITC or Dig is added in modification.Its interior Quality Control forward primer sequence modifies and Biotin is added as shown in SEQ ID NO:3；Reversely
Primer sequence is modified and TAMRA is added as shown in SEQ ID NO:4.
Table 1
Kit described in the present embodiment for mycobacterium tuberculosis TB quick diagnosis includes above-mentioned upstream primer and downstream
Primer, the kit further include recombinase, polymerase, DNA binding protein, buffer solution, the positive in Quality Control, ddH2O and solid
Phase chromogenic detection reagents.The recombinase is T4 uvsX/uvsY, and the polymerase is Bsu archaeal dna polymerase, and the DNA is combined
Albumen is T4gp32；The buffer contains the component of following concentration: Tris-HCl buffer 50mM, potassium acetate 100mM, acetic acid
Magnesium 14mM, 2mM, dNTPs200 μM of dithiothreitol (DTT), ATP3mM, phosphocreatine 50mM, creatine kinase 100ng/ μ L,
Carbowax20M5wt%；The pH value of the Tris-HCl buffer is 7.9.The solid phase chromogenic detection reagents include that solid phase is aobvious
Color test strips and color developing detection buffer, the color developing detection buffer are made of the component of following concentration: BSA 5wt% and
PBS buffer solution 10mM.
Embodiment 2: detection mycobacterium tuberculosis TB
The method that the present embodiment uses the detection mycobacterium tuberculosis TB detection of kit described in embodiment 1 to use is recombinase
Polymerase constant temperature gene amplification method simultaneously combines solid phase developing technology, the specific steps are as follows:
1, clinical sample prepares
Collect sputum, collection condition are as follows: check through laboratory and be confirmed as after (quantitative fluorescent PCR) with mycobacterium tuberculosis
The positive clinical sample (49) of TB, and it is confirmed as the negative clinical sample (51) of nontuberculous mycobacteria TB after inspection.
Clinical sample is placed in -80 DEG C of preservations.
2, the nucleic acid extraction of sample
The present embodiment is extracted or is automated sample nucleic acid extraction apparatus using DNA col-umn chromatography, carries out the nucleic acid of clinical sample
It extracts.
3, prepared by positive reference product nucleic acid
Respectively using primer shown in SEQ ID NO:3 and SEQ ID NO:4 as upstream and downstream primer, with people's GAPDH gene
CDNA recombinates its PCR product genetic fragment into plastid (pMD-20T), to filter out list with antibiotic (ampicillin)
One bacterium colony, then Plasmid DNA is extracted with Plasmid DNA Preparation Mini Kit (QIAGEN, Valencia, CA), it surveys
The correct recombinant plasmid of sequence is Quality Control in the positive.
4, reaction system configures
By 2 proportional arrangement reaction system of table:
Table 2
5, concussion mixes made from step 4 after reaction system, sample in 39 DEG C isothermal reaction 20 minutes, it is poly- to carry out recombinase
Synthase isothermal duplication.
6, a certain number of solid phase colour developing test strips are taken, are marked for different detection samples.Each detection sample
50 μ L color developing detection buffers (5wt%BSA in PBS) are taken, are added in reaction tube.The resulting amplification of 20 μ L steps (5) is taken to produce
Object mixes in centrifuge tube, amplified production and color developing detection buffer totally 70 μ L.Test strips sample application zone is added in above-mentioned reaction solution,
It is incubated for 5 minutes.
7, after being incubated for, naked eyes are observed immediately.If test strips reaction zone shows test paper quality control point (Marker) and matter
It controls point (Internal Control), then this reaction is effecting reaction；If test strips reaction zone shows mycobacterium tuberculosis TB
Reflecting point, then test sample is the positive；If test strips reaction zone does not show mycobacterium tuberculosis TB reflecting point, sample is detected
For feminine gender.
8, result interpretation
The primer sets designed by the present invention are used for quickly detecting mycobacterium tuberculosis TB, through laboratory quantitative fluorescent PCR
The sample of 49 M. tuberculosis-positive TB of method identification has 48 display tuberculosis branch bars in solid phase colour developing test strips
Bacterium TB reflecting point, all negative samples do not show this reflecting point (Fig. 2, table 3).It can thus be appreciated that wherein sample 1-6 is positive sample
This, sample 7 is negative sample.
Table 3
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of primer for detecting mycobacterium tuberculosis TB, which is characterized in that the primer is made of forward primer and reverse primer,
The forward primer is marked with biotin, and the reverse primer is marked with FITC or Dig,
The forward primer is nucleotide sequence SEQ ID NO:1:
5'Biotin-GCG TAG GCG TCG GTG ACA AAG GCC ACG TAG3'；
The reverse primer is nucleotide sequence SEQ ID NO:2:
5’FITC/DIG-TCC CGC CGA TCT CGT CCA GCG CCG CTT CGG3’。
2. a kind of kit for detecting mycobacterium tuberculosis TB, which is characterized in that the kit includes described in claim 1 draws
Object.
3. a kind of kit for detecting mycobacterium tuberculosis TB according to claim 2, which is characterized in that the kit
Further include recombinase, polymerase, DNA binding protein, buffer solution, the positive in Quality Control, ddH2O and solid phase chromogenic detection reagents.
4. a kind of kit for detecting mycobacterium tuberculosis TB according to claim 3, which is characterized in that the recombinase
For T4 uvsX/uvsY；
The polymerase is Bsu archaeal dna polymerase；
The DNA binding protein is T4 gp32；
The buffer solution includes Tris-HCl buffer, potassium acetate, magnesium acetate, dithiothreitol (DTT), dNTPs, ATP, phosphoric acid flesh
Acid, creatine kinase and Carbowax20M.
5. a kind of kit for detecting mycobacterium tuberculosis TB according to claim 4, which is characterized in that the Tris-
HCl buffer concentration is 50mM, and the pH value of the Tris-HCl buffer is 7.9, and the acetic acid potassium concn is 100mM, described
Acetic acid magnesium density is 14mM, and the dithiothreitol (DTT) concentration is 2mM, and the dNTPs concentration is 200 μM, and the ATP concentration is
3mM, the phosphocreatine concentration are 50mM, and the creatine kinase concentration is 100ng/ μ L, and the Carbowax20M concentration is
5wt%.
6. a kind of kit for detecting mycobacterium tuberculosis TB according to claim 3, which is characterized in that in the positive
Quality Control includes the primer pair being made of the nucleotide sequence as shown in SEQ ID NO:3 and SED ID NO:4.
7. a kind of kit for detecting mycobacterium tuberculosis TB according to claim 3, which is characterized in that the solid phase is aobvious
Color detection reagent includes solid phase colour developing test strips and color developing detection buffer.
8. a kind of kit for detecting mycobacterium tuberculosis TB according to claim 7, which is characterized in that the colour developing inspection
The PBS buffer solution that the BSA and concentration that survey buffer is 5wt% by concentration are 10mM forms.
9. a kind of kit for detecting mycobacterium tuberculosis TB according to claim 3, which is characterized in that the positive
Interior Quality Control is the recombinant plasmid for inserting people's GAPDH gene.
10. a kind of method using the described in any item kit detection mycobacterium tuberculosis TB of claim 2~9, feature
It is, comprising the following steps:
(1) DNA of sample to be tested is extracted as detection template；
(2) detection template is expanded, obtains DNA amplification sequence；
(3) amplified production is detected using solid phase colour developing test strips；
(4) point is presented according to test strips reaction zone and judges testing result.
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| CN201910155026.6A CN109880922A (en) | 2019-03-01 | 2019-03-01 | A kind of primer, kit and detection method detecting mycobacterium tuberculosis TB |
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| CN201910155026.6A CN109880922A (en) | 2019-03-01 | 2019-03-01 | A kind of primer, kit and detection method detecting mycobacterium tuberculosis TB |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110499377A (en) * | 2019-09-11 | 2019-11-26 | 深圳市芯思微生物科技有限公司 | A kind of primer and probe, kit and application detecting mycobacterium tuberculosis and its drug resistant gene |
| CN111187857A (en) * | 2020-02-14 | 2020-05-22 | 深圳市芯思微生物科技有限公司 | Primer pair, kit and preparation method and application of kit for detecting novel coronavirus through isothermal amplification |
| CN111690626A (en) * | 2020-07-02 | 2020-09-22 | 南京诺唯赞生物科技股份有限公司 | Fusion type Taq DNA polymerase and preparation method and application thereof |
-
2019
- 2019-03-01 CN CN201910155026.6A patent/CN109880922A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110499377A (en) * | 2019-09-11 | 2019-11-26 | 深圳市芯思微生物科技有限公司 | A kind of primer and probe, kit and application detecting mycobacterium tuberculosis and its drug resistant gene |
| CN111187857A (en) * | 2020-02-14 | 2020-05-22 | 深圳市芯思微生物科技有限公司 | Primer pair, kit and preparation method and application of kit for detecting novel coronavirus through isothermal amplification |
| CN111690626A (en) * | 2020-07-02 | 2020-09-22 | 南京诺唯赞生物科技股份有限公司 | Fusion type Taq DNA polymerase and preparation method and application thereof |
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Application publication date: 20190614 |