CN109655613A - A kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis - Google Patents
A kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis Download PDFInfo
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- CN109655613A CN109655613A CN201910117260.XA CN201910117260A CN109655613A CN 109655613 A CN109655613 A CN 109655613A CN 201910117260 A CN201910117260 A CN 201910117260A CN 109655613 A CN109655613 A CN 109655613A
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- mycobacterium tuberculosis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The present invention relates to detection technique fields, more particularly to a kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis, the kit is used to test mycobacterium tuberculosis, its reagent preparation step is as follows: taking sputum sample to put into sodium hydroxide solution, shakes and remove the mucin components of sputum sample surface；Sputum sample is subjected to high speed centrifugation, extracts required Mycobacterium tuberculosis cell；The Mycobacterium tuberculosis cell of extraction is crushed, is centrifuged, and is washed with distillation, obtained precipitating film lysate dissolution can be obtained outer membrane protein；The outer albumen of seperation film, obtains three kinds of outer membrane proteins, and three kinds of outer membrane proteins are purified and recombinated, three after being recombinated kind recombinant protein；Three kinds of recombinant protein fliud flushings after purification are diluted and use ELISA Plate；It is washed after re-closed ELISA Plate；ELISA Plate is put into sterile vacuum drier after drying and is packed into reagent bottle；The present invention can detect the source of antibody and antibody in sample.
Description
Technical field
The present invention relates to detection technique fields, and in particular to a kind of kit for detection sensitivity mycobacterium tuberculosis
And its detection method.
Background technique
China is one of 22 tuberculosis high burden countries in the whole world, and annual new cases account for the 16% of global sum.China
Tuberculosis control work directly affects global Tubercufosis control implementation and result.According to the estimation of WHO, China is sent out every year
Raw activity tuberculosis patient 1,300,000, China become one of global tuberculosis high burden country, occupy 22 high burden country
Second.The whole nation has nearly half (5.5 hundred million) number population to infect tulase, is obviously higher by the level of global 1/3 population infection；Entirely
State-owned active tuberculosis 5,000,000, wherein positive 1,500,000, especially the Midwest epidemic situation lungy of sputum smear is very tight
Weight；In addition China's resistance problems are extremely serious, and the prevalence of AIDS makes tuberculosis make the matter worse in addition, floating population's tuberculosis
Number obviously increases, and increases the difficulty of city Tubercufosis control.In the fight of confrontation pulmonary tuberculosis, the maximum that faces now
Challenge is can not to be quickly and accurately diagnosed to be patient in a large amount of crowd.There is no reliable quick diagnosis technology, patient is just
It can not be by the antituberculosis therapy of early detection and regularization, so as to cause the deterioration of the state of an illness, drug resistant generation and pathogen
Diffusion.Timely diagnosis can help clinician to select early stage targeted therapy, reduce case fatality rate, meanwhile, reduce empirical use
Medicine reduces medical burden.
At present to the detection of tuberculosis, no matter to animal body or on human body, all using classical tuberculin PPD
(Purifiedprotein derivative) skin test method, according to the journey of skin turgor (erythema) after inoculation tuberculin 3-4 days
Degree judges whether to infect.But this mode cannot distinguish between antibody caused by natural infection and vaccine inoculation, and Spain scholar uses
PCR method detects tuberculosis (Parra et al., 2008) from confirmed cases, and recall rate is only 80.64%.But this method pair
The pre-processing of sample is very harsh, and it is extremely difficult to extract from sample mycobacteria DNA；In addition, mycobacteria base
Because a group G/C content is up to 66%, target fragment itself is expanded, it may have certain difficulty.Therefore, the selection of diagnostic antigen is always
Key link in Diagnosis of Tuberculosis.
More than 80 outer membrane proteins (Song et al., 2008) is identified from mycobacterium tuberculosis at present, and is passed through
Proteinase K experiment, the experiment of full cell ELISA and Western blot analytical proof Rv0899 (OmpA), Rv1973
It is not exposed only on cell surface, and the recombinant protein of 1ng can be with intracorporal antibody response.Mce4B is also identified 80
More than one of memebrane protein, which is only just expressed in the later period of infection or active stage.
Summary of the invention
In view of the deficienciess of the prior art, the technical problem to be solved by the invention is to provide one kind for detect it is quick
The kit and its detection method of perceptual mycobacterium tuberculosis, so as to it is quick, sensitive, accurately to sensibility knot in sample to be tested
Core mycobacteria is detected.
In order to solve the above technical problems, the technical solution adopted by the present invention is that: one kind being used for detection sensitivity tuberculosis branch
The kit and its detection method of bacillus, the kit are used for Enzyme-linked Immunosorbent Assay and immune colloid gold to mycobacterium tuberculosis
Test, reagent preparation step are as follows:
A. sputum sample is taken to put into the sodium hydroxide solution equipped with l~2 times own vol, concussion makes sputum in 1 minute
Sample is sufficiently reacted with sodium hydroxide solution, removes the mucin components of sputum sample surface, and stands 18~19 at room temperature
Minute；
B. sputum sample is subjected to high speed centrifugation, abandons supernatant liquor, physiological saline is added and relaunders, washes away in sample
Impurity extracts required Mycobacterium tuberculosis cell；
C. the Mycobacterium tuberculosis cell of extraction is put into ultrasonic disruption device to be crushed, addition sonification medium is water, is surpassed
Sound is 15 minutes broken, by the ultrasonic energy that medium is propagated, destroys tuberculosis and divides skill bacilli-cell structure, low-speed centrifugal removing is not split
The thallus and matrix of solution, supernatant ultracentrifugation, precipitating are washed 3 times with 100 times of distillation, will be centrifuged obtained precipitating for the last time
It is dissolved with film lysate, 2000r/min is centrifuged 30min, can be obtained the supernatant for outer membrane protein；
D. the outer albumen of seperation film, can be obtained three kinds of outer membrane proteins, and three kinds of outer membrane proteins are purified and recombinated, i.e.,
Three kinds of recombinant proteins after being recombinated；
E, three kinds of recombinant proteins after purification are diluted with carbonate buffer solution, concentration is 3ng-8 μ g/ μ L, with recombination egg
White dilution coated elisa plate；
F, ELISA Plate is closed with 4% casein, bovine serum albumin(BSA), gelatin or skimmed milk power；Then slow with phosphate
Fliud flushing washing；
G, ELISA Plate is put into sterile vacuum drier after drying and is packed into reagent bottle.
Three kinds of outer membrane proteins are respectively Rv0899, Rv1973, Mce4B.
Three kinds of outer membrane proteins are respectively ESAT-6, Rv1973, Mce4B.
Three kinds of outer membrane proteins are respectively Rv0899, MPT64, Mce4B.
The test strips of the immune colloidal gold test are the recombinant protein warps with Rv0899, Rv1973 or Mce4B of purifying
The following steps preparation:
A, gold chloride is reduced into the particle of 20-30nm with sodium citrate；
B, preparation and reorganization staphylococcal protein A/G and colloid gold label object；
C, preparation blank PVC backing colloidal gold detection test strips, one end of test strips PVC backing successively adheres to sample
Pad, bonding pad；The intermediate adhesion nitrocellulose filter or pvdf membrane of PVC backing；The PVC backing other end adheres to water absorption pad；Wherein
There are two lines, respectively detection line and control line on nitrocellulose filter or pvdf membrane；
D, albumin A and colloid gold label object are sprayed on to the bonding pad of PVC backing colloidal gold detection test strips with film machine
On；The recombinant protein segment of Rv0899, Rv1973 or Mce4B are sprayed in the detection line of nitrocellulose filter or pvdf membrane；It will
The anti-protein A antibody of chicken or corresponding secondary antibody are sprayed on the control line of film；
E, the PVC backing colloidal gold detection test strips for being sprayed with sample drying is sealed, is refrigerated spare.
Described detection method the following steps include: with purifying three kinds of mycobacterium tuberculosis outer membrane protein Rv0899,
The recombinant protein of Rv1973 and Mce4B prepares the reagent of Enzyme-linked Immunosorbent Assay and immune colloid gold reagent box as antigen, utilizes
Tuberculosis antibody in the kit detection sample to be tested.
Described detection method the following steps include:
A. packaging is opened at 25 DEG C of room temperature using preceding take test strips；
B. serum to be checked is added into well, result is determined according to detection line and the color of control line.
C. if aubergine occur in detection line and control line, illustrate the tuberculosis antibody positive；If detection line is colourless, and control
There is aubergine in line processed, illustrates tuberculosis antibody feminine gender；If control line is colourless, illustrate that test strips are invalid；
D. if serum to be checked and the reaction of recombinant protein Rv0899 test strips, illustrate that there are tuberculosis antibodies in serum to be checked；
If serum to be checked respectively with recombinant protein Rv1973, Mce4BRv1973 test strips react, illustrate serum to be checked antibody be by
In causing naturally；If serum to be checked and recombinant protein Rv1973, Mce4BRv1973 and Mce4B test strips are reacted, illustrate this
Contain sensibility mycobacterium tuberculosis to current in serum to be checked.
Beneficial effects of the present invention are as follows:
The present invention is centrifugated out the outer albumen of three kinds of films of mycobacterium tuberculosis by sampling, removal of impurities, and pure by separating
The mode changed and recombinated, the outer albumen of three kinds of films after being recombinated, to prepare reagent using albumen outside three kinds of special films
Corresponding reagent in box, between the recombinant protein by three kinds of serum and Rv1973, Mce4BRv1973 and Mce4B to be checked
Whether reaction, there is sensibility mycobacterium tuberculosis and antibody in test sample, and by reacting with three kinds of recombinant proteins, can be really
The reason of making the formation of antibody；The present invention has good practicability, and the reagent of detection mycobacterium tuberculosis is prepared for people
Provide one well selection.
Specific embodiment
Below with reference to embodiment, the present invention is described in more detail.The following example is only for the skill of this professional skill field
Art personnel more fully understand the present invention, but do not limit the invention in any way.
The kit is used for Enzyme-linked Immunosorbent Assay and immune colloidal gold test to mycobacterium tuberculosis, reagent preparation step
It is rapid as follows:
A. sputum sample is taken to put into the sodium hydroxide solution equipped with l~2 times own vol, concussion makes sputum in 1 minute
Sample is sufficiently reacted with sodium hydroxide solution, removes the mucin components of sputum sample surface, and stands 18~19 at room temperature
Minute, it is layered sputum sample, so as to subsequent centrifugation, improves the effect of centrifuge separation；
B. sputum sample is subjected to high speed centrifugation, abandons supernatant liquor, physiological saline is added and relaunders, washes away in sample
Impurity extracts required Mycobacterium tuberculosis cell；By centrifugation and multiple washing, a large amount of impurity can be removed, to obtain
More pure Mycobacterium tuberculosis cell, going on smoothly in reagent preparation after being conducive to；
C. the Mycobacterium tuberculosis cell of extraction is put into ultrasonic disruption device to be crushed, addition sonification medium is water, is surpassed
Sound is 15 minutes broken, by the ultrasonic energy that medium is propagated, destroys tuberculosis and divides skill bacilli-cell structure, low-speed centrifugal removing is not split
The thallus and matrix of solution, supernatant ultracentrifugation, precipitating are washed 3 times with 100 times of distillation, will be centrifuged obtained precipitating for the last time
It is dissolved with film lysate, 2000r/min is centrifuged 30min, can be obtained the supernatant for outer membrane protein；By it is damaged, be centrifuged, remove
Multiple link steps of miscellaneous and multiple cleaning, dissolution, centrifugation can get the higher outer membrane protein of purity；
D. the outer albumen of seperation film, can be obtained three kinds of outer membrane proteins, and three kinds of outer membrane proteins are purified and recombinated, i.e.,
Three kinds of recombinant proteins after being recombinated；Required reagent can be prepared by three kinds of recombinant proteins；
E, three kinds of recombinant proteins after purification are diluted with carbonate buffer solution, concentration is 3ng-8 μ g/ μ L, with recombination egg
White dilution coated elisa plate；
F, ELISA Plate is closed with 4% casein, bovine serum albumin(BSA), gelatin or skimmed milk power；Then slow with phosphate
Fliud flushing washing；
G, ELISA Plate is put into sterile vacuum drier after drying and is packed into reagent bottle.
Three kinds of outer membrane proteins are respectively Rv0899, Rv1973, Mce4B.
Three kinds of outer membrane proteins are respectively ESAT-6, Rv1973, Mce4B.
Three kinds of outer membrane proteins are respectively Rv0899, MPT64, Mce4B.It can be prepared by replacing different outer membrane proteins
The reagent of similar effects is provided, so that reagent of the invention can be with abundantization.
The test strips of the immune colloidal gold test are the recombinant protein warps with Rv0899, Rv1973 or Mce4B of purifying
The following steps preparation:
A, gold chloride is reduced into the particle of 20-30nm with sodium citrate；
B, preparation and reorganization staphylococcal protein A/G and colloid gold label object；
C, preparation blank PVC backing colloidal gold detection test strips, one end of test strips PVC backing successively adheres to sample
Pad, bonding pad；The intermediate adhesion nitrocellulose filter or pvdf membrane of PVC backing；The PVC backing other end adheres to water absorption pad；Wherein
There are two lines, respectively detection line and control line on nitrocellulose filter or pvdf membrane；
D, albumin A and colloid gold label object are sprayed on to the bonding pad of PVC backing colloidal gold detection test strips with film machine
On；The recombinant protein segment of Rv0899, Rv1973 or Mce4B are sprayed in the detection line of nitrocellulose filter or pvdf membrane；It will
The anti-protein A antibody of chicken or corresponding secondary antibody are sprayed on the control line of film；
E, the PVC backing colloidal gold detection test strips for being sprayed with sample drying is sealed, is refrigerated spare.
Described detection method the following steps include: with purifying three kinds of mycobacterium tuberculosis outer membrane protein Rv0899,
The recombinant protein of Rv1973 and Mce4B prepares the reagent of Enzyme-linked Immunosorbent Assay and immune colloid gold reagent box as antigen, utilizes
Tuberculosis antibody in the kit detection sample to be tested.
Described detection method the following steps include:
A. packaging is opened at 25 DEG C of room temperature using preceding take test strips；
B. serum to be checked is added into well, result is determined according to detection line and the color of control line.
C. if aubergine occur in detection line and control line, illustrate the tuberculosis antibody positive；If detection line is colourless, and control
There is aubergine in line processed, illustrates tuberculosis antibody feminine gender；If control line is colourless, illustrate that test strips are invalid；
D. if serum to be checked and the reaction of recombinant protein Rv0899 test strips, illustrate that there are tuberculosis antibodies in serum to be checked；
If serum to be checked respectively with recombinant protein Rv1973, Mce4BRv1973 test strips react, illustrate serum to be checked antibody be by
In causing naturally；If serum to be checked and recombinant protein Rv1973, Mce4BRv1973 and Mce4B test strips are reacted, illustrate this
Contain sensibility mycobacterium tuberculosis to current in serum to be checked.
It because Rv1973 is existed only in virulent mycobacterium tuberculosis, is not present in BCG vaccine (vaccine strain), therefore can be with
By recombinant protein Rv1973 be used as differential test sample in antibody be due to being vaccinated with vaccine or nature caused by.Mce4B
Albumen only just expressed by the later period caused by nature or active stage, therefore the recombinant protein of Mce4B can be used as to survey
Sample is originally by stages.According to the analysis to Rv0899 (OmpA), Rv1973 or Mce4B transbilayer helix and epitope as a result, this hair
Recombinant protein in bright eliminates respective interminable non-essential amino acid sequence, and its epitope is spliced, and every
The C-terminal of a albumen is added to 6xHis label and codon is optimized.The present invention using purifying recombinant protein as
Antigen, the enzyme-linked immunosorbent assay (ELISA) and immune colloid gold reagent of preparation, can be used to detect the tuberculosis in test sample
Antibody；Enzyme-linked immunosorbent assay kit with corresponding reagent can provide testing result in 1.5 hours, be mainly used for
Laboratory test sample can determine result by microplate reader.And the immune colloid gold reagent box with corresponding reagent then can be with
Testing result is provided in 6-10 minutes, is not necessarily to any instrument, it is easy to use.
Embodiment 1
Detect the preparation of Enzyme-linked Immunosorbent Assay (ELISA) reagent of tuberculosis antibody
1) recombinant protein of Rv0899 or Rv1973 or Mce4B after purification are diluted to concentration with carbonate buffer solution by
For 3ng-6 μ g/ μ L, it is coated with 96 hole elisa Plates, every 90 μ L of hole；37 DEG C are reacted 50 minutes；The carbonate buffer solution is 8mm
Na2CO3 and NaHCO3 solution, pH9；
2) closes ELISA Plate, every hole using 3.5-4.5% casein, bovine serum albumin(BSA), gelatin or skimmed milk power
200μL；37 DEG C are reacted 50 minutes；
3) is washed 3 times with PBST, and the PBST is 60mM Na2HPO4,15mM NaH2PO4,80mM NaCl, 0.12%
The mixed liquor of Tween-20, pH7.3-7.4；
4) is by ELISA Plate sterile vacuum, dry packing.Deepfreeze saves, and storage life is more than half a year；When use according to
Specification operation, as described in step 5-9；The kit can detecte the tuberculosis antibody in a variety of serum samples；
5) ELISA Plate is taken room temperature using preceding by, opens packaging.Sample to be tested is diluted with PBST or without dilute
It releases, is added in ELISA Plate, the sample is serum to be detected.Negative control sera is normal serum, and positive serum is immune
The serum that the recombinant protein of Rv0899 or Rv1973 or Mce4B obtains；Every 90 μ L of hole；37 DEG C are reacted 50 minutes；Should 8SL3 with
Test experiments related with associated serum sample are carried out in upper rank laboratory or Biohazard Safety Equipment；
6) washs ELISA Plate 4-5 times with PBST；
7) Recombinant Staphylococal Protein A/G antibody of the diluted horseradish peroxidase of PBST (HRP) label is added in, should
Antibody can be reacted with the IgG in a variety of serum samples.Also the antibody in the serum of detection, Selection utilization be can according to need
Antibody in the laboratory sample of HRP label is as secondary antibody；Every 90 μ L of hole；37 DEG C are reacted 25 minutes；
8) washs ELISA Plate 4-5 times with PBST；
9) o-phenylenediamine or TMB colour developing liquid is added in；Result is determined according to color reaction.It is whole that 1M sulfuric acid can also be used
It only reacts, reads 490nm absorption value using microplate reader.If color is colourless in ELISA Plate negative serum hole, and sample positive hole
Turn yellow with liquid in serum hole to be checked, illustrates that serum to be checked is that tuberculosis antibody is positive.By the numerical value interpretation of microplate reader 490nm according to
According to as follows: if the absorption value of positive serum is more than or equal to 4, and measuring samples serum divided by the absorption value of negative serum
Absorption value also greater than or equal to 4, is then determined as blood serum sample tuberculosis antibody to be checked for sun divided by the absorption value of feminine gender serum
Property.If the recombinant protein of serum to be checked and Rv0899 react, illustrate that there are tuberculosis antibodies in vivo.If serum to be checked and
The recombinant protein of Rv0899 or Rv1973 reacts, and illustrates that intracorporal antibody is due to causing naturally.If serum to be checked and
The recombinant protein of Rv0899 or Rv1973 or Mce4B reacts, and prompts the tuberculosis branch bar contained in the corresponding serum of the sample
Bacterium is in tuberculosis active stage or advanced stage.
Embodiment 2
Detect the preparation of the colloidal gold strip reagent of tuberculosis antibody
1) sodium citrate of 1-1.5% is added in 0.02% gold chloride (HAuCl4), and stirring while adding, 90 DEG C are boiled 20
Minute, obtain the colloidal gold solution of 20-30nm particle；
2) it preparation and reorganization staphylococcal protein A/G and colloid gold label object: will be cooled to 0.02-0.0.09M potassium carbonate
The colloidal gold solution of room temperature is transferred to pH5.5-6, and Recombinant Staphylococal Protein A/G is slowly added into colloidal gold solution and is mixed, room
Temperature reaction 0.6-0.8h.Then the bovine serum albumin(BSA) of final concentration 0.08% is added into reaction solution as stabilizer, 4 DEG C of high speeds
It is centrifuged 25 minutes and is concentrated.Recombinant Staphylococal Protein A/the G and colloidal gold mark that 4-5 times is purified are washed with 0.08% BSA
Remember object.Marker is suspended in the 0.08%BSA and 0.009% Sodium azide of 3-6mm；
3) prepares blank PVC backing colloidal gold and detects test strips: one end of test strips PVC backing successively adheres to sample
Product pad, bonding pad；The intermediate adhesion nitrocellulose filter or pvdf membrane of PVC backing；The PVC backing other end adheres to water absorption pad；Its
Middle nitrocellulose filter or pvdf membrane have two lines, respectively detection line and control line；
4) albumin A and colloid gold label object are sprayed on the bonding pad of PVC backing colloidal gold detection test strips with point film machine by
On；The recombinant protein segment of the Rv0899 of purifying or Rv1973 or Mce4B is sprayed on to the detection of nitrocellulose filter or pvdf membrane
On line；The anti-protein A antibody of chicken is sprayed on the control line of film, also can according to need a variety of antibody to be detected of detection, selection
Different antibodies are sprayed on the control line of film；The site being not associated on film is closed using the skimmed milk power of 3-4.5%, gelatin etc.；
Film is washed 4-5 times with PBST, is dried in vacuo；
5) wherein step 3 and 4 in no particular order sequence, can first adhere to and spray sample afterwards, be adhered to after can also first spraying sample；
6) the PVC backing colloidal gold detection test strips for being sprayed with sample drying is sealed by, is refrigerated spare.
Test strips are taken into room temperature opening packaging using preceding.Serum to be checked is added into well, according to detection line and control
The color of line processed determines result.If aubergine occur in detection line and control line, illustrate the tuberculosis antibody positive；If detection line
It is colourless, and there is aubergine in control line, illustrates tuberculosis antibody feminine gender；If control line is colourless, illustrate that test strips are invalid.If to
Serum and the reaction of recombinant protein Rv0899 test strips are examined, illustrates that there are tuberculosis antibodies in vivo.If serum to be checked respectively and
The recombinant protein test strips of Rv0899 or Rv1973 are reacted, and illustrate that the antibody detected in sample is due to causing naturally.If to
The recombinant protein test strips of inspection serum and Rv0899 or Rv1973 or Mce4B are reacted, then can detect that the knot of the sample centering
Core mycobacteria is likely to be at tuberculosis active stage or advanced stage.
Sequence table
<110>Anhui University of Science and Technology
<120>a kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis
<130>a kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis
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tattgccgcg tgccgcagga tagcccgttt aacgtgcgcg gcgcgcgcaa cattccgtgc 1020
gaaaccgtgc cgggcaaacg cgcgccgacc gtgaaactgt gcgaaagcga tgcgccgtat 1080
ctgccgctga acgatggcta taactggaaa ggcgatccga acgcgaccgt gccgggcctg 1140
ggcagcggcc aggatattcc gcagacctgg cagaccatgc tgctgccgcc gggcagcgcg 1200
catcatcatc atcatcatta a 1221
<210> 3
<211> 1635
<212> DNA
<213>artificial synthesized (Artificial synthesis)
<400> 3
atggcgagcc gcctgaaagc gggccagaaa gtgcgcattg cgggcgtgcc ggtgggcagc 60
gtgaaagcgg tgaaactgaa cccggatcat agcattgatg tggcgtttgc gattgatcgc 120
agctataccc tgtatagcag cacccgcgcg gtgattcgct atgaaaacct ggtgggcgat 180
cgctttctgg aaattaccag cggcccgggc gaactgcgca aactgccgcc gggcggcacc 240
attaacgtgg cgcataccca gccggcgctg gatctggatg cgctgctggg cggcctgcgc 300
ccggtgctga aaggctttga tgcggataaa attaacacca ttaccagcgc ggtgattgaa 360
ctgctgcagg gccagggcgg cccgctggcg aacgtgctgg cggataccgg cgcgtttagc 420
gcggcgctgg gcgcgcgcga tcagctgatt ggcgaagtga ttaccaacct gaacgcggtg 480
ctggcgaccg tggatgcgaa aagcgcgcag tttagcgcga gcgtggatca gctgcagcag 540
ctggtgagcg gcctggcgaa aaaccgcgat ccgattgcgg gcgcgattag cccgctggcg 600
agcaccacca ccgatctgac cgaactgctg cgcaacagcc gccgcccgct gcagggcatt 660
ctggaaaacg cgcgcccgct ggcgaccgaa ctggataacc gcaaagcgga agtgaacaac 720
gatattgaac agctgggcga agattatctg cgcctgagca gcgcgaacct ggatcagacc 780
ctgggcaccc tgaaccaggc gctgagcgat attcgcggct ttctgcgcga aaacaacagc 840
accctgattg aaaccgtgaa ccagctgaac gattttgcgc agaccctgag cgatcagagc 900
gaaaacattg aacaggtgct gcatgtggcg ggcccgggca ttaccaactt ttataacatt 960
tatgatccgg cgcagggcac cctgaacggc ctgctgagca ttccgaactt tgcgaacccg 1020
gtgcagttta tttgcggcgg cagctttgat accgcggcgg gcccgagcgc gccggattat 1080
tatcgccgcg cggaaatttg ccgcgaacgc ctgggcccgg tgctgcgccg cctgaccgtg 1140
aactatccgc cgattatgtt tcatccgctg aacaccatta ccgcgtataa aggccagatt 1200
atttatgata ccccggcgac cgaagcgaaa agcgaaaccc cggtgccgga actgacctgg 1260
gtgccggcgg gcggcgtgcc gcaggatgaa ggcgcgaaac tggattttaa aattgatctg 1320
catgatccgc cgccgtgcat gaccggcttt ctgccgccgc cgctggtgcg cagcccggcg 1380
gatgaaagcg tgcgcgaaat tccgcgcgat atgtattgca aaaccgcgca gaacgatccg 1440
agcaccgtgc gcggcgcgcg caactatccg tgccaggaat ttccgggcaa acgcgcgccg 1500
accgtgcagc tgtgccgcga tccgcgcggc tatgtgccgg tgggcaccaa cccgtggcgc 1560
ggcccgccga ttccgtatgg caccgaagtg accgatggcc gcaacattct gcatcatcat 1620
catcatcatc attaa 1635
Claims (7)
1. a kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis, it is characterised in that: the kit
For the Enzyme-linked Immunosorbent Assay and immune colloidal gold test to mycobacterium tuberculosis, reagent preparation step is as follows:
A. sputum sample is taken to put into the sodium hydroxide solution equipped with l~2 times own vol, concussion makes sputum sample in 1 minute
It is sufficiently reacted with sodium hydroxide solution, removes the mucin components of sputum sample surface, and stand 18~19 minutes at room temperature；
B. sputum sample is subjected to high speed centrifugation, abandons supernatant liquor, physiological saline is added and relaunders, washes away miscellaneous in sample
Matter extracts required Mycobacterium tuberculosis cell；
C. the Mycobacterium tuberculosis cell of extraction is put into ultrasonic disruption device to be crushed, addition sonification medium is water, and ultrasound is broken
It broken 15 minutes, by the ultrasonic energy that medium is propagated, destroys tuberculosis and divides skill bacilli-cell structure, low-speed centrifugal removes uncracked
Thallus and matrix, supernatant ultracentrifugation, precipitating are washed 3 times with 100 times of distillation, will be centrifuged obtained precipitating film for the last time
Lysate dissolution, 2000r/min are centrifuged 30min, can be obtained the supernatant for outer membrane protein；
D. the outer albumen of seperation film, can be obtained three kinds of outer membrane proteins, and three kinds of outer membrane proteins are purified and recombinated, can obtain
Three kinds of recombinant proteins after to recombination；
E, three kinds of recombinant proteins after purification are diluted with carbonate buffer solution, concentration is 3ng-8 μ g/ μ L, dilute with recombinant protein
Release liquid coated elisa plate；
F, ELISA Plate is closed with 4% casein, bovine serum albumin(BSA), gelatin or skimmed milk power；Then phosphate buffer is used
Washing；
G, ELISA Plate is put into sterile vacuum drier after drying and is packed into reagent bottle.
2. a kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis according to claim 1,
It is characterized by: three kinds of outer membrane proteins are respectively Rv0899, Rv1973, Mce4B.
3. a kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis according to claim 1,
It is characterized by: three kinds of outer membrane proteins are respectively ESAT-6, Rv1973, Mce4B.
4. a kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis according to claim 1,
It is characterized by: three kinds of outer membrane proteins are respectively Rv0899, MPT64, Mce4B.
5. a kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis according to claim 2,
It is characterized by: the test strips of the immune colloidal gold test are the recombinant proteins with Rv0899, Rv1973 or Mce4B of purifying
It is prepared through the following steps:
A, gold chloride is reduced into the particle of 20-30nm with sodium citrate；
B, preparation and reorganization staphylococcal protein A/G and colloid gold label object；
C, preparation blank PVC backing colloidal gold detection use test strips, one end of test strips PVC backing successively adhere to sample pad,
Bonding pad；The intermediate adhesion nitrocellulose filter or pvdf membrane of PVC backing；The PVC backing other end adheres to water absorption pad；Wherein nitric acid
There are two lines, respectively detection line and control line on cellulose membrane or pvdf membrane；
D, albumin A and colloid gold label object are sprayed on the bonding pad of PVC backing colloidal gold detection test strips with film machine；It will
The recombinant protein segment of Rv0899, Rv1973 or Mce4B are sprayed in the detection line of nitrocellulose filter or pvdf membrane；By the anti-egg of chicken
White A antibody or corresponding secondary antibody are sprayed on the control line of film；
E, the PVC backing colloidal gold detection test strips for being sprayed with sample drying is sealed, is refrigerated spare.
6. a kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis according to claim 2,
It is characterized by: described detection method the following steps include: with purifying three kinds of mycobacterium tuberculosis outer membrane protein Rv0899,
The recombinant protein of Rv1973 and Mce4B prepares the reagent of Enzyme-linked Immunosorbent Assay and immune colloid gold reagent box as antigen, utilizes
The kit detects sample to be tested tuberculosis antibody.
7. a kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis according to claim 6,
It is characterized in that, described detection method the following steps include:
A. packaging is opened at 25 DEG C of room temperature using preceding take test strips；
B. serum to be checked is added into well, result is determined according to detection line and the color of control line.
C. if aubergine occur in detection line and control line, illustrate the tuberculosis antibody positive；If detection line is colourless, and control line
There is aubergine, illustrates tuberculosis antibody feminine gender；If control line is colourless, illustrate that test strips are invalid；
D. if serum to be checked and the reaction of recombinant protein Rv0899 test strips, illustrate that there are tuberculosis antibodies in serum to be checked；If
Serum to be checked is reacted with recombinant protein Rv1973, Mce4BRv1973 test strips respectively, illustrates that the antibody of serum to be checked is due to certainly
So cause；If serum to be checked and recombinant protein Rv1973, Mce4BRv1973 and Mce4B test strips are reacted, illustrate that this is to be checked
Contain sensibility mycobacterium tuberculosis to current in serum.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172036A (en) * | 2019-12-24 | 2020-05-19 | 光明乳业股份有限公司 | Method for separating thallus from yoghourt and application of thallus in genome DNA extraction |
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| CN111172036A (en) * | 2019-12-24 | 2020-05-19 | 光明乳业股份有限公司 | Method for separating thallus from yoghourt and application of thallus in genome DNA extraction |
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