CN107202894A - Purposes of the Rv1078 albumen in diagnosis latency/active tuberculosis - Google Patents
Purposes of the Rv1078 albumen in diagnosis latency/active tuberculosis Download PDFInfo
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Abstract
The present invention relates to purposes of the mycobacterium tuberculosis Rv1078 albumen in diagnosis latency/active tuberculosis, described Rv1078 albumen is the protein of the amino acid sequence composition shown in the SEQ ID No.1 in sequence table；The invention further relates to detection chip, the detection kit of diagnosis latency/active tuberculosis using the Rv1078 albumen as active component；And pharmacy, the detection application of the antibody of anti-Rv1078 albumen.
Description
Technical field
The invention belongs to biomedicine field, and in particular to mycobacterium tuberculosis Rv1078 albumen is in diagnosis latency/work
Purposes in dynamic property pulmonary tuberculosis.
Background technology
Since multiple centuries, tuberculosis continues to turn into a public health problem that can not be ignored in the whole world.It is global at present
Existing 1/3rd population carries mycobacterium tuberculosis, just increases cases of tuberculosis 8,800,000, dead 145 newly within only 1 year 2010
Ten thousand, average to have a people to die from pulmonary tuberculosis less than 22 seconds, tuberculosis is in first of Death of Infectious Diseases number.And China is the whole world
22 tuberculosis height bear one of country, and tuberculosis patient number height is ranked the second in the world, and infected number is more than 500,000,000, only
Just there are new cases 90~1,200,000 within 1 year 2010, occupy about the 12% of global total new cases, such as take not in time effectively
Measure, may there is 30,000,000 people morbidity in following 10 years, it will cause serious public health problem and social concern, so
It must be realized as early as possible to phthisical effective control from national strategy aspect.
Scientific evidence shows, after tuberculosis infection human body, in most cases, can be controlled by immune system into the human body
System, so as in a latent infection state, be likely to be converted into latent tuberculosis disease at any time.In view of whole world knot
Core infects population, or even in China is all a very huge numeral, with reference to transmissibility lungy and social harm,
The latent patient of examination tuberculosis is just particularly important in healthy population.Confirmation latent tuberculosis infection is studied
The prophylactic treatment of (1atent tuberculosis infection, LTBI) can reduce HIV/TB double infections crowd progress
For latency TB risk (Bucher HC, Griffith LE, Guyatt GH, et al：Isoniazid prophylaxis
for tuberculosis in HIV infection：a meta-analysis of randomized controlled
Trails [J] .AIDS, 1999,13：501-508).But, it is domestic at present to diagnose latent infection Main Basiss tuberculin skin test
The result of (PPD skin tests), it is considered that PPD strong positives or to switch to positive from feminine gender in a short time be latent tuberculosis infection
Person.But because PPD skin tests cannot be distinguished by the immune response and latent infection that BCG inoculations are produced being widely popularized in China
The responsing reaction of generation.And the serodiagnosis based on antigen-antibody reaction, it is important dive due to its simplicity, rapidity
The clinical complementary diagnosis means of volt property Pulmonary Tuberculosis Infection.Therefore, from a long-term perspective, it should be directed to finding Sensitivity and Specificity
Preferable tuberculosis mark.
Preferable Diagnosis of Tuberculosis mark should meet following condition：(1) sensitiveness is high；(2) specificity is high；(3) it is present in
In body fluid, particularly blood, it is easy to detect.But the targeted antigen such as antigen of Mycobacterium tuberculosis serodiagnosis at present
5th, 38KD antigens, 30/31KD antigens and antigen 60 etc., have that positive rate is low, exempt from intersecting for other mycobacteriums
The problems such as epidemic disease, although there is certain value in curative effect monitoring, prompting recurrence, judging prognosis and the generaI investigation of people at highest risk,
But making a definite diagnosis for latent tuberculosis infection is still cannot be used at present.In order to realize the high sensitivity infected latent tuberculosis,
The diagnosis of high specific, urgent need searches out more sensitive, more special latent tuberculosis infection biological mark on a molecular scale
Thing.
Rv1078 GFPs name Pra, belongs to coding Pro-rich GFP family.Current Unknown Function..
The content of the invention
Discriminating, diagnosis/auxiliary diagnosis, examination/auxiliary examination target detection sample are being prepared the present invention relates to Rv1078 albumen
Whether this comes from the application in the product of latency/active tuberculosis patient.
The Rv1078 albumen is following albumen a) or b)：
A) protein of the amino acid sequence composition shown in the SEQ ID No.1 in sequence table；
SEQ ID No.1：
>M.tuberculosis H37Rv|Rv1078|pra
MTEQPPPGGSYPPPPPPPGPSGGHEPPPAAPPGGSGYAPPPPPSSGSGYPPPPPPPGGGA
YPPPPPSAGGYAPPPPGPAIRTMPTESYTPWITRVLAAFIDWAPYVVLVGIGWVIMLVTQ
TSSCVTSISEYDVGQFCVSQPSMIGQLVQWLLSVGGLAYLVWNYGYRQGTIGSSIGKSVL
KFKVVSETTGQPIGFGMSVVRQLAHFIDAIICFVGFLFPLWDAKRQTLADKIMTTVCVPI
B) amino acid sequence of SEQ ID No.1 in sequence table is passed through to the substitution of one or several amino acid residues
And/or missing and/or addition and the protein with albumen shown in SEQ ID No.1 with identical function.
Described detection sample is whole blood sample, blood serum sample, tissue sample, pathological tissue, preferably blood serum sample.
The present invention also provide it is a kind of be used to differentiating, diagnosis/auxiliary diagnosis, examination/auxiliary examination target detection sample whether
Come from the label of latency/active tuberculosis patient, the antibody that described label is anti-Rv1078,
It is preferred that, the antibody is IgG antibody,
It is furthermore preferred that be the IgG antibody in the in vitro serum of people,
The Rv1078 is following albumen a) or b)：
A) protein of the amino acid sequence composition shown in the SEQ ID No.1 in sequence table；
B) amino acid sequence of SEQ ID No.1 in sequence table is passed through to the substitution of one or several amino acid residues
And/or missing and/or addition and the protein with albumen shown in SEQ ID No.1 with identical function.
Described detection sample is whole blood sample, blood serum sample, tissue sample, pathological tissue, preferably blood serum sample.
It is also another object of the present invention to provide serum moderate resistance Rv1078 antibody as mark exploitation, design and/or
Prepare with whether discriminating, diagnosis/auxiliary diagnosis, examination/auxiliary examination target detection sample come from latency/activity lung
Application in the product of tubercular；
It is preferred that, the antibody is IgG antibody,
It is furthermore preferred that be the IgG antibody in the in vitro serum of people,
The Rv1078 is following albumen a) or b)：
A) protein of the amino acid sequence composition shown in the SEQ ID No.1 in sequence table；
B) amino acid sequence of SEQ ID No.1 in sequence table is passed through to the substitution of one or several amino acid residues
And/or missing and/or addition and the protein with albumen shown in SEQ ID No.1 with identical function.
Described detection sample is whole blood sample, blood serum sample, tissue sample, pathological tissue, preferably blood serum sample.
Whether the discriminating, diagnosis/auxiliary diagnosis, examination/auxiliary examination target detection sample come from latency/activity
Property lunger refers specifically to,
(1) whether the source individual of discriminating, diagnosis and/or auxiliary diagnosis sample to be tested suffers from latency/activity lung knot
Core；
Or the feelings of latency/active tuberculosis are suffered from (2) examination and/or auxiliary examination sample to be tested source crowd
Condition.
Described detection sample is whole blood sample, blood serum sample, tissue sample, pathological tissue, preferably blood serum sample.
A further object of the present invention be to provide it is a kind of by bioactive molecule of the Rv1078 albumen be used for differentiate, examine
The kit of disconnected, auxiliary diagnosis, examination and/or auxiliary examination latency/active tuberculosis, described kit includes,
(1) test point of connection Rv1078 albumen is provided with detection chip, the detection chip, each test point is independent
In adjacent test point, the Rv1078 is following albumen a) or b)：
A) protein of the amino acid sequence composition shown in the SEQ ID No.1 in sequence table；
B) amino acid sequence of SEQ ID No.1 in sequence table is passed through to the substitution of one or several amino acid residues
And/or missing and/or addition and the protein with albumen shown in SEQ ID No.1 with identical function；
(2) reagent that detection chip is used together is coordinated, the reagent includes sample diluting liquid, cleaning solution, confining liquid；
(3) it is used for the label for showing testing result, the label is preferably the anti-human secondary antibody of fluorescence labeling.
Described sample diluting liquid is the PBS solutions of pH 7.4, consisting of：
Described cleaning solution is pH 7.4 PBST solution, consisting of：
Described confining liquid is the PBS solutions of pH 7.4 containing BSA：
The anti-human secondary antibody of described fluorescence labeling is preferably anti-human IgG secondary antibodies.
The kit also includes specification, and the specification is described below content：
(1) compared with normal person, in the detection sample of latency/active tuberculosis infected patient, anti-Rv1078 antibody
Level is significantly raised；
(2) the detection sample described in is whole blood sample, blood serum sample, tissue sample, pathological tissue, preferably whole blood
Product；
(3) antibody is the antibody of IgG types.
The invention further relates to the preparation method of the detection chip in the kit, described method includes,
(1) by the solution point sample containing Rv1078 albumen on carrier slide, the solution containing Rv1078 albumen
It is formulated and is：
(2) biochip point sample instrument point sample is used, the spotting solution volume that each point sample is used is 0.3-1nl；It is preferred that
0.5-1nl；Most preferably 1nl；
(3) after point sample, carrier slide is maintained in 30%RH-40%RH humidity, 4 DEG C of environment overnight, it is preferred that humidity
For 35%RH；
(4) carrier slide is positioned over -80 DEG C of Cord bloods of sealing in plastic casing.
In the solution containing Rv1078 albumen, still further comprise：Glycerine, volume hundred that percent by volume is 25%
Divide the NaN of the BSA and 0.1g/L than Tween20,0.05mg/ml for 0.02%3。
As needed, between point sample step (2) and step (3), it can be sticked on each carrier slide every for isolating
The plastics fence of individual test point, forms loading wells on slide.
The carrier slide is three-dimensional H carriers slide.
The method whether testing sample comes from resting form/active type lunger is detected the invention further relates to a kind of,
Described method is：
Treated using the Rv1078 albumen or the detection chip by bioactive molecule of Rv1078 albumen/detection kit detection
Test sample product, judge whether anti-Rv1078 antibody levels significantly raise, and described determination methods are：The testing result of testing sample
In, anti-Rv1078 antibodies positives, then it is that latency/active tuberculosis is positive to judge the testing sample, otherwise for latency/
Active tuberculosis is negative；
If the snr value that the positive specific criterion is each antibody is between following value, then it is assumed that be that this resists
Body is positive：
The snr value of Rv1078 antibody is between 7.15 and 8.70, including 7.15 and 8.70；
The calculation formula of the snr value is：
In formula, S and B represent the signal value and non-sample point region that scanner is directly displayed in the detection chip respectively
Background value, and σ is then the standard deviation for representing the primary signal；
The corresponding experiment number of the σ is 3 times；S the and B values are obtained with scanner in 532nm Channel scans；Institute
It is GenePix to state scanner to be preferably.
The anti-Rv1078 antibody is preferably IgG antibody.
Final object of the present invention is to provide any of the above-described kit and prepared with discriminating, diagnosis, auxiliary
Application in the product of diagnosis, examination and/or auxiliary examination latency/active tuberculosis purposes.
Brief description of the drawings
Fig. 1 is prepared antigen protein Rv1078 silver staining quantitative result figure.
Fig. 2 is prepared antigen protein Rv1078 Western-Blotting qualification result figures.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The preparation of embodiment 1, protein-chip
(1) Mycobacterium tuberculosis antigen protein Rv1078 preparation
1st, express
The saccharomyces cerevisiae that genetic engineering was transformed induces overexpression using galactolipin, and detailed process is：
First from mycobacterium mycobacterium tuberculosis (Mycobacterium M.tuberculosis) (Beijing Strain),
The gene code fragment for obtaining albumen Rv1078 is from the beginning cloned by PCR, is connected fragment by the BP enzymes of Invitrogen companies
It is connected on pDONR221 carriers (being purchased from Invitrogen), is transformed into bacillus coli DH 5-Alpha and expands, extracts carrier and lead to again
Crossing the pEGH-A carriers that can express GST labels that LR enzymes (Invitrogen) shift to by transformation, (carrier is in " Jian
Zhu, Heng Zhu, et al：Crossed disclosed in J.Virol.May 2009vol.83no.10 5219-5231 ") on, convert again
Expanded into bacillus coli DH 5-Alpha, extraction plasmid is transformed into Pep4 Wine brewing yeast strains, and (bacterial strain is in document " Heng
Zhu, Michael Snyder, et al：Nature Genetics 26,283–289(2000)doi:10.1038/81576 " in
It is disclosed) in.Cultivated in inducing culture, when its OD600 is 0.6-0.8, add final concentration of 2g/L galactolipin, induction
Bacterium, -80 DEG C of preservations is collected by centrifugation in 6h, 4000rpm.
PCR from the beginning clone used in PCR primer sequence it is as follows：
Expand the primer pair of the gene code fragment of Rv1078 albumen：
Sense primer：GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACCGAACAGCCGC
Anti-sense primer：GGGGACCACTTTGTACAAGAAAGCTGGGTATCAGATCGGCACGCACAC
The component contained in per 1L inducing cultures (solvent is water) is as shown in table 1：
Table 1
2nd, purify
1) lysate, is prepared：
Add 50 μ l mercaptoethanols, 125 μ lPMSF and two panels Roche protein inhibitors in 50ml lysates；
2), in the bacterium (bacterium being collected into from 120ml inducing culture cultures that the collection of above-mentioned steps 1 is taken out in -80 DEG C of refrigerators
Body), plus 400 μ l zirconium oxide beads and 400 μ l lysates shake 30s, rearmounted 2min on ice in 4 DEG C of environment, are repeated four times；
3) 11,000rpm centrifugations 2min, takes supernatant in a new 15ml centrifuge tubes after, taking out；
4), repeat 2 and 3 step four times, supernatant is collected in same centrifuge tube；
5), addition lysate to 12ml be the 1/10 of original inducing culture volume, while with the cracking for not having inhibiting
Liquid cleans glutathione beads 3 times.300 μ l beads is added in 12ml lysates；
6), the lysate added after beads is incubated 2h in 4 DEG C；
7), 11,000rpm takes supernatant to be stored in 4 DEG C after centrifuging 2min.Beads respectively washes 3 with cleaning fluid Ι and cleaning fluid II
It is secondary；
8), plus after 300 μ l eluents incubation 15min, centrifuging and taking supernatant is collected in a new centrifuge tube, is repeated once.
Obtained eluent is i.e. dissolved with the albumen.
The composition of used buffer solution (solvent is water) is shown in Table 2- tables 5. in above-mentioned purge process
The lysate of table 2 (1L)
The cleaning fluid I (1L) of table 3
The cleaning fluid II (1L) of table 4
The eluent of table 5 (1L)
3rd, identify
In following experimentations, described solvent or the percentage of liquid are percentage by volume.
1), prepared by material
The SDS-PAGE glue of two piece 12% of configuration, 1.0mm, 15 holes.One piece of silver staining, one piece of Western Blotting.Take
Above-mentioned each 20 μ l of purified albumen, add 4 μ l 6X Loading buffer, while preparing the BSA samples of specification concentration gradient
Product boil sample 5min as silver staining quantitative control.
2) glue, is run
The above-mentioned samples prepared of 12 μ l, BSA gradient samples, 2.5 μM of arker (Takara) records are added per hole successively
Sequence.80V 30min, 140V 1h.
3) silver staining operating procedure：
It is fixed：30min or the longer time glacial acetic acid of 40% ethanol 10% add water to 250ml
Sensitization：The ethanol 17g sodium acetate 28.2g sodium acetate trihydrate 0.5g sodium thiosulfate of 30min 75ml ethanol 30% is (big
Soda) add water to final volume 250ml
Washing：3x 10min
Silver staining：The formaldehyde of 100 μ l of 20min 0.625g AgNO3 37% (using preceding addition) adds water to final volume 250ml
Washing：2x 1min
Colour developing：Time depends on the circumstances 6.25g Na2CO3The formaldehyde of 50 μ l 37% (using preceding addition) adds water to whole body
Product 250ml
Terminate：10min 1g glycine adds water to final volume 250ml
Preserve：1% glacial acetic acid, 4 DEG C
4) Western-Blotting steps：
Transferring film：15V 40min (half-dried turn, Bio-Rad).Transferring film buffer solution：Glycine 2.9g；Tris 5.8g；SDS
0.37g；Methanol 200ml；Plus ddH2O is settled to 1000ml
Closing：5% skim milk (Bio-Rad) 1h.
First antibody is incubated：Anti-GST mouse resist the μ g/ml 1h of (NovaGen) final concentration 1
Secondary antibody is incubated：The anti-μ g/ml 1h of the passage of fluorescence 800 (Odyssey) final concentration 1 of sheep mouse
Odyssey scanner scannings, preserve picture.
Carry out quantitative and Western-Blotting identifications the result difference of silver staining as depicted in figs. 1 and 2.Fig. 1 result tables
The amount of bright prepared Rv1078 albumen is 50 μ g/ml；Fig. 2 results prove that prepared Rv1078 albumen is correct.
Through sequencing, prepared Rv1078 protein components have the amino acid sequence shown in sequence table SEQ ID No.1.
(2) preparation of the protein-chip of pre- point antigen
In the eluate solution of the Rv1078 antigen proteins of the 50 μ g/ml containing above-mentioned preparation, final concentration of 25% is added
The NaN of the glycerine of (percent by volume), Tween20,0.05mg/ml of 0.02% (percent by volume) BSA and 0.1g/L3。
Using biochip point sample instrument, in carrier slide, (carrier slide is the three-dimensional H carriers slide of commercialization, purchase by above-mentioned mixed liquor point
From CapitalBio Corporation) on, every point sample about 1nL, 2 parallel points.Using biochip point sample instrument
(being purchased from CapitalBio Corporation) point system.
It is used as IgG (1mg/ml) standard items or Cy3 mark anti-human antibody's secondary antibody (20 μ g/ml) of positive control, mixed liquor
In other components and final concentration it is constant, prepare the mixed liquor that IgG standard items or Cy3 mark anti-human antibody's secondary antibody.With above-mentioned phase
Standard items containing IgG respectively or Cy3 are marked the mixed liquor point of anti-human antibody's secondary antibody in above-mentioned identical carrier glass by same spotting methods
On piece, microarray is formed, 12 repetition parallel seals can be put per slide interval.Point sample sticks 12 holes plastics after terminating enclose
Column, and be maintained in 4 DEG C of environment of 35%RH humidity after 16h, slide is positioned over -80 DEG C of Cord bloods of sealing in plastic casing.
, should herein it should be noted that the present embodiment only records the cloning expression method of a specific Rv1078 albumen
Related host and carrier in method, which have no effect on the application, needs the realization of technical scheme to be protected, is used only as helping this area
Technical staff understands the clonal expression process of the albumen.Those skilled in the art can use conventional point with apparent completely
Sub- biological experimental method, by other common carrier, hosts, can also complete the clonal expression of target protein.
Embodiment 2, using protein-chip auxiliary diagnosis latency/active tuberculosis
(1) preparation of test serum sample
Whole blood sample is placed in room temperature and centrifuged 20 minutes or so after 1000g overnight for 2 hours or 4 DEG C, takes supernatant to stand
Detect；Or dispensed, and sample is put in -20 DEG C or -80 DEG C preservations, but multigelation should be avoided.Sample after 4 DEG C of defrostings
Product should be centrifuged again, then be detected.
(2) preparation of involved various buffer solutions and reagent during diagnosing
(1) sample diluting liquid (being shown in Table 6)：The PBS solutions of pH 7.4
Table 6
(2) cleaning solution (being shown in Table 7)：PH 7.4 PBST solution
Table 7
(3) chip confining liquid (being shown in Table 8)：PH 7.4PBS solution containing BSA
Table 8
(4) Cy3 marks the concentrate of anti-human secondary antibody：Use commercially available anti-human igg-Cy3 fluorescence labeling secondary antibodies, dilution
It is 1mg/ml to concentration, is sub-packed in lucifuge tubule.
(3) method of application protein-chip auxiliary diagnosis latent tuberculosis
The protein-chip prepared using embodiment 1 can detect that antigen Rv1078 is corresponding in tested person's blood sample
The level of IgG antibody, analyzes the testing result of the corresponding antibodies of this protein, it can be determined that whether tested person is latency lung
Tuberculosis.
1st, concrete operation step
1) 1 point of embodiment is made into sealed chip from -80 DEG C of taking-ups, room temperature rewarming 10 minutes.
2) close：Chip is put into sink, about 50ml chips confining liquid (table 8), shaking table 50rpm, room temperature 1h is added.
3) liquid unnecessary on chip quickly is got rid of, be placed in wet box.
4) dilution of testing sample and sample-adding：By test serum sample by volume 1:100 is dilute with the sample of above-mentioned preparation
Liquid (table 6) dilution is released, takes the solution containing test serum after 30 μ L dilutions to be added in the fence-enclosing space of closing.Reaction
1h, room temperature.Detected sample is prepared in 15 minutes before use.
5) chip is taken out from wet box, is placed in sink, add the cleaning solution of the above-mentioned preparations of about 50ml, shaking table 50rpm,
Room temperature 5min, is repeated 3 times.
6) liquid unnecessary on chip quickly is got rid of, be placed in wet box.
7) each closing space adds 30 μ L and has been diluted to the μ g/ml of the final concentration 1 anti-human secondary antibody of Cy3 fluorescence in chip
On, lucifuge reaction 1h.
8) chip is taken out from wet box, is placed in sink, add the cleaning solution of the above-mentioned preparations of about 50ml, shaking table 50rpm,
Room temperature 5min, is repeated 3 times.Again with the ultrapure washings of about 50ml once, 5min.
9) centrifugal drying, data are read with Genepix scanners under 532nm passages.
2nd, the judgement of testing sample respectively to 3 kinds of antigen protein resistances
Entered with Genepix scanners the result that 532nm Channel scans are obtained is the signal to noise ratio (SNR) by point of sample
Row is weighed, and signal to noise ratio (SNR) calculation formula of point of sample is：
In formula, S and B represent signal value (being the numerical value that scanner is directly displayed) and background value original in chip respectively
(signal value in non-sample point region), and σ is then to represent the standard deviation of the primary signal (the corresponding experiment numbers of σ are 3
It is secondary).
By interval (the signal to noise ratio interval bag of the snr value of point of sample on protein-chip and the signal to noise ratio of table 9 below
Include the interval endpoints thereof of given signal to noise ratio) contrasted, to carry out testing sample to the positive, negative of corresponding antigens resistance
As a result judgement：
Anti- Rv1078 (being shown in Table 9)：
Table 9
3rd, the judgement of testing sample assay
Criterion：
Compared with not suffering from the people of any disease normally, in latent tuberculosis infection people, anti-Rv1078 antibody levels show
Rise is write, is embodied in：
If in the testing result of testing sample, anti-Rv1078 antibodies positives (judge whether antibody is positive standard：
When 2 parallel point of samples of same antigen are all determined as the positive on protein-chip prepared by embodiment 1, the antibody is the positive),
It is that activity/latent tuberculosis is positive then to judge the testing sample, otherwise negative for activity/latent tuberculosis.
(4), using protein-chip auxiliary diagnosis activity/latent tuberculosis method specificity and sensitiveness
Use 200 parts of activity/latent tuberculosis associated patient serum (clinical diagnosis for active tuberculosis trouble
100 parts of person, 100 parts of latent tuberculosis patient；Serum sample is provided by Guangdong Province Tubercufosis control center, and serum sample takes
Obtain the informed consent by patient and examinee.) specificity is carried out to the protein-chip of embodiment 1 and sensitiveness is examined
Survey, judge person under test whether as activity/latent tuberculosis positive with above-mentioned criterion.
Testing result is shown, is the protein that sensitiveness/(1- specificity) calculates embodiment 1 according to positive likelihood ratio
The specificity of the best operating point of chip auxiliary diagnosis activity/latent tuberculosis is 72%, and sensitiveness is 74%, significantly
Higher than the index of activity in the prior art/latent tuberculosis diagnosis.
In 100 parts of active tuberculosis patients, there are 74 parts to be detected the positive；In 100 parts of latent tuberculosis patients, have
28 parts are detected the positive.
(5), using protein-chip auxiliary diagnosis active tuberculosis method specificity and sensitiveness
Use 200 parts of active tuberculosis associated patients serum (clinical diagnosis for active tuberculosis patient 100
Part, 100 parts of Healthy People；Serum sample is provided by Guangdong Province Tubercufosis control center, the acquirement of serum sample by patient and
The informed consent of examinee.) specificity and sensitivity Detection have been carried out to the protein-chip of embodiment 1, with above-mentioned judgement mark
Whether standard judges person under test as the active tuberculosis positive..
Testing result is shown, is the protein that sensitiveness/(1- specificity) calculates embodiment 1 according to positive likelihood ratio
The specificity of the best operating point of chip auxiliary diagnosis active tuberculosis is 94%, and sensitiveness is 80%, is both significantly higher than existing
There is the index that active tuberculosis is diagnosed in technology.
In 100 parts of active tuberculosis patients, there are 80 parts to be detected the positive；In 100 parts of Healthy Peoples, there are 6 to be detected
It is positive.
Finally it should be noted that above example is used only as helping skilled in the art to understand technical solution of the present invention
Essence, rather than limiting the scope of the present invention.
Claims (10)
- Whether 1.Rv1078 albumen comes from preparation discriminating, diagnosis/auxiliary diagnosis, examination/auxiliary examination target detection sample Application in the product of latency/active tuberculosis patient,The Rv1078 albumen is following albumen a) or b)：A) protein of the amino acid sequence composition shown in the SEQ ID No.1 in sequence table；B) by the amino acid sequence of the SEQ ID No.1 in sequence table by one or several amino acid residues substitution and/or Missing and/or addition and the protein with albumen shown in SEQ ID No.1 with identical function.
- 2. application according to claim 1, it is characterised in that described detection sample is whole blood sample, blood serum sample, group Tissue samples, pathological tissue, preferably blood serum sample.
- 3. anti-Rv1078 antibody as mark exploitation, design and/or prepare with discriminating, diagnosis/auxiliary diagnosis, examination/ Whether auxiliary examination target detection sample comes from the application in the product of latency/active tuberculosis patient, described Rv1078 is following albumen a) or b)：A) protein of the amino acid sequence composition shown in the SEQ ID No.1 in sequence table；B) by the amino acid sequence of the SEQ ID No.1 in sequence table by one or several amino acid residues substitution and/or Missing and/or addition and the protein with albumen shown in SEQ ID No.1 with identical function.
- 4. application according to claim 3, it is characterised in thatThe antibody is IgG antibody, it is preferred that the IgG antibody in the in vitro serum of the antibody behaviour；Described detection sample is whole blood sample, blood serum sample, tissue sample, pathological tissue, preferably blood serum sample；Whether the discriminating, diagnosis/auxiliary diagnosis, examination/auxiliary examination target detection sample come from latency/activity lung Tubercular refers specifically to：(1) whether the source individual of discriminating, diagnosis and/or auxiliary diagnosis sample to be tested suffers from latent tuberculosis；Or the situation of latent tuberculosis is suffered from (2) examination and/or auxiliary examination sample to be tested source crowd.
- 5. it is a kind of by bioactive molecule of the Rv1078 albumen be used for differentiate, diagnose, auxiliary diagnosis, examination and/or auxiliary sieve The kit of latency/active tuberculosis is looked into, described kit includes,(1) test point of connection Rv1078 albumen is provided with detection chip, the detection chip, each test point is independently of phase Adjacent test point, the Rv1078 is following albumen a) or b)：A) protein of the amino acid sequence composition shown in the SEQ ID No.1 in sequence table；B) by the amino acid sequence of the SEQ ID No.1 in sequence table by one or several amino acid residues substitution and/or Missing and/or addition and the protein with albumen shown in SEQ ID No.1 with identical function；(2) reagent that detection chip is used together is coordinated, the reagent includes sample diluting liquid, cleaning solution, confining liquid；(3) it is used for the label for showing testing result, the label is preferably the anti-human secondary antibody of fluorescence labeling.
- 6. kit according to claim 5, it is characterised in thatDescribed sample diluting liquid is pH 7.4PBS solution, consisting of：Described cleaning solution is pH 7.4 PBST solution, consisting of：Described confining liquid is the pH 7.4PBS solution containing BSA：The anti-human secondary antibody of described fluorescence labeling is anti-human IgG secondary antibodies.
- 7. the preparation method of the detection chip in kit described in claim 5 or 6, described method comprises the following steps：(1) by the solution point sample containing Rv1078 albumen on carrier slide, the formula of the solution containing Rv1078 albumen For：(2) biochip point sample instrument point sample is used, the spotting solution volume that each point sample is used is 0.3-1nl；It is preferred that 0.5- 1nl；Most preferably 1nl；(3) after point sample, carrier slide is maintained at after being stayed overnight in 4 DEG C of environment of 30%RH-40%RH humidity, it is preferred that humidity is 35%RH；(4) carrier slide is positioned over -80 DEG C of Cord bloods of sealing in plastic casing.
- 8. preparation method according to claim 7, it is characterised in that in the solution containing Rv1078 albumen, also enter One step includes：Tween20,0.05mg/ml that glycerine that percent by volume is 25%, percent by volume are 0.02% BSA and 0.1g/L NaN3。
- 9. the preparation method according to claim 7 or 8, it is characterised in thatBetween point sample step (2) and step (3), stick and enclosed for isolating the plastics of each test point on each carrier slide Column, forms loading wells on slide；The carrier slide is three-dimensional H carriers slide.
- 10. kit, the detection chip prepared according to any described methods of claim 7-9 described in claim 5 or 6 In the product with discriminating, diagnosis, auxiliary diagnosis, examination and/or auxiliary examination latency/active tuberculosis purposes is prepared Application.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002040517A2 (en) * | 2000-11-17 | 2002-05-23 | St. George's Enterprises Limited | M. tuberculosis chaperonin 60.1 and uses thereof |
| CN101124335A (en) * | 2004-07-01 | 2008-02-13 | 加州大学评议会 | High throughput proteomics |
| CN103698512A (en) * | 2013-11-25 | 2014-04-02 | 广东体必康生物科技有限公司 | Application of mycobacterium tuberculosis proteins in preparation of products used for diagnosis of latent tuberculosis infection |
| CN103884847A (en) * | 2014-03-14 | 2014-06-25 | 中国科学院生物物理研究所 | Mycobacterium M. tuberculosis holoprotein chip and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002040517A2 (en) * | 2000-11-17 | 2002-05-23 | St. George's Enterprises Limited | M. tuberculosis chaperonin 60.1 and uses thereof |
| CN101124335A (en) * | 2004-07-01 | 2008-02-13 | 加州大学评议会 | High throughput proteomics |
| CN103698512A (en) * | 2013-11-25 | 2014-04-02 | 广东体必康生物科技有限公司 | Application of mycobacterium tuberculosis proteins in preparation of products used for diagnosis of latent tuberculosis infection |
| CN103884847A (en) * | 2014-03-14 | 2014-06-25 | 中国科学院生物物理研究所 | Mycobacterium M. tuberculosis holoprotein chip and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 未知: "UniProtKB-P9WIM7", 《UNIPROT数据库》 * |
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