CN108872610A - The application of tuberculosis infection diagnostic reagent kit, screening system and the kit - Google Patents
The application of tuberculosis infection diagnostic reagent kit, screening system and the kit Download PDFInfo
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The present invention relates to medical sciences, in particular to the application of tuberculosis infection diagnostic reagent kit, screening system and the kit.The kit includes mitogenesis original and antigenic stimulus object；The antigenic stimulus object is at least three kinds of in following tuberculin pure protein or derivatives thereof：ESAT6, CFP10, TB7.7, MPT64, CFP21, EsxV, Rv1985C and Rv0222.The skin experiment of novel screening tuberculosis latent infection and active tuberculosis provided by the present invention has the advantages that high specificity (identify BCG vaccination and tuberculosis infection), susceptibility high (limited a variety of antigen protein Co stituations), difference is immunized by individual primary influenced it is small etc..
Description
Technical field
The present invention relates to medical sciences, in particular to tuberculosis infection diagnostic reagent kit, screening system and the examination
The application of agent box.
Background technique
There are about 1/3 population infection mycobacterium tuberculosis in the whole world at present, and wherein only having 5-10% to develop into has
Communicable active tuberculosis patient, therefore, how discovery active tuberculosis sufferer fast and convenient in latent infection person
Person is particularly important.And in the country of newborns' bcg vaccination such as China, due to the base of BCG vaccine and mycobacterium tuberculosis
Because group similitude is more than 95%, thus it is distinguished in reality firstly the need of by latent infection person and vaccine recipient.Tuberculosis skin
Skin tests the advantage that (TST) has economical and convenient, and in the whole world, many countries still have certain use value at present.TST warp
The transformation for being changed into injection purified protein derivative of tuberculin (PPD) by intracutaneous injection tuberculin (OT), tuberculosis sieve are gone through
The specificity looked into improves, but still mycobacterium tuberculosis cannot be infected and be infected with non-tuberculous mycobacteria,
BCG vaccination etc. is distinguished.Therefore, that there is also sensibility and specificities is not high enough, indistinguishable for current TST test
The shortcomings that latent infection and active tuberculosis and deficiency, the additionally tremendous influence big by immune state difference between individual.
In view of this, the present invention is specifically proposed.
Summary of the invention
Disadvantages mentioned above existing for tuberculosis skin experiment for current PPD, we use bcg bacteria genomic deletion area
Antigen protein identify mycobacterium tuberculosis and BCG vaccination to improve specificity, and use a variety of different missing areas anti-
Former albumen joint intracutaneous injection can improve sensibility to a certain extent[2].Identification for latent infection and active tuberculosis
It is the fact high compared with latent infection person according to the tuberculosis immunity active state of doubtful active tuberculosis patient, by using plant blood
Element (PHA) is coagulated to correct and judge that the fundamental immunity of individual is horizontal.ESAT6,CFP10,TB7.7,MPT64,CFP21,EsxV,
This 8 kinds of tuberculosis antigen albumen of Rv1985C and Rv0222 are it has been reported that being located at the albumen in BCG vaccine genomic deletion area[1-3],
Their application values in the test of tuberculosis skin test are not yet clear.The reasons why based on above-mentioned three aspect, the present invention devise a kind of new
The screening tuberculosis latent infection of type and the skin experimental approach of active tuberculosis.
Specifically, the present invention relates to a kind of tuberculosis infection diagnostic reagent kits comprising mitogenesis is former and antigen pierces
Swash object；
The antigenic stimulus object is at least three kinds of in following tuberculin pure protein or derivatives thereof：
ESAT6, CFP10, TB7.7, MPT64, CFP21, EsxV, Rv1985C and Rv0222.
According to an aspect of the present invention, the invention further relates to a kind of tuberculosis infection diagnostic system, the system comprises：
Data sink, Assignment Analysis module and reporting system main interface；
The received data of data sink include：Skin test is carried out to patient using kit as described above to be presented
The antigenic stimulus object caused by allergic immune reaction scleroma size DTBAgAnd it is immunized caused by the phytohemagglutin phytolectin
The scleroma size D of allergyPHA；
If DTBAg>=5mm, and DPHAWhen >=10mm, data are passed to the Assignment Analysis module, are otherwise transferred directly to institute
State reporting system main interface；
The Assignment Analysis module is for calculating Ratio=DTBAg/DPHA, and then tuberculosis infection type is analyzed, and be in
Pass the reporting system main interface.
The invention further relates to kit as described above in preparation for detecting latent tuberculosis, Active infection tuberculosis,
Or for distinguishing the application in the latent diagnosticum with Active infection of tuberculosis.
The skin experiment of novel screening tuberculosis latent infection and active tuberculosis provided by the present invention has specificity
By force (identify BCG vaccination and tuberculosis infection), susceptibility high (limited a variety of antigen protein Co stituations), exempted from by individual primary
Epidemic disease difference influences the advantages of small etc..
[1]Chen J1,Wang S,Zhang Y,Su X,Wu J,Shao L,Wang F,Zhang S,Weng X,Wang
H,Zhang W.Rv1985c,promising novel antigen for diagnosis of tuberculosis
infection from BCG-vaccinatedcontrols.BMC Infect Dis.2010Sep 17；10:273.
[2]Arlehamn CS1,Sidney J,Henderson R,Greenbaum JA,James EA,Moutaftsi
M,Coler R,McKinney DM,Park D,Taplitz R,Kwok WW,Grey H,Peters B,Sette
A.Dissecting mechanisms of immunodominance to the common tuberculosis
antigens ESAT-6,CFP10,Rv2031c(hspX),Rv2654c(TB7.7),and Rv1038c(EsxJ).J
Immunol.2012,188(10):5020-31.
[3]Fu R,Wang C,Shi C,Lu M,Fang Z,Lu J,Wang F,Fan X.An improved whole-
blood gamma interferon assay based on the CFP21-MPT64fusion protein.Clin
Vaccine Immunol.2009,16(5):686-91.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the principle and process schematic of tuberculosis Skin-test.
Specific embodiment
The present invention relates to a kind of tuberculosis infection diagnostic reagent kits comprising mitogenesis original and antigenic stimulus object；
3,4,5,6,7 or 8 kind of the antigenic stimulus object in following tuberculin pure protein or derivatives thereof：
ESAT6, CFP10, TB7.7, MPT64, CFP21, EsxV, Rv1985C and Rv0222.
Kit provided by the invention has good clinical applicability in Diagnosis of Tuberculosis, and specificity is good, sensitive
Property it is high, it is high with the coincidence rate of other diagnostic techniques.
The kit can be used for detecting screening latent tuberculosis, Active infection tuberculosis, or latent for distinguishing tuberculosis
Volt and Active infection.
Preferably, kit as described above, the antigenic stimulus object are selected from following tuberculosis antigen albumen or its segment group
It closes：
ESAT6,CFP10,TB7.7,MPT64；
Or CFP21, EsxV, Rv1985C, Rv0222；
Or MPT64, Rv1985C, Rv0222；
Or ESAT6, CFP10, TB7.7, MPT64, CFP21, EsxV, Rv1985C, Rv0222.
Preferably, kit as described above, in the antigenic stimulus object, every kind of tuberculin pure protein or derivatives thereof
It mixes；Preferably equimolar is than mixing.
Preferably, the concentration of kit as described above, the antigenic stimulus object is the 2 μ g/0.1ml of μ g~4, can also be selected
Select 3 μ g/0.1ml.
Preferably, kit as described above, the mitogenesis original include phytolectin, concanavalin A, american pokeweed root leaf and seed
Agglutinin；
Optionally, the concentration of the mitogenesis original is 0.3mg~0.8mg/2ml, also can choose 0.5mg/2ml.
Preferably, kit as described above, positive control is also contained in the kit, and the positive control is human-like
Purified protein derivative of tuberculin.
According to an aspect of the present invention, the invention further relates to a kind of tuberculosis infection diagnostic system, the system comprises：
Data sink, Assignment Analysis module and reporting system main interface；
The received data of data sink include：Skin test is carried out to patient using kit as described above to be presented
The antigenic stimulus object caused by allergic immune reaction scleroma size DTBAgAnd it is immunized caused by the phytohemagglutin phytolectin
The scleroma size D of allergyPHA；
If DTBAg>=5mm, and DPHAWhen >=10mm, data are passed to the Assignment Analysis module, are otherwise transferred directly to institute
State reporting system main interface；
The Assignment Analysis module is for calculating Ratio=DTBAg/DPHA, and then tuberculosis infection type is analyzed, and be in
Pass the reporting system main interface.
Tuberculin skin reaction is delayed cell mediated hypersensitivity.It is that antigen (tulase or BCG vaccine) enters body
Make the immune T lymphocyte sensitization of body, and a large amount of differentiation and proliferations.When the body of sensitization is invaded by antigen again,
Sensitized lymphocyte will be in combination, causes allergic inflammation.Show that tuberculin injection site forms scleroma very
To generation bubble, necrosis.The tuberculin test positive shows that body once by tubercle bacillus affection or inoculated BCG vaccine, also illustrated that machine
Body has certain immunity to tulase.But also there is people's (about 5%) of a small number of hypoimmunities to be negative or be in because of technical reason
Existing false negative.After usual bcg vaccination, if PPD skin test is negative, illustrate inoculation failure.
The skin experiment of novel screening tuberculosis latent infection and active tuberculosis is needed using at least three kinds of above BCG vaccines
Gene delection area tuberculosis antigen albumen carries out the scleroma size (D of intracutaneous injection observation allergic immune reactionTBAg)；Individual primary is exempted from
Epidemic disease state then uses the scleroma size (D of phytohemagglutin phytolectin antigen progress intracutaneous injection observer allergic immune reactionPHA).The two
Between ratio R atio=DTBAg/DPHAIt is more big, it is bigger that possibility is obtained for tuberculosis, it is on the contrary then smaller.
The system can be used for detecting screening latent tuberculosis, Active infection tuberculosis, or latent for distinguishing tuberculosis
And Active infection.
Preferably, system as described above, the Assignment Analysis module is when analyzing tuberculosis infection type, by with lower section
Formula carries out assignment：
If antigenic stimulus object is selected from ESAT6, CFP10, TB7.7, MPT64, and Ratio=DTBAg/DPHAWhen >=0.83,
Latent sensibility >=90.5% with Active infection of tuberculosis is distinguished, specificity is >=87.1%；
If antigenic stimulus object is selected from CFP21, EsxV, Rv1985C, Rv0222, and Ratio=DTBAg/DPHAWhen >=0.82,
It distinguishes latent sensibility >=89.3% with Active infection of tuberculosis, and specificity is >=85.8%；
If antigenic stimulus object is selected from MPT64, Rv1985C, Rv0222, and Ratio=DTBAg/DPHAWhen >=0.85, distinguish
Latent sensibility >=88.7% with Active infection of tuberculosis, specificity are >=86.4%；
If antigenic stimulus object is selected from ESAT6, CFP10, TB7.7, MPT64, CFP21, EsxV, Rv1985C, Rv0222, and
Ratio=DTBAg/DPHAWhen >=0.91, distinguish that tuberculosis is latent and sensibility >=91.2% of Active infection, specificity for >=
87.0%.
Preferably, system as described above, the received data of data sink further include：Whether inoculated card is situated between
Seedling, if there is inoculation is then collected inoculation times and/or time, the photo of detected object, age, gender, weight, occupation, medication
History, smoking history, history of drinking history, diabetic history.
Preferably, system as described above, the information that the reporting system main interface is shown include one in following content
, it is multinomial or whole：
1) record of date information and modification function；
The date information includes：The skin test time, instrument detection the date, user information date of entry, the audit report date,
The printed report date and send reporting day interim one or more；
2) calls the detected object information in Assignment Analysis module, is inserted in evaluation text template and shows, and provides the power of amendment
Limit；
3) printing and establish customized report template that is reported；Custom item includes detected object number, report
Head, detected value, reference value, report picture, Health & Fitness Tip, auditor, printing people.
The invention further relates to kit as described above in preparation for detecting latent tuberculosis, Active infection tuberculosis,
Or for distinguishing the application in the latent diagnosticum with Active infection of tuberculosis.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment 1
One, research object
1. cavy：SPF grades of cavys, the age 6~9 weeks, are ground by Chinese Academy of Medical Sciences medical experiment animal by 400~500 grams
Study carefully and is provided, animal licensing：SCXK (capital) 2014-0004.75 cavys are randomly divided into 3 groups, respectively tuberculosis branch bar
Bacterium (H37Rv ATCC27294) viable bacteria sensitization group 25, BCG vaccine (Pasteur strain) sensitization group 25 and saline control group
25.Every group of sensitized guinea pig is made a skin test with four kinds of different amalgamation and expression protein combinations and PPD respectively, and every group each 5.
Method of sensitization is to infect above-mentioned mycobacterium tuberculosis and each 100CFU of BCG vaccine respectively.Sensitization group cavy is raised in negative pressure animal house
It supports.
2. volunteer, due to there are four types of recombinant antigen mixing, randomly selects following each group and wherein half volunteer while using
The two kinds therein skin tests for carrying out left arm are tested, and right arm then uses PHA to carry out skin test experiment, and physiological saline is as control.
1) healthy population (HBCG-) of not inoculated BCG vaccine, 90, negative through IGRAs verification experimental verification, no card trace.
2) healthy population (HBCG+) of inoculated BCG vaccine, 96 negative through IGRAs verification experimental verification, there is card trace.
3) mycobacterium tuberculosis latent infection crowd (LTBI), 102, be the positive through IGRAs verification experimental verification.
4) the active tuberculosis patient (PTB) that outpatient service is gone to a doctor for the first time, 92, medication is no more than 2 weeks.
The equal non-diabetic of all research objects, autoimmune disease and other chronic infectious diseases.Age 18-50
Year, male or female.Show each group there are no significant in terms of age, gender difference through statistical analysis.
Two, material and reagent
The recombinant bacillus Calmette-Guerin vaccine of purifying lacks area's antigen protein：His-ESAT6-CFP10-TB7.7 amalgamation and expression albumen, His-
CFP21-EsxV amalgamation and expression albumen, His-MPT64 recombinant protein, His-Rv1985C and His-Rv0222 recombinant protein by
The preparation of this research department.Research be divided into His-ESAT6-CFP10-TB7.7+His-MPT64 group (four kinds albumen equal proportions mixing),
His-CFP21-EsxV+His-Rv1985C+His-Rv0222 group (four kinds of albumen equal proportion mixing), His-MPT64+His-
Rv1985C+His-Rv0222 group (three kinds of albumen equal proportion mixing) and His-ESAT6-CFP10-TB7.7+His-MPT64+
His-CFP21-EsxV+His-Rv1985C+His-Rv0222 group (eight kinds of albumen equal proportion mixing).Every kind of mixed protein is through fixed
3 μ g/0.1ml are taken to carry out intracutaneous injection after amount.
Human-like PPD is provided by Nat'l Pharmaceutical & Biological Products Control Institute, specification 100IU/2ml.
Phytohemagglutin phytolectin (PHA) is produced by Guangzhou Medicine Industry Inst, specification 10mg/2ml, using preceding through 1:20 is dilute
It releases and is made into solution for skin test.
Three, research method
1. skin test method：
Cavy：After sensitized animal 4 weeks, by guinea pig back backbone two sides unhairing, respectively at 5 μ g/ml's of two sides intracutaneous injection
Each 0.1ml of the PPD of recombinant protein and 50IU/ml.The vertical diameter that the observation of 48 hours double-blind studys and record locally harden after injection with
Transverse diameter, and it is averaged (vertical diameter is added with transverse diameter divided by 2), criterion is that average Callosity reaction diameter is sentenced not less than 5mm
For the positive, feminine gender is judged to less than 5mm.
Volunteer：It is operated by special messenger, the recombinant protein (3 μ g) of 0.1ml purifying, human-like PPD (is contained into 5 tuberculosis elements respectively
Unit) solution for skin test accurately injects 1/3 on left forearm palmar, in lower 1/3 intersection it is intradermal；It injects PHA dilution and (contains PHA 33
μ g), each 0.1ml of physiological saline 1/3 on right forearm palmar, in it is lower 1/3 intradermal, make skin mound diameter 6-10mm (Fig. 1).
2. stress prepare：Anaphylactic shock can occur in Skin-test for only a few tubercular, often the several seconds after injection
Start in 5 minutes, first pruitus, numb limb, after then out of breath, uncomfortable in chest, cyanosis, palpitating speed, arteries and veins be thin, blood pressure decline,
A large amount of perspirations etc..Therefore rescue should be carried out and prepared, such as standing adrenalin hydrochloride, hydrocortisone, central stimulant and antiallergy
Medicine.
3. result judges：Part scleroma or scleroma diameter are measured by special messenger, and recorded, 24 hours observation PHA after injection
As a result, hardening diameter DPHA >=10mm as positive reaction using part.48 hours after injection, recombinant protein is observed respectively within 72 hours
PPD, human-like PPD result：Diameter 5mm≤DTBAg≤the 15mm that hardens is the positive, and DTBAg > 15mm is the superpower positive.In addition to scleroma
Still having blister, bad the dead is the superpower positive.
Four, result of study
1. different antigen combinations skin test in sensitized guinea pig reacts
The scleroma diameter of sensitized guinea pig is measured at the 4th week, as a result BCG vaccine inoculation group is for four different recombinant proteins
It is negative for group, scleroma size is 0；And PPD skin test group is positive in BCG vaccination group, scleroma diameter difference
For 13.18 ± 1.33mm.For Mycobacterium tuberculosis H37Rv infected group, at this time four recombinant protein groups and PPD group (12.14 ±
The positive is presented in all cavys 1.71mm), and wherein the Callosity reaction of His-ESAT6-CFP10-TB7.7+His-MPT64 group is flat
Mean value is 16.33 ± 2.92mm, and the Callosity reaction average value of His-CFP21-EsxV+His-Rv1985C+His-Rv0222 group is
The Callosity reaction average value of 16.09 ± 2.51mm, His-MPT64+His-Rv1985C+His-Rv0222 group be 16.18 ±
2.76mm, His-ESAT6-CFP10-TB7.7+His-MPT64+His-CFP21-EsxV+His-Rv198 5C+His-Rv0222
The Callosity reaction average value of group is 18.23 ± 3.08mm.Its diameter that hardens of the skin test experiment results proved of 8 kinds of mixed reorganization albumen
It is better than the mixture of four kinds or three kinds recombinant proteins, and the average scleroma diameter of recombinant protein group is all larger than PPD skin test group.It says
The test results of bright recombinant protein group are better than PPD group.
2. the skin test reaction result (table 1) of different tests crowd
The skin test effect compared between four kinds of recombinant antigen protein combinations is only observed in this test, does not do the comparison of PPD
Test, because PPD does not identify the ability of active tuberculosis.In four groups of crowds, have on a small quantity enter a group individual occur PHA reaction it is lower
The case where, but the less (p of the otherness in each group crowd>0.05).As a whole, healthy population whether has
BCG is inoculated with history, and the Skin-test that four groups of recombinant antigen protein combinations carry out all is feminine gender, illustrates that these types of antigen is tuberculosis branch
Specific to bacillus, therefore, the specificity of skin test is high.
Either LTBI crowd or tuberculosis patient crowd, the reaction of skin test positive caused by PHA proportion are more than
92%, illustrating, which just has PHA to be used as, judges that immune system health degree has feasibility.
The ratio for the number that the people of recombination tuberculosis antigen skin test positive accounts for the PHA positive is very high, and recombinates in four groups of differences
It is all close between antigen mixture group, between 92.2% and 95.5%, illustrate the selected several groups of antigen proteins tool of this research
It is representative, special type Ⅳ allergy can be caused, can be used as the antigen of skin test test.
For four groups of skin test tests with four kinds or three kinds antigen combinations, either in LTBI group, or in PTB group, it
Between ratio R atio=DTBAg/DPHAAll difference is not significant, illustrates that this four groups of recombinant antigen proteins all have carry out skin test
The potential value of test, effect are approximate.
From ratio R atio=DTBAg/DPHAFrom the point of view of, latent infection group is lower than lunger's group, in the difference model of LTBI group
Enclose between 0.73-0.82, PTB group disparity range between 1.97-1.21, the two significant difference (p<0.05).
In the skin test test of four groups of difference antigen combinations, this trend is all identical, illustrates that the ratio can identify to a certain extent
Tuberculosis latent infection and active tuberculosis.
The skin test experiment of 1 four kinds of table different antigen combinations is compared with the experiment of PHA skin test
3. the size between different antigen combinations compares
1) except there is no a external of positive reaction individually, for all latent infection crowds, His-ESAT6-
Scleroma size the average out to 9.12 ± 2.86mm, His-CFP21-EsxV+His- of CFP10-TB7.7+His-MPT64 antigen group
Scleroma size the average out to 9.81 ± 2.99mm, His-MPT64+His-Rv1985C+ of Rv1985C+His-Rv0222 antigen group
Scleroma size the average out to 9.11 ± 3.49mm, His-ESAT6-CFP10-TB7.7+His-MPT64+ of His-Rv0222 antigen group
10.12 ± 3.26mm of scleroma size average out to of His-CFP21-EsxV+His-Rv1985C+His-Rv02222 antigen group.
2) except there is no a external of positive reaction individually, for all active tuberculosis crowds, His-
Scleroma size the average out to 13.56 ± 3.11mm, His-CFP21-EsxV of ESAT6-CFP10-TB7.7+His-MPT64 antigen group
Scleroma size the average out to 12.89 ± 2.77mm, His-MPT64+His- of+His-Rv1985C+His-Rv0222 antigen group
Scleroma size the average out to 13.14 ± 3.89mm, His-ESAT6-CFP10-TB7.7+ of Rv1985C+His-Rv0222 antigen group
The scleroma size average out to 15.12 of His-MPT64+His-CFP21-EsxV+His-Rv1985C+His-Rv02222 antigen group ±
3.76mm。
3) except there is no a external of positive reaction individually, difference of the scleroma between different crowd caused by PHA is not
Greatly, the size of HBCG- group, HBCG+ group, LTBI group and PTB group is respectively 11.12 ± 4.79mm, 11.26 ± 4.35mm, 12.03
± 5.05mm and 11.99 ± 5.13mm.
4. skin test tests ratio R atio=DTBAg/DPHAFor identify latent infection person and active tuberculosis.
In order to further analyze Ratio=DTBAg/DPHARatio is latent in terms of identifying latent infection and active tuberculosis
It being worth, we analyze the threshold value of Ration first, as a result, it has been found that：Different antigen groups is combined with different threshold values, but
It is that its distinguishing ability is essentially identical, judges that the accuracy of latent infection and lunger are above 85%.For His-
ESAT6-CFP10-TB7.7+His-MPT64 antigen group distinguishes latent infection and active tuberculosis when threshold value is 0.83
Sensibility be 90.5%, specificity be 87.1%；For His-CFP21-EsxV+His-Rv1985C+His-Rv0222 antigen
Group, when threshold value is 0.82, the sensibility for distinguishing latent infection and active tuberculosis is 89.3%, and specificity is
85.8%；For His-MPT64+His-Rv1985C+His-Rv0222 antigen group, when threshold value is 0.85, latent sense is distinguished
Dye and the sensibility of active tuberculosis are 88.7%, and specificity is 86.4%；For His-ESAT6-CFP10-TB7.7+
His-MPT64+His-CFP21-EsxV+His-Rv1985C+His-Rv02222 antigen group, when threshold value is 0.91, difference
The sensibility of latent infection and active tuberculosis is 91.2%, and specificity is 87.0%.
5. the adverse reaction of skin test test
This is mainly shown as pruitus researches show that the adverse reaction for having 3 experimenters that the test of PHA skin test has occurred,
Wherein 1 breathing has slight acceleration.His-ESAT6-CFP10-TB7.7+His-MPT64+His-CFP21-EsxV+His-
4 adverse reactions have occurred in Rv1985C+His-Rv0222 group, but only pruitus and big bubble fracture phenomena.Remaining is each
1-2 not equal adverse reactions have occurred in mixed antigen group.Thus speculate, the mixed species for increasing tuberculosis antigen may be with not
The phenomenon that good reaction incidence increases.Therefore, it is suggested that being made a skin test using appropriate more antigen type.
Five, it discusses and suggests
1) BCG vaccine gene delection area is obtained according to gene order-checking, by a variety of antigen combinations in BCG vaccine missing area one
It rises and carries out skin test experiment, can effectively identify BCG vaccination bring interference problem.
2) not reacting in the experiment of tuberculosis antigen skin test can be significantly reduced using the preparatory assessment that PHA carries out immune system
Or the probability of low reaction individual, the accuracy of enhancing skin test experiment.Importantly, although the test results of PHA are without tuberculosis
Specificity, but be conducive to the Skin-test that specificity is further carried out using tuberculosis antigen, the ratio R atio=of the two
DTBAg/DPHAWith the preferable ability for identifying latent infection and active tuberculosis.
3) ratio R atio=D is usedTBAg/DPHAIdentify latent infection and active tuberculosis, when ratio is greater than threshold value,
It is judged as active tuberculosis, is judged as latent infection when less than threshold value.Using different recombination tuberculosis antigen protein combination institutes
The threshold value of use slightly has difference, should further determine that threshold value and its specificity and sensibility according to its corresponding clinical test results
Etc. indexs.
Although 4) type for increasing tuberculosis antigen theoretically can improve the size hardened in skin test experiment, it is contemplated that
Adverse reaction and sensibility and its specificity, it is not recommended that together using too many antigen combination.
5) this research uses skin test method for intracutaneous injection, and side arm intracutaneous injection recombinates tuberculosis antigen albumen, another
Side intracutaneous injection PHA.According to the principle that skin test is tested, recombinant antigen protein is injected simultaneously using the same side arm and PHA is also
It is feasible.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that：Its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Claims (10)
1. a kind of tuberculosis infection diagnostic reagent kit comprising mitogenesis original and antigenic stimulus object；
The antigenic stimulus object is at least three kinds of in following tuberculin pure protein or derivatives thereof：
ESAT6, CFP10, TB7.7, MPT64, CFP21, EsxV, Rv1985C and Rv0222.
2. kit according to claim 1, which is characterized in that the antigenic stimulus object is selected from following tuberculosis antigen albumen
Or its fragment combination：
ESAT6,CFP10,TB7.7,MPT64；
Or CFP21, EsxV, Rv1985C, Rv0222；
Or MPT64, Rv1985C, Rv0222；
Or ESAT6, CFP10, TB7.7, MPT64, CFP21, EsxV, Rv1985C, Rv0222.
3. kit according to claim 1, which is characterized in that in the antigenic stimulus object, the pure egg of every kind of tuberculin
It is white or derivatives thereof to mix；Preferably equimolar is than mixing.
4. described in any item kits according to claim 1~3, which is characterized in that the concentration of the antigenic stimulus object is 2 μ g
~4 μ g/0.1ml.
5. described in any item kits according to claim 1~3, which is characterized in that the mitogenesis original includes plant
Agglutinin, concanavalin A, american pokeweed root leaf and seed agglutinin；
Preferably, the concentration of the mitogenesis original is 0.3mg~0.8mg/2ml.
6. described in any item kits according to claim 1~3, which is characterized in that also containing positive right in the kit
According to the positive control is human-like purified protein derivative of tuberculin.
7. a kind of tuberculosis infection diagnostic system, which is characterized in that the system comprises：
Data sink, Assignment Analysis module and reporting system main interface；
The received data of data sink include：Patient is carried out using claim 1~6 described in any item kits
The scleroma size D of allergic immune reaction caused by the antigenic stimulus object that skin test is presentedTBAgAnd the phytohemagglutin phytolectin
The scleroma size D of caused allergic immune reactionPHA；
If DTBAg>=5mm, and DPHAWhen >=10mm, data are passed to the Assignment Analysis module, are otherwise transferred directly to the report
Announcement system main interface；
The Assignment Analysis module is for calculating Ratio=DTBAg/DPHA, and then tuberculosis infection type is analyzed, and be presented to
The reporting system main interface.
8. according to the system as claimed in claim 7 for being subordinated to any one of claim 2,4,5,6, which is characterized in that the tax
It is worth analysis module when analyzing tuberculosis infection type, carries out assignment in the following manner：
If antigenic stimulus object is selected from ESAT6, CFP10, TB7.7, MPT64, and Ratio=DTBAg/DPHAWhen >=0.83, knot is distinguished
Latent sensibility >=90.5% with Active infection of core, specificity are >=87.1%；
If antigenic stimulus object is selected from CFP21, EsxV, Rv1985C, Rv0222, and Ratio=DTBAg/DPHAWhen >=0.82, distinguish
Latent sensibility >=89.3% with Active infection of tuberculosis, specificity are >=85.8%；
If antigenic stimulus object is selected from MPT64, Rv1985C, Rv0222, and Ratio=DTBAg/DPHAWhen >=0.85, tuberculosis is distinguished
Latent and Active infection sensibility >=88.7%, specificity are >=86.4%；
If antigenic stimulus object is selected from ESAT6, CFP10, TB7.7, MPT64, CFP21, EsxV, Rv1985C, Rv0222, and Ratio
=DTBAg/DPHAWhen >=0.91, distinguish that tuberculosis is latent and sensibility >=91.2% of Active infection, specificity for >=
87.0%.
9. system according to claim 7, which is characterized in that the received data of data sink further include：Whether
Inoculated BCG vaccine, if there is inoculation is then collected inoculation times and/or time, the photo of detected object, the age, gender, weight,
Occupation, medication history, smoking history, history of drinking history, diabetic history；
Preferably, the information that the reporting system main interface is shown includes one in following content, multinomial or whole：
1) record of date information and modification function；
The date information includes：Skin test time, instrument detection date, user information date of entry, audit report date, printing
It reports the date and sends reporting day interim one or more；
2) calls the detected object information in Assignment Analysis module, is inserted in evaluation text template and shows, and provides modification authority；
3) printing and establish customized report template that is reported；Custom item includes detected object number, report head, inspection
Measured value, reference value, report picture, Health & Fitness Tip, auditor, printing people.
10. the described in any item kits of claim 1~6 are being prepared for detecting latent tuberculosis, Active infection tuberculosis,
Or for distinguishing the application in the latent diagnosticum with Active infection of tuberculosis.
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