CN107653308B - One group is combined and kit for distinguishing active tuberculosis patient with the primer pair of non-tuberculous pneumonia patient - Google Patents
One group is combined and kit for distinguishing active tuberculosis patient with the primer pair of non-tuberculous pneumonia patient Download PDFInfo
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Abstract
The invention discloses combine with the primer pair of non-tuberculous pneumonia patient and kit for distinguishing active tuberculosis patient.The primer pair combination is respectively SEQ ID NO:1-2, SEQ ID NO:3-4 and SEQ ID NO:5-6.Wherein SEQ ID NO:1-2 corresponds to CD157 gene；SEQ ID NO:3-4 corresponds to IL-1b gene；SEQ ID NO:5-6 corresponds to MS4A6A gene.RT-PCR extension is carried out using primer of the invention, in the formula for then establishing the substitution of relevant gene expression dose, active tuberculosis patient and non-tuberculous pneumonia patient can be distinguished, so that Diagnosis of Tuberculosis is more accurate, quick.
Description
Technical field
The present invention relates to field of biotechnology more particularly to one group for distinguishing active tuberculosis patient and non-tuberculous lung
The primer pair of scorching patient combines and kit.
Background technique
Tuberculosis (Tuberculosis, TB) is chronic infectious disease caused by being infected by mycobacterium tuberculosis, tuberculosis
Mycobacteria can not only cause pulmonary tuberculosis (85%), but also can cause the tuberculosis of a variety of organs outside lung.Although have has at present
The antituberculotic of effect, but tuberculosis is still the number one killer of current infectious diseases, and annual about 2,000,000 people in the whole world die of knot
Core disease；The population of the whole world about one third infects mycobacterium tuberculosis, is referred to as tulase latent infection (LTBI) at these
In crowd, there is about 10% will finally progress to active tuberculosis.
Due to lacking effective tuberculosis vaccine at present, prevention and control lungy depend on early detection, treatment,
Active tuberculosis patient is isolated.However, there are wretched insufficiencies for diagnostic activities detection technique lungy now, it is not able to satisfy and faces
The requirement of bed and tuberculosis prophylaxis control.The goldstandard of diagnosis of tuberculosis is phlegm mycobacterium tuberculosis microorganism checking (phlegm picture
Or Sputum culturing), although detection technique specificity is high, it is low (less than 40%) that there are sensibility, (tulase culture consumption that time-consuming
When 1-2 months), the demanding disadvantage of Laboratory biosafety.In other words, the tuberculosis of 60%-70% sputum bacteria feminine gender at present
The diagnosis of people clinically needs other detections and data to support.In fact, clinically the diagnosis of the tuberculosis patient of the type is big
Majority is clinical diagnosis, i.e., is diagnosed by clinical manifestation, iconography or antituberculosis therapy effect.This diagnostic method is maximum
The shortcomings that be cannot by caused by tuberculosis and its tulase he pulmonary disease distinguish.
Summary of the invention
The technical problem to be solved in the present invention is to provide one group for distinguishing active tuberculosis patient and non-tuberculous pneumonia
The primer pair of patient combines and kit.
To achieve the above object, the present invention provides a kind of CD157, IL-1b and MS4A6A combination conduct differentiation activity knot
The purposes of the molecular marker of core patient and non-tuberculous pneumonia patient.
It is combined for distinguishing active tuberculosis patient with the primer pair of non-tuberculous pneumonia patient the present invention also provides one group,
It is characterized in that, the primer pair combination is respectively SEQ ID NO:1-2, SEQ ID NO:3-4 and SEQ ID NO:5-6；Its
Middle SEQ ID NO:1-2 corresponds to CD157 gene；SEQ ID NO:3-4 corresponds to IL-1b gene；SEQ ID NO:5-6 is corresponding
MS4A6A gene.
The present invention also provides a kind of for distinguishing the kit of active tuberculosis patient Yu non-tuberculous pneumonia patient, special
Sign is, combines containing primer pair as claimed in claim 2.
The primer pair combination and the kit are used to distinguish active tuberculosis patient and non-tuberculous pneumonia patient's
Purposes.
The primer pair combination and the kit are used to distinguish active tuberculosis patient and non-tuberculous pneumonia patient's
Method, which is characterized in that
SEQ ID NO:1-2, SEQ ID NO:3-4 is respectively adopted or with SEQ ID NO:5-6 as primer, to disease to be measured
The DNA of human PBMC carries out RT-PCR reaction；
Calculate the relative expression quantity of each gene: according to real-time quantitative PCR as a result, detecting the glimmering of 3 genes respectively
Light quantitive CT value establishes machine learning model lasso trick/elasticity pessimistic concurrency control LASSO by R linguistic algorithm packet Glmnet, then
To protein combination conversion as a result, being indicated with R；
As a result judge: as R > 0.5, being then judged as active tuberculosis；Conversely, being then non-tuberculous pneumonia patient.
Further, the reaction system of the RT-PCR reaction are as follows:
Further, the response procedures of RT-PCR reaction are, 95 DEG C 30 seconds；95 DEG C 5 seconds, 60 DEG C 30 seconds, 40 circulation.
Method of the inventor in the experiment of early period through genetic chip has detected draws including tuberculosis patient and its tulase
The expression of more than the 6000 a genes in his pulmonary disease human peripheral risen, discovery have multiple genes specific in tuberculosis patient
Expression, can be used as Diagnosis of Tuberculosis marker.On this basis to the assortment of genes of differential expression in multiple tuberculosis patients, discovery
The assortment of genes below: the expression that CD157/IL-1b/MS4A6A can be specific in tuberculosis patient.Pass through the operation of foundation
Formula distinguishes tuberculosis and the non-tuberculous pneumonia patient of lung, to establish diagnosis new method lungy.
Using kit of the invention, the expression of patient's CD157/IL-1b/MS4A6A gene can detecte, then
Using the operational formula model of foundation, to diagnose whether patient suffers from active tuberculosis.
In the present invention, term " primer " refers to a kind of oligonucleotides, can be natural being also possible to synthesis, it can
To induce synthesize the primer complementary with nucleic acid chains under suitable conditions as the starting point for inducing DNA synthesis under certain condition
Amplified production, i.e., in four kinds of different triphosphoric acid dezyribonucleosides and a kind of polymerization agent (i.e. archaeal dna polymerase or reverse transcription
Enzyme) in the presence of, it carries out amplification reaction in a kind of suitable buffer and at a suitable temperature.Preferred primer is sub-thread widow
Deoxyribonucleotide.The appropriate length of primer depend on the primer designed use, but generally 15~25 nucleotide it
Between.
In the present invention, the probe can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives.The spy
The length of needle there is no limit as long as completing specific hybrid, specifically binding with purpose nucleotide sequence, all may be used by any length
With.
The experiment proved that the expression of the CD157/IL-1b/MS4A6A primer detection related gene designed using the present invention,
Then relevant gene expression dose is substituted into the formula established, discovery calculates above-mentioned gene by means of the present invention
Expression of the group expression in tuberculosis patient is substantially less than non-tuberculous pneumonia patient.Therefore CD157/IL-1b/MS4A6A base
Because group can be used as the specific marker gene of diagnosis of tuberculosis, keep Diagnosis of Tuberculosis more accurate, quick.
Specific embodiment
The embodiment of the present invention is described below in detail, in which the same or similar labels are throughly indicated same or like
Element or element with the same or similar functions.It is exemplary below by the embodiment of description, it is intended to for explaining
The present invention, and be not considered as limiting the invention.In the examples where no specific technique or condition is specified, according in the art
Document described in technology or conditions or carried out according to product description.Production firm is not specified in agents useful for same or instrument
Person, being can be with conventional products that are commercially available.
Embodiment 1: the preparation of peripheral blood mononuclear cells (PBMC) suspension
Lymphocyte separation medium (Fresenius Kabi NOrge As:LYS3773) 5ml is added in centrifuge tube；It takes
The phosphate buffer (PBS) for stating each 2ml of anticoagulant heparin venous blood and equivalent 1M of different type crowd, which mixes well, to be mixed
Mixed liquor is slowly superimposed on lymphocyte separation medium liquid level by liquid with pipettor along tube wall, keeps clearly interface, and 2000
Rev/min centrifugation 20 minutes；Intermediate cloud and mist stratification, which is drawn, with suction pipe enters the 1M PBS that 5 times of volumes are followed by added in another centrifuge tube,
1500 revs/min of centrifugations, 10 minutes washing cells abandon supernatant, and the same terms repeated washing cell is primary, is subsequently added into containing small ox blood
RPMI1640 (Thermo scientific:SH30807.01b) 1ml that clear percent by volume is 10%, is resuspended cell, obtains
To PBMC suspension；Every takes the PBMC suspension of 20 μ l on blood counting chamber, counts cell concentration.
Embodiment 2:RNA extracting
Using the RNeasy Mini Kit (article No. 74106) of Qiagene company to the PBMC of three groups of crowds obtained above
Suspension carries out RNA extracting.Concrete operations are: taking and above-mentioned contain 1 × 106The PBMC suspension of a cell in go DNA enzymatic and RNA enzyme from
In heart pipe, 3000 revs/min are centrifuged 10 minutes, abandon supernatant；350 μ l × Buffer RLT are added in cell precipitation, mix well
Cracking；250 μ l dehydrated alcohols are added, mixes, liquid is transferred in RNeasy pillar, waste liquid is abandoned in 8,000g centrifugations 30 seconds；Add
Enter 350 μ l Buffer RW1 with 8,000g centrifugation 30 seconds, abandons waste liquid；80 μ l DNase solution (10 μ l DNase+70 μ l are added
Buffer RDD), 15min is digested on column, waste liquid is abandoned in 8,000g centrifugations 30 seconds；Be added 350 μ l Buffer RW1,8,000g from
The heart 30 seconds, abandon waste liquid；500 μ l Buffer RPE are added, waste liquid is abandoned in 8,000g centrifugations 30 seconds；Sky is got rid of, 8,000g 1 point of centrifugations
Clock；Posts transfer is removed the 1.5ml centrifuge tube of DNA enzymatic and RNA enzyme to one, is added ddH20 of the 40 μ l without RNase, 10,000g
Centrifugation 1 minute, each 20 RNA for collecting three groups of crowds are saved in -80 DEG C, are stand-by.
Embodiment 3: reverse transcription
Using the reverse transcription reagent box (DRR047) of TAKARA company, the 0.5 μ g of RNA for taking step 2 to obtain is inverted
The step of record, the more traditional reverse transcription reagent box of the kit increases removal genomic DNA, it can guarantee RNA to the full extent
Purity and amplification specificity.
Substep is as follows:
(1) the removal reaction of genomic DNA
The removal reaction system table of 1 genomic DNA of table
| Reagent | Usage amount |
| 5 × gDNA Eraser buffer | 2μl |
| gDNA Eraser | 1μl |
| Total serum IgE | 0.5μg |
| Rnase Free ddH20 | It mends to 10 μ l |
After preparing reaction system according to table 1, in 42 DEG C of warm bath 2min, gained is the RNA reaction for removing genomic DNA
Liquid saves at 4 DEG C.
(2) reverse transcription reaction
Reaction system preparation carries out on ice, and specific system is as follows:
2 reverse transcription reaction system table of table
| Reagent | Usage amount |
| 5 × Prime Script buffer 2 | 4μl |
| PrimeScript RT enzymatic mixture I | 1μl |
| RT primer mixture | 1μl |
| Remove the RNA reaction solution of genomic DNA | 10μg |
| Rnase Free ddH2O | It mends to 20 μ l |
After preparing reaction system according to table 2, in 37 DEG C of warm bath 15min, 85 DEG C are placed 5 seconds, have reacted to obtain reverse transcription production
Object puts 4 DEG C of preservations.
Embodiment 4: quantitative fluorescent PCR reaction
Template: the template that above-mentioned gained reverse transcription product is reacted as quantitative fluorescent PCR, template consumption are 1 μ l.It utilizes
The primer of CD157/IL-1b/MS4A6A gene (NG_032578.1) sequence, design table 3 (is had by Invitrogen (Shanghai) trade
Limit company synthesis).
3 primer sequence table of table
The system of PCR reaction:
PCR reaction is using TAKARA companyPremix Ex TaqTMII (article No.: DRR081D), this product energy
Enough inhibit nonspecific reaction, is carried out in broad range more quantitative.This Buffer and improvement after
HotStart method is applied in combination with archaeal dna polymerase TaKaRa Ex Taq HS, can carry out the Real that reproducibility is good, with a high credibility
TimePCR parsing.
The system table of table 4PCR reaction
According to upper table prepare fluorescent quantitation reaction solution, and using instrument be 7500 real-time fluorescence quantitative PCR instrument of ABI into
The reaction of row real-time quantitative PCR.
Real-time quantitative PCR reaction use two-step method PCR, expand standardization program: 95 DEG C 30 seconds；95 DEG C 5 seconds, 60 DEG C 30 seconds,
40 circulations.
Embodiment 5: the judgement of Data Processing in Experiment and testing result
According to real-time quantitative PCR as a result, detecting the CT value of each gene respectively.Once by taking 6 PBMC as an example (wherein
Sample 1-3 is non-tuberculous patients with pneumonia, sample 4-6 active tuberculosis patient), the fluorescent quantitation of 3 genes is detected respectively
CT value establishes machine learning model lasso trick/elasticity pessimistic concurrency control (LASSO), then obtains egg by R linguistic algorithm packet Glmnet
The result (being indicated with R) of white combination conversion.
The type of interpretation sample.Judgment criteria are as follows: as R > 0.5, be then judged as active tuberculosis patient.Conversely, being then
Non-tuberculous pneumonia patient.
The result table of CT value and the protein combination conversion of 56, table, three, sample gene
| CD157 | IL1-B | MS4A6A | R value | |
| Sample 1 | 18.81699944 | 19.85099983 | 17.49799919 | 0.051979883 |
| Sample 2 | 20.47800064 | 23.38599968 | 20.77799988 | 0.097066745 |
| Sample 3 | 17.62899971 | 12.74400043 | 17.79700089 | 0.049131244 |
| Sample 4 | 19.14900017 | 9.87100029 | 22.14100075 | 0.792579128 |
| Sample 5 | 19.84499931 | 13.24600029 | 20.78700066 | 0.876919261 |
| Sample 6 | 17.90699959 | 16.66600037 | 19.44300079 | 0.972986521 |
As can be seen from Table 5, the R value of sample 1-3 is non-tuberculous pneumonia patient less than 0.5；The R value of sample 4-6 is greater than
0.5, it is active tuberculosis patient.It is complied fully with actual conditions.
Embodiment 6: of the invention in the sensibility and specificity for differentiating active tuberculosis
Crowd is divided into two groups by the present embodiment: active tuberculosis patient (24), non-tuberculous pneumonia patient (26),
By detecting CD157/IL-1b/MS4A6A gene C T in every Patients with Peripheral blood mononuclear cell (PBMC), counted after finally substituting into formula
The result and judging result of each sample are calculated, result value is greater than 0.5 and is diagnosed as active tuberculosis.
It the results are shown in Table 6,
6 different type clinical sample testing result table of table
As can be seen from Table 6, it is detected in 24 active tuberculosis patients using this kit, 21 are judged as activity knot
Core, true positive rate 87.5% illustrate that the protein chip detection method of this discovery is specific well；26 of detection are inactive
In property tuberculosis patient, 2 are judged as active tuberculosis, and false positive rate 7.69% illustrates the protein chip detection side of this discovery
The good sensibility of method.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.
SEQUENCE LISTING
<110>Shenzhen's galaxy Biotechnology Co., Ltd
<120>a kind of to be combined with the primer pair of non-tuberculous pneumonia patient and reagent for distinguishing active tuberculosis patient
Box
<130> 10001
<160> 6
<170> PatentIn version 3.5
21
<210> 1
<211> 20
<212> DNA
<213>artificial synthesized
<400> 1
ccagtgaagc tcttacagtg 20
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized
<400> 2
cactgtaaga gcttcactgg 20
<210> 3
<211> 21
<212> DNA
<213>artificial synthesized
<400> 3
tgggataacg aggcttatgt g 21
<210> 4
<211> 21
<212> DNA
<213>artificial synthesized
<400> 4
acaaaggaca tggagaacac c 21
<210> 5
<211> 21
<212> DNA
<213>artificial synthesized
<400> 5
atcgccacag agaaaaggtt a 21
<210> 6
<211> 21
<212> DNA
<213>artificial synthesized
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Claims (2)
1. one group is combined for distinguishing active tuberculosis patient with the primer pair of non-tuberculous pneumonia patient, which is characterized in that institute
Stating primer pair combination is respectively SEQ ID NO:1-2, SEQ ID NO:3-4 and SEQ ID NO:5-6；Wherein SEQ ID NO:1-
2 corresponding CD157 genes；SEQ ID NO:3-4 corresponds to IL-1b gene；SEQ ID NO:5-6 corresponds to MS4A6A gene.
2. a kind of for distinguishing the kit of active tuberculosis patient Yu non-tuberculous pneumonia patient, which is characterized in that containing having the right
Benefit require 1 described in primer pair combination.
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| CN201710684735.4A CN107653308B (en) | 2017-08-11 | 2017-08-11 | One group is combined and kit for distinguishing active tuberculosis patient with the primer pair of non-tuberculous pneumonia patient |
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| CN107653308B true CN107653308B (en) | 2019-06-07 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112143796A (en) * | 2020-09-30 | 2020-12-29 | 中国医学科学院病原生物学研究所 | CARD16 as molecular marker for diagnosis and identification of tuberculosis |
| CN112481370A (en) * | 2020-12-03 | 2021-03-12 | 中国医学科学院病原生物学研究所 | Application of BST1 as tuberculosis diagnosis molecular marker |
| CN114381509B (en) * | 2021-12-27 | 2022-10-25 | 深圳大学 | Plasma miRNA marker related to non-tuberculous pneumonia and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060446A (en) * | 2012-12-28 | 2013-04-24 | 深圳市第三人民医院 | Use of CD157 gene |
| CN103074422A (en) * | 2012-12-29 | 2013-05-01 | 深圳市第三人民医院 | MS4A6A gene application |
| CN104823052A (en) * | 2012-07-31 | 2015-08-05 | 蛋白逻辑有限责任公司 | Biomarkers for diagnosing and/or monitoring tuberculosis |
-
2017
- 2017-08-11 CN CN201710684735.4A patent/CN107653308B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104823052A (en) * | 2012-07-31 | 2015-08-05 | 蛋白逻辑有限责任公司 | Biomarkers for diagnosing and/or monitoring tuberculosis |
| CN103060446A (en) * | 2012-12-28 | 2013-04-24 | 深圳市第三人民医院 | Use of CD157 gene |
| CN103074422A (en) * | 2012-12-29 | 2013-05-01 | 深圳市第三人民医院 | MS4A6A gene application |
Non-Patent Citations (2)
| Title |
|---|
| "CD157在结核患者体内的表达水平、作用及其机制的初步探讨;张弛;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20160415;E061-51 |
| Transcriptional and inflammasome-mediated pathways for the induction of IL-1b production by Mycobacterium tuberculosis;Johanneke Kleinnijenhuis et al.;《Eur. J. Immunol.》;20090706;第39卷(第7期);1914-1922 |
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Effective date of registration: 20190726 Address after: 518000 Guangdong Province Longgang District Yuanshan Street Longgang Avenue 8288 Dayun Software Town 35 4th floor G Patentee after: Shenzhen Orange Moon Biotechnology Co., Ltd. Address before: 518112 Colente Research and Development Building 705, No. 1 Ganli Wu Road, Buji Science and Technology New City, Bulan Road, Longgang District, Shenzhen City, Guangdong Province Patentee before: Shenzhen galactic biological science and Technology Co., Ltd. |