CN107653308A - One group is used to distinguishing active tuberculosis patient and is combined with the primer pair of non-tuberculous pneumonia patient and kit - Google Patents
One group is used to distinguishing active tuberculosis patient and is combined with the primer pair of non-tuberculous pneumonia patient and kit Download PDFInfo
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Abstract
The invention discloses combined for distinguishing active tuberculosis patient with the primer pair of non-tuberculous pneumonia patient and kit.The primer pair combination is respectively SEQ ID NO:12, SEQ ID NO:34 and SEQ ID NO:5‑6.Wherein SEQ ID NO:12 corresponding CD157 genes;SEQ ID NO:34 corresponding IL 1b genes;SEQ ID NO:56 corresponding MS4A6A genes.RT PCR extensions are carried out using the primer of the present invention, are then substituted into the gene expression dose of correlation in the formula established, 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 biological technical field, more particularly to one group is used to distinguish active tuberculosis patient and non-tuberculous lung
The primer pair combination of scorching patient and kit.
Background technology
Tuberculosis (Tuberculosis, TB) is the chronic infectious disease as caused by infecting mycobacterium tuberculosis, tuberculosis
Mycobacteria can not only cause pulmonary tuberculosis (85%), and can cause the tuberculosis of a variety of organs outside lung.Although have 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 from knot
Core disease;The population infection mycobacterium tuberculosis in the whole world about 1/3rd, is referred to as tulase latent infection (LTBI) at these
In crowd, there is about 10% most to progress to active tuberculosis at last.
Due to lacking effective tuberculosis vaccine at present, prevention and control lungy depend on early detection, treatment,
Isolate active tuberculosis patient.However, now there is wretched insufficiency in diagnostic activities detection technique lungy, it is impossible to which satisfaction is faced
Bed and the requirement of 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, (the tulase culture consumption that has that sensitiveness is low (less than 40%), and time-consuming
When 1-2 months), Laboratory biosafety requires the shortcomings that high.In other words, the negative tuberculosis of 60%-70% sputum bacterias 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 can not by caused by tuberculosis and its tulase he PUD D distinguish.
The content of the invention
It is used to distinguish active tuberculosis patient and non-tuberculous pneumonia the technical problem to be solved in the present invention is to provide one group
The primer pair combination of patient and kit.
To achieve the above object, the present invention provides a kind of CD157, IL-1b and MS4A6A combination as differentiation activity knot
Core patient and the purposes of the molecular marker of non-tuberculous pneumonia patient.
The present invention also provides one group and combined for distinguishing active tuberculosis patient with the primer pair of non-tuberculous pneumonia patient,
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 genes;SEQ ID NO:3-4 corresponds to IL-1b genes;SEQ ID NO:5-6 is corresponding
MS4A6A genes.
The present invention also provides a kind of kit for being used to distinguish active tuberculosis patient and non-tuberculous pneumonia patient, and it is special
Sign is, contains the primer pair combination described 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, it is characterised in that
SEQ ID NO are respectively adopted:1-2, SEQ ID NO:3-4 or with SEQ ID NO:5-6 is as primer, to disease to be measured
The DNA of human PBMC carries out RT-PCR reactions;
Calculate the relative expression quantity of each gene:According to the result of real-time quantitative PCR, the glimmering of 3 genes is detected respectively
Light quantitive CT value, by R linguistic algorithm bag Glmnet, machine learning model lasso trick/elastic pessimistic concurrency control LASSO is established, then
To the result of protein combination conversion, represented with R;
As a result judge:Work as R>When 0.5, then it is judged as active tuberculosis;Conversely, it is then non-tuberculous pneumonia patient.
Further, the reaction system of the RT-PCR reactions is:
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.
Inventor have detected in the experiment of early stage by the method for genetic chip to draw including tuberculosis patient and its tulase
The expression of more than 6000 individual genes in his the PUD D human peripheral risen, discovery have multiple genes specific in tuberculosis patient
Expression, Diagnosis of Tuberculosis mark can be used as.On this basis to the assortment of genes of differential expression in multiple tuberculosis patients, find
The following assortment of genes:CD157/IL-1b/MS4A6A being capable of the specific expression in tuberculosis patient.Pass through the computing of foundation
Formula distinguishes tuberculosis and the non-tuberculous pneumonia patient of lung, so as to establish diagnosis new method lungy.
Using the kit of the present invention, the expressions of patient's CD157/IL-1b/MS4A6A genes can be detected, then
Using the operational formula model of foundation, so as to diagnose whether patient suffers from active tuberculosis.
In the present invention, term " primer " refers to a kind of oligonucleotides, can be it is natural can also be synthesis, it can
As the starting point for inducing DNA synthesis under certain condition, to induce the synthesis primer complementary with nucleic acid chains under suitable conditions
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, carry out in a kind of suitable buffer solution and at a suitable temperature amplified reaction.Preferable primer is sub-thread widow
Deoxyribonucleotide.The appropriate length of primer depends on the designed use of the primer, but typically between 15~25 nucleotides.
In the present invention, the probe can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives.The spy
The length of pin does not limit, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence, any length can.
It the experiment proved that, the expression of the CD157/IL-1b/MS4A6A primer detection related genes designed using the present invention,
Then the gene expression dose of correlation is substituted into the formula established, finds to calculate above-mentioned gene by the method for the present invention
Expression of the group expression in tuberculosis patient is substantially less than non-tuberculous pneumonia patient.Therefore CD157/IL-1b/MS4A6A bases
Because group can make Diagnosis of Tuberculosis more accurate, quick as the specific marker gene of diagnosis of tuberculosis.
Embodiment
Embodiments of the invention are described below in detail, wherein same or similar label represents same or like from beginning to end
Element or with same or like function element.Embodiment below by description is exemplary, 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 by technology or condition or carried out according to product description.Agents useful for same or the unreceipted production firm of instrument
Person, being can be by the conventional products of acquisition purchased in market.
Embodiment 1:The preparation of PMNC (PBMC) suspension
Lymphocyte separation medium (Fresenius Kabi NOrge As are added in centrifuge tube:LYS3773)5ml;Take
The phosphate buffer (PBS) for stating each 2ml of anticoagulant heparin venous blood and equivalent 1M of different type crowd is fully uniformly mixed so as to obtain and mixed
Liquid, mixed liquor is slowly superimposed on lymphocyte separation medium liquid level along tube wall with pipettor, keeps clearly interface, 2000
Rev/min centrifugation 20 minutes;With suction pipe draw among cloud and mist stratification enter the 1M PBS that another centrifuge tube followed by adds 5 times of volumes,
1500 revs/min of centrifugations, 10 minutes washing cells, abandon supernatant, the same terms repeated washing cell once, is subsequently added into containing small ox blood
RPMI1640 (the Thermo scientific that clear percent by volume is 10%:SH30807.01b) 1ml, cell is resuspended, obtains
To PBMC suspensions;Every takes 20 μ l PBMC suspensions to count cell concentration on blood counting chamber.
Embodiment 2:RNA is extracted
Using the RNeasy Mini Kit (article No. 74106) of Qiagene companies to the PBMC of three groups of crowds obtained above
Suspension carries out RNA extractings.Concrete operations are:Take and above-mentioned contain 1 × 106The PBMC suspensions of individual cell in go DNA enzymatic and RNase from
In heart pipe, 3000 revs/min centrifuge 10 minutes, abandon supernatant;350 μ l × Buffer RLT are added in cell precipitation, are fully mixed
Cracking;250 μ l absolute ethyl alcohols are added, mixes, liquid is transferred in RNeasy pillars, 8,000g centrifugations 30 seconds, abandon waste liquid;Add
Enter 350 μ l Buffer RW1 to centrifuge 30 seconds with 8,000g, abandon waste liquid;Add 80 μ l DNase solution (10 μ l DNase+70 μ l
Buffer RDD), 15min is digested on post, 8,000g centrifugations 30 seconds, abandons waste liquid;Add 350 μ l Buffer RW1,8,000g
Centrifugation 30 seconds, abandons waste liquid;500 μ l Buffer RPE are added, 8,000g centrifugations 30 seconds, abandon waste liquid;Sky is got rid of, 8,000g 1 point of centrifugations
Clock;Posts transfer to a 1.5ml centrifuge tube for removing DNA enzymatic and RNase, ddH20s of the 40 μ l without RNase is added, 10,
000g is centrifuged 1 minute, and the RNA of each 20 for collecting three groups of crowds is preserved in -80 DEG C, be stand-by.
Embodiment 3:Reverse transcription
Using the reverse transcription reagent box (DRR047) of TAKARA companies, the μ g of RNA 0.5 for taking step 2 to obtain are inverted
Record, the more traditional reverse transcription reagent box of the kit add the step of removing genomic DNA, can ensure 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 the genomic DNA of table 1
Reagent | Usage amount |
5 × gDNA Eraser buffer solutions | 2μl |
gDNA Eraser | 1μl |
Total serum IgE | 0.5μg |
Rnase Free ddH20 | Mend to 10 μ l |
After preparing reaction system according to table 1, in 42 DEG C of warm bath 2min, gained is the RNA reactions for removing genomic DNA
Liquid, preserved at 4 DEG C.
(2) reverse transcription reaction
Reaction system is prepared and carried out on ice, and specific system is as follows:
The reverse transcription reaction diagram of system of table 2
Reagent | Usage amount |
5 × Prime Script buffer solutions 2 | 4μl |
PrimeScript RT enzymatic mixtures I | 1μl |
RT primer mixtures | 1μl |
Remove the RNA reaction solutions of genomic DNA | 10μg |
Rnase Free ddH2O | Mend 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
Thing, put 4 DEG C of preservations.
Embodiment 4:Quantitative fluorescent PCR reacts
Template:The template that above-mentioned gained reverse transcription product reacts as quantitative fluorescent PCR, template consumption are 1 μ l.Utilize
CD157/IL-1b/MS4A6A genes (NG_032578.1) sequence, the primer of design table 3 (are had by Invitrogen (Shanghai) trade
Limit company synthesizes).
The primer sequence table of table 3
The system of PCR reactions:
PCR reactions are using TAKARA companiesPremix Ex TaqTMII (article No.s:DRR081D), this product energy
Enough suppress nonspecific reaction, carried out in the scope of broadness more quantitative.This Buffer and Hot after improvement
Start methods are applied in combination with archaeal dna polymerase TaKaRa Ex Taq HS, can carry out the Real that repeatability is good, with a high credibility
Time PCR are parsed.
The diagram of system of table 4PCR reactions
The reaction solution of fluorescent quantitation is prepared according to upper table, and the use of instrument is that the real-time fluorescence quantitative PCR instrument of ABI 7500 enters
Row real-time quantitative PCR reacts.
Real-time quantitative PCR reaction uses two-step method PCR, expands 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 the result of real-time quantitative PCR, the CT values of each gene are detected respectively.Once by taking the PBMC of 6 as an example (wherein
Sample 1-3 is non-tuberculous patients with pneumonia, sample 4-6 active tuberculosis patient), the fluorescent quantitations of 3 genes is detected respectively
CT values, by R linguistic algorithm bag Glmnet, machine learning model lasso trick/elastic pessimistic concurrency control (LASSO) is established, then obtains egg
The result (being represented with R) of white combination conversion.
The type of interpretation sample.Criterion is:Work as R>When 0.5, then it is judged as active tuberculosis patient.Conversely, it is then
Non-tuberculous pneumonia patient.
The result table of CT values and the protein combination conversion of 56, table, three, sample gene
CD157 | IL1-B | MS4A6A | R values | |
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, sample 1-3 R values are less than 0.5, are non-tuberculous pneumonia patient;Sample 4-6 R values are more than
0.5, it is active tuberculosis patient.Complied fully with actual conditions.
Embodiment 6:The present invention's is differentiating the Sensitivity and Specificity of active tuberculosis
Crowd is divided into two groups by the present embodiment:Active tuberculosis patient (24), non-tuberculous pneumonia patient (26),
By CD157/IL-1b/MS4A6A gene C T in detecting per Patients with Peripheral blood mononuclear cell (PBMC), counted after finally substituting into formula
The result and judged result of each sample are calculated, result value is diagnosed as active tuberculosis more than 0.5.
It the results are shown in Table 6,
The different type clinical sample testing result table of table 6
As can be seen from Table 6, detected using this kit in 24 active tuberculosis patients, 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, false positive rate 7.69%, illustrate the protein chip detection side of this discovery
The good sensitiveness of method.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art is not departing from the principle and objective of the present invention
In the case of above-described embodiment can be changed within the scope of the invention, change, replace and modification.
SEQUENCE LISTING
<110>Galaxy bio tech ltd of Shenzhen
<120>It is a kind of be used to distinguishing active tuberculosis patient combined with the primer pair of non-tuberculous pneumonia patient and kit
<130> 10001
<160> 6
<170> PatentIn version 3.5
21
<210> 1
<211> 20
<212> DNA
<213>It is artificial synthesized
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<210> 2
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 2
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<210> 3
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 3
tgggataacg aggcttatgt g 21
<210> 4
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 4
acaaaggaca tggagaacac c 21
<210> 5
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 5
atcgccacag agaaaaggtt a 21
<210> 6
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<213>It is artificial synthesized
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Claims (7)
1.CD157, IL-1b and MS4A6A combination are as the molecule mark for distinguishing active tuberculosis patient and non-tuberculous pneumonia patient
The purposes of will thing.
2. one group is combined for distinguishing active tuberculosis patient with the primer pair of non-tuberculous pneumonia patient, it is characterised in that institute
It is respectively SEQ ID NO to state primer pair combination: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 genes;SEQ ID NO:5-6 corresponds to MS4A6A genes.
3. a kind of kit for being used to distinguish active tuberculosis patient and non-tuberculous pneumonia patient, it is characterised in that containing having the right
Profit requires the primer pair combination described in 2.
4. described in claim 2 primer pair combination and claim 3 described in kit be used for distinguish active tuberculosis patient with it is non-
The purposes of tuberculous pneumonia patient.
5. described in claim 2 primer pair combination and claim 3 described in kit be used for distinguish active tuberculosis patient with it is non-
The method of tuberculous pneumonia patient, it is characterised in that
SEQ ID NO are respectively adopted:1-2, SEQ ID NO:3-4 or with SEQ ID NO:5-6 is as primer, to patient to be measured
PBMC DNA carries out RT-PCR reactions;
Calculate the relative expression quantity of each gene:According to the result of real-time quantitative PCR, detect that the fluorescence of 3 genes is determined respectively
CT values are measured, by R linguistic algorithm bag Glmnet, machine learning model lasso trick/elastic pessimistic concurrency control LASSO is established, then obtains egg
The result of white combination conversion, is represented with R;
As a result judge:Work as R>When 0.5, then it is judged as active tuberculosis;Conversely, it is then non-tuberculous pneumonia patient.
6. claim 5 methods described, it is characterised in that the reaction system of RT-PCR reaction is:
7. claim 5 methods described, it is characterised in that the response procedures of RT-PCR reaction are, 95 DEG C 30 seconds;95℃5
Second, 60 DEG C 30 seconds, 40 circulation.
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Cited By (3)
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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 |
CN114381509A (en) * | 2021-12-27 | 2022-04-22 | 深圳大学 | Plasma miRNA marker related to non-tuberculous pneumonia and application thereof |
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CN103074422A (en) * | 2012-12-29 | 2013-05-01 | 深圳市第三人民医院 | MS4A6A gene application |
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Cited By (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 |
CN114381509A (en) * | 2021-12-27 | 2022-04-22 | 深圳大学 | Plasma miRNA marker related to non-tuberculous pneumonia and application thereof |
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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. |