CN107190075A - For the mRNA reagents detected and purposes - Google Patents
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- CN107190075A CN107190075A CN201710498661.5A CN201710498661A CN107190075A CN 107190075 A CN107190075 A CN 107190075A CN 201710498661 A CN201710498661 A CN 201710498661A CN 107190075 A CN107190075 A CN 107190075A
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Abstract
The present invention is provided to the reagent of mRNA detections and purposes, the reagent of the mRNA detections includes gene primer and internal control primer；The present invention purposes be：Prepare the product for differentiating and combining latent infection and active tuberculosis.Beneficial technique effect：Product of the present invention can distinguish active tuberculosis infection and latent infection, healthy population and other non-tuberculosis infected studentses, and detection sensitivity and specificity are more than 85%；Sensitivity, which is more than 80%, to be checked to the patient of AIDS virus co-infection；Positive child patient detection sensitivity is cultivated to tulase and is more than 70%.Easy to operate, detection time is less than 8 hours.
Description
Technical field
The invention belongs to detection technique field, particularly for the mRNA reagents detected and purposes.
Background technology
Tuberculosis (Tuberculosis, TB) is the chronic infectious disease caused by mycobacterium tuberculosis infection, according to generation
Boundary's health organization (WHO) estimates that the whole world in 2014 is new to send out tuberculosis patient 9,600,000, every year because of the dead patient of tuberculosis about
1500000 people, current China is the second largest tuberculosis high burden country for being only second to India in the world, the annual kainogenesis activity in the whole nation
Lunger is more than 1,200,000 for property, and death toll is up to 130,000.Although having effective antituberculotic at present, tuberculosis is still
It is so the number one killer of current infectious diseases.
Due to combining patient's atypical symptom, prevention and control lungy depend on the discovery and treatment of early stage.
However, lacking the method for effective early screening and quick diagnosis at present.Phlegm mycobacterium tuberculosis microorganism checking specificity is high,
It is the goldstandard of Current Diagnostic active tuberculosis, but has that susceptibility is low, the shortcoming that time-consuming；M. tuberculosis genes are examined
Survey, although time-consuming short, but the genetic test sensitivity carried out from sputum specimen differs widely；Cellular immunology method tuberculin
Skin test (TST) and tulase interferon release test (IGRA) can not effectively distinguish active tuberculosis patient and tulase is latent
Lie prostrate the infected.
There is wretched insufficiency for the detection technique lungy of diagnostic activities now, it is impossible to meet clinical and tuberculosis pre-
The requirement of anti-control, WHO proposes new Diagnosis of Tuberculosis product technology suggestion in December, 2010：1) to improve Detection accuracy,
It is recommended that being detected using non-sputum sample (such as blood).2) patient of reply AIDS co-infection checks that sensitivity is more than
80%, positive child patient detection sensitivity is cultivated tulase more than 66%3) simple to operate.
In view of the above circumstances, it is wide present inventor has performed some in order to meet the requirement that clinical and tuberculosis prophylaxis is controlled
General research there is provided GBP1 genes, DUSP3 genes, KLF2 genes, ID3 genes, P2RY14 genes, IFITM3 genes,
9 kinds of FCGR1A genes, GBP5 genes or KLRB1 genes can as blood combination diagnosis marker gene.Detection can be passed through
The expression of 9 kinds of genes effectively distinguishes active tuberculosis patient and tulase latent infection person.
The content of the invention
, can be to above-mentioned 9 kinds of genes (GBP1 genes, DUSP3 the technical problem to be solved in the present invention is to provide a kind of reagent
Gene, KLF2 genes, ID3 genes, P2RY14 genes, IFITM3 genes, FCGR1A, GBP5 gene or KLRB1 genes) adopted
Sample and detection, that is, provide a kind of molecular marker for combining latent infection and activity combination as differentiating.
In order to solve the above technical problems, the present invention is achieved through the following technical solutions：
The present invention is provided to the reagent of mRNA detections, including gene primer, gene primer includes：Specific amplification GBP1
Primer, the primer of specific amplification DUSP3 genes, the primer of specific amplification KLF2 genes, the specific amplification ID3 bases of gene
Primer, the primer of specific amplification P2RY14 genes, primer, the specific amplification of specific amplification IFITM3 genes of cause
The primer of the primers of FCGR1A genes, specific amplification GBP5 gene primers or specific amplification KLRB1 genes.
The detection sensitivity and specificity of this reagent pair are more than 85%；Easy to operate, detection time is less than 8 hours.
Furtherly, the specific amplification GBP1 gene primer sequences are：
GBP1-F 5’-TGGCAGAGCAACAGAAAATG-3’
GBP1-R 5’-GCTCACTGAGAAGGCTTCTATTT-3’；
The specific amplification DUSP3 gene primer sequences are：
DUSP3-F 5’-CCTCAGCGCTTACTTTGAAAGG-3’
DUSP3-R 5’-GAGCACCCGGCCATTCT-3’；
The specific amplification KLF2 gene primer sequences are：
KLF2-F 5’-CGGCAAGACCTACACCAAGAG-3’
KLF2-R 5’-CGTCTGAGCGCGCAAACT-3’；
The specific amplification ID3 gene primer sequences are：
ID3-F 5’-CCCTGGACCCCCTGATG-3’
ID3-R 5’-CCTTTTGTCGTTGGAGATGACA-3’；
The specific amplification P2RY14 gene primer sequences are：
P2RY14-F 5’-AATCTTAAAAGGCCTCTGCCTT-3’
P2RY14-R 5’-AGGTTCTGAGAGCAGGATTCAT-3’；
The specific amplification IFITM3 gene primer sequences are：
IFITM3-F 5’-CGTCTGGTCCCTGTTCAACAC-3’
IFITM3-R 5’-CCATCTTCCTGTCCCTAGACTTC-3’；
The specific amplification FCGR1A gene primer sequences are：
FCGR1A-F 5’-AAGCGCAGCCCTGAGTTG-3’
FCGR1A-R 5’-TGCCAGATAGAAAAGGACATGAAA-3’；
The specific amplification GBP5 gene primer sequences are：
GBP5-F 5’-GCTGGCAGAGCAACAGAAAA-3’
GBP5-R 5’-CGTGCTGGAGCTCACTGAGA-3’
The specific amplification KLRB1 gene primer sequences are：
KLRB1-F 5’-TCTTCCTCGGGATGTCTGTCA-3’
KLRB1-R 5’-GACAAGGAGAATAATCCCAGCACAG-3’。
Furtherly, this is used for the reagent of real-time fluorescence quantitative PCR diagnostic method, in addition to 4 kinds of internal control primers, is respectively：
Specific amplification GAPDH internal control primers, specific amplification ACTB internal control primers, specific amplification B2M internal control primers and specificity
Expand RPLP0 internal control primers.
Furtherly, the specific amplification GAPDH internal control primers are：
GAPDH-F 5’-TTTGGTATCGTGGAAGGACT-3’
GAPDH-R 5’-CATCACGCCACAGTTTCCC-3’
The specific amplification ACTB internal control primers are：
ACTB-F 5’-CCTGGCACCCAGCACAAT-3’
ACTB-R 5’-GCCGATCCACACGGAGTACT-3’
The specific amplification B2M internal control primers are：
B2M-F 5’-TCTCGCTCCGTGGCCTTAG-3’
B2M-R 5’-CTGCTGGATGACGTGAGTAAACC-3’
The specific amplification RPLP0 internal control primers are：
RPLP0-F 5’-TGAAGTGCTTGATATCACAGAGGAA-3’
RPLP0-R 5’-AGACAGACACTGGCAACATTGC-3’。
Furtherly, the purposes for the mRNA reagents detected：Differentiate for preparing with reference to latent infection and activity
The product of tuberculosis.
Furtherly, the purposes for the mRNA reagents detected：For preparing using real-time fluorescence quantitative PCR diagnostic method
Product.
Furtherly, the purposes for the mRNA reagents detected：For preparing using real-time fluorescence quantitative PCR diagnostic method
Kit, the kit can detect GBP1 genes, DUSP3 genes, KLF2 genes, ID3 genes, P2RY14 genes, IFITM3
The expression of gene, GBP5 genes or KLRB1 genes, so as to whether be provided for diagnosis with active tuberculosis with reference to letter
Breath.
Beneficial technique effect
, can be accurately and simultaneously to GBP1 genes, DUSP3 genes, KLF2 genes, ID3 the invention provides a kind of reagent
Gene, P2RY14 genes, IFITM3 genes, FCGR1A genes, GBP5 genes or KLRB1 genes are sampled, characterized and locked
Specific marker gene, makes Diagnosis of Tuberculosis more accurate quick.It can be prepared using the reagent of the present invention and combine latent sense for differentiating
The product that dye and activity are combined.
Product of the present invention can distinguish active tuberculosis infection and latent infection, healthy population and other non-tuberculosis infections
Person, detection sensitivity and specificity are more than 85%；Sensitivity, which is more than 80%, to be checked to the patient of AIDS virus co-infection；It is right
The positive child patient detection sensitivity of tulase culture is more than 70%.Easy to operate, detection time is less than 8 hours.
The product being made of the present invention is included：Differentiated with real-time fluorescence quantitative PCR detection and combine latent infection and work
The product that dynamic property is combined.
Prepared using (and using real time fluorescence quantifying PCR method) of the invention and differentiate that combining latent infection and activity combines
During the product of disease, at least preferably include the primers of a pair of specific amplification GBP1 genes, specific amplification DUSP3 genes and draw
Thing, the primer of specific amplification KLF2 genes, the primers of specific amplification ID3 genes, specific amplification P2RY14 genes draw
Thing, the primer of specific amplification IFITM3 genes, the primer of specific amplification FCGR1A genes, specific amplification GBP5 genes or
Specific amplification KLRB1 genes.
Using the present invention kit, can detect mankind GBP1 genes, DUSP3 genes, KLF2 genes, ID3 genes,
P2RY14 genes, IFITM3 genes, the expression of GBP5 genes or KLRB1 genes, so that whether diagnosing human is with activity
Property tuberculosis.
The experiment proved that, GBP1 genes of the present invention, DUSP3 genes, KLF2 genes, ID3 genes, P2RY14 genes,
IFITM3 genes, GBP5 genes or KLRB1 genes can as diagnosis of tuberculosis specific marker gene, make Diagnosis of Tuberculosis more accurate
Quickly.
Brief description of the drawings
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.
Fig. 1 be the GBP1 genes of the embodiment of the present invention 1, DUSP3 genes, KLF2 genes, ID3 genes, P2RY14 genes,
The quantitative RT-PCR result figure of IFITM3 genes, GBP5 genes or KLRB1 the genes differential expression in human blood.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described further：
Example 1：
Crowd is divided into three groups by the present embodiment：Tuberculosis patient, latent infection crowd and healthy population (each 20), pass through
Detect GBP1 genes, DUSP3 genes, KLF2 genes, ID3 genes, P2RY14 genes, IFITM3 genes, GBP5 genes in blood
It is in obvious up-regulated expression trend in tuberculosis patient.KLRB1 genes become in tuberculosis patient in substantially downward expression
Gesture.
The present embodiment with quantitative RT-PCR method detect GBP1 genes, DUSP3 genes, KLF2 genes, ID3 genes,
P2RY14 genes, IFITM3 genes, the expression change of GBP5 genes or KLRB1 genes.Comprise the following steps that
Step one：Whole blood RNA extraction；
Using (the article No. of Qiagene companies：DP433) to being stored in RNA stabilizer (article No.s：DP440 blood sample)
Carry out RNA extractings.Concrete operations are：Go bail for and be stored in the blood sample room temperature placement of RNA stabilizers, it is then every with 6600 turns of rotating speed
Minute centrifugation 10 minutes, then uses pipettor supernatant, takes precipitation to enter next step, and purification storage first is stable in blood rna
The blood sample of agent：First blood sample room temperature is placed or 37 DEG C of water-baths, it is warmed to room temperature；Then with the revolutions per minute of rotating speed 6600
Zhongli's heart 10min；Then inhaled with pipettor and abandon supernatant, take precipitation to enter next step；1mL is added into precipitation without RNase's
ddH2O, precipitation is completely dissolved with pipettor piping and druming；With 6600 rpms of centrifugation 10min of rotating speed, inhaled with pipettor and abandon supernatant
Liquid；240 μ l suspension RSB are slowly added to, being blown and beaten repeatedly with pipettor makes precipitation dissolving complete；Added into the sample after dissolving
200 μ l lysate RL, then add 20 μ l Proteinase K solution, fully reverse to mix, 55 DEG C of incubation 10min, run therebetween
Mix 2-3 times, until solution becomes limpid；All solution are transferred in Filter column CS, 12,000 rpms of centrifugation 3min,
The supernatant in collecting pipe is drawn into the new centrifuge tube without RNase；220 μ l absolute ethyl alcohol is added, is mixed, it is molten by what is obtained
Liquid and precipitation are transferred in adsorption column CR2 together, 12,000 rpms of centrifugation 1min, outwell the waste liquid in collecting pipe；To absorption
350 μ l protein liquid removals RW1,12000rpm rpms of centrifugation 15-30sec are added in post CR2, the waste liquid in collecting pipe is outwelled；
80 μ l DNase I (10 μ l DNase I+70 μ l Buffer RDD) working solution is added to adsorption column CR2 centers, room temperature is placed
15min；350 μ l protein liquid removals RW1,12,000 rpms of centrifugation 15-30sec are added into adsorption column CR2, collecting pipe is outwelled
In waste liquid；500 μ l rinsing liquid RW are added into adsorption column CR2,2min, 12,000 rpms of centrifugation 2min are stored at room temperature,
The waste liquid in collecting pipe is outwelled, the operation repeated the above steps 1 to 4 times；12,000 rpms of centrifugation 2min, outwell waste liquid；
Adsorption column CR2 is placed in room temperature and places 3min, thoroughly to dry rinsing liquid remaining in sorbing material；Adsorption column CR2 is transferred to
In one new RNase-Free centrifuge tube, add ddH2O room temperatures of the 30-50 μ l without RNase and place 2min, 12,000 revolutions per minute
Zhongli heart 2min, obtains RNA solution.
Step 2：Reverse transcription
Using Promega reverse transcription reagent box (article No.：A5001), the μ g of RNA 1 for taking step 2 to obtain are inverted
Record, substep is as follows
(1) each component and brief centrifugation are first dissolved before, is configured according to table 1：
| Component | Volume |
| RNA | 1μg |
| 0.5μg/μl Primer(Oligo dT) | 1μl |
The system configured according to table 1, then 70 DEG C of warm bath are taken out and are placed at least 5min on ice immediately after 5 minutes,
Centrifuge 10s.
(2) reverse transcription system is configured according to table 2：
| Component | Volume |
| GoScript 5×Reaction Buffer | 4.0μl |
| 2.5mM MgCl2 | 2.0μl |
| 0.5mM PCR Nucleotide Mix | 1.0μl |
| 20unit RNasin | 0.5μl |
| GoScript Reverse Transcriptase | 1.0μl |
The solution of table 1 and the solution of table 2 are mixed and the water of no RNase is mended to 20 μ l.Reverse transcription reaction is carried out in PCR instrument kind,
Response procedures are 25 DEG C of 5min；42℃1h；70℃15min；4 DEG C of preservations.
Step 3：Quantitative fluorescent PCR reacts
Template：The template that above-mentioned reverse transcription product reacts as quantitative fluorescent PCR, template consumption is 5 μ l.Utilize GBP1 bases
Because of (NM_002053.2), DUSP3 genes (NM_004090.3), KLF2 genes (NM_016270.3), ID3 genes (NM_
002167.4), P2RY14 genes (NM_001081455.1), IFITM3 genes (NM_021034.2), GBP5 genes (NM_
052942.3), KLRB1 genes (NM_002258.2) and GADPH genes (NM_002046.4), ACTB genes (NM_
001101.3), B2M genes (NM_004048.2), RPLP0 genes (NM_001002.3) sequence, with Primer Premier 5
Software Design primers.
Primer is synthesized by Invitrogen (Shanghai) Trading Co., Ltd., design such as table 3：
The system of PCR reactions：
PCR reactions use the PowerUp of ABI companiesTM SYBRTMGreen Master Mix (article No.s：A25742)
Table 4
| SYBR 2×PowerUpTM SYBRTM Green Master Mix | 10μl |
| PCR forward primers | 0.4μl |
| PCR reverse primers | 0.4μl |
| CDNA templates | 5μl |
| ddH2O | 4.2μl |
| Total | 20μl |
The reaction solution of fluorescent quantitation is prepared according to upper table, and is reacted using instrument for the real-time fluorescence quantitative PCRs of ABI 7500.
Real-time quantitative PCR reaction uses two-step method PCR, expands standardization program：50℃2min；95℃2min；95 DEG C 15 seconds,
58 DEG C 30 seconds, 45 circulation.
According to the result of real-time quantitative PCR, Treatment Analysis is carried out to result with ABI7500software v2.0.6, with
GADPH genes, ACTB genes, B2M genes, the geometric average of RPLP0 genes and for reference gene, utilize 2-ΔΔCTMethod meter
Tubercular and latent infection crowd are calculated relative to Healthy People.As a result as shown in figure 1, wherein abscissa represents different people
Group, ordinate represents relative expression quantity, and ordinate shows that more greatly its expression is higher, as a result shown, GBP1 genes, DUSP3
The expression of gene, KLF2 genes, ID3 genes, P2RY14 genes, IFITM3 genes, GBP5 genes in tuberculosis patient is bright
The aobvious expression for being higher than latent infection and healthy population, significant difference (P<0.05).KLRB1 genes are in tuberculosis patient
Expression is significantly lower than latent infection and the expression of healthy population, significant difference (P<0.05).In Fig. 1, " TB "
Refer to active tuberculosis the infected, " LTBI " refers to latent tuberculosis infects person, and " HC " refers to normal healthy controls.According to above-mentioned experimental result,
GBP1 genes, DUSP3 genes, KLF2 bases can be designed by fluorescence quantitative RT-RCR diagnosis of tuberculosis latent infection and active tuberculosis
This 9 kinds in cause, ID3 genes, P2RY14 genes, IFITM3 genes, GBP5 genes, the PCR primer of KLRB1 genes, detection blood
The expression quantity of gene, if GBP1 genes, DUSP3 genes, KLF2 genes, ID3 genes, P2RY14 genes, IFITM3 genes,
The expression quantity of GBP5 genes is significantly raised, and the expression quantity of KLRB1 genes is significantly reduced, then illustrates that tuberculate possibility is high, instead
It is then low, preferably to distinguish active tuberculosis the infected and latent tuberculosis infects person.
Claims (7)
1. the reagent detected for mRNA, including gene primer, it is characterised in that gene primer includes：Specific amplification GBP1
Primer, the primer of specific amplification DUSP3 genes, the primer of specific amplification KLF2 genes, the specific amplification ID3 bases of gene
Primer, the primer of specific amplification P2RY14 genes, primer, the specific amplification of specific amplification IFITM3 genes of cause
The primer of the primers of FCGR1A genes, specific amplification GBP5 gene primers or specific amplification KLRB1 genes；This reagent pair
Detection sensitivity and specificity are more than 85%；Easy to operate, detection time is less than 8 hours.
2. the reagent according to claim 1 detected for mRNA, it is characterised in that
The specific amplification GBP1 gene primer sequences are：
GBP1-F 5’-TGGCAGAGCAACAGAAAATG-3’
GBP1-R 5’-GCTCACTGAGAAGGCTTCTATTT-3’；
The specific amplification DUSP3 gene primer sequences are：
DUSP3-F 5’-CCTCAGCGCTTACTTTGAAAGG-3’
DUSP3-R 5’-GAGCACCCGGCCATTCT-3’；
The specific amplification KLF2 gene primer sequences are：
KLF2-F 5’-CGGCAAGACCTACACCAAGAG-3’
KLF2-R 5’-CGTCTGAGCGCGCAAACT-3’；
The specific amplification ID3 gene primer sequences are：
ID3-F 5’-CCCTGGACCCCCTGATG-3’
ID3-R 5’-CCTTTTGTCGTTGGAGATGACA-3’；
The specific amplification P2RY14 gene primer sequences are：
P2RY14-F 5’-AATCTTAAAAGGCCTCTGCCTT-3’
P2RY14-R 5’-AGGTTCTGAGAGCAGGATTCAT-3’；
The specific amplification IFITM3 gene primer sequences are：
IFITM3-F 5’-CGTCTGGTCCCTGTTCAACAC-3’
IFITM3-R 5’-CCATCTTCCTGTCCCTAGACTTC-3’；
The specific amplification FCGR1A gene primer sequences are：
FCGR1A-F 5’-AAGCGCAGCCCTGAGTTG-3’
FCGR1A-R 5’-TGCCAGATAGAAAAGGACATGAAA-3’；
The specific amplification GBP5 gene primer sequences are：
GBP5-F 5’-GCTGGCAGAGCAACAGAAAA-3’
GBP5-R 5’-CGTGCTGGAGCTCACTGAGA-3’
The specific amplification KLRB1 gene primer sequences are：
KLRB1-F 5’-TCTTCCTCGGGATGTCTGTCA-3’
KLRB1-R 5’-GACAAGGAGAATAATCCCAGCACAG-3’。
3. the reagent according to claim 1 detected for mRNA, it is characterised in that including 4 kinds of internal control primers, difference
For：It is specific amplification GAPDH internal control primers, specific amplification ACTB internal control primers, specific amplification B2M internal control primers, special
Property amplification RPLP0 internal control primers.
4. the reagent according to claim 3 detected for mRNA, it is characterised in that
The specific amplification GAPDH internal control primers are：
GAPDH-F 5’-TTTGGTATCGTGGAAGGACT-3’
GAPDH-R 5’-CATCACGCCACAGTTTCCC-3’
The specific amplification ACTB internal control primers are：
ACTB-F 5’-CCTGGCACCCAGCACAAT-3’
ACTB-R 5’-GCCGATCCACACGGAGTACT-3’
The specific amplification B2M internal control primers are：
B2M-F 5’-TCTCGCTCCGTGGCCTTAG-3’
B2M-R 5’-CTGCTGGATGACGTGAGTAAACC-3’
The specific amplification RPLP0 internal control primers are：
RPLP0-F 5’-TGAAGTGCTTGATATCACAGAGGAA-3’
RPLP0-R 5’-AGACAGACACTGGCAACATTGC-3’。
5. the purposes for the mRNA reagents detected, it is characterised in that differentiate for preparing with reference to latent infection and activity
The product of tuberculosis.
6. the purposes for the mRNA reagents detected, it is characterised in that for preparing using real-time fluorescence quantitative PCR diagnostic method
Product.
7. the purposes for the mRNA reagents detected, it is characterised in that for preparing using real-time fluorescence quantitative PCR diagnostic method
Kit, the kit can detect patient GBP1 genes, DUSP3 genes, KLF2 genes, ID3 genes, P2RY14 genes,
The expression of IFITM3 genes, GBP5 genes and KLRB1 genes, so as to whether provide ginseng with active tuberculosis for diagnosis
Examine information.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110527721A (en) * | 2019-09-10 | 2019-12-03 | 深圳市优圣康生物科技有限公司 | A kind of oldness tuberculosis marker and its application |
| CN111440866A (en) * | 2019-01-17 | 2020-07-24 | 中山大学附属第六医院 | Application of DUSP3 gene methylation detection reagent in preparation of colorectal cancer prognosis diagnosis reagent |
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| CN111440866B (en) * | 2019-01-17 | 2023-08-11 | 中山大学附属第六医院 | Application of DUSP3 gene methylation detection reagent in preparation of colorectal cancer prognosis diagnosis reagent |
| CN110527721A (en) * | 2019-09-10 | 2019-12-03 | 深圳市优圣康生物科技有限公司 | A kind of oldness tuberculosis marker and its application |
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