CN109825508A - A kind of biomarker for assessing AIDS merging tuberculosis infection - Google Patents
A kind of biomarker for assessing AIDS merging tuberculosis infection Download PDFInfo
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Abstract
The present invention relates to technical field of bioengineering, specifically provide the biomarker and application thereof for merging tuberculosis infection for assessing AIDS, merge the product of tuberculosis infection, a kind of Primer composition and application thereof the present invention also provides a kind of for assessing AIDS, on this basis, invention further provides the kits that assessment AIDS merges tuberculosis infection, and screening AIDS to merge the system of the biological sample of tuberculosis infection.Experiments have shown that, relative expression quantity in PBMC of the biomarker provided according to the present invention in human blood can be used for the risk size for judging that AIDS is caused to merge tuberculosis infection, therefore the combination of these biomarkers can be used as the early warning factor that evaluation AIDS merges tuberculosis infection, keep clinical treatment more personalized, this all has directive significance to the diagnosing and treating of disease.
Description
Technical field
The present invention relates to technical field of bioengineering, specially a kind of biology for assessing AIDS merging tuberculosis infection
Marker.
Background technique
AIDS is referred to as the No.1 infectious disease killer in the world, so far from U.S.'s report first case AIDS case in 1981,
The whole world has added up more than 6,000 ten thousand people of aids infection virus, has resulted in more than 2,700 ten thousand people death.Annual about 2,000,000 people
Die of AIDS and related disease (WHO 2013).According to statistics China HIV infection number in 2013 about 780,000 or so.It cuts
In the only end of the year 2014, Shenzhen HIV/AIDS patient of surviving is 8092, wherein patient AIDS 2651, and year by year with 15~
20% or so speed is increasing, and AIDS not only has become the public health problem for seriously threatening city's people's health, and
Influence economic development and social stability.
Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) infection is AIDS (Acquired
Immunodeficiency Syndrome, AIDS) most common opportunistic infections and underlying cause of death.China's investigation display, HIV/
The double infection person that AIDS merges tuberculosis (HIV/TB) is about 20,000, infection rate 14.44%.HIV/TB infection seriously affects me
State's AIDS patient's prognosis and life quality.However, the clinical manifestation of HIV/TB concurrent infection lacks specific, difficult diagnosis,
Ineffective, case fatality rate is higher.Therefore, the specific immune response spectrum for understanding HIV/TB infection, further develops new morning
Phase diagnosis index and specific treatment target spot become the task of top priority of the current HIV/AIDS prevention in China.
Summary of the invention
The technical problem to be solved in the present invention is to provide it is a kind of assessment AIDS merge tuberculosis infection biomarker, with
And the biomarker is applied in the product that assessment AIDS merges tuberculosis infection.The present invention also provides a kind of primer sets
Close object and purposes and a kind of system of the biological sample of screening AIDS merging tuberculosis infection.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
First aspect present invention provides a kind of biomarker, and the biomarker includes CDKN1C gene.
In a preferred example, the biomarker also includes SOCS3 gene.
In a preferred example, the biomarker also includes SFXN1 gene.
Second aspect of the present invention provides a kind of biomarker, the biomarker be CDKN1C gene and/or
The albumen of mRNA expressed by SOCS3 gene and/or SFXN1 gene or its coding.
Third aspect present invention provides the production that above-mentioned biomarker merges tuberculosis infection in preparation detection AIDS
Application in product.
Fourth aspect present invention provides a kind of product, and the product includes for detecting above-mentioned biomarker
The reagent of the real-time quantitative PCR of expression quantity.
Fifth aspect present invention provides a kind of examination of the real-time quantitative PCR of expression quantity for detecting above-mentioned biomarker
Agent is used to assess the purposes on the product that AIDS merges tuberculosis infection in preparation.
Sixth aspect present invention provides a kind of Primer composition, which includes to expand SFXN1 gene extremely
Few pair of primers, at least pair of primers for expanding SOCS3 gene and at least one of the pair of primers for expanding CDKN1C gene
Or a variety of combinations with the primer of amplification β-Actin reference gene.
In a preferred example, the primer of the amplification SFXN1 gene includes SEQ ID NO.1 and SEQ ID NO.2 institute
The nucleotide sequence shown.
In a preferred example, the primer of the amplification SOCS3 gene includes SEQ ID NO.3 and SEQ ID NO.4 institute
The nucleotide sequence shown.
In a preferred example, the primer of the amplification CDKN1C gene includes SEQ ID NO.5 and SEQ ID NO.6 institute
The nucleotide sequence shown.
In a preferred example, the primer of the amplification β-Actin reference gene includes SEQ ID NO.7 and SEQ ID
Nucleotide sequence shown in NO.8.
Seventh aspect present invention provides above-mentioned Primer composition and merges tuberculosis infection for assessing AIDS in preparation
Purposes on kit.
Eighth aspect present invention provides a kind of kit, and the kit includes Primer composition described above.
In a preferred example, the kit also includes quantitative fluorescent PCR reagent.
In a preferred example, the quantitative fluorescent PCR reagent comes from Takara company SYBR Premix Ex Taq II
Reagent in RR820 kit.
In a preferred example, the kit also includes Reverse Transcription.
In a preferred example, the Reverse Transcription comes from Takara company PrimeScriptTMII 1st strand
Reagent in cDNA Synthesis Kit 6210A kit.
In a preferred example, the kit also includes that RNA extracts reagent.
In a preferred example, it is that Trizol method extracts reagent that the RNA, which extracts reagent,.
In a preferred example, the kit also includes PBMC separation agent.
In a preferred example, the PBMC separation agent is drenched from the Ficoll that GE company article No. is #17-1440-03
Bar cell separating liquid.
Ninth aspect present invention provides a kind of system of the biological sample of screening AIDS merging tuberculosis infection, described
System includes:
Real-time quantitative PCR amplification device is provided with SFXN1 gene, SOCS3 base in the real-time quantitative PCR amplification device
The specific primer of cause, CDKN1C gene and β-Actin reference gene, to utilize the specific primer, to biological sample
The sample of nucleic acid of middle extraction carries out real-time quantitative PCR amplification, respectively obtain SFXN1 gene, SOCS3 gene, CDKN1C gene and
The Ct value of β-Actin reference gene and be calculated based on Ct value 2-ΔΔCtValue；
Judgment means, the judgment means and the real-time quantitative PCR amplification module are connected, by SFXN1 gene,
The expression of SOCS3 gene and CDKN1C gene is judged, the biological sample of tuberculosis infection is merged for screening AIDS；
In a preferred example, the judgement for merging the biological sample of tuberculosis infection for screening AIDS in the judgment means
Benchmark are as follows: the mrna expression amount times ratio > 2.5 and/or the SOCS3 gene in biological sample of the SFXN1 gene in biological sample
Mrna expression amount times ratio > 3 and/or CDKN1C gene mrna expression amount times ratio < 0.2, wherein in biological sample
SFXN1 gene/SOCS3 gene/CDKN1C gene mrna expression amount times ratio=(SFXN1 gene in biological sample/
SOCS3 gene/CDKN1C gene m RNA relative expression quantity)/(SFXN1 gene/SOCS3 gene in control sample/
The mRNA relative expression quantity of CDKN1C gene), SFXN1 gene/SOCS3 gene/CDKN1C gene mRNA in biological sample
Relative expression quantity=(the SFXN1 gene of biological sample/SOCS3 gene/CDKN1C gene 2-ΔΔCtThe β-of)/biological sample
The 2 of Actin reference gene-ΔΔCt。
In a preferred example, the SFXN1 gene-specific primer includes SEQ ID NO.1 and SEQ ID NO.2 institute
The nucleotide sequence shown.
In a preferred example, the SOCS3 gene-specific primer includes SEQ ID NO.3 and SEQ ID NO.4 institute
The nucleotide sequence shown.
In a preferred example, the CDKN1C gene-specific primer includes SEQ ID NO.5 and SEQ ID NO.6 institute
The nucleotide sequence shown.
In a preferred example, the β-Actin reference gene specific primer includes SEQ ID NO.7 and SEQ ID
Nucleotide sequence shown in NO.8.
In a preferred example, the system also includes post transcription cloning device, for converting the RNA of biological sample to
CDNA, the post transcription cloning module are connected with real-time quantitative PCR amplification device.
In a preferred example, the system also includes nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus and described
Post transcription cloning device is connected, for extracting the sample of nucleic acid in the biological sample.
" expression quantity of biomarker " refers to the mRNA amount or albumen of biomarker gene expression in the present invention
Amount.
" SFXN1 " is also referred to as three carboxyls and carries albumen TCC in the present invention, is one of sideroflexin family member, fixed
It is a kind of Eukaryotic evolution conservative protein on mitochondrial membrane.
" SOCS3 " is that cytokine signaling inhibits (suppressor of cytokine in the present invention
Signaling, SOCS) molecule families important component, by multiple inflammatory factors and anti-inflammatory factors inducing expression (such as IL-6, IL-
10, IFN-γ, LPS etc.), and inhibit the signal transduction of panimmunity molecule.
" CDKN1C " belongs to Cyclin kinase inhibitor (cyclin-dependent in the present invention
Kinases inhibitors, CDKI) family important member.
The invention has the following advantages:
The present invention provides a kind of marker for assessing AIDS merging tuberculosis infection, the experiment proved that, institute of the present invention
PBMC (peripheral blood mononuclear cells) of the specific gene in the human blood that AIDS merges tuberculosis infection in the marker of column
In the relative expression quantity risk size that can be used for judging that AIDS is caused to merge tuberculosis infection, therefore the combination of these genes can be made
The early warning factor for merging tuberculosis infection for evaluation AIDS, keeps clinical treatment more personalized, this diagnosing and treating to disease
All there is directive significance.The present invention also provides Primer composition and include the kit of Primer composition, the Primer composition
With the relative expression quantity that can be used for detecting CDKN1C gene, SOCS3 gene and SFXN1 gene with kit, experiments have shown that this draws
Each pair of primer pair in compositions and while carrying out multiple fluorescence quantitative PCR respectively with kit, have high sensitivity and specificity
Strong feature can accurately measure the relative expression quantity of CDKN1C gene, SOCS3 gene and SFXN1 gene.
Detailed description of the invention
Fig. 1: three kinds of biomarker multiple mutation analysis figures.Control: 4,50 physical examination of healthy population samples of group；HIV/
TD: 1,100 HIV/TD clinical samples of group；HIV: 2,100 simple HIV infection clinical samples of group；TD: 3,100 tuberculosis of group
Patient's sample.Scheme the mrna expression amount multiple mutation analysis figure of the SFXN1 gene of A: four groups of samples；B: four groups of samples of figure
The mrna expression amount multiple mutation analysis figure of SOCS3 gene；Scheme the mrna expression amount multiple of the CDKN1C gene of C: four groups of samples
Mutation analysis figure.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail, it should be noted that these embodiments are only
It is illustrative, and is not considered as limiting the invention.Unless otherwise specified, technological means employed in embodiment is
Conventional means well-known to those skilled in the art, used reagent, product and instrument are also available commercial.Not in detail
The various processes and method carefully described are conventional method as known in the art, such as Sambrook molecular cloning experiment handbook
(Sambrook J&Russell DW, Molecular cloning:a laboratory manual, 2001), or according to manufacture
The condition of manufacturers instruction suggestion.
Biological sample used in embodiment is taken from The Third People's Hospital of Shenzhen in the present invention.The regulation of RNA-Seq
And its follow-up test has obtained the approval of the Hospital Ethical Committee.All patients fill in Written informed consent, award
Power uses their sample.
Embodiment 1
Present embodiments provide marker, primer sets and its application that assessment AIDS merges tuberculosis infection.
Assessment AIDS merges the marker of tuberculosis infection: commenting to preferably merge tuberculosis infection to AIDS
Estimate, using Healthy People, the sample of HIV/TD patient, simple HIV infection patient and tuberculosis patient (TD), for multiple markers
A large amount of experiment screening is carried out, by comparing the difference of expression quantity of multiple markers in different types of biological sample, most
Three SFXN1 gene, SOCS3 gene and CDKN1C gene genes have been filtered out eventually as evaluation AIDS merges tuberculosis infection
Biomarker (while being detected for SFXN1 gene, SOCS3 gene and CDKN1C gene, as a result mutual reference, more can
The crowd that AIDS merges tuberculosis infection occurs and does not occur for effective and efficient distinguish).
It assesses the primer sets that AIDS merges tuberculosis infection: being directed to SFXN1 gene, SOCS3 gene, CDKN1C gene respectively
A pair of of fluorescence quantification PCR primer is respectively devised with β-Actin reference gene, wherein β-Actin reference gene is quantitative fluorescent PCR
Reference gene.
Particular sequence is shown in Table 1:
Table 1: primer sets sequence
Above-mentioned primer is synthesized by BGI Technology Solutions Co., Ltd. and passes through regular-PCR specificity verification.
Above-mentioned primer is after determining using SFXN1 gene, SOCS3 gene and CDKN1C gene as marker use in conjunction
It is obtained by repeatedly screening, it is demonstrated experimentally that these primers have the characteristics that high specificity, high sensitivity and reproducible, and
SFXN1 gene, SOCS3 gene, CDKN1C gene primer mixed respectively with the primer of β-Actin reference gene carry out it is multiple glimmering
It,, can be more accurate when carrying out quantitative fluorescent PCR using these primers without interfering with each other between primer when Fluorescent Quantitative PCR expands
Carry out the relative quantification of target gene.
Above-mentioned biomarker and Primer composition can be respectively used to the product that preparation detection AIDS merges tuberculosis infection,
It such as can be used for preparing the kit that detection AIDS merges tuberculosis infection, which includes for detecting above-mentioned biomarker
The reagent of the real-time quantitative PCR of the expression quantity of object.
Embodiment 2
It present embodiments provides a kind of kit and its application, the kit includes:
(1) primer sets in embodiment 1: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4,
SEQ ID NO.5,SEQ ID NO.6,SEQ ID NO.7,SEQ ID NO.8；
(2) quantitative fluorescent PCR reagent: the examination in Takara company SYBR Premix Ex Taq II RR820 kit
Agent；
(3) Reverse Transcription: Takara company PrimeScriptTM II 1st strand cDNA Synthesis Kit
Reagent in 6210A kit；
(4) RNA extracts reagent: Trizol method extracts reagent；
(5) PBMC separation agent: the Ficoll lymphocyte separation medium #17-1440-03 of GE company.
It is demonstrated experimentally that the reagent of above-mentioned (1), (2), (3), (4) and (5) is used in combination, effect is more superior, embodies
Time saving, economic, the repeatable high, high sensitivity and high specificity the characteristics of.And randomly selected using primer-design software
SFXN1 gene, SOCS3 gene, CDKN1C gene and the primer of β-Actin reference gene are combined, and more or less deposits
Specificity is strong, sensitivity is not high and the various defects such as repeatability is bad.
Mentioned reagent box can be used for merging for assessing AIDS the biological sample of tuberculosis infection.
Embodiment 3
The present embodiment merges the clinical samples of tuberculosis infection and the gene expression product mRNA of other samples to AIDS
Expression quantity is determined, it is determined that AIDS merges three kinds of biomarkers of tuberculosis infection, and qualification process is as follows:
One, samples selection
Four group objects are had chosen altogether is used as detection samples sources, as follows:
1:100 HIV/TD patients of group (HIV/TD: AIDS merges tuberculosis infection)；
2:100 simple HIV infection patients of group；
3:100 tuberculosis patients (TD) of group；
Group 4: control group, 50 physical examination of healthy population.
Two .mRNA are extracted
1.PBMC separation
Peripheral blood about 5mL is respectively adopted to the group 1- group 4 in one, using Ficoll density-gradient centrifugation method, utilizes GE company goods
Number for #17-1440-03 Ficoll lymphocyte separation medium separate PBMC peripheral blood lymphocytes, separate the specific step of PBMC
Rapid following (ratio involved in following steps refers both to volume ratio unless otherwise specified):
(1) after 5ml heparin tube collect specimen, accession designation number, date.
(2) 5ml blood specimen and equivalent PBS are mixed into (blood: PBS=1:1).
(3) it takes sterile 15ml centrifuge tube to mark, Ficoll lymphocyte separation medium (GE company, article No. #17- is added
1440-03) 5ml is rolled, and the mixed liquor of the PBS+ blood for the 10ml for being slowly added to mix in (2) along tube wall keeps liquid level layering
(PBS: blood: lymphocyte separation medium=1:1:1).
(4) 2 DEG C of centrifugation 2000rpm, 20min (rising g is Max, and decline G is slow) i.e. P3 program.
(5) liquid is divided into four layers after being centrifuged: being from top to bottom followed successively by blood plasma+PBS, PBMC, lymphocyte separation medium and blood
Cell, drawing second layer PBMC, (i.e. peripheral blood mononuclear cells includes lymphocyte, monocyte, Dendritic Cells and other
A small amount of cell) it is placed in that new label is good to be added in the sterile 15ml centrifuge tube of 10ml PBS.
(6) cell is cleaned, P6 program (2000rpm, 5min, 25 DEG C) centrifugation after centrifugation (if having red precipitate can after mixing
With erythrocyte cracked liquid (ACK) 5ml splitting erythrocyte 5min, 10ml PBS cleaning is added).
(7) after being centrifuged, supernatant liquor is removed, tube bottom is the PBMC deposited；
(8) 2ml frozen stock solution is added with capillary or sample loading gun in the PBMC of deposition, piping and druming mixes；
(9) cell suspension mixed is separately added into cell cryopreservation tube, every each 1ml of pipe, mark number, date, by cryopreservation tube
The program temperature reduction box for being reduced to room temperature is gone to, is put into -80 DEG C of refrigerators, cryopreservation tube is gone into 9*9 freezing storing box again after at least 2 hours
Middle Liquid nitrogen storage is spare.
2. Total RNAs extraction
PBMC is cracked using Trizol method, extract total serum IgE and is dissolved in 20ul H2In O, obtain concentration be 100~
The RNA solution of 300ng/ul takes 1.5ul RNA solution for post transcription cloning respectively.Total RNAs extraction concrete operation step is such as
Under:
(1) above-mentioned 1 pipe is taken to freeze PBMC, 37 DEG C of water-bath dissolutions are added 1ml Trizol, shake 1 minute on oscillator.
It is placed at room temperature for 5min, decomposes ribosome completely.
(2) 12000rpm is centrifuged 5min, abandons precipitating.
(3) it takes supernatant that 200ul chloroform is added, is placed at room temperature for 10min after acutely shaking 15s.
(4) 12000g is centrifuged 15min under the conditions of 4 DEG C, and liquid in pipe is divided into three layers, water sample layer is transferred to new centrifugation
500ul isopropanol is added in Guan Zhong, and after being mixed by inversion, room temperature stands 10min.
(5) under the conditions of 4 DEG C, 12000g is centrifuged 10min, abandons supernatant, stays RNA precipitate in tube bottom.75% (v/ of 1ml is added
V) ethanol water, shakes 5s on the oscillator, and washing precipitating is primary.
(6) under the conditions of 4 DEG C, 8000g is centrifuged 5min, abandons supernatant, and DEPC water 20ul dissolution is added in drying at room temperature 5min
RNA precipitate.
Three .qRT-PCR mRNA and analysis
1. reverse transcription is cDNA
Use Takara company PrimeScriptTMThe examination of II 1st strand cDNA Synthesis Kit 6210A
Agent box, the RNA1.5ml that step 2 is extracted carry out post transcription cloning, and concrete operation step and reaction system are as follows:
(1) with reaction mixture shown in tabulation 2 in no RNA enzyme EP pipe.
Table 2: reaction mixture
| Reagent | Usage amount |
| Random 6mers | 1μl |
| dNTP Mixture | 1μl |
| Template ribonucleic acid | 1.5μl |
| RNase free dH2O | 6.5μl |
It is cooling rapidly on ice after (2) 65 DEG C of heat preservation 5min, the time > 1min.
(3) with inverse transcription reaction liquid shown in tabulation 3, total amount 20ul in above-mentioned EP pipe.
Table 3: inverse transcription reaction liquid
| Reagent | Usage amount |
| Reaction mixture after step (2) processing | 10μl |
| 5×PrimeScript II Buffer | 4.0μl |
| RNase Inhibitor(40U/μl) | 0.5μl |
| PrimeScript II RTase | 1.0μl |
| RNase free dH2O | 4.5μl |
(4) inverse transcription reaction liquid is slowly mixed, carries out reverse transcription reaction by following condition:
30 DEG C, 10min；42 DEG C, 60min；75 DEG C, 15min；1 μ l of RNase H (5U/ μ l) then is added, 37 DEG C
20min。
The DNA profiling for taking 1ul reaction product to react as next step real-time fluorescence quantitative PCR.
2. real-time fluorescence quantitative PCR (qPCR)
QPCR reaction is carried out in strict accordance with Takara company SYBR Premix Ex Taq II RR820A operational manual,
Its reaction system is as shown in table 4.
Table 4:qPCR reaction system
Illustrate: forward primer and reverse primer be respectively SFXN1 gene in table 1, SOCS3 gene, CDKN1C gene just
It mixes with the forward primer of reference gene β-Actin and reverse primer to primer and reverse primer and matches according to table 4 respectively
Set reaction system.
QPCR reaction condition is as follows: 95 DEG C of initial denaturation, 30s；(95 DEG C of denaturation 5s；60 DEG C of annealing 30s) × 40 circulations；75℃
Extend 15s；60 DEG C, 1min；95 DEG C, 15s；4℃forever.Each each gene of sample does 3 repetitions, and uses 2-ΔΔCt
Method carries out relative quantitative assay.Multiple mutation analysis is carried out according to qRT-PCR result is measured, with the mean of control group quantitative result
As denominator, the quantitative result of each biological sample obtains times ratio of each biological sample as molecule, i.e. biological sample
Gene mRNA expression amount times ratio=biological sample gene mRNA relative expression quantity/control sample gene mRNA relative expression
Amount, the 2 of gene mRNA relative expression quantity=biological sample gene of biological sample-ΔΔCtThe 2 of/reference gene β-Actin-ΔΔCt。
Four, results
Multiple mutation analysis result is using the statistical method of quartile to SFXN1 gene, SOCS3 gene, CDKN1C base
Because the relative expression levels in four groups of crowds count, statistical result see the table below 5:
The relative expression levels of table 5:SFXN1 gene, SOCS3 gene, CDKN1C gene in four groups of crowds
Note: median (quartile)
Multiple mutation analysis the result is shown in Figure 1, concrete outcome are as follows:
It can be seen that SFXN1 gene obviously increases in simple HIV infection person compared with Healthy People from 1-A in figure, and tuberculosis infection
No significant difference between person and Healthy People.When using 2-ΔΔCtWhen the relative expression quantity that method carries out SFXN1 gene calculates, SFXN1 base
HIV infection or HIV/TB suprainfection possibility are higher when times ratio of the mRNA of cause is greater than 2.5.Statistical analysis uses
Kruskal-Wallis test analysis method carries out global analysis comparison, statistical result are as follows: HIV/TB suprainfection to group
The SFXN1 gene expression dose of person is respectively relative to Healthy People (P < 0.01), simple HIV infection person (P < 0.05) and simple
The SFXN1 gene expression dose of tuberculosis infected students (P < 0.01) is significantly increased.
1-B can be seen that the expression of SOCS3 gene in Healthy People, simple HIV infection person, simple tuberculosis sense from figure
Dye person, HIV/TB suprainfection person successively rise.When using 2-ΔΔCtWhen the relative expression quantity that method carries out SOCS3 gene calculates,
Times ratio of the mRNA of SOCS3 gene, which is greater than 3, can determine whether that TB infection or HIV/TB suprainfection possibility are higher.Statistical analysis is adopted
With Kruskal-Wallis test analysis method, global analysis comparison, statistical result are as follows: bis- double infection of HIV/TB are carried out to group
The SOCS3 gene expression dose of dye person is respectively relative to Healthy People (P < 0.01), simple HIV infection person (P < 0.01) and list
The SOCS3 gene expression dose ascensional range of pure tuberculosis infected students (P < 0.01) all has statistics difference.
It can be seen that CDKN1C gene is expressed in simple tubercular without substantially changeing from 1-C in figure, feel in simple HIV
Decline is expressed in dye person, and expression is remarkably decreased in HIV/TB suprainfection person, fall substantially exceeds simple HIV
The infected.When using 2-ΔΔCtWhen the relative expression quantity that method carries out CDKN1C gene calculates, times ratio of the mRNA of CDKN1C gene
Can determine whether less than 0.2 for HIV/TB suprainfection possibility it is higher.Statistical analysis uses the analysis side Kruskal-Wallis test
Method carries out global analysis comparison, statistical result to group are as follows: the CDKN1C gene expression dose of HIV/TB suprainfection person is distinguished
Relative to Healthy People (P < 0.01), simple HIV infection person (P < 0.01) and simple tuberculosis infected students (P < 0.01)
CDKN1C gene expression dose is decreased significantly, and has statistics difference.
It can be seen that SFXN1 gene, SOCS3 gene and CDKN1C base from Kruskal-Wallis test statistic analysis result
Because merging the expression in tuberculosis infected students in AIDS compared to Healthy People, simple HIV infection person and simple tuberculosis sense
Expression significant difference in dye person can be used as AIDS and merge the new biomarker of tuberculosis infection.Comprehensive 3 genes
Mrna expression amount times ratio result can be learnt: as the mrna expression amount times ratio > 2.5 and/or SOCS3 gene of SFXN1 gene
Mrna expression amount times ratio > 3 and/or CDKN1C gene mrna expression amount times ratio < 0.2 when, patient be HIV/TB bis-
A possibility that double infection contaminates is larger.Above-mentioned conclusion is subsequent also have been obtained largely facts have proved its reliability.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
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Claims (10)
1. a kind of biomarker, which is characterized in that the biomarker includes CDKN1C gene.
2. biomarker according to claim 1, which is characterized in that the biomarker also includes SOCS3 base
Cause；
Optional, the marker also includes SFXN1 gene.
3. a kind of biomarker, which is characterized in that the biomarker be CDKN1C gene and/or SOCS3 gene and/
Or the albumen of mRNA expressed by SFXN1 gene or its coding.
4. biomarker described in any claim merges tuberculosis infection in preparation detection AIDS in claim 1-3
Application in product.
5. a kind of product, which is characterized in that including the expression quantity for detecting biomarker described in claims 1 or 22
Real-time quantitative PCR reagent.
6. including being made for the reagent of the real-time quantitative PCR for detecting the expression quantity of biomarker described in claim 1
Standby assessment AIDS merges the purposes on the product of tuberculosis infection.
7. a kind of Primer composition, which is characterized in that at least pair of primers, amplification SOCS3 gene comprising expanding SFXN1 gene
At least pair of primers and expand CDKN1C gene at least one of pair of primers or it is a variety of with amplification β-Actin internal reference base
The combination of the primer of cause；
Optional, the primer of the amplification SFXN1 gene includes nucleotide shown in SEQ ID NO.1 and SEQ ID NO.2
Sequence；
Optional, the primer of the amplification SOCS3 gene includes nucleotide shown in SEQ ID NO.3 and SEQ ID NO.4
Sequence；
Optional, the primer of the amplification CDKN1C gene includes nucleotide shown in SEQ ID NO.5 and SEQ ID NO.6
Sequence；
Optional, the primer of the amplification β-Actin reference gene includes shown in SEQ ID NO.7 and SEQ ID NO.8
Nucleotide sequence.
8. use of the Primer composition according to claim 7 on the kit that preparation assessment AIDS merges tuberculosis infection
On the way.
9. a kind of kit, which is characterized in that the kit includes Primer composition as claimed in claim 7；
Optional, the kit also includes quantitative fluorescent PCR reagent；
Optional, the quantitative fluorescent PCR reagent comes from Takara company SYBR Premix Ex Taq II RR820 reagent
Reagent in box；
Optional, the kit also includes Reverse Transcription；
Optional, the Reverse Transcription comes from Takara company PrimeScriptTMII 1st strand cDNA
Reagent in the kit of Synthesis Kit 6210A；
Optional, the kit also includes that RNA extracts reagent；
Optional, it is that Trizol method extracts reagent that the RNA, which extracts reagent,；
Optional, the kit also includes PBMC separation agent；
Optional, the Ficoll separation of lymphocytes that the PBMC separation agent is #17-1440-03 from GE company article No.
Liquid.
10. the system that a kind of screening AIDS merges the biological sample of tuberculosis infection, which is characterized in that the system includes:
Real-time quantitative PCR amplification device, be provided in the real-time quantitative PCR amplification device SFXN1 gene, SOCS3 gene,
The specific primer of CDKN1C gene and β-Actin reference gene, to utilize the specific primer, to being mentioned in biological sample
The sample of nucleic acid taken carries out real-time quantitative PCR amplification, respectively obtains SFXN1 gene, SOCS3 gene, CDKN1C gene and β-
The Ct value of Actin reference gene and be calculated based on Ct value 2-ΔΔCtValue；
Judgment means, the judgment means are connected with real-time quantitative PCR amplification module, by SFXN1 gene, SOCS3
The expression of gene and CDKN1C gene is judged, the biological sample of tuberculosis infection is merged for screening AIDS；
Optional, the judgement benchmark for merging the biological sample of tuberculosis infection for screening AIDS in the judgment means are as follows: raw
The mrna expression amount times ratio > 2.5 of SFXN1 gene in object sample and/or the mRNA table of the SOCS3 gene in biological sample
Up to the amount times ratio > 3 and/or mrna expression amount times ratio < 0.2 of CDKN1C gene, wherein the SFXN1 base in biological sample
Cause/SOCS3 gene/CDKN1C gene mrna expression amount times ratio=(SFXN1 gene/SOCS3 gene in biological sample/
The m RNA relative expression quantity of CDKN1C gene)/(SFXN1 gene/SOCS3 gene/CDKN1C gene in control sample
MRNA relative expression quantity), SFXN1 gene/SOCS3 gene/CDKN1C gene mRNA relative expression quantity in biological sample=
(the SFXN1 gene of biological sample/SOCS3 gene/CDKN1C gene 2-ΔΔCtβ-Actin the reference gene of)/biological sample
2-ΔΔCt；
Optional, the SFXN1 gene-specific primer includes nucleotide shown in SEQ ID NO.1 and SEQ ID NO.2
Sequence；
Optional, the SOCS3 gene-specific primer includes nucleotide shown in SEQ ID NO.3 and SEQ ID NO.4
Sequence；
Optional, the CDKN1C gene-specific primer includes nucleotide shown in SEQ ID NO.5 and SEQ ID NO.6
Sequence；
Optional, the β-Actin reference gene specific primer includes shown in SEQ ID NO.7 and SEQ ID NO.8
Nucleotide sequence；
Optional, the system also includes post transcription cloning device, described for converting cDNA for the RNA of biological sample
Post transcription cloning device be connected with real-time quantitative PCR amplification device；
Optional, the system also includes nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus and the post transcription cloning
Device is connected, for extracting the sample of nucleic acid in the biological sample.
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| CN201711183449.6A CN109825508A (en) | 2017-11-23 | 2017-11-23 | A kind of biomarker for assessing AIDS merging tuberculosis infection |
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