CN107227343A - It is a kind of to detect the kit and its primer pair and probe for including incubation period tuberculosis infection - Google Patents
It is a kind of to detect the kit and its primer pair and probe for including incubation period tuberculosis infection Download PDFInfo
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- CN107227343A CN107227343A CN201710335197.8A CN201710335197A CN107227343A CN 107227343 A CN107227343 A CN 107227343A CN 201710335197 A CN201710335197 A CN 201710335197A CN 107227343 A CN107227343 A CN 107227343A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention discloses a kind of kit and its primer pair and probe for detecting and including incubation period tuberculosis infection.The kit includes at least one reaction solution, and the reaction solution includes a primer pair and a probe, the gene order design for downstream MIG, CXCL11 or the CXCL13 that described primer pair and probe are secreted for mycobacterium tuberculosis specific T-cells.This kit is preferred to use quantitative real-time PCR, it is easy to operate, take short, high specificity, sensitivity is high and can quantify, original tuberculosis detection method is avoided to be caused false positive and result is inaccurate by extraneous factor interference, while each phase, particularly preclinical tuberculosis infection can accurately, delicately be detected.
Description
Technical field
The present invention relates to a kind of kit of tuberculosis infection, more particularly to use Fluorescent quantitative PCR
Technology is come the kit and its primer pair and probe of the whole tuberculosis infection including detecting comprising incubation period.
Background technology
In the world, tuberculosis be still up to now in infectious disease morbidity and mortality highest disease it
One, it is estimated that annual new cases about 900,0000 people there are about 200,0000 people and dies from tuberculosis every year.Tuberculosis is by tuberculosis
A kind of infectious diseases common to human beings and animals caused by bacillus, is that single pathogenic infection causes death rate highest infectious diseases.China
It is one of tuberculosis high burden country, national tuberculosis epidemiological random sampling survey data is shown within 2000：Annual age group tuberculosis
Infection rate is 44.5%, active tuberculosis patient 4,510,000, and annual death toll is up to 130,000, and tuberculosis death accounts for various infectious diseases,
The 65.1% of parasitic disease death, 2 times of the dead summation of almost other various infectious diseases, parasitic disease.Due to human immunity
The wide-scale distribution of defective virus and Drug-Resistant Mycobacterium tuberculosis it is increasing so that this ancient disease causes people again
Extensive concern.Tuberculosis turns into one of Important Infectious Diseases of harm human health.
Because the detection method progress for diagnosing latent tuberculosis infects is slow, cause the control of tuberculosis epidemic situation not good.Mesh
Before, whole blood gamma interferon release test (IGRA) makes extensively in multiple low tuberculosis epidemic situation countries such as European and American areas and Japan
With IGRA is used in the tulase pathogenic bacteria lacked originating from BCG vaccination bacterium and most of non-tuberculous mycobacterias
Differential stimulus albumen such as ESAT-6 and CFP-10 coded by RD1- areas are detected that such stimulant has had commercialization
IGRA reagents, such as QuantiFERON TB Gold test and T SPOT.TB test.There is researcher to be measured in IGRA detections
Change interferon-γ (IFN-γ) mRNA expressions, as IFN-γ indicant, but IFN-γ RT-PCR specificity and
Sensitivity is poor, and can not differentiate active period and incubation period tuberculosis infection (LTBI).
In China, TST is based on to the delayed metamorphosis produced by purifying protein derived from tulase (PPD) instead as a kind of
The detection method answered is still the main method for diagnosis of tuberculosis bacterium latent infection (LTBI).TST susceptibility and special
Degree is all relied on depending on the different cutoff values defined in a special group.If it is special to improve reaction scleroma diameter cutoff value
Degree will increase (while susceptibility decline).The specificity change of the experiment greatly and is relied primarily on and Nontuberculosis mycobacteria very much
Between cross reaction possibility depending on.In addition, TST cannot distinguish between outmoded infection with infecting recently, with BCG vaccine and environment
There is cross immunity between Nontuberculosis mycobacteria and cause false positive, more due to the testing result presence in immunosuppressed humanses
False negative possibly even causes mistaken diagnosis.Boosting effect may be caused to cause quick by repeating TST reactions or two-step method TST detections
The increase of sensitivity.
TST and IGRAs can not differentiate incubation period and active stage tuberculosis, and one of key of eradication of tuberculosis is to differentiate latent
Volt property tuberculosis infection.Therefore, the detection method of the high diagnosis latent tuberculosis infects of a kind of high specificity of exploration, sensitivity is extremely closed
It is important.
Real-time fluorescence quantitative PCR (FQ-PCR) is the nucleic acid detection method grown up in recent years on the basis of regular-PCR, it
The accumulation of fluorescent value is combined with the amplification procedure of PCR primer, it is to be measured to calculate by measuring the fluorescent value of exponential amplification phase
The original vol of sample amplifying nucleic acid.It has it is easy to operate, time-consuming it is short, the unrivaled advantage of conventional method such as can quantify.In addition,
Chemotactic factor (CF) is that a class formation function is similar, and the small molecular weight protein with chemotactic attractability, its primary structure is quite similar, when
Chemotactic factor (CF) is after its corresponding acceptor is combined, by activated G protein-coupled acceptor, cascade signal conduction is produced, to a variety of inflammation
Property cell chemotaxis and activation, played an important role in the diseases such as respiratory system, cardiovascular and liver kidney.Such as, the lymph by activating
The downstream chemotactic factor (CF) gamma interferon of cell secretion in tuberculosis infection detection susceptibility up to 80%.
The content of the invention
The technical problems to be solved by the invention be for overcome present in prior art be directed to tuberculosis infection detection side
There is provided one kind detection for the defect such as method can not differentiate latent tuberculosis infects and existing detection method specificity or susceptibility is not strong
Kit comprising the full course of disease tuberculosis infection including incubation period, particularly fluorescence quantitative polymerase chain reaction (FQ-PCR) reagent
Box and its primer pair and probe.The kit has easy to operate, high specificity, sensitivity are high, it is short to take and can quantify etc.
Advantage, is particular enable to detect the tuberculosis infection of incubation period and/or early stage.
It is special when the T cell of activation is activated when the present inventor selects tuberculosis infection, particularly latent tuberculosis infection first
Different target molecule can be raised, and be had been surprisingly found that more than one or both of MIG, CXCL11 and CXCL13
Combination, which is for latent tuberculosis infection, preferable sensitivity and specificity.Then conjunction is selected further directed to above-mentioned chemotactic factor (CF)
Suitable detection method.Due to, FQ-PCR different from based on the traditional PCR method that single end point determination is carried out after the completion of amplified reaction
Method can monitor the generation of amplified production at any time during amplified reaction is carried out, so as to greatly improve quantitative detection
Accuracy and precision, therefore the present invention preferably FQ-PCR methods.Real-time fluorescence quantitative PCR (Taqman sonde methods) is PCR's
In amplification, occur group and fluorescent quenching group while adding primer and being marked with fluorescence respectively in 5 ' ends and 3 ' ends
Specific oligonucleotide probe.If probe is not combined with target sequence complementation, probe sequence keeps complete, and group occurs for fluorescence
The fluorescence signal of transmitting is quenched group absorptions, therefore the generation without fluorescence signal, and when probe and target sequence can complementation tie
During conjunction, during PCR amplifications are carried out, archaeal dna polymerase its circumscribed enzyme activity in 5 ' -3 ' end while guiding DNA sequence dna to replicate
Property will cut off fluorescence probe, cause fluorescence to occur the separation of group and fluorescent quenching group, so that fluorescence detecting system can connect
Receive and record fluorescence signal.It is the generation for having a fluorescence signal often to expand a DNA, and accumulation and the PCR of fluorescence signal are produced
The formation Complete Synchronization of thing, therefore whole PCR courses of reaction can be monitored in real time, and can be constant by standard curve or expression quantity
Internal reference product absolute or relative quantitative analysis is carried out to unknown target polynucleotide.Therefore, the design of probe and primer
For realizing that the quantitative detection of target polynucleotide is most important.The present inventor is this repetition test and research, specific and quick
It is balanced in terms of sensitivity, has filtered out the relevant primer pair for determining MIG, CXCL11 and CXCL13 and visited
Pin.
The present invention solves one of technical scheme of above-mentioned technical problem：It is a kind of to detect the examination for including incubation period tuberculosis infection
Agent box, it includes at least one reaction solution, and the reaction solution includes the primer pair and a probe of target gene, described primer
Pair and probe downstream MIG, CXCL11 or the CXCL13 base secreted for mycobacterium tuberculosis specific T-cells
Because of sequences Design；Wherein,
The forward primer (primer 1) of the primer pair designed for MIG is as shown in SEQ ID NO.1
The nucleic acid of sequence, reverse primer (primer 2) is the nucleic acid of the sequence as shown in SEQ ID NO.2, the nucleosides of the probe (probe 1)
Acid sequence is as shown in SEQ ID NO.9；
Forward primer (primer 3) for the Chemokines CC XCL11 primer pairs designed is such as SEQ ID NO.3 institutes
Show the nucleic acid of sequence, reverse primer (primer 4) is the nucleic acid of the sequence as shown in SEQ ID NO.4, the core of the probe (probe 2)
Nucleotide sequence is as shown in SEQ ID NO.10；Or
Forward primer (primer 5) for the Chemokines CC XCL13 primer pairs designed is such as SEQ ID NO.5 institutes
Show the nucleic acid of sequence, reverse primer (primer 6) is the nucleic acid of the sequence as shown in SEQ ID NO.6, the core of the probe (probe 3)
Nucleotide sequence is as shown in SEQ ID NO.11.
Wherein, primer 1 is located at the 197-221 positions of the CXCL9 gene orders of people, and primer 2 is located at 289-265, expands piece
Segment length is 93bp, and corresponding probe is located at 234-260；Primer 3 is located at the 113-136 of the CXCL11 gene orders of people
Position, primer 4 is located at 255-230, and expanding fragment length is 143bp, and corresponding probe is located at 184-211；Primer 5 is located at
The 199-224 positions of the CXCL13 gene orders of people, primer 6 is located at 314-293, and expanding fragment length is 116bp, corresponding
Probe is located at 226-249.
It is preferred that making reliable results, antijamming capability is stronger, and as expression quantity reference, the present invention is made from house-keeping gene
For internal reference, preferably people β-globin (betaglobulin) gene, described reaction solution also includes being directed to people's β-globin bases
Because of the primer pair and a probe of the house-keeping gene of design；In the primer pair, forward primer (primer 7) is such as SEQ ID
The nucleic acid of sequence shown in NO.7, reverse primer (primer 8) is the nucleic acid of the sequence as shown in SEQ ID NO.8, the probe (probe
4) nucleotide sequence is as shown in SEQ ID NO.12.Wherein, primer 7 is located at the HBB gene of people's β-globin genome sequences
The 62627-62647 positions of (62137-63742), primer 8 is located at 62756-62775, and expanding fragment length is 149bp, and institute is right
The probe answered is located at the 62731-62755 positions of β-globin gene order reverse sequences.
It is preferred that described reaction solution also includes PCR reaction buffers and 2 '-deoxynucleoside triphosphate；It is preferred that institute
The concentration for stating 2 '-deoxynucleoside triphosphate is 100mM.
Kit of the present invention is preferably real-time fluorescence quantitative PCR (FQ-PCR) kit.
It is preferred that mentioned reagent box can also include regular-PCR, fluorescent PCR, especially FQ-PCR other conventional reagents：
EZ Taq mixed liquors and negative quality-control product etc.；The EZ Taq mixed liquors include hot start Taq polymerase, the preferred 1-5U/ μ l of concentration.
More preferably, be improve blood sample in chemotactic factor (CF) content, correspondingly improve kit detection sensitivity and
Specificity, the kit can also be included in tuberculosis antigen pipe and Background control pipe, the combination antigen pipe comprising antigen thorn
Swash thing or its composition；It is the antigenic stimulus thing or its composition preferred antigens CFP10 derived peptides, antigen ESAT6 derived peptides, anti-
At least one of former PPE derived peptides and antigen A g85A derived peptides.The amino acid sequence of the antigens c FP10 derived peptides is more
It is preferred that as shown in SEQ ID NO.13, the amino acid sequence of the antigen ESAT6 derived peptides is more preferably such as SEQ ID NO.14 institutes
Show, the amino acid sequence of the antigen PPE derived peptides is more preferably as shown in SEQ ID NO.15, the antigen A g85A derived peptides
Amino acid sequence more preferably as shown in SEQ ID NO.16.
What the present invention solved the technical scheme of above-mentioned technical problem two is：It is a kind of to detect the spy for including incubation period tuberculosis infection
Pin, nucleotide sequence such as SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or the SEQ ID of the probe
Shown in NO.12.
It is preferred that above-mentioned probe is the fluorescence probe for being applicable FQ-PCR detections, what the two ends of its above-mentioned sequence were connected is
Conventional fluorescent reporter group and quenching group.Wherein, target gene (SEQ ID NO.9-11) reporter group：FAM；House keeper's base
Because of reporter group (SEQ ID NO.12)：VIC；The quenching group of target gene and house-keeping gene：BHQ MGB).
What the present invention solved the technical scheme of above-mentioned technical problem three is：A kind of detect includes drawing for incubation period tuberculosis infection
Thing pair, one of the primer pair is the nucleic acid of the sequence as shown in SEQ ID NO.1, and another is as shown in SEQ ID NO.2
The nucleic acid of sequence；One of the primer pair is the nucleic acid of the sequence as shown in SEQ ID NO.3, and another is such as SEQ ID
The nucleic acid of sequence shown in NO.4；One of the primer pair is the nucleic acid of the sequence as shown in SEQ ID NO.5, another be as
The nucleic acid of sequence shown in SEQ ID NO.6；Or one of the primer pair is the nucleic acid of the sequence as shown in SEQ ID NO.7,
Another be the sequence as shown in SEQ ID NO.8 nucleic acid.
What the present invention solved the technical scheme of above-mentioned technical problem four is：A kind of reaction solution, it includes a primer pair and one
Probe, described primer pair and probe for mycobacterium tuberculosis specific T-cells secrete downstream MIG,
CXCL11 or CXCL13 gene order design；Wherein,
The forward primer of the primer pair designed for MIG is the sequence as shown in SEQ ID NO.1
Nucleic acid, reverse primer is the nucleic acid of the sequence as shown in SEQ ID NO.2, the nucleotide sequence such as SEQ ID NO.9 of the probe
It is shown；
Forward primer for the Chemokines CC XCL11 primer pairs designed is the sequence as shown in SEQ ID NO.3
Nucleic acid, reverse primer is the nucleic acid of the sequence as shown in SEQ ID NO.4, the nucleotide sequence such as SEQ ID NO.10 of the probe
It is shown；Or
Forward primer for the Chemokines CC XCL13 primer pairs designed is the sequence as shown in SEQ ID NO.5
Nucleic acid, reverse primer is the nucleic acid of the sequence as shown in SEQ ID NO.6, the nucleotide sequence such as SEQ ID NO.11 of the probe
It is shown.
It is preferred that the reaction solution also includes as expression quantity with reference to the pipe for detecting house-keeping gene people's β-globin genes
A primer pair and a probe for family's gene；In the primer pair, forward primer is the nucleic acid of the sequence as shown in SEQ ID NO.7,
Reverse primer is the nucleic acid of the sequence as shown in SEQ ID NO.8, and the nucleotide sequence of the probe is as shown in SEQ ID NO.12.
What the present invention solved the technical scheme of above-mentioned technical problem four is：Described probe, described primer pair or institute
Application of the reaction solution stated in preparing detection to include the kit of incubation period tuberculosis infection, preferably FQ-PCR kits.Preferably
Ground, the application is that mentioned reagent box is used for the detection of blood sample.
On the basis of common sense in the field is met, above-mentioned each optimum condition can be combined, and produce each preferable reality of the present invention
Example.
Agents useful for same and raw material of the present invention are commercially available.
The positive effect of the present invention is：As described above, in order to detect whether clinical blood sample is latent knot
Core infects sample, and MIG, CXCL11, CXCL13 nucleotide sequence are analyzed, is specifically designed suitable by the present invention
With the Oligonucleolide primers and probe of detection kit of the present invention.The Real_time quantitative detection completed using these primer and probes,
Not only easy to operate, time-consuming short, high specificity, and drastically increase tuberculosis detection sensitivity, it is to avoid BCG vaccine is outmoded
The interference of infection etc. causes false positive and result is inaccurate, reduces template usage amount.It is demonstrated experimentally that the present invention is to tuberculosis of hiding
The sensitivity of detection is more than 97%.Latent tuberculosis infection can be found early using the kit of the present invention and takes reply
Measure, to mitigate patient suffering, improves tuberculosis cure rate.
Brief description of the drawings
Fig. 1 is the sample that " S " type or Ct values ＞ 35 is not presented in amplification curve.
Fig. 2 is Ct value dummies.
Fig. 3 is that " S " type is presented in amplification curve and tuberculosis stimulates sample CT values<Background control sample CT values；Wherein, Fig. 1-3
In " a " and " b " refer respectively to " target gene " and " house-keeping gene ".
Embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to described reality
Apply among a scope.The experimental method of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product specification is selected.
The preparation of each constituent of the kit of embodiment 1 and the assembling of kit
The each component title of table 1., concentration and content
Above-mentioned reaction buffer is from commercial goods (being purchased from Shanghai Duo Ze bio tech ltd), and dNTP is (purchased from upper
Extra large Katherine Victoria development in science and technology Co., Ltd), EZ Taq enzymes or mixed liquor (are purchased from Shanghai Duo Ze bio tech ltd), primer and
Probe (is commercially synthesized, Invitrogen (Shanghai) Trading Co., Ltd. (ABI)).
(1) preparing includes the kit of following composition：
Reaction solution 1 (650 μ l/ pipes) 1 is managed：
PCR reaction buffers, 2 '-deoxynucleoside triphosphate, the forward primer (primer 1) for expanding CXCL9 and reversely
Primer (primer 2), oligonucleotide probe is probe 1；For drawing that house-keeping gene people's β-globin genes target polynucleotide is expanded
Thing 7 and primer 8, oligonucleotide probe are probe 4.
Reaction solution 2 (650 μ l/ pipes) 1 is managed：
Reaction solution 2 is made up of following component：It is PCR reaction buffers, 2 '-deoxynucleoside triphosphate, many for CXCL11 targets
The forward primer (primer 3) and reverse primer (primer 4) of amplification oligonucleotide, oligonucleotide probe are probe 2；For house-keeping gene
The primer 7 and primer 8 of people's β-globin genes target polynucleotide amplification, oligonucleotide probe are probe 4.
Reaction solution 3 (650 μ l/ pipes) 1 is managed：
Reaction solution 3 is made up of following component：It is PCR reaction buffers, 2 '-deoxynucleoside triphosphate, many for CXCL13 targets
The forward primer (primer 5) and reverse primer (primer 6) of amplification oligonucleotide, oligonucleotide probe are probe 3；For house-keeping gene
The forward primer (primer 7) and reverse primer (primer 8) of people's β-globin genes target polynucleotide amplification, oligonucleotide probe is
Probe 4.
EZ Taq enzymes mixed liquor (150 μ l/ pipes) 1 is managed：Taq enzyme.
The EZ Taq enzymes mixed liquor of table 2 is constituted
Titer：Negative quality-control product (80 μ l/ pipes)：Purified water.
Tuberculosis antigen pipe (heparin tube 1)：Antigenic stimulus thing is put into heparin tube, and every heparin tube contains antigenic stimulus thing 20
μ g (0.5mg/ml) and liquaemin dry powder 30IU (900IU/ml).
Background control pipe (heparin tube 2)：Liquaemin dry powder 30IU (900IU/ml) is comprised only in heparin tube.
Reaction solution 1 is used as system I with EZ Taq enzymes mixed liquor, titer and tuberculosis antigen pipe and Background control pipe；
Reaction solution 2 is used as system II with EZ Taq enzymes mixed liquor, titer and tuberculosis antigen pipe and Background control pipe；
Reaction solution 3 is used as system III with EZ Taq enzymes mixed liquor, titer and tuberculosis antigen pipe and Background control pipe.
Wherein, primer 1-8 constitutes the nucleic acid amplification system in each system, and probe 1-4 constitutes the fluorescence in each system
Detection architecture；Each primer and probe concrete composition is as follows：
Each primer and probe concrete composition of table 3
| Primer | Nucleotides is constituted | Sequence number |
| Primer 1 | 5’-CCACCTACAATCCTTGAAAGACCTT-3’ | SEQ ID NO.1 |
| Primer 2 | 5’-TGAACTCCATTCTTCAGTGTAGCAA-3’ | SEQ ID NO.2 |
| Primer 3 | 5’-TCAATTTCCTTTCATGTTCAGCAT-3’ | SEQ ID NO.3 |
| Primer 4 | 5’-ACACAATATCACAGCCAAGGCTATAG-3’ | SEQ ID NO.4 |
| Primer 5 | 5’-GTCTTTATCCCTAGACGCTTCATTGA-3’ | SEQ ID NO.5 |
| Primer 6 | 5’-GGGTCCACACACACAATTGACT-3’ | SEQ ID NO.6 |
| Primer 7 | 5’-TTCCTCCTCCTGAGCAGT-3’ | SEQ ID NO.7 |
| Primer 8 | 5’-AAGGTCATAACCTGGTTCATC-3’ | SEQ ID NO.8 |
| Probe 1 | 5’-CCAAGCCCTTCCTGCGAGAAAATTGAA-3’ | SEQ ID NO.9 |
| Probe 2 | 5’-CACCAGCAGCAACAGCAAAAAACAAACA-3’ | SEQ ID NO.10 |
| Probe 3 | 5’-CGAATTCAAATCTTGCCCCGTGGG-3’ | SEQ ID NO.11 |
| Probe 4 | 5’-CCCTGGCGTCGTGATTAGTGATGATGAAC-3’ | SEQ ID NO.12 |
5 ' ends of above-mentioned probe 1~3 are connected with reporter group FAM, and 3 ' ends are connected with quenching group BHQ MGB；Probe 4
5 ' ends are connected with reporter group VIC, and 3 ' ends are connected with quenching group BHQ MGB.
(2) assembling of kit
According to demand, can be by the Reagent Tubes such as EZ Taq enzymes mixed liquor, titer and tuberculosis antigen pipe and Background control pipe
Respectively with equipped with above-mentioned reaction solution 1~3 single centrifuge tube comparting packaging composition respectively determine MIG, CXCL11,
CXCL13 kit；Above-mentioned each Reagent Tube and heparin tube and more than 2 can also be respectively provided with different above-mentioned reactions
The centrifuge tube of liquid 1~3 packs the kit of target spot in pairs or three target spots.
The application method of the kit of embodiment 2
1) sample drawn blood is into Background control pipe and tuberculosis antigen pipe
Sample collection, transport and preservation：Disposal vacuum hemostix is used, sterile working takes individual blood sample to be checked, done
The blood sample of acquisition is placed in freezen protective in low temperature refrigerator (- 70 DEG C or -20 DEG C) after good sign.Such as test, sample
It need to be transported and in environment in being sent to laboratory in 24 hours below 0 DEG C.
Clinic extracts 2ml sample bloods and added in Background control pipe, labeled as Background control sample；Separately extract same individual
Sample blood 2ml, add containing 20 μ g antigenic stimulus things (comprising CFP10, ESAT6, PPE4 and Ag85A derived peptide segment (mole
Than for 1:1:1:1), synthesize by Jin Sikang scientific and technological (Nanjing) Co., Ltds) tuberculosis antigen pipe in being stimulated at 35 DEG C after 6h, mark
It is designated as tuberculosis antigen sample.Wherein, the sequence of 4 kinds of antigen derived-peptides is：
CFP10 derived peptides：QGQWRGAAGTAA (as shown in SEQ ID NO.13)；
ESAT6 derived peptides：ELNNAL (as shown in SEQ ID NO.14)；
PPE derived peptides：AGWQTLSAALDAQAVELTAR (shown in SEQ ID NO.15)；
Ag85A derived peptides：RHVKPTGSAVVGL (as shown in SEQ ID NO.16).
2) RNA in Background control pipe and tuberculosis antigen pipe blood sample is extracted
Taking each 300 μ l of blood sample to be measured in two pipes, (blood to be measured refers to need to detect whether as tuberculosis infection of hiding
Clinical blood sample), extract (extracts reagent is purchased from the invincible Science and Technology Ltd. of Shenzhen's peace) blood sample with batch with paramagnetic particle method
This mRNA, is cDNA using RNA Reverse Transcriptase kits (purchased from precious bioengineering (Dalian) Co., Ltd) reverse transcription, uses light splitting light
Degree meter or similar instrument confirm cDNA quality (OD260/280 is between 1.8-2.0).
3) cDNA is added in reaction solution, carries out expanding instead by nucleic acid amplification system, with real-time fluorescence PCR instrument
Should, the target polynucleotide in cyclic amplification testing sample；The fluorescence in the fluorescent detection system is set to occur group with being amplified
Target polynucleotide sequence combine indirectly.
The every part of tuberculosis antigen pipe cDNA and Background control pipe cDNA of reverse transcription are respectively placed in 3 PCR reaction tubes or PCR
In 3 holes of plate, per the μ l of hole 2.Then, a kind of PCR reaction solutions (reaction solution is respectively added in 3 holes for adding same DNA sample
1st, reaction solution 2, reaction solution 3) 15 μ l, then add in each hole 3 μ l EZ Taq enzyme mixed liquors.Mix after centrifugation, PCR is anti-
System is answered to be placed on automatic fluoroscopic examination thermal cycler (ABI7500), selection FAM passages (Reporter:FAM,Quencher:
None specific amplified signal) is collected；Select VIC passages (Reporter:VIC,Quencher:None internal standard) is detected；Reference is glimmering
Light (Passive Reference) is set to none；It is 20 to set Sample Volume.Phase is carried out according to instrumentation explanation
Start experiment after should setting.
Specific pcr amplification reaction system, reaction condition are shown in Table 4 and table 5.
The pcr amplification reaction system of table 4
| Component | Content |
| Reaction solution 1 or 2 or 3 | 15μl |
| EZ Taq enzyme mixed liquors | 3μl |
| Template | 2μl |
| Amount to | 20μl |
The pcr amplification reaction condition of table 5
4) by compare measuring samples and standard items cycle threshold judge fluorescence occur group produced by fluorescence volume, from
And determine the presence of target polynucleotide.
Reaction is preserved after terminating can check that each PCR reacts after detection data file, the data analysis option for clicking instrument
The result of system, such as PCR amplification curves, CT values of sample and master sample to be checked etc..
Computational methods：
10 times of standard deviations of the fluorescent value of using before amplification procedure 3~15 circulations are super with fluorescent value as threshold value (threshold)
The period for crossing threshold value is threshold cycle number (value) Ct values.If house-keeping gene Ct values ＞ 35, as a result unreliable, please redeterminate or
Observe blood and whether there is problem in itself.If carrying out following calculate during house-keeping gene Ct Zhi≤35：
Background control pipe：△ Ct A=Ct values (target gene)-Ct is worth (house-keeping gene)
Tuberculosis antigen pipe：△ Ct B=Ct values (target gene)-Ct is worth (house-keeping gene)
As a result interpretation：T values=△ Ct B-△ Ct A.
1. null result judges：With the oligonucleotides fluorescence probe 1 being present in system I, II, III, probe 2, probe 3 and
The related amplification curve of probe 4 " S " type or Ct values blank is not presented and or Ct values ＞ 35 sample, be reported as it is invalid (see Fig. 1,
Fig. 2)；
2. positive findings judges：Meet the oligonucleotide probe 1 with system I or in system II or system III, probe 2, visit
" S " type and T values≤- 1.04 are presented in the amplification curve related to probe 4 of pin 3, are reported as positive (see Fig. 3)；
3. negative findings judges：Belong to null result to judge and the situation and T values ＞ -1.04 beyond positive findings judgement.
Then it is reported as negative (negative controls are exactly not expand, and illustrate that this experimental system is not affected by outside contamination).
Result and analysis of the embodiment 3 using kit 30 clinical blood samples of detection of the present invention
Using single blind experiment method, it (is needs to select 30 from from 500 blood samples of (Nanjing chest hospital)
Detect whether as the clinical blood sample for tuberculosis infection of hiding) tested with the kit of the present invention, with clinical diagnosis to reality
Result is tested to be checked.
Experimentation：Clinic extracts 2ml sample bloods into Background control pipe, labeled as Background control sample；It is another to extract
2ml blood samples, are added in the tuberculosis antigen pipe containing 20 μ g antigenic stimulus things, are stimulated in 35 DEG C after 6h, labeled as tuberculosis
Antigen sample.
Blood sample to be detected is taken into 300 μ l, blood sample mRNA is extracted with batch with paramagnetic particle method, buys commercially available RNA reverse transcriptions
Kit reverse transcription is cDNA, and with the quality of spectrophotometer or similar instrument confirmation cDNA, (OD260/280 is in 1.8-2.0
Between), every part of reverse transcription sample cDNA and standard items cDNA to be checked are respectively placed in 3 of 3 PCR reaction tubes or PCR plate
Kong Zhong, per the μ l of hole 2.Then, a kind of PCR reaction solutions (reaction solution 1, reaction are respectively added in 3 holes for adding same DNA sample
Liquid 2, reaction solution 3) 15 μ l, then add in each hole 3 μ l EZ Taq enzyme mixed liquors.Specific pcr amplification reaction system is shown in Table
3.Mix after centrifugation, PCR reaction systems are placed on automatic fluoroscopic examination thermal cycler, selection FAM passages (Reporter:
FAM,Quencher:None specific amplified signal) is collected；Select VIC passages (Reporter:VIC,Quencher:None) examine
Survey internal standard；Reference fluorescent (Passive Reference) is set to none；It is 20 to set Sample Volume.Grasped according to instrument
Explain to carry out and start experiment after relative set.The used reaction condition (as shown in table 5) of this kit is：(1)95℃
Pre-degeneration 5 minutes；(2) 95 DEG C 15 seconds；(3) 58 DEG C 35 seconds；(4) 72 DEG C 20 seconds；Circulate 40 times step (2)-(4).
Test data and data analysis：
The testing result of 6 reaction solution of table 1
The testing result of 7 reaction solution of table 2
The testing result of 8 reaction solution of table 3
The testing result of 93 kinds of reaction solutions of table compares
Sensitivity when the differential responses liquid of table 10 is combined
Experimental result shows that sensitivity of the present invention to tuberculosis detection of hiding is up to more than 97%.
<110>Suzhou Chuan Lan bio tech ltd
<120>It is a kind of to detect the kit and its primer pair and probe for including preclinical tuberculosis infection
<130> P1611497C
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer 1
<400> 1
ccacctacaa tccttgaaag acctt 25
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer 2
<400> 2
tgaactccat tcttcagtgt agcaa 25
<210> 3
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<212> DNA
<213> Artificial Sequence
<220>
<223>Primer 3
<400> 3
tcaatttcct ttcatgttca gcat 24
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer 4
<400> 4
acacaatatc acagccaagg ctatag 26
<210> 5
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer 5
<400> 5
gtctttatcc ctagacgctt cattga 26
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer 6
<400> 6
gggtccacac acacaattga ct 22
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer 7
<400> 7
ttcctcctcc tgagcagt 18
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer 8
<400> 8
aaggtcataa cctggttcat c 21
<210> 9
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe 1
<400> 9
ccaagccctt cctgcgagaa aattgaa 27
<210> 10
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe 2
<400> 10
caccagcagc aacagcaaaa aacaaaca 28
<210> 11
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe 3
<400> 11
cgaattcaaa tcttgccccg tggg 24
<210> 12
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe 4
<400> 12
ccctggcgtc gtgattagtg atgatgaac 29
<210> 13
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223>CFP10 derived peptide segments
<400> 13
Gln Gly Gln Trp Arg Gly Ala Ala Gly Thr Ala Ala
1 5 10
<210> 14
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223>ESAT6 derived peptide segments
<400> 14
Glu Leu Asn Asn Ala Leu
1 5
<210> 15
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223>PPE derived peptide segments
<400> 15
Ala Gly Trp Gln Thr Leu Ser Ala Ala Leu Asp Ala Gln Ala Val Glu
1 5 10 15
Leu Thr Ala Arg
20
<210> 16
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223>Ag85A derived peptide segments
<400> 16
Arg His Val Lys Pro Thr Gly Ser Ala Val Val Gly Leu
1 5 10
Claims (10)
1. a kind of detect the kit for including incubation period tuberculosis infection, it is characterised in that it includes at least one reaction solution, described
Reaction solution includes a primer pair and a probe, and described primer pair and probe are secreted for mycobacterium tuberculosis specific T-cells
Downstream MIG, CXCL11 or CXCL13 gene order design；Wherein,
The forward primer of the primer pair designed for MIG is the nucleic acid of the sequence as shown in SEQ ID NO.1,
Reverse primer is the nucleic acid of the sequence as shown in SEQ ID NO.2, and the nucleotide sequence of the probe is as shown in SEQ ID NO.9；
For Chemokines CC XCL11 design the primer pair forward primer be the sequence as shown in SEQ ID NO.3 core
Acid, reverse primer is the nucleic acid of the sequence as shown in SEQ ID NO.4, the nucleotide sequence such as SEQ ID NO.10 institutes of the probe
Show；Or
For Chemokines CC XCL13 design the primer pair forward primer be the sequence as shown in SEQ ID NO.5 core
Acid, reverse primer is the nucleic acid of the sequence as shown in SEQ ID NO.6, the nucleotide sequence such as SEQ ID NO.11 institutes of the probe
Show.
2. kit as claimed in claim 1, it is characterised in that the reaction solution also includes the primer pair of a house-keeping gene
With a probe；In the primer pair, forward primer is the nucleic acid of the sequence as shown in SEQ ID NO.7, and reverse primer is such as SEQ
The nucleic acid of sequence shown in ID NO.8, the nucleotide sequence of the probe is as shown in SEQ ID NO.12.
3. kit as claimed in claim 1, it is characterised in that the reaction solution also include PCR reaction buffers and 2 '-
Deoxynucleoside triphosphate；It is preferred that the concentration of the 2 '-deoxynucleoside triphosphate is 100mM.
4. the kit as described in any one of claims 1 to 3, it is characterised in that described kit is real time fluorescent quantitative
PCR kit；And/or the kit also includes EZ Taq mixed liquors and negative quality-control product；The EZ Taq mixed liquors include
Hot start Taq polymerase, the preferred 1-5U/ μ l of concentration；More preferably, the kit also includes tuberculosis antigen pipe and Background control pipe, institute
State and antigenic stimulus thing or its composition are included in tuberculosis antigen pipe；The antigenic stimulus thing or its composition preferred antigens CFP10
At least one of derived peptide, antigen ESAT6 derived peptides, antigen PPE derived peptides and antigen A g85A derived peptides；The antigen
The amino acid sequence of CFP10 derived peptides is preferably as shown in SEQ ID NO.13, the amino acid sequence of the antigen ESAT6 derived peptides
It is preferred that as shown in SEQ ID NO.14, the amino acid sequence of the antigen PPE derived peptides is preferably as shown in SEQ ID NO.15, institute
The amino acid sequence of antigen A g85A derived peptides is stated preferably as shown in SEQ ID NO.16.
5. a kind of detect the probe for including incubation period tuberculosis infection, it is characterised in that the nucleotide sequence of the probe such as SEQ
ID NO.9, SEQ ID NO.10, shown in SEQ ID NO.11 or SEQ ID NO.12.
6. a kind of detect the primer pair for including incubation period tuberculosis infection, it is characterised in that one of the primer pair is such as SEQ
The nucleic acid of sequence shown in ID NO.1, another be the sequence as shown in SEQ ID NO.2 nucleic acid；One of the primer pair is
The nucleic acid of sequence as shown in SEQ ID NO.3, another be the sequence as shown in SEQ ID NO.4 nucleic acid；The primer pair
One be the sequence as shown in SEQ ID NO.5 nucleic acid, another be the sequence as shown in SEQ ID NO.6 nucleic acid；Or institute
State primer pair one is the nucleic acid of the sequence as shown in SEQ ID NO.7, another be the sequence as shown in SEQ ID NO.8 core
Acid.
7. a kind of reaction solution, it is characterised in that it includes a primer pair and a probe, described primer pair and probe are directed to tuberculosis
The gene order design for downstream MIG, CXCL11 or the CXCL13 that mycobacteria specific T cell is secreted；Wherein,
The forward primer of the primer pair designed for MIG is the nucleic acid of the sequence as shown in SEQ ID NO.1,
Reverse primer is the nucleic acid of the sequence as shown in SEQ ID NO.2, and the nucleotide sequence of the probe is as shown in SEQ ID NO.9；
For Chemokines CC XCL11 design the primer pair forward primer be the sequence as shown in SEQ ID NO.3 core
Acid, reverse primer is the nucleic acid of the sequence as shown in SEQ ID NO.4, the nucleotide sequence such as SEQ ID NO.10 institutes of the probe
Show；Or
For Chemokines CC XCL13 design the primer pair forward primer be the sequence as shown in SEQ ID NO.5 core
Acid, reverse primer is the nucleic acid of the sequence as shown in SEQ ID NO.6, the nucleotide sequence such as SEQ ID NO.11 institutes of the probe
Show.
8. reaction solution as claimed in claim 7, it is characterised in that the reaction solution also includes the primer pair of a house-keeping gene
With a probe；In the primer pair, forward primer is the nucleic acid of the sequence as shown in SEQ ID NO.7, and reverse primer is such as SEQ
The nucleic acid of sequence shown in ID NO.8, the nucleotide sequence of the probe is as shown in SEQ ID NO.12.
9. the reaction described in probe as claimed in claim 5, primer pair as claimed in claim 6 or claim 7 or 8
Application of the liquid in kit of the detection comprising incubation period tuberculosis infection is prepared.
10. application as claimed in claim 9, it is characterised in that the kit is real-time fluorescence quantitative PCR kit；Compared with
Goodly, the kit is used for the detection of blood sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184362A (en) * | 2018-02-23 | 2019-08-30 | 山东诺信检测有限公司 | A kind of kit using Taqman probe quantitative detection M tuberculosis complex |
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| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhao Zhiqiang Inventor after: Xiao Weiming Inventor after: Sun Zuyong Inventor before: Xiao Weiming |
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Application publication date: 20171003 |