CN109402250A - It is a kind of for the primer of SCID genetic screening, detection kit and detection method - Google Patents
It is a kind of for the primer of SCID genetic screening, detection kit and detection method Download PDFInfo
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Abstract
The present invention relates to for the primer of SCID genetic screening, detection kit and detection method, kit includes DNA extracting solution, the upstream and downstream primer and probe of TREC gene magnification, the upstream and downstream primer and probe of internal reference C α gene magnification, the upstream and downstream primer and probe of external control C α gene magnification, PCR MIX reaction solution；The upstream and downstream primer sequence of TREC gene magnification is as shown in SEQ ID NO.1 and SEQ ID NO.2, and TREC gene probe sequence is as shown in SEQ ID NO.3；The upstream and downstream primer sequence of internal reference C α gene magnification is as shown in SEQ ID NO.4 and SEQ ID NO.5, and internal reference C α gene probe sequence is as shown in SEQ ID NO.6；The upstream and downstream primer sequence of external control C α gene magnification is as shown in SEQ ID NO.4 and SEQ ID NO.5, and external control C α gene probe sequence is as shown in SEQ ID NO.7.This method is based on fluorescent quantitative PCR technique, deletes ring using the method qualitative detection T cell receptor that chelex100 method processing dried blood spot directly expands, establishes SCID screening technical system quickly, efficiently, sensitive.This method is easy to operate, low in cost, easy to spread.
Description
Technical field
The invention belongs to neonatal hereditary diease gene screening fields, and in particular to a kind of drawing for SCID genetic screening
Object, detection kit and detection method.
Background technique
Severe combined immunodeficiency (severe combined immunodeficiency disease, SCID) be by
T lymphocyte caused by a variety of inherent causes, bone-marrow-derived lymphocyte, NK cell quantity lacks and/or dysfunction, and body fluid is caused to be exempted from
Epidemic disease, cellular immunity exist simultaneously major defect, and this primary immunodeficiency is often defined as T cells shortage.SCID's
Incidence is about 1/,100,000 life birth newborns, but because SCID infant is often dead before clarifying a diagnosis, which may be serious
Underestimate, untreated, the case fatality rate of SCID is 100%.95% infant is boy, is in sex-linked recessive inheritance more, occasionally there is normal dye
Colour solid recessive inheritance and Sporadic cases.In the initial several months of being born primary or repeated infection, serious prestige occur for clinical manifestation for infant
Coerce infant life and quality of life.
Chan and Puck first reported in 2005 detects T cell receptor resecting loop (T- using quantifying PCR method
cell receptor excisioncircles,TRECs).TREC is the encoding T cell receptor in thymus development maturation
Gene recombinated, the free cyclic DNA of small fragment is generated in regrouping process.TRECs is not multiple in T cell fission process
System, therefore can be used as the reliability index for judging that thymus gland exports initial T lymphocyte function.If TRECs is reduced or missing, height
Suspect SCID, further row fluidic cell and gene diagnosis are made a definite diagnosis.
The common treatment method of SCID is hematopoietic stem cell transplantation, and in certain type SCID, (such as ADA gene is prominent for gene therapy
Become caused by adenosine deaminase deficiency) in also have successful application.Studies have shown that receives HSCT survival rate before 3.5 monthly ages
94%, and carrying out HSCT survival rate after 3.5 monthly ages generation chronic diarrhea, severe infections is only 69%.If birth when fail and
When screening go out SCID, infant will face repeated infection, life quality decline；In addition, metainfective stem cell transplantation needs to adjust
Medication reduces transplanting success rate, illustrates that early diagnosis, early treatment can significantly improve survival rate, reduces case fatality rate.However,
Since SCID patient lacks clinical symptoms before infecting, how before infection EARLY RECOGNITION becomes key.
Currently, newborn's SCID screening, U.S.'s disease have been carried out in the developed countries such as U.S. and China Taiwan
This project is also incorporated examination of newborn infant diseases External quality evaluation system with prevention center by disease control, but country SCID is new
Raw youngster's screening and corresponding gene diagnosis detection work are also in the elementary step.There has been no carry out newborn crowd's large sample for the existing country
The report of SCID screening does not set up specific newborn SCID screening technical system yet, therefore, establishes quick, efficient, sensitivity
SCID screening technical system has very important significance.
Summary of the invention
For technological gap in the prior art, the object of the present invention is to provide a kind of drawing for SCID genetic screening
Object, detection kit and detection method are based on fluorescent quantitative PCR technique, are directly expanded using chelex100 method processing dried blood spot
Method qualitative detection T cell receptor delete ring, establish SCID screening technical system quickly, efficiently, sensitive.This method operation
It is convenient, it is low in cost, it is easy to spread.
The present invention is achieved by the following technical solutions:
The first object of the present invention is the primer for proposing to be used for SCID genetic screening, including TREC gene magnification
Draw the upstream and downstream of upstream and downstream primer and probe, the upstream and downstream primer and probe of internal reference C α gene magnification, external control C α gene magnification
Object and probe；The upstream and downstream primer sequence of the TREC gene magnification is described as shown in SEQ ID NO.1 and SEQ ID NO.2
TREC gene probe sequence is as shown in SEQ ID NO.3；The upstream and downstream primer sequence such as SEQ ID of the internal reference C α gene magnification
Shown in NO.4 and SEQ ID NO.5, the internal reference C α gene probe sequence is as shown in SEQ ID NO.6；The external control C α gene
The upstream and downstream primer sequence of amplification is as shown in SEQ ID NO.4 and SEQ ID NO.5, and the external control C α gene probe sequence is such as
Shown in SEQ ID NO.7.
The second object of the present invention is to propose a kind of gene detecting kit for SCID genetic screening, the examination
Agent box includes DNA extracting solution, the upstream and downstream primer and probe of TREC gene magnification, the upstream and downstream primer of internal reference C α gene magnification
And probe, the upstream and downstream primer and probe of external control C α gene magnification；The upstream and downstream primer sequence of the TREC gene magnification is such as
Shown in SEQ ID NO.1 and SEQ ID NO.2, the TREC gene probe sequence is as shown in SEQ ID NO.3；The internal reference C α
The upstream and downstream primer sequence of gene magnification is as shown in SEQ ID NO.4 and SEQ ID NO.5, the internal reference C α gene probe sequence
Column are as shown in SEQ ID NO.6；The upstream and downstream primer sequence of the external control C α gene magnification such as SEQ ID NO.4 and SEQ ID
Shown in NO.5, the external control C α gene probe sequence is as shown in SEQ ID NO.7.
Further, the kit further includes PCR MIX reaction solution and deionized water.
Further, the PCR MIX reaction solution includes Taq archaeal dna polymerase, buffer, dNTPs and MgCl2；It is described
The concentration of Taq archaeal dna polymerase is 0.2~2U/reaction；The dNTPs includes dATP, dGTP, dCTP, dTTP, they
Concentration is 2.5mM；The MgCl2Concentration be 250mM.
Further, the Taq archaeal dna polymerase is chemical modification hot start Taq polymerase, and concentration is 5U/ μ l.
Third object of the present invention is to propose a kind of SCID genetic screening method, include the following steps:
(1) two groups of PCR reaction solutions are prepared, including TREC detection liquid and external control detect liquid；The TREC detection liquid includes PCR
Draw the upstream and downstream of MIX reaction solution, deionized water, the upstream and downstream primer and probe of TREC gene magnification, internal reference C α gene magnification
Object and probe；The external control C α detection liquid includes PCR MIX reaction solution, deionized water, the upstream and downstream primer of external control gene magnification
And probe；The upstream and downstream primer sequence of the TREC gene magnification is described as shown in SEQ ID NO.1 and SEQ ID NO.2
TREC gene probe sequence is as shown in SEQ ID NO.3；The upstream and downstream primer sequence such as SEQ ID of the internal reference C α gene magnification
Shown in NO.4 and SEQ ID NO.5, the internal reference C α gene probe sequence is as shown in SEQ ID NO.6；The external control C α gene
The upstream and downstream primer sequence of amplification is as shown in SEQ ID NO.4 and SEQ ID NO.5, and the external control C α gene probe sequence is such as
Shown in SEQ ID NO.7；
(2) sample genomic dna is extracted；
(3) template DNA obtained in the step (2) is carried out respectively using two groups of PCR reaction solutions in step (1)
PCR amplification obtains two groups of pcr amplification products；
(4) two groups of pcr amplification products obtained in step (4) are analyzed, according to sample genomic dna to be detected
In target amplification Ct value judge a possibility that infant suffers from SCID.
Further, sample genomic dna is extracted method particularly includes:
(1) piece blood card is played from blood card using punch and is put into 1.5mlEP pipe；
(2) 1000 μ l ddH2O are added into the EP pipe equipped with blood card, stand 15min after the concussion that is vortexed, 12000rpm from
Heart 2min abandons supernatant；
(3) 200 μ l 5%chelex100 solution are added into step (2) EP pipe, put 56 DEG C of incubations after concussion centrifugation
30min is centrifuged after taking out concussion 1min, puts 100 DEG C of processing 8min, takes out 12000rpm centrifugation 2min after acutely concussion, takes out
Put 4 DEG C it is spare.
Further, in the step (1) PCR MIX reaction solution include Taq archaeal dna polymerase, buffer, dNTPs and
MgCl2；The Taq archaeal dna polymerase is chemical modification hot start Taq polymerase, and concentration is 5U/ μ l；The dNTPs include dATP,
DGTP, dCTP, dTTP, their concentration are 2.5mM；The MgCl2Concentration be 250mM.
Further, it is 12.5 μ L (2 × Mix) that TREC, which detects the total volume of liquid, and amplification system includes 55 × Buffer of μ L
(TrispH8.0, KCl), 0.25 μ L MgCl2, 2 μ L dNTP Mix (10mM), 1 μ L TREC gene magnification upstream primer SEQ
ID NO1,1 μ L TREC gene magnification downstream primer SEQ ID NO2,0.5 μ L TREC gene probe SEQ ID NO3,0.125 μ
L internal reference C α gene magnification upstream primer SEQ ID NO4,0.125 μ L internal reference C α gene magnification downstream primer SEQ ID NO5,
0.25 μ L internal reference C α gene probe SEQ ID NO6,0.2 μ L chemical modification hot start Taq polymerase and 2.05 μ L deionized waters.
Further, the total volume of external control detection liquid is 12.5 μ L (2 × Mix), and amplification system includes 55 × Buffer of μ L
(TrispH8.0, KCl), 0.25 μ L MgCl2, 2 μ L dNTP Mix (10mM), 1 μ L external control C α gene magnification upstream primer SEQ
ID NO4,1 μ L external control C α gene magnification downstream primer SEQ ID NO5,0.5 μ L external control C α gene probe SEQ ID NO7,0.2 μ
L chemical modification hot start Taq polymerase and 2.05 μ L deionized waters.
Compared with prior art, the advantages of the present invention are as follows:
(1) technical solution of the present invention is based on fluorescent quantitative PCR technique, by the high target primer of design specificity, interior
Reference object and external control primer are reconfigured to easy to use, the reliable kit of testing result, it is anti-to design scientific and reasonable PCR
Answer system；Ring is deleted using the method qualitative detection T cell receptor that chelex100 method processing dried blood spot directly expands, is established fast
Speed, SCID screening technical system efficiently, sensitive.So that the present invention is high for newborn SCID hereditary disease spectrum sieve accuracy, it is special
Anisotropic strong, quickly, easy, cost performance is high, is particularly suitable for the popularization and application in clinical examination work.
(2) kit of the invention and detection method are compareed provided with external control, can effectively be assessed this PCR amplification system and be built
It is vertical whether reasonability, while being added to internal reference C α gene primer in detection reaction solution, can be effective by the amplification of reference gene
The appearance of false positive results is avoided, to guarantee the reliability of data, finally ensures that kit test result is accurate.
(3) sample acquisition, storage are convenient in detection method of the invention, and DNA is extracted simply, and blood using amount is seldom, very suitable
Close the neonatal screening that should not measure blood greatly.Meanwhile blood card sample properties stablize the transport for being conducive to the long period and long-term guarantor
It deposits, considerably reduces the appearance of sample problem in acquisition, storage and all links of transport, be the accuracy of last inspection result
And stability has established good basis.
Detailed description of the invention
Fig. 1 is SY01709088 sample amplification curve in embodiment 3.
Fig. 2 is SY01801001 sample amplification curve in embodiment 3.
Fig. 3 is SY01803012 sample amplification curve in embodiment 3.
Fig. 4 is SY01806056 sample amplification curve in embodiment 3.
Specific embodiment
Applicant is in conjunction with specific embodiments described in further details technical solution of the present invention below, so that this field
Technical staff may be better understood the present invention and can be practiced, but range is claimed not as the present invention in illustrated embodiment
Restriction.
Embodiment 1: specific primer, gene detecting kit for SCID genetic screening
One, the design of primer and probe and synthesis are somebody's turn to do:
According to UCSCHumanGeneSorter on the website UCSC inquire TRECs and internal reference C α gene order (http: //
Genome.ucsc.edu/cgi-bin/hgNear), quantitative fluorescent PCR is designed on TRECs and C α gene using Primer3.0
Upstream and downstream primer and probe.Selected primer is combined with good specificity for gene, there is higher PCR amplification efficiency.
5 ' ends of TRECs probe are FAM fluorophor, and 3 ' ends are TAMRA fluorophor；5 ' ends of C α gene probe are VIC fluorescent base
Group, 3 ' ends are TAMRA fluorophor.Meanwhile the present invention is also provided with the primer and probe of external control control.The following institute of primer sequence
Show.
TRECs target expands upstream primer SEQ ID NO.1:
5`-gtttttgtaaaggtgcccactcc-3`
TRECs target expands downstream primer SEQ ID NO.2:
5`-acggtgaatgaagagcagacag-3`
TRECs targeting probe SEQ ID NO.3:
5`-FAM-cacctgcaccccgtgcctaaacc-TAMRA-3`
Internal reference C α expands upstream primer SEQ ID NO.4:
5`-aaatgagatcatgtcctaaccctg-3`
Internal reference C α expands downstream primer SEQ ID NO.5:
5`-tggatttagagtctctcagctggt-3`
Internal reference C α probe SEQ ID NO.6:
5`-VIC-acagatatccagaaccctgaccctgcc-TAMRA-3`
External control C α expands upstream primer SEQ ID NO.4
5`-aaatgagatcatgtcctaaccctg-3`
External control C α expands downstream primer SEQ ID NO.5
5`-tggatttagagtctctcagctggt-3`
External control C alpha specific probe SEQ ID NO7:
5`-FAM-acagatatccagaaccctgaccctgcc-TAMRA-3`
Two, two groups of PCR reaction solutions are prepared
PCR reaction solution includes TREC detection liquid and external control detection liquid in the present embodiment.
1, TREC detects liquid
Including PCR MIX reaction solution, deionized water, the upstream and downstream primer and probe of TREC gene magnification, C α gene magnification
Upstream and downstream primer and probe
The total volume that TREC detects liquid system is 12.5 μ L (2 × Mix), and the component of amplification system, concentration or content are as follows:
TREC detects liquid:
Wherein amplimer, probe mixed liquor include 1 μ L TREC gene magnification upstream primer SEQ ID NO1,1 μ L
TREC gene magnification downstream primer SEQ ID NO2,0.5 μ L TREC gene probe SEQ ID NO3,0.125 μ L C α gene expand
Increase upstream primer SEQ ID NO4,0.125 μ L C α gene magnification downstream primer SEQ ID NO5,0.25 μ L C α gene probe
SEQ ID NO6。
2, external control detects liquid
Including PCR MIX reaction solution, deionized water, the upstream and downstream primer and probe of external control gene magnification.
The total volume that external control detects liquid system is 12.5 μ L (2 × Mix), and the component of amplification system, concentration or content are as follows:
External control detects liquid:
Wherein amplimer, probe mixed liquor include 1 μ L external control gene magnification upstream primer SEQ ID NO4,1 μ L external control
Gene magnification downstream primer SEQ ID NO5,0.5 μ L external control gene probe SEQ ID NO7.
Embodiment 2: the use of kit in embodiment 1
1, sample genomic dna is extracted
(1) 3 blood cards are played from blood card using aperture 3.0mm punch be put into 1.5mlEP pipe.
(2) 1000 μ l ddH2O are added into the above-mentioned EP pipe equipped with blood card, stand 15min after the concussion that is vortexed,
12000rpm is centrifuged 2min, abandons supernatant, repeatable to washed once if Blood piece color is still very deep.
(3) 200 μ l 5%chelex100 (sigma) solution are added in Xiang Shangshu EP pipe, put 56 DEG C of incubations after concussion centrifugation
30min is centrifuged after taking out concussion 1min, puts 100 DEG C of processing 8min, takes out 12000rpm centrifugation 2min after acutely concussion, takes out
Put 4 DEG C it is spare.
2, PCR amplification
PCR expansion is carried out respectively to template DNA obtained in the step 1 using two groups of PCR reaction solutions in embodiment 1
Increase, obtains two groups of pcr amplification products.Fluorescence detection channel selects FAM, TAMRA sense channel.
PCR program are as follows: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 40s) × 45cycles, 60 DEG C are collected simultaneously 2 kinds of fluorescence letters
Number.
3, real-time fluorescence PCR detection
Two groups of pcr amplification products obtained in step 2 are analyzed, according in sample genomic dna to be detected
Target expands Ct value to judge a possibility that infant suffers from SCID.
Embodiment 3: pattern detection
1,4 infant's dried blood spots of clinically known diagnosis result are chosen, sample DNA is extracted, are operated as follows:
(1) each from 4 dried blood spots using aperture 3.0mm punch to play 3 blood cards and be put into 4 corresponding 1.5mlEP pipes
It is interior.
(2) 1000 μ l ddH2O are added into the above-mentioned EP pipe equipped with blood card, stand 15min after the concussion that is vortexed,
12000rpm is centrifuged 2min, abandons supernatant, repeatable to washed once if blood card color is still very deep.
(3) 200 μ l 5%chelex100 (sigma) solution are added into above-mentioned each EP pipe, puts 56 DEG C after concussion centrifugation and incubates
30min is educated, is centrifuged after taking out concussion 1min, puts 100 DEG C of processing 8min, 12000rpm centrifugation 2min after acutely concussion is taken out, takes
Put out 4 DEG C it is spare.
1 sample information of table
| Subject's number | Age | Gender | Clinical diagnosis |
| SY01709088 | 8 months | Male | Normally |
| SY01801001 | 4 months | Male | SCID |
| SY01803012 | 5 months | Male | Normally |
| SY01806056 | 3 months | Female | Normally |
2, PCR is detected
Two kinds of detection liquid premix Mix (2 ×) prepared is taken out from -20 DEG C of refrigerators, puts room-temperature dissolution, is waited completely molten
Centrifugation is slightly mixed by inversion after solution, every kind of template is detected with two kinds of detection liquid respectively simultaneously, and two kinds of detection liquid respectively prepare 16 instead
Mix is answered, every hole dispenses 23 μ l Mix, is then respectively adding each 2 μ l of template supernatant that 4 kinds have prepared, and every kind of template repeats 3
2 μ l ddH2O are added in hole, the hole NTC.Reaction system is formulated as follows:
2 reaction system of table
Thermal cycle conditions:
95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 40s) × 45cycles, 60 DEG C are collected simultaneously 2 kinds of fluorescence signals
3, amplification is analyzed
Testing result interpretation standard:
(1) channel NTC target detection liquid FAM and the channel TAMRA are without amplification Ct value or without sigmoid curve；The detection of NTC external control
The channel liquid FAM is also without amplification Ct value or without sigmoid curve.
(2) sample external control detects value≤28 liquid Ct, then sample form amount is met the requirements, and can further be analyzed；Sample external control
Liquid Ct value > 30 is detected, then sample form amount deficiency should handle sample again；Sample external control detects 28 value≤30 < Ct of liquid Ct value,
The only high sample of TREC content can detect.
(3) sample internal control Ct value meets value≤36 Ct, there is normal S-shaped amplification curve.
When meeting conditions above, Ct value is expanded according to sample target and judges a possibility that infant suffers from SCID.
Table 3
Remarks: △ Ct=CtTREC-Ct external control
The amplification curve of (4) 4 samples to be tested is as shown in Figure 1, Figure 2, Figure 3, Figure 4, and amplification curve exports Ct value result such as
Shown in table 4:
Table 4
| Sample | CtTREC | CtExternal control | CtTREC-CtExternal control | Result judgement |
| SY01709088 | 36.43 | 27.78 | 8.65 | Normally |
| SY01801001 | - | 27.13 | - | It is abnormal |
| SY01803012 | 33.97 | 28.01 | 5.96 | Normally |
| SY01806056 | 33.16 | 27.08 | 6.08 | Normally |
Therefore, TREC curve is expanded by the fluorescence PCR detection reagent kit in the present embodiment and result judgement standard obtains
Sample results information it is consistent with sample to be tested known results.
This detection kit is only used for SCID screening use, it is intended that illustrates a possibility that newborn suffers from SCID, cannot function as most
Whole clinical diagnosis result.As a result normally illustrate that a possibility that subject suffers from SCID is little, results abnormity illustrates the possibility for suffering from SCID
Property is bigger, need to further make fluidic cell and gene diagnosis is made a definite diagnosis.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Sequence table
<110>scientific and technological (Wuhan) Co., Ltd of good training gene biological
<120>a kind of for the primer of SCID genetic screening, detection kit and detection method
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Claims (10)
1. being used for the primer of SCID genetic screening, which is characterized in that including the upstream and downstream primer of TREC gene magnification and spy
Needle, the upstream and downstream primer and probe of internal reference C α gene magnification, the upstream and downstream primer and probe of external control C α gene magnification；It is described
The upstream and downstream primer sequence of TREC gene magnification is as shown in SEQ ID NO.1 and SEQ ID NO.2, the TREC gene probe
Sequence is as shown in SEQ ID NO.3；The upstream and downstream primer sequence of the internal reference C α gene magnification such as SEQ ID NO.4 and SEQ
Shown in ID NO.5, the internal reference C α gene probe sequence is as shown in SEQ ID NO.6；The external control C α gene magnification it is upper and lower
Primer sequence is swum as shown in SEQ ID NO.4 and SEQ ID NO.5, the external control C α gene probe sequence such as SEQ ID NO.7
It is shown.
2. a kind of gene detecting kit for SCID genetic screening, which is characterized in that the kit includes that DNA is extracted
Liquid, the upstream and downstream primer and probe of TREC gene magnification, the upstream and downstream primer and probe of internal reference C α gene magnification, external control C α base
The upstream and downstream primer and probe of gene-amplification；The upstream and downstream primer sequence of the TREC gene magnification such as SEQ ID NO.1 and SEQ
Shown in ID NO.2, the TREC gene probe sequence is as shown in SEQ ID NO.3；The upstream and downstream of the internal reference C α gene magnification
Primer sequence is as shown in SEQ ID NO.4 and SEQ ID NO.5, the internal reference C α gene probe sequence such as SEQ ID NO.6 institute
Show；The upstream and downstream primer sequence of the external control C α gene magnification is as shown in SEQ ID NO.4 and SEQ ID NO.5, the external control
C α gene probe sequence is as shown in SEQ ID NO.7.
3. a kind of gene detecting kit for SCID genetic screening according to claim 2, which is characterized in that institute
Stating kit further includes PCR MIX reaction solution and deionized water.
4. a kind of gene detecting kit for SCID genetic screening according to claim 3, which is characterized in that institute
Stating PCR MIX reaction solution includes Taq archaeal dna polymerase, buffer, dNTPs and MgCl2；The concentration of the Taq archaeal dna polymerase
For 0.2~2U/reaction；The dNTPs includes dATP, dGTP, dCTP, dTTP, their concentration is 2.5mM；It is described
MgCl2Concentration be 250mM.
5. a kind of gene detecting kit for SCID genetic screening according to claim 4, which is characterized in that institute
Stating Taq archaeal dna polymerase is chemical modification hot start Taq polymerase, and concentration is 5U/ μ l.
6. a kind of SCID genetic screening method, which comprises the steps of:
(1) two groups of PCR reaction solutions are prepared, including TREC detection liquid and external control detect liquid；The TREC detection liquid includes PCR MIX
Reaction solution, deionized water, the upstream and downstream primer and probe of TREC gene magnification, the upstream and downstream primer of internal reference C α gene magnification and
Probe；The external control C α detection liquid includes PCR MIX reaction solution, deionized water, the upstream and downstream primer of external control gene magnification and spy
Needle；The upstream and downstream primer sequence of the TREC gene magnification is as shown in SEQ ID NO.1 and SEQ ID NO.2, the TREC base
Because probe sequence is as shown in SEQ ID NO.3；The upstream and downstream primer sequence such as SEQ ID NO.4 of the internal reference C α gene magnification
With shown in SEQ ID NO.5, the internal reference C α gene probe sequence is as shown in SEQ ID NO.6；The external control C α gene magnification
Upstream and downstream primer sequence as shown in SEQ ID NO.4 and SEQ ID NO.5, the external control C α gene probe sequence such as SEQ
Shown in ID NO.7；
(2) sample genomic dna is extracted；
(3) PCR expansion is carried out respectively to template DNA obtained in the step (2) using two groups of PCR reaction solutions in step (1)
Increase, obtains two groups of pcr amplification products；
(4) two groups of pcr amplification products obtained in step (4) are analyzed, according in sample genomic dna to be detected
Target expands Ct value to judge a possibility that infant suffers from SCID.
7. a kind of SCID genetic screening method according to claim 6, which is characterized in that extract sample genomic dna
Method particularly includes:
(1) 3 blood cards are played from blood card using punch and are put into 1.5mlEP pipe；
(2) 1000 μ l ddH2O are added into the EP pipe equipped with blood card, 15min, 12000rpm centrifugation are stood after the concussion that is vortexed
2min abandons supernatant；
(3) 200 μ l 5%chelex100 solution are added into step (2) EP pipe, puts 56 DEG C of incubation 30min after concussion centrifugation, takes
Out shake 1min after be centrifuged, put 100 DEG C of processing 8min, take out acutely concussion after 12000rpm be centrifuged 2min, take out put 4 DEG C it is standby
With.
8. a kind of SCID genetic screening method according to claim 6, which is characterized in that PCR in the step (1)
MIX reaction solution includes Taq archaeal dna polymerase, buffer, dNTPs and MgCl2；The Taq archaeal dna polymerase is chemical modification heat
Start Taq enzyme, concentration is 5U/ μ l；The dNTPs includes dATP, dGTP, dCTP, dTTP, their concentration is 2.5mM；Institute
State MgCl2Concentration be 250mM.
9. a kind of SCID genetic screening method according to claim 8, which is characterized in that the total volume of TREC detection liquid
For 12.5 μ L (2 × Mix), amplification system includes 55 × Buffer of μ L (TrispH8.0, KCl), 0.25 μ L MgCl2, 2 μ L
DNTP Mix (10mM), 1 μ L TREC gene magnification upstream primer SEQ ID NO1,1 μ L TREC gene magnification downstream primer SEQ
ID NO2,0.5 μ L TREC gene probe SEQ ID NO3,0.125 μ L internal reference C α gene magnification upstream primer SEQ ID NO4,
0.125 μ L internal reference C α gene magnification downstream primer SEQ ID NO5,0.25 μ L internal reference C α gene probe SEQ ID NO6,0.2 μ L
Chemical modification hot start Taq polymerase and 2.05 μ L deionized waters.
10. a kind of SCID genetic screening method according to claim 8, which is characterized in that the totality of external control detection liquid
Product is 12.5 μ L (2 × Mix), and amplification system includes 55 × Buffer of μ L (TrispH8.0, KCl), 0.25 μ L MgCl2, 2 μ L
DNTP Mix (10mM), 1 μ L external control C α gene magnification upstream primer SEQ ID NO4,1 μ L external control C α gene magnification downstream primer
SEQ ID NO5,0.5 μ L external control C α gene probe SEQ ID NO7,0.2 μ L chemical modification hot start Taq polymerase and 2.05 μ L go from
Sub- water.
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| CN109852687A (en) * | 2019-03-26 | 2019-06-07 | 普文博泰生物科技(佛山)有限公司 | A kind of composite amplification primer sets, kit and method for genetic screening |
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