CN108998511A - It is a kind of detect TRECs and KRECs gene real-time fluorescence quantitative PCR kit and its application - Google Patents
It is a kind of detect TRECs and KRECs gene real-time fluorescence quantitative PCR kit and its application Download PDFInfo
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Abstract
The present invention relates to a kind of for detecting the real-time fluorescence quantitative PCR kit of TRECs and KRECs gene, include: in the kit 1) DNA extract reagent, 2) standard items comprising TRECs gene, KRECs gene and TRAC gene inserts, 3) comprising the Fluorescence PCR liquid I of TRECs gene, the fluorescence PCR primer of TRAC gene and detection probe, 4) include KRECs gene, the fluorescence PCR primer of TRAC gene and detection probe Fluorescence PCR liquid II.Kit high sensitivity of the invention, specificity is good, detection method is simple and fast, experimental result is reliable, it not only can be with the immunologic deficiency disease based on screening SCID and antibody deficiency, and clinically relevant indication can be provided for other primary immunodeficiency disease relevant to T cell and B cell development or other systems disease, be conducive to early diagnose and treated.
Description
Technical field
The invention belongs to beyond body nucleic acid diagnostic fields, are related to a kind of detection T cell receptor resecting loop (T-cell
Receptor excision circles, TRECs) and K- deletion recombination resecting loop (K-deleting recombination
Excision circles, KRECs) gene Fluorescent quantitative PCR kit and its application.
Background technique
Severe combined immunodeficiency (severe combined immunodeficiency disease, SCID) is connection
The type of most serious in immunologic deficiency disease is closed, patient is usually dead in 1 year after birth.It is obtained according to external a wide range of screening
The incidence of SCID is about 1/58000 life birth newborn, is significantly higher than 1/100000 thought in the past.SCID is by a variety of heredity
T lymphocyte caused by factor, bone-marrow-derived lymphocyte, NK cell quantity lacks and (or) dysfunction, leads to humoral immunity, cell
Immune to exist simultaneously major defect, this primary immunodeficiency is often defined as T cells shortage.It is untreated, SCID
Case fatality rate be 100%.Currently used treatment method is hematopoietic stem cell transplantation (hematopoietic stem cell
Transplantation, HSCT), gene therapy (gene therapy, GT) or enzyme replacement therapy (enzyme
Replacement therapy, ERT) carry out immunologic reconstitution.Pai etc. passes through the SCID patient after follow-up 240 transplanting, it is believed that <
The survival rate of the SCID graft at 3.5 monthly ages is significantly higher than the infant of > 3.5 monthly ages or secondary infection, and the survival after transplanting
Rate is mainly to infant whether there is or not infection is related, and the source with graft is without obvious relation between persistence.Therefore, SCID patient is early diagnosed
And early treatment, the prognosis of infant can be significantly improved.
SCID infant majority there are T cell shortage, therefore lymphocyte absolute counting can be used as SCID screening means it
One.Chan and Puck first reported in 2005 detects T cell receptor resecting loop (T-cell using quantifying PCR method
receptor excision circles,TRECs).TRECs is the non-functional DNA fragmentation generated in TCR forming process, about
70% α β TCR can generate TRECs simultaneously in generating process, these DNA fragmentations are not replicated with the division of T cell, stablize
It is present in into the cell, therefore TRECs can quantitatively reflect the initial T lymphocyte quantity of thymus gland output indirectly.If TRECs subtracts
It less or lacks, then strong suspicion SCID, further progress fluidic cell and gene diagnosis are made a definite diagnosis.It can by TRECs screening SCID
To share the dry blood scraps of paper with other newborn's routine screening projects, be conducive to the formal a wide range of popularization of the screening method.In addition to
Outside SCID, TRECs detection can also find include trisomy 21, DiGeorge syndrome, idiopathic T lymphocyte reduction disease etc. other
Cause the disease of the serious reduction of t subset lymphocyte count.
TRECs detection is cheap, including expense required for equipment, labor service and reagent etc. is about 4.25 dollars/baby,
It is suitable or lower with other screening item prices；Method is highly sensitive, special, and the sensitivity of screening test can have close to 100%
Effect detection SCID infant；Therefore public health service can be effectively applied to by carrying out SCID neonatal screening using TRECs, be considered
It is the U.S.'s " neonatal screening project of national standard ".
It may only be omitted by TRECs detection when some non-specific, phenotypic delay SCID patients are born, K- is needed to delete
Except other detection means such as recombination resecting loop, association is examined.KRECs is in B cell maturation, when encoding B cells receptor gene rearrangements
The small fragment DNA product of the not replicated of generation can be used as the sieve of the bone-marrow-derived lymphocyte immune deficiency with B cell quantity reduction
Checking method.Such as in retardance ADA patient, T cell division may be preferable to the increased tolerance of toxic metabolic products, and B is thin
Born of the same parents are then easier to crack, and KRECs is conducive to the early detection of such patient；KRECs is in X- linked agammaglobulinemia
(XLA) it is also played an important role in screening.Ideally, it should combine TRECs-KRECs and carry out neonatal immunity
The screening of defect.
It is estimated that SCID and the common disease incidence of agammaglobulinemia are 1/50000~1/30000, detect simultaneously
TRECs and KRECs can not only expand disorder in screening spectrum, but also the cost-effectiveness of the examination of newborn infant diseases can be improved on the whole.
One research of France to 2006 to 2010 SCID screening costs analysis shows that, the early detection of SCID can make every trouble
The treatment cost of person reduces by 50000~100000 Euros；Assuming that the unit cost of test is 5 Euros, needed for SCID balance between revenue and expenditure
Disease incident be about 1/20000；However the survival advantage that stem cell transplantation is once carried out in 3 monthly ages is proved, Ren Qun great
Range disorder in screening can obviously save cost.Most of all, the postnatal detection of SCID case allows infant to obtain in time
Treatment avoids long-term, expensive Intensive Care Therapy and the medical treatment cost dramatically increased after complication occurs.Early screening can be improved
Cure rate substantially improves quality of life, reduces burden on society, identifies that SCID seems most important as early as possible.
Application No. is 201210470713.5 Chinese patent applications to provide quantitative detection TRECs and KRECs gene
Real-time fluorescence quantitative PCR kit and application.The patent is used to be expanded including nested PCR amplification and real-time fluorescence quantitative PCR
Two-step method, it is complicated for operation it is cumbersome, time-consuming, without specific reference threshold, in particular by DNA extraction method extract
DNA purity is not high, nest-type PRC bring expands the pipetting bring sample contamination after error, nest-type PRC, all polycyclic
A possibility that accumulative error of section eventually leads to missing inspection is big.As missing inspection occurs during screening, infant will miss excellent diagnostics
And therapic opportunity.
In view of the above-mentioned problems, the present invention first improves DNA extraction method, reduces operating procedure, shortens time-consuming,
The DNA for obtaining higher purity, is then also optimized primer and probe, is reduced using a step real-time fluorescence quantitative PCR amplification
Error and sample pollution are expanded, there is TRECs and KRECs gene detecting kit more highly sensitive and specific, grasp simultaneously
Make easy to be quick.
Summary of the invention
It is higher and fast and convenient the main purpose of the present invention is to provide a kind of sensitivity and specificity, it is new to be used to screening
The real-time fluorescence quantitative PCR reagent for being used to detect TRECs and KRECs gene of raw youngster's primary T cell and B cell immune deficiency
Box, the kit are suitable for all types of fluorescent quantitative PCR instrument currently on the market.The present invention is able to detect newborn T
The level of cell and B cell judges whether neonatal immune system function is normal, before there is good application in clinical examination field
Scape.
It is another object of the present invention to provide a kind of above-mentioned real-time fluorescence quantitative PCR kits in screening newborn T
Application in cell and B cell immune deficiency, sample acquisition, transport needed for mentioned reagent box, storage are convenient, sensitivity and spy
Anisotropic high, detection method is easy, quick, and experimental result is accurate and reliable.
To achieve the goals above, the present invention provides the following technical scheme that
It is a kind of for detecting the real-time fluorescence quantitative PCR kit of TRECs and KRECs gene, include: 1) in the kit
DNA extraction reagent, 2) standard items comprising TRECs gene, KRECs gene and TRAC gene inserts, 3) it include TRECs
The Fluorescence PCR liquid I of gene, the fluorescence PCR primer of TRAC gene and detection probe, 4) it include KRECs gene, TRAC base
The fluorescence PCR primer of cause and the Fluorescence PCR liquid II of detection probe.
In a concrete scheme of the invention, the fluorescence PCR primer of the TRECs gene includes TRECs fluorescent PCR
Forward primer and reverse primer；The fluorescence PCR primer of the TRAC gene includes TRAC fluorescent PCR forward primer and reversely draws
Object；The fluorescence PCR primer of the KRECs gene includes KRECs fluorescent PCR forward primer and reverse primer；The detection probe
For TRECs detection probe, TRAC detection probe or KRECs detection probe.
In a concrete scheme of the invention, the Fluorescence PCR liquid I be by TRECs fluorescent PCR forward primer,
Reverse primer and TRECs detection probe, TRAC fluorescent PCR forward primer, reverse primer and TRAC detection probe, 2X real
Time Mix and sterile ultrapure water composition；The content of the TRECs fluorescent PCR forward primer and reverse primer is respectively 300-
1000nM, preferably 400nM；The content of the TRAC fluorescent PCR forward primer and reverse primer is respectively 300-1000nM,
Preferably 400nM；The content of the TRECs detection probe and TRAC detection probe is respectively 50-250nM, preferably 100nM.
In a concrete scheme of the invention, the sequence of the TRECs fluorescent PCR forward primer such as SEQ ID NO.1
Shown, the sequence of reverse primer is as shown in SEQ ID NO.2, and the sequence of TRECs detection probe is as shown in SEQ ID NO.3；
The sequence of TRAC fluorescent PCR forward primer as shown in SEQ ID NO.7, the sequence of reverse primer as shown in SEQ ID NO.8,
The sequence of TRAC detection probe is as shown in SEQ ID NO.9.
In a concrete scheme of the invention, the Fluorescence PCR liquid II be by KRECs fluorescent PCR forward primer,
Reverse primer and KRECs detection probe, TRAC fluorescent PCR forward primer, reverse primer and TRAC detection probe, 2X
Realtime Mix and sterile ultrapure water composition.The content of the KRECs fluorescent PCR forward primer and reverse primer is respectively
300-1000nM, preferably 400nM；The content of the TRAC fluorescent PCR forward primer and reverse primer is respectively 300-
1000nM, preferably 400nM；The KRECs detection probe, TRAC detection probe content are respectively 50-250nM, preferably
100nM。
In a concrete scheme of the invention, the sequence of the KRECs fluorescent PCR forward primer such as SEQ ID NO.4
Shown, the sequence of reverse primer is as shown in SEQ ID NO.5, and the sequence of KRECs detection probe is as shown in SEQ ID NO.6；
The sequence of TRAC fluorescent PCR forward primer as shown in SEQ ID NO.7, the sequence of reverse primer as shown in SEQ ID NO.8,
The sequence of TRAC detection probe is as shown in SEQ ID NO.9.
The TRECs in a concrete scheme of the invention, in Fluorescence PCR liquid I and Fluorescence PCR liquid II
Detection probe, KRECs detection probe and TRAC detection probe are Taqman probe, and label Taqman probe 5 ' end is fluorescence
Luminophore is one of FAM, VIC, TET, JOE, ROX, CY3, CY5, HEX；The end of label 3 ' is fluorescent quenching base
Group, is one of TAMRA, BHQ1.TRECs and TRAC in reaction system of the invention, in fluorescent PCR reaction solution I
Identical fluorescence radiation group not can be used for the specific probe of gene；KRECs and TRAC base in fluorescent PCR reaction solution II
Identical fluorescence radiation group not can be used for the specific probe of cause.
In a concrete scheme of the invention, 5 ' ends of TRECs detection probe label are HEX fluorophor, 3 '
End is TAMRA fluorescent quenching group；5 ' ends of the KRECs detection probe label are HEX fluorophor, and 3 ' ends are that TAMRA is glimmering
Optical quenching group；5 ' ends of the TRAC detection probe label are FAM fluorophor, and 3 ' ends are TAMRA fluorescent quenching group.
In a concrete scheme of the invention, the DNA extracts reagent and includes: 1) 0.5% (V/V) Triton X-
100,100-200 μ l；2) PBS:100-200 μ l；3) Generation DNA Elution Solu II, 120-300 μ l,
In, first step dosage: 100-200 μ l, second step dosage: 20-100 μ l；The standard items are by 138 cores of TRECs gene
80 nucleotide fragments of acid fragments, 90 nucleotide fragments of KRECs gene and TRAC gene connect after being stitched together
Enter pUC57 vector plasmid；The method that droplet type digital pcr is used in standard items digestion after purification measures copy number, and storage concentration is
1.0x104copies/μl、1.0x103copies/μl、1.0x102copies/μl、2.0x101copies/μl、
1.0x1016 copies/ μ l, 2.0copies/ μ l concentration gradients.
In a concrete scheme of the invention, the insetion sequence of the TRECs gene as shown in SEQ ID NO.10,
The insetion sequence of KRECs gene is as shown in SEQ ID NO.11, and the insetion sequence of TRAC gene is as shown in SEQ ID NO.12.
In another aspect of the invention, a kind of above-mentioned real-time fluorescence quantitative PCR kit is additionally provided for screening
Application in newborn's T cell and B cell immune deficiency, including the following steps: with kit in dried blood spot sample to be measured
DNA extract；The DNA of extraction is subjected to real-time fluorescence quantitative PCR amplification and detection；By comparing sample to be tested and standard
The cycle threshold of product calculates starting TRECs, KRECs and TRAC gene copy number of sample to be tested according to standard curve, and uniformly
It is scaled to copies/ μ l blood.
In a concrete scheme of the invention, the negative controls of the sample to be tested be containing TRECs, KRECs and
The recombinant plasmid of tri- genetic fragments of TRAC, concentration copy are 1.0x103copies/μl。
In a concrete scheme of the invention, the blank control product of the sample to be tested are sterile nuclease-free water.
Beneficial effects of the present invention:
1) sample acquisition, transport, storage are convenient.Kit of the present invention only needs punching to drill through a piece of diameter 3mm's
Round dried blood spot (blood volumes of about 3 μ l), which extracts DNA, can be completed the detection of the project, and blood using amount is seldom, be very suitable to unsuitable big
Measure the neonatal screening of blood.Meanwhile dried blood spot sample properties are stablized conducive to prolonged transport and long-term preservation, greatly
Reduce the appearance of sample problem in the links such as acquisition, storage and transport, is the accuracy and stability of last inspection result
Good basis is established.
2) sensitivity and specificity are high.DNA abundance, the concentration extracted using kit of the present invention are high, normal sample
The copy number of this TRECs and KRECs gene is high, TRECs the and KRECs numerical value of the positive sample through clinical diagnosis verifying is 0.Sun
Property result and negative findings significant difference, be highly susceptible to being judged.Also extremely efficient reduce false negative result simultaneously
Occur, to avoid the generation of missing inspection.
3) present invention effectively reduces going out for false positive results by selecting the TRAC gene of sample itself as internal reference
It is existing, to guarantee the reliability of data.
4) at low cost.Usual 1 genetic test needs 1 hole, and 3 genetic tests need 3 holes, and present invention only requires 2
3 genes are detected in a hole, efficiently reduce reacting hole, reduce cost, are highly convenient for promoting and applying in hospital and laboratory.
5) the method for the present invention is simple, and detection time-histories is short, and testing result is accurate and reliable, and it is very clear to determine, is highly convenient for
Hospital and laboratory promote and apply.
6) kit of the present invention had both been applicable to manual experiment, also easily expands to automation equipment, is conducive to height
The screening project of flux is carried out.
7) defining for critical value integrates with international standard.The threshold value of detection method is one of most important parameter.
In SCID screening field, the research level of American-European study group maintains the leading position.Each study group TRECs and KRECs gene screening
PCR detection method it is not identical, and the threshold value for the TRECs and KRECs gene that different experimental methods obtains is also different.It is comprehensive
The threshold value that the threshold value of TRECs is scheduled on 25copies/ μ l blood, KRECs is scheduled on by analysis, most America and Europe study groups
15copies/ μ l blood, including in the several well-known study groups in this field.The threshold value for the TRECs that the present invention obtains is
The threshold value of 25copies/ μ l blood, KRECs are 15copies/ μ l blood, consistent with most American-European results of study group.
In short, the method that the present invention takes real time fluorescent quantitative detection TRECs and KRECs gene, is extracting DNA circle section,
Approach is extracted using different DNA and the DNA of stable purity is high, directly progress real-time fluorescence can be obtained after repeatedly improving
Quantitative pcr amplification.Process is simple, and the time is short, and reagent cost is low, while reducing bring due to more wheel nested PCR amplifications
Expand error and liquid relief pollution.Link is expanded in real-time fluorescence quantitative PCR, gropes to have adjusted PCR reaction system, so that negative knot
Fruit and positive findings comparison are significant, are easy to determine, achieve the purpose that reducing false negative improves specificity, this faces in detection later
It is verified in the test of bed positive sample.Kit high sensitivity of the invention, specificity is good, and detection method is simply fast
Victory, experimental result is reliable, not only can be with the immunologic deficiency disease based on screening SCID and antibody deficiency, but also can be other and T
Cell and B cell develop relevant primary immunodeficiency disease or other systems disease provides clinically relevant indication, are conducive to morning
Phase diagnosis and treatment.
Detailed description of the invention
Figure 1A and 1B is respectively standard items TRECs real time PCR amplification curve and standard curve；
Fig. 2A and 2B is respectively that standard items TRECs detects corresponding internal reference TRAC real time PCR amplification curve and standard curve；
Fig. 3 A and 3B are respectively standard items KRECs real time PCR amplification curve and standard curve；
Fig. 4 A and 4B are respectively that standard items KRECs detects corresponding internal reference TRAC real time PCR amplification curve and standard curve；
Fig. 5 A to 5D is respectively negative controls TRECs, TRAC, KRECs and TRAC real time PCR amplification curve；
Fig. 6 A to 6D is respectively blank control product TRECs, TRAC, KRECs and TRAC real time PCR amplification curve；
Fig. 7 is the real-time PCR TRECs and TRAC amplification curve of normal child's DNA sample；
Fig. 8 is the real-time PCR TRECs amplification curve of SCID positive children's DNA sample；
Fig. 9 is the real-time PCR TRAC amplification curve of SCID positive children's DNA sample；
Figure 10 is the real-time PCR KRECs and TRAC amplification curve of normal child's DNA sample；
Figure 11 is the KRECs amplification curve of XLA positive children's DNA sample；
Figure 12 is the TRAC amplification curve of XLA positive children's DNA sample.
Specific embodiment
Invention is further explained with reference to the accompanying drawings of the specification.
Following material or reagent are unless stated otherwise commercially available.
Embodiment 1: the preparation of kit
1, the design and synthesis of primer and probe
According to UCSC query site TRECs, KRECs and TRAC gene order, using Primer 3.0 TRECs,
The forward and reverse primer and probe of quantitative fluorescent PCR is designed on KRECs and TRAC gene.Selected primer has higher PCR amplification efficiency,
Selected probe is combined with good specificity for gene.Primer and probe entrust the Suzhou gold only limited public affairs of intelligence biotechnology
Department carries out chemical synthesis, and wherein primer is PAGE purifying, and probe is HPLC purifying.5 ' ends of TRECs detection probe label are HEX
Fluorophor, 3 ' ends are TAMRA quenching group；5 ' ends of KRECs detection probe label are HEX fluorophor, and 3 ' ends are
TAMRA quenching group；5 ' ends of TRAC detection probe label are FAM fluorophor, and 3 ' ends are TAMRA quenching group.Primer sequence
Column such as table 1.
Table 1 is inserted into gene, specific primer and detection probe sequence
2, the building of standard items
Standard items containing tri- genetic fragments of TRECs, KRECs and TRAC by Suzhou Jin Weizhi Biotechnology Co., Ltd into
Row gene chemical synthesis, carrier are pUC57 carrier, and recipient bacterium is bacillus coli DH 5 ɑ bacterial strain；Concrete operations are, by TRECs gene
80 nucleotide fragments of 138 nucleotide fragments, 90 nucleotide fragments of KRECs gene and TRAC gene are stitched together
After connect into pUC57 vector plasmid.Standard items measure concentration and purity with Nano, and magnetic beads for purifying is used after HindIII digestion,
Be diluted to after the concentration of pg rank with the copy number of the method measurement standard product of droplet type digital pcr, according to the concentration of measurement into
Row dilution, obtains 1.0x104copies/μl、1.0x103copies/μl、1.0x102copies/μl、2.0x101 copies/μ
l、1.0x101The plasmid standard of 6 copies/ μ l, 2.0copies/ μ l concentration gradients are placed in -20 DEG C of preservations.
3, DNA extracts the preparation of reagent
DNA extracts reagent: 1) 0.5% (V/V) Triton X-100；2)PBS；3)Generation DNA
Elution Solu II.TritonX-100 stoste, PBS and Generation DNA Elution Solu II solution are city
Purchase.The configuration method of 0.5% (V/V) Triton X-100 are as follows: 20% (V/V) Triton X-100 storing liquid of configuration first, tool
Body step is that 10ml Triton X-100 stoste is stored for a long time after mixing well with 40ml PBS solution；Then 2ml is taken
20% (V/V) Triton X-100 storing liquid is mixed well with 78ml PBS solution, obtains 0.5% (V/V) Triton X-100
Extraction for DNA in this kit.
4, the preparation of negative controls
Weight containing tri- genetic fragments of TRECs, KRECs and TRAC prepared by Suzhou Jin Weizhi Biotechnology Co., Ltd
The bacillus coli DH 5 ɑ bacterial strain of group plasmid, extracts plasmid, measures concentration and purity using Nano, uses after HindIII digestion
Magnetic beads for purifying is diluted to after the concentration of pg rank with the copy number of the method measurement standard product of droplet type digital pcr, according to measurement
Concentration be diluted, obtain 1.0x103The negative controls of copies/ μ l are saved in -20 DEG C.
5, the preparation of blank control product
Blank control is commercially available sterile nuclease-free water.
6, the composition of Fluorescence PCR liquid I, such as table 2.
The composition of 2 Fluorescence PCR liquid I of table
Wherein 2X real time Mix is purchased from Bioline company, ddH2O is sterile ultrapure water.
7, the composition of Fluorescence PCR II, such as table 3.
The composition of 3 Fluorescence PCR liquid II of table
Embodiment 2: the verifying and use of kit
1, the acquisition of sample is verified after kit developing
With the dried blood spot sample (being gifted by Beijing Obstetrical and Gynecological Hospital) for through clinical definite being positive SCID and XLA children source
Positive controls as kit verifying, it is contemplated that inspection result should be positive.
It (is given by Beijing Obstetrical and Gynecological Hospital with clinic without phenotype, through the dried blood spot sample that clinical judgment is normal children source
With) it is used as sample to be examined, it is contemplated that inspection result should be negative.Positive controls dried blood spot and sample to be examined dried blood spot are all in 4 DEG C
It saves.2, in dried blood spot sample DNA extraction
Operating procedure is as follows:
A. the dried blood spot of a piece of diameter 3mm is taken using clean punch, and is put into 1.5mlEP pipe, and 100 μ l 0.5% are added
(V/V) Triton X-100,800rpm shake 10min, suck solution as far as possible；
B. 100 μ l PBS (commercially available) are added, 12000rpm is centrifuged 1min, sucks solution as far as possible；
C. 100 μ l Generation DNA Elution Solu II (commercially available) are added, 800rpm shakes 10min, as far as possible
Suck solution；
D. 30 μ l Generation DNA Elution Solu II are added, after 99 degree of water-bath water-bath 30min,
12000rpm is centrifuged 1min, and transfer solution to new pipe saves.
3, pattern detection
1) copy number of real-time fluorescence quantitative PCR detection TRECs gene and TRACs gene
It is formed according to Fluorescence PCR liquid I, prepares real-time fluorescence quantitative PCR reaction system, the DNA of sample to be examined is taken to mention
Take liquid, the DNA extracting solution of positive control, negative controls and blank control product, the plasmid standard of 6 concentration gradients
As template detection architecture is respectively configured, system ingredient such as the following table 4 in each 5 μ l:
Table 4
Real-time fluorescence quantitative PCR response procedures:
The double fluorescence detection channels for collecting FAM and HEX fluorescence signal are set, reaction tube is put into Bio-rad fluorescent quantitation
Start to expand in PCR instrument, response procedures such as the following table 5:
5 real-time fluorescence quantitative PCR response procedures of table
2) copy number of real-time fluorescence quantitative PCR detection KRECs gene and TRACs gene
It is formed according to Fluorescence PCR liquid II, prepares real-time fluorescence quantitative PCR reaction system, take the DNA of sample to be examined
Extracting solution, the DNA extracting solution of positive control, negative controls and blank control product, the plasmid control of 6 concentration gradients
As template detection architecture is respectively configured, system ingredient such as the following table 6 in each 5 μ l of product:
Table 6
Real-time fluorescence quantitative PCR response procedures:
The double fluorescence detection channels for collecting FAM and HEX fluorescence signal are set, reaction tube is put into Bio-rad fluorescent quantitation
Start to expand in PCR instrument, response procedures are the same as table 5.
3) result judges
The C of baseline rangeTValue (recurring number) is 6-15 or is automatically selected by software that given threshold is more than that random amplification is bent
The peak of line.Fluorescent PCR instrument is different, the C of gained baseline rangeTIt is worth different.
4) result calculates
As a result calculate and be divided into two steps: 1) gene copy number calculates in quantitative fluorescent PCR reaction system, which is used for
Quality Control judgement；2) it is former to per unit volume (μ l) to continue gene copy number in quantitative fluorescent PCR reaction system for sample to be tested
The conversion of gene copy number in beginning blood, the step results are final sample testing result, are judged for sample results.It is specific
Method is described as follows:
The plasmid of three kinds of gene nucleotide fragment assemblies as standard items through amplification make standard curve, work as related coefficient >
0.99 can be used for the quantitative analysis of TRECs, KRECs and TRAC copy number, otherwise test again.Sample to be examined, positive control
The copy number of TRECs, KRECs and TRAC gene in the reaction system is according to mark in sample, negative controls and blank control product
The standard curve of quasi- product converts, and basic skills refers to normal condition such as: fine works Molecular Biology, F.M. Ao Sibai
Deng chief editor, Science Press, 1995, Molecular Cloning:A Laboratory guide (third edition)；D.L. Spector etc., Science Press,
2001, cell experiment guide etc..If quantitative fluorescent PCR reacts ineffective amplified signal, then it is considered as corresponding gene copy number
It is 0.
The conversion of gene copy number gene copy number into original blood in the quantitative fluorescent PCR reaction system of sample to be tested
Formula is as follows: gene copies/ μ l blood=(each response gene copy number/each reaction sample volume) * (sample elution
Volume/dried blood spot contains blood volume).
In this embodiment, the extraction of DNA uses the dried blood spot of a piece of diameter 3mm (containing about 3 μ l in sample to be examined
Blood volume), elution volume is 30 μ l, and each reaction sample volume is 5 μ l, according to calculation formula: gene copies/ μ l blood=
(each response gene copy number/5) * (30/3)=each response gene copy number * 2.
5) quality control standard
All kinds of control quality-control product judging result such as the following table 7:
7 quality-control product standard testing result of table
Figure 1A is the amplification curve for recombinating plasmid standard TRECs, and Figure 1B is the standard curve obtained according to the curve.
Fig. 2A is the amplification curve for recombinating plasmid standard TRECs and detecting corresponding internal reference TRAC, and Fig. 2 B is according to the curve
Obtained standard curve.
Fig. 3 A is the amplification curve for recombinating plasmid standard KRECs, and Fig. 3 B is the standard curve obtained according to the curve.
Fig. 4 A is the amplification curve for recombinating plasmid standard KRECs and detecting corresponding internal reference TRAC, and Fig. 4 B is according to the curve
Obtained standard curve.
Fig. 5 A to 5D is respectively negative controls TRECs, TRAC, KRECs and TRAC real time PCR amplification curve, and fluorescence is fixed
The quantitative result of gene copy number is each about 5.0x10 in amount PCR reaction system3Copies, it was demonstrated that testing result is qualified.
Fig. 6 A to 6D is respectively blank control product TRECs, TRAC, KRECs and TRAC real time PCR amplification curve, each result
Without amplified signal, the copy number for being considered as corresponding gene is 0.
6) result is reported
The judgment criteria of sample results such as the following table 8:
8 pattern detection result of table
Fig. 7 is multiple clinics without phenotype, is judged as the TRECs and internal reference TRAC real time fluorescent quantitative of normal child's sample
PCR amplification curve, the C of institute this TRECs of test sample and TRACTValue is between 20-35, fixed within the range of linearity of standard items
Amount result is TRECs gene copy number >=25copies/ μ l blood, TRAC gene copy number >=1.0x103copies/μl
Blood, then it is negative sample.
Fig. 8 is the TRECs real-time fluorescence quantitative PCR amplification curve for the infant sample that clinical definite is SCID, as the result is shown
Without amplified signal, it is computed and thinks that copy number is 0, then it is positive sample.
Fig. 9 is the TRAC real-time fluorescence quantitative PCR amplification curve for the infant sample that clinical definite is SCID, institute's test sample sheet
CTValue is between 20-35, and within the range of linearity of standard items, quantitative result is TRAC gene copy number >=1.0x103
Copies/ μ l blood, it was demonstrated that sample extraction and amplification procedure are normal.
Figure 10 is multiple clinics without phenotype, is judged as KRECs the and TRAC real-time fluorescence quantitative PCR of normal child's sample
Amplification curve, the C of institute this KRECs of test sample and TRACTValue is between 20-35, quantitative to tie within the range of linearity of standard items
Fruit is KRECs gene copy number >=15copies/ μ l blood, TRAC gene copy number >=1.0x103copies/μl
Blood, then it is negative sample.
Figure 11 is the KRECs real-time fluorescence quantitative PCR amplification curve that clinical definite is XLA infant sample, as the result is shown without
Amplified signal, then it is positive sample.
Figure 12 is the TRAC real-time fluorescence quantitative PCR amplification curve that clinical definite is XLA infant sample, this C of institute's test sampleTValue
Between 20-35, within the range of linearity of standard items, quantitative result is TRAC gene copy number >=1.0x103copies/ul
Blood, it was demonstrated that sample extraction and amplification procedure are normal.
This kit clinical sample testing result and the sample use the T cell in flow cytomery sample blood, B
Cell quantity, gene sequencing result and clinical symptoms are consistent, illustrate that the method result is reliable, and high sensitivity is reproducible, is
Clinical screening newborn SCID and XLA provide effective diagnostic means.
Embodiment described above be only for absolutely prove the present invention and for embodiment, protection scope of the present invention is unlimited
In this.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in protection of the invention
Within the scope of.Protection scope of the present invention is subject to claims.
Sequence table
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Claims (13)
1. it is a kind of for detecting the real-time fluorescence quantitative PCR kit of TRECs and KRECs gene, it include: 1) in the kit
DNA extraction reagent, 2) standard items comprising TRECs gene, KRECs gene and TRAC gene inserts, 3) it include TRECs base
Because of the Fluorescence PCR liquid I of the, fluorescence PCR primer of TRAC gene and detection probe, 4) comprising KRECs gene, TRAC gene
The Fluorescence PCR liquid II of fluorescence PCR primer and detection probe.
2. real-time fluorescence quantitative PCR kit as described in claim 1, which is characterized in that the fluorescence of the TRECs gene
PCR primer includes TRECs fluorescent PCR forward primer and reverse primer；The fluorescence PCR primer of the TRAC gene includes TRAC glimmering
Light PCR forward primer and reverse primer；The fluorescence PCR primer of the KRECs gene include KRECs fluorescent PCR forward primer and
Reverse primer；The detection probe is TRECs detection probe, TRAC detection probe or KRECs detection probe.
3. real-time fluorescence quantitative PCR kit as claimed in claim 2, which is characterized in that the Fluorescence PCR liquid I is
By TRECs fluorescent PCR forward primer, reverse primer and TRECs detection probe, TRAC fluorescent PCR forward primer, reverse primer and
TRAC detection probe, 2X real time Mix and sterile ultrapure water composition；The TRECs fluorescent PCR forward primer and anti-
It is respectively 300-1000nM to the content of primer, preferably 400nM；The TRAC fluorescent PCR forward primer and reverse primer
Content is respectively 300-1000nM, preferably 400nM；The content of the TRECs detection probe and TRAC detection probe is respectively
50-250nM, preferably 100nM.
4. real-time fluorescence quantitative PCR kit as claimed in claim 3, which is characterized in that the TRECs fluorescent PCR is positive
The sequence of primer is as shown in SEQ ID NO.1, and the sequence of reverse primer is as shown in SEQ ID NO.2, the sequence of TRECs detection probe
Column are as shown in SEQ ID NO.3；The sequence of TRAC fluorescent PCR forward primer is as shown in SEQ ID NO.7, the sequence of reverse primer
As shown in SEQ ID NO.8, the sequence of TRAC detection probe is as shown in SEQ ID NO.9.
5. real-time fluorescence quantitative PCR kit as claimed in claim 2, which is characterized in that the Fluorescence PCR liquid II is
By KRECs fluorescent PCR forward primer, reverse primer and KRECs detection probe, TRAC fluorescent PCR forward primer, reverse primer and
TRAC detection probe, 2X realtime Mix and sterile ultrapure water composition；The KRECs fluorescent PCR forward primer and reversed
The content of primer is respectively 300-1000nM, preferably 400nM；The TRAC fluorescent PCR forward primer and reverse primer contain
Amount is respectively 300-1000nM, preferably 400nM；The KRECs detection probe, TRAC detection probe content are respectively 50-
250nM, preferably 100nM.
6. real-time fluorescence quantitative PCR kit as claimed in claim 5, which is characterized in that the KRECs fluorescent PCR is positive
The sequence of primer is as shown in SEQ ID NO.4, and the sequence of reverse primer is as shown in SEQ ID NO.5, the sequence of KRECs detection probe
Column are as shown in SEQ ID NO.6；The sequence of TRAC fluorescent PCR forward primer is as shown in SEQ ID NO.7, the sequence of reverse primer
As shown in SEQ ID NO.8, the sequence of TRAC detection probe is as shown in SEQ ID NO.9.
7. real-time fluorescence quantitative PCR kit as claimed in claim 2, which is characterized in that Fluorescence PCR liquid I and fluorescence
The TRECs detection probe, KRECs detection probe and TRAC detection probe in PCR reaction solution II are Taqman probe, mark
Remember that the end of Taqman probe 5 ' is fluorescence radiation group, is one of FAM, VIC, TET, JOE, ROX, CY3, CY5, HEX；
The end of label 3 ' is fluorescent quenching group, is one of TAMRA, BHQ1.
8. real-time fluorescence quantitative PCR kit as claimed in claim 7, which is characterized in that the 5 ' of the TRECs detection probe
End is HEX fluorophor, and 3 ' ends are TAMRA fluorescent quenching group；5 ' ends of the KRECs detection probe are HEX fluorophor,
3 ' ends are TAMRA fluorescent quenching group；5 ' ends of the TRAC detection probe are FAM fluorophor, and 3 ' ends are that TAMRA fluorescence is quenched
Go out group.
9. real-time fluorescence quantitative PCR kit as described in claim 1, which is characterized in that the DNA extracts reagent and includes:
1) 0.5% (V/V) Triton X-100,100-200 μ l；2) PBS:100-200 μ l；3)Generation DNA Elution
Solu II, 120-300 μ l, wherein first step dosage: 100-200 μ l, second step dosage: 20-100 μ l；The standard items are
By 138 nucleotide fragments of TRECs gene, 80 nucleotide of 90 nucleotide fragments of KRECs gene and TRAC gene
Fragment assembly connects afterwards together into pUC57 vector plasmid；The method that droplet type digital pcr is used in standard items digestion after purification measures
Copy number, storage concentration are 1.0x104copies/μl、1.0x103copies/μl、1.0x102copies/μl、
2.0x101copies/μl、1.0x1016 copies/ μ l, 2.0copies/ μ l concentration gradients.
10. real-time fluorescence quantitative PCR kit as described in claim 1, which is characterized in that the insertion of the TRECs gene
Sequence is as shown in SEQ ID NO.10, and the insetion sequence of KRECs gene is as shown in SEQ ID NO.11, the insertion sequence of TRAC gene
Column are as shown in SEQ ID NO.12.
11. a kind of if the described in any item real-time fluorescence quantitative PCR kits of claim 1-10 are thin for screening newborn T
Application in born of the same parents and B cell immune deficiency, characterized in that it comprises the following steps: with kit in dried blood spot sample to be measured
DNA extract；The DNA of extraction is subjected to real-time fluorescence quantitative PCR amplification and detection；By comparing sample to be tested and standard
The cycle threshold of product calculates starting TRECs, KRECs and TRAC gene copy number of sample to be tested according to standard curve, and uniformly
It is scaled to copies/ μ l blood.
12. application as claimed in claim 11, which is characterized in that the negative control of the dried blood spot sample to be measured be containing
The recombinant plasmid of tri- genetic fragments of TRECs, KRECs and TRAC, concentration copy are 1.0x103copies/μl。
13. application as claimed in claim 11, which is characterized in that the blank control of the dried blood spot sample to be measured is sterile
Nuclease-free water.
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