CN108913770A - It is a kind of to utilize newborn's TRECs and KRECs gene copy number detection kit of digital pcr technology and its application - Google Patents
It is a kind of to utilize newborn's TRECs and KRECs gene copy number detection kit of digital pcr technology and its application Download PDFInfo
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Abstract
The present invention relates to a kind of newborn's TRECs and KRECs gene copy number detection kits using digital pcr technology, which is characterized in that the kit includes：For detecting the upstream primer, downstream primer and TRECs genetic test probe of TRECs gene, for detecting the upstream primer, downstream primer and KRECs genetic test probe of KRECs gene, and upstream primer, downstream primer and reference gene TRAC detection probe for detecting reference gene TRAC；The content of the upstream primer and downstream primer is respectively 400~1000nM；The TRECs genetic test probe, KRECs genetic test probe, reference gene TRAC detection probe content be respectively 200~400nM.The round dried blood spot (blood volumes of about 3 μ l) that the present invention only needs punching to drill through a piece of diameter 3mm extracts DNA the detection of the project can be completed, and blood using amount is seldom, is very suitable to that the neonatal screening of blood should not be measured greatly.
Description
Technical field
The present invention relates to genetic test field, be specifically related to a kind of newborn TRECs using digital pcr technology and
KRECs gene copy number detection kit and its answering in severe combined immunodeficiency and agammaglobulinemia screening
With.
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 is reduced
Or missing, then strong suspicion SCID, further progress fluidic cell and gene diagnosis are made a definite diagnosis.It can be with by TRECs screening SCID
The dry blood scraps of paper are shared with other newborn's routine screening projects, are 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 progress neonatal immunity and lack
Sunken screening.
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.
Digital pcr is the third generation round pcr after regular-PCR, fluorescent quantitative PCR technique.DdPCR refers to droplet type number
PCR is the digital pcr technology of current most mainstream.DdPCR system combines Water-In-Oil emulsification microdrop technique and micro-fluidic technologies.
Each sample is generated into 20000 uniform nanoliter level droplets first on drop generators, target fragment and background sequence exist
Random distribution in droplet, each droplet are an independent reactors.Subsequent droplet is transferred in 96 hole PCR plates, general
Amplification is completed in logical PCR instrument.After PCR amplification, fluorescence detection is carried out one by one to droplet with droplet analyzer.It is micro- compared to negative
The fluorescence signal intensity of drop, the positive droplet comprising at least one target dna or RNA molecule increases.Finally according to Poisson distribution,
The absolute copy number of target molecule is provided by the ratio of positive droplet.Digital pcr method has sensitivity superelevation, accuracy
It is high, can absolute quantitation and good to complex samples adaptability, the advantages that PCR mortifier relative tolerance.
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, nest-type PRC bring amplification error, the pipetting after nest-type PRC are brought
Sample contamination, a possibility that accumulative error of all too many levels eventually leads to missing inspection is big.Meanwhile real time fluorescent quantitative amplification master
Relative quantification, the Stability and veracity of testing result are carried out to target sequence copy number by the curve of standard quality-control product
It is affected by standard items, eventually leads to higher false positive and false negative.As missing inspection occurs during screening, infant will be wrong
Cross excellent diagnostics and therapic opportunity.
In view of the above-mentioned problems, especially two-step method missing inspection positive sample possibility is larger and quantitative fluorescent PCR can not be absolute
Quantitative problem, the purpose of the present invention is to provide one kind can be reduced amplification error and sample pollution, have it is more highly sensitive and
Specificity can carry out copy number absolute quantitation, while quick TRECs and KRECs gene inspection easy to operate to target sequence
Test agent box.It is characterized in that easy to operate, time-consuming short, at low cost, absolute quantitation, experimental result are accurate and reliable.
Summary of the invention
In order to overcome the above-mentioned deficiency of the prior art, the main object of the present invention is to provide a kind of utilization digital pcr technology
Detect newborn's TRECs and KRECs gene copy number detection kit of newborn's TRECs and KRECs gene copy number.
It is a further object of the present invention to provide above-mentioned newborn TRECs and KRECs gene copy number detection kit in weight
Application in disease combined immunodeficiency disease and agammaglobulinemia screening.
The first aspect of the invention provides a kind of newborn's TRECs and KRECs gene using digital pcr technology
Copy number detection kit, the kit include：For detecting the upstream primer, downstream primer and TRECs base of TRECs gene
Because of detection probe, for detecting the upstream primer, downstream primer and KRECs genetic test probe of KRECs gene, and for detecting
Upstream primer, downstream primer and the reference gene TRAC detection probe of reference gene TRAC；The upstream primer and downstream primer
Content be respectively 400~1000nM, preferably 900nM；The TRECs genetic test probe, KRECs genetic test probe,
The content of reference gene TRAC detection probe is respectively 200~400nM, preferably 250nM.
Further, described for detecting the sequence such as SEQ ID NO of the upstream primer of TRECs gene:Shown in 1, under
Swim the sequence such as SEQ ID NO of primer:Shown in 2, the nucleotide sequence of the TRECs genetic test probe such as SEQ ID NO:3
It is shown；The sequence such as SEQ ID NO of the upstream primer for being used to detect KRECs gene:Shown in 4, the sequence of primer downstream
Such as SEQ ID NO:Shown in 5, the nucleotide sequence of the KRECs genetic test probe such as SEQ ID NO:Shown in 6；It is described to be used for
Detect the sequence such as SEQ ID NO of the upstream primer of reference gene TRAC:Shown in 7, the sequence of primer such as SEQ ID downstream
NO:Shown in 8, the nucleotide sequence of the TRAC genetic test probe such as SEQ ID NO:Shown in 9.
Further, the TRECs genetic test probe, KRECs genetic test probe, TRAC genetic test probe are
Taqman probe, 5 ' ends of the TRECs genetic test probe label are HEX fluorophor, and 3 ' ends are BHQ1 quenching group；Institute
5 ' the ends for stating KRECs genetic test probe label are HEX fluorophor, and 3 ' ends are BHQ1 quenching group；The TRAC gene inspection
5 ' ends of probing needle label are FAM fluorophor, and 3 ' ends are BHQ1 quenching group.
Further, also containing universal 2X ddPCR amplification premixed liquid and operation instructions in the kit.
The second aspect of the invention provides a kind of application of kit as described in first aspect of the present invention, (a)
For detecting or tentatively judging newborn with the presence or absence of severe combined immunodeficiency；(b) newborn for detection or preliminary judgement
Youngster whether there is agammaglobulinemia.
Further, the application of the kit, includes the following steps：
1) DNA of dried blood spot sample to be checked is extracted；
2) DNA obtained using step 1) is template, by the DNA profiling to be measured, the upstream and downstream for being used to detect TRECs gene
Primer, TRECs genetic test probe, the upstream and downstream primer for detecting TRAC gene, TRAC genetic test probe and 2X
DdPCR expands premixed liquid mixing, prepares ddPCR mixed liquor I；The DNA obtained using step 1) is template, by the DNA mould to be measured
Plate, KRECs genetic test probe, draws for detecting the upstream and downstream of TRAC gene at the upstream and downstream primer for detecting KRECs gene
Object, TRAC genetic test probe and 2X ddPCR amplification premixed liquid mixing, prepare ddPCR mixed liquor I I；
3) ddPCR mixed liquor I and ddPCR mixed liquor I I are used, the micro- reaction drop of ddPCR is made on drop generators；
4) it after preparing the micro- reaction drop of ddPCR, is transferred in 96 hole PCR plates, and pcr amplification reaction is carried out in PCR instrument；
5) product after pcr amplification reaction reads instrument using droplet and carries out signal collection, according to fluorescence signal type interpretation
Copy number containing TRAC gene, TRECs gene or KRECs gene in sample to be tested, and equivalent is to copies/ μ l
blood。
Further, the volume of DNA profiling is 2~8 μ l in the ddPCR mixed liquor I and ddPCR mixed liquor I I.
Further, the volume of DNA profiling is 5 μ l in the ddPCR mixed liquor I and ddPCR mixed liquor I I.
Further, it is used to detect TRAC gene, TRECs gene in the ddPCR mixed liquor I and ddPCR mixed liquor I I
Or the upstream primer of KRECs gene and the content of downstream primer are 400~1000nM, TRAC genetic test probe, TRECs base
Because the content of detection probe or KRECs genetic test probe is 200~400nM.
Further, it is used to detect TRAC gene, TRECs base in the ddPCR mixed liquor I and ddPCR mixed liquor I I
Because or the upstream primer of KRECs gene and the content of downstream primer be 900nM, TRAC detection probe, TRECs detection probe or
The content of KRECs detection probe is 250nM.
Further, in step 3), the micro- reaction of ddPCR is made using the ddPCR mixed liquor I and ddPCR mixed liquor I I
The method of drop is：Drop generator is added in ddPCR mixed liquor I and ddPCR mixed liquor I I, it is a micro- to generate 10000-20000
React drop.
Further, in step 4), the pcr amplification reaction condition is：93-97 DEG C initial denaturation 3-15 minutes；93-95℃
Denaturation 5-50 seconds, 55-60 DEG C of annealing/extension 30-70 seconds carry out 35-45 circulation altogether；95-98 DEG C of enzyme inactivates 10 minutes；2-12
DEG C terminate reaction.
Further, in step 4), the pcr amplification reaction condition is：95 DEG C initial denaturation 10 minutes；94 DEG C of denaturation 30
Second, 60 DEG C annealing/extension 1 minute, altogether carry out 40 circulation；98 DEG C of enzymes inactivate 10 minutes；12 DEG C of termination reactions.
Compared with prior art, the beneficial effects of the present invention are：
(1) the design high specificity of primer and probe of the invention, high sensitivity is (in sample when applied to ddPCR detection
Can be detected when containing a target gene TRECs or KRECs), it avoids generating false negative result；
(2) the round dried blood spot (blood volumes of about 3 μ l) that the present invention only needs punching to drill through a piece of diameter 3mm extracts DNA
The detection of the project is completed, blood using amount is seldom, is very suitable to that the neonatal screening of blood should not be measured greatly.
(3) method of the invention is strong to the tolerance of protein, polysaccharide contained in template, salt ion residual etc., as a result
Repeated high, stability is good.
(4) the method for the present invention carries out the calculating and Quality Control of copy number using the synchronous experiment of reference gene, by target gene
TRECs or KRECs and reference gene TRAC are detected simultaneously, avoid the false positive results generated due to sample quality；Operating process
Simplicity greatly reduces clinical detection cost and workload.
Detailed description of the invention
Fig. 1 is that TRECs the and TRAC gene ddPCR of blank reference substance in the embodiment of the present invention detects two-dimension analysis figure；
Fig. 2 is that KRECs the and TRAC gene ddPCR of blank reference substance in the embodiment of the present invention detects two-dimension analysis figure；
Fig. 3 is the testing result reference value figure of normal sample TRECs of the present invention and TRAC gene；
Fig. 4 is the testing result reference value figure of normal sample KRECs of the present invention and TRAC gene；
Fig. 5 is the testing process schematic diagram of the method for the present invention；
Fig. 6 is that TRECs the and TRAC gene ddPCR of sample 1 in the embodiment of the present invention detects two-dimension analysis figure；
Fig. 7 is that KRECs the and TRAC gene ddPCR of sample 1 in the embodiment of the present invention detects two-dimension analysis figure；
Fig. 8 is that TRECs the and TRAC gene ddPCR of sample 2 in the embodiment of the present invention detects two-dimension analysis figure；
Fig. 9 is that KRECs the and TRAC gene ddPCR of sample 2 in the embodiment of the present invention detects two-dimension analysis figure；
Figure 10 is that TRECs the and TRAC gene ddPCR of sample 3 in the embodiment of the present invention detects two-dimension analysis figure；
Figure 11 is that KRECs the and TRAC gene ddPCR of sample 3 in the embodiment of the present invention detects two-dimension analysis figure.
Specific embodiment
It shows that example illustrates certain embodiments of the present invention, and should not be construed as limiting model of the invention
It encloses.Present disclosure can be improved from material, method and reaction condition simultaneously, all these improvement should all
It falls within the spirit and scope of the present invention.
Following material or reagent are unless stated otherwise commercially available.
Term definition
" TRECs genetic test probe " refers to the probe for detecting TRECs gene.
" KRECs genetic test probe " refers to the probe for detecting KRECs gene.
" reference gene TRAC detection probe " or " TRAC genetic test probe " is referred to for detecting TRAC gene
Probe.
Embodiment 1：DdPCR technology detect TRECs gene, KRECs gene and reference gene TRAC copy number primer and
The design and synthesis of probe
For TRECs gene, KRECs gene and reference gene TRAC, using the gene order of UCSC as source, use
3.0 software design PCR primer of Primer and Taqman probe therefrom select optimal combination as follows：
For detecting the upstream and downstream primer of TRECs gene：
Upstream primer SEQ ID NO:1：5'-CCATGCTGACACCTCTGGTT-3'
Downstream primer SEQ ID NO:2：5'-TCGTGAGAACGGTGAATGAAG-3'
When carrying out PCR amplification using above-mentioned primer, amplified fragments 138bp；
For detecting the upstream and downstream primer of KRECs gene：
Upstream primer SEQ ID NO:4：5'-TCCCTTAGTGGCATTATTTGTATCACT-3'
Downstream primer SEQ ID NO:5：5'-AGGAGCCAGCTCTTACCCTAGAGT-3'
When carrying out PCR amplification using above-mentioned primer, amplified fragments 90bp；
For detecting the upstream and downstream primer of TRAC gene：
Upstream primer SEQ ID NO:7：5'-TGGCCTAACCCTGATCCTCTT-3'
Downstream primer SEQ ID NO:8：5'-GGATTTAGAGTCTCTCAGCTGGTACAC-3'
When carrying out PCR amplification using above-mentioned primer, amplified fragments 80bp；
The genetic test probe of design is by fluorescent marker, and 5 ' ends are marked with fluorophor, and 3 ' ends are marked with
Quenching group, sequence are as follows：
TRECs genetic test probe is SEQ ID NO:3：5'-HEX-CACGGTGATGCATAGGCACCTGC-BHQ1-3'
KRECs genetic test probe is SEQ ID NO:6：5'-HEX-TCTGCACGGGCAGCAGGTTGG-BHQ1-3'
TRAC genetic test probe is SEQ ID NO:9：5'-FAM-TCCCACAGATATCCAGAACCCTGACCC-
BHQ1-3’
The design high specificity of primer and probe of the invention is applied to high sensitivity when ddPCR detection, can be from DNA
It detects low-abundance target gene, plays an important role for the judgement of newborn SCID and XLA；What is provided in the present embodiment draws
Object and the probe application copy number of energy while testing goal gene and reference gene when SCID and XLA is detected, operating process letter
Just, clinical detection cost and workload are substantially reduced.
Embodiment 2：The detection (1) of TRECs gene, KRECs gene and reference gene TRAC copy number in dried blood spot sample
The extraction of dried blood spot DNA：
A. the dried blood spot to be measured of diameter 3mm is taken (to be given in this concrete case by Beijing Obstetrical and Gynecological Hospital using clean punch
With), and it is put into 1.5mlEP pipe, the PBS (commercially available) that 100 μ l contain 0.5% (V/V) Triton X-100,800rpm concussion is added
10min sucks 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.
(2) ddPCR mixed liquor I is prepared according to following proportion in PCR plate：2X ddPCR amplification 10 μ l of premixed liquid, it is used for
Detect TRECs gene upstream and downstream primer (900nM) and TRECs genetic test probe (250nM), for detecting TRAC gene
Upstream and downstream primer (900nM) and TRAC genetic test probe (250nM) and DNA profiling to be measured (5 μ l) or blank control product (5 μ l
Nuclease-free water) mixing, 20 μ l of final volume is complemented to nuclease-free water, is configured to ddPCR mixed liquor I.
(3) ddPCR mixed liquor I I is prepared according to following proportion in PCR plate：2X ddPCR amplification 10 μ l of premixed liquid, it is used for
Detect KRECs gene upstream and downstream primer (900nM) and KRECs genetic test probe (250nM), for detecting TRAC gene
Upstream and downstream primer (900nM) and TRAC genetic test probe (250nM) and DNA profiling to be measured (5 μ l) or blank control product (5 μ l
Nuclease-free water) mixing, 20 μ l of final volume is complemented to nuclease-free water, is configured to ddPCR mixed liquor I I.
(4) prepared 20 μ l ddPCR mixed liquor I and ddPCR mixed liquor I I are added to one among DG8cartridge
In 8 holes of row.70 μ l ddPCR droplets are respectively added in DG8cartridge most 8 holes of bottom next row, oil occurs, covers glue
DG8cartridge is lightly steadily placed in droplet and generated in instrument by pad, starts to generate droplet.Droplet is created on DG8
Cartridge topmost in a round, the droplet of generation is transferred in 96 new hole PCR plates.
(5) the 96 hole PCR plates equipped with the micro- reaction drop of ddPCR are sealed with sealing plate film, micro- reaction drop of generation
Quantity is 10000~20000.
(6) 96 hole PCR plates PCR instrument is put into carry out amplification reaction according to following condition：95 DEG C initial denaturation 10 minutes；94℃
Denaturation 30 seconds, 60 DEG C annealing/extension 1 minute, altogether carry out 40 circulation；98 DEG C of enzymes inactivate 10 minutes；12 DEG C of termination reactions.
(7) PCR reaction plate is placed in the micro- reaction drop signal-obtaining instrument of ddPCR after pcr amplification reaction and carries out signal
It collects, detects the fluorescence signal of FAM and HEX.
(8) result calculates
Data analysis is carried out with QuantaSoft software, as a result as shown in Fig. 6, Fig. 7, Fig. 8, Fig. 9, Figure 10, Figure 11.It will be to
Gene copy number gene copy number into per unit volume (μ l) original blood changes in sample this progress ddPCR reaction system
It calculates, which is final sample testing result, is determined for sample results.
The reduction formula of gene copy number gene copy number into original blood is such as in the ddPCR reaction system of sample to be tested
Under：Gene copies/ μ l blood=(each response gene copy number/each reaction sample volume) * (sample elution volume/
Dried blood spot contains blood volume).
In the present embodiment, in sample to be examined DNA extraction use a diameter 3mm dried blood spot (containing about 3 μ l blood volumes),
Elution volume is 30 μ l, and each reaction sample volume is 5 μ l, according to calculation formula：Gene copies/ μ l blood=is (each
Response gene copy number/5) * (30/3)=each response gene copy number * 2.
(9) quality control standard
All kinds of control quality-control product judging results are as shown in table 1 below：
1 quality-control product standard testing result of table
Blank control product are commercially available sterile nuclease-free water.
Fig. 1 is blank control product TRECs and the ddPCR of TRAC gene detects two-dimension analysis figure, and corresponding gene copy number is
0。
Fig. 2 is blank control product KRECs and the ddPCR of TRAC gene detects two-dimension analysis figure, and corresponding gene copy number is
0。
(10) result judgement
Sample results decision procedure is as shown in table 2 below：
Table 2
Sample 1-3 is three representational dried blood spot samples (being gifted by Beijing Obstetrical and Gynecological Hospital), wherein 1 is healthy children
The dried blood spot in source, 2 be the dried blood spot that the children source of SCID is diagnosed as through clinical diagnosis, and 3 be to be diagnosed as XLA through clinical diagnosis
Children source dried blood spot.
Fig. 3 is the testing result reference value figure of normal sample TRECs of the present invention and TRAC gene, and horizontal axis is in original blood
TRECs gene copy number, the longitudinal axis are TRAC gene copy number in original blood, delimit normal sample detection threshold value line according to this figure
For TRAC copy number >=5000copies/ μ l blood, TRECs copy number >=10copies/ μ l blood.According to the threshold of delimitation
It is worth line, obtains four quadrants.When pattern detection result is fallen into the quadrant of the upper left corner, then the sample is SCID positive；When sample is examined
It surveys result to fall into top right quadrant, then the sample is normal sample；When pattern detection result fall into the lower left corner and the lower right corner this
In two quadrants, then sample is unqualified, need to extract sample DNA again, re-starts detection.
Fig. 4 is the testing result reference value figure of normal sample KRECs of the present invention and TRAC gene, and horizontal axis is in original blood
KRECs gene copy number, the longitudinal axis are TRAC gene copy number in original blood, delimit normal sample detection threshold value line according to this figure
For TRAC copy number >=5000copies/ μ l blood, KRECs copy number >=10copies/ μ l blood.According to the threshold of delimitation
It is worth line, obtains four quadrants.When pattern detection result is fallen into the quadrant of the upper left corner, then the sample is XLA positive；Work as pattern detection
As a result it falls into top right quadrant, then the sample is normal sample；When pattern detection result fall into the lower left corner and the lower right corner this two
In a quadrant, then sample is unqualified, need to extract sample DNA again, re-starts detection.
Fig. 5 is the testing process schematic diagram of the method for the present invention, after extracting dried blood spot DNA, carries out two single hole detections respectively,
One of single hole detects TRECs and reference gene TRAC, another single hole detects KRECs and reference gene TRAC.In single hole
In TRECs and reference gene TRAC detection, if normal (the present embodiment middle finger TRAC copy number >=5000copies/ μ of reference gene
L blood), TRECs is extremely low or is not detected (the present embodiment middle finger TRECs copy number is less than 10copies/ μ l blood), then sentences
Determine the SCID positive；If TRECs and reference gene TRAC detect it is normal (the present embodiment middle finger TRAC copy number >=
5000copies/ μ l blood, and TRECs copy number >=10copies/ μ l blood), then determine SCID feminine gender；If internal reference base
Because TRAC is extremely low or (the present embodiment middle finger TRAC copy number is less than 5000copies/ μ l blood) is not detected, then determine this time
It detects unqualified, need to examine again.In single hole KRECs and reference gene TRAC detection, if normal (the present embodiment middle finger of reference gene
TRAC copy number >=5000copies/ μ l blood), KRECs is extremely low or is not detected that (the present embodiment middle finger KRECs copy number is small
In 10copies/ μ l blood), then determine the XLA positive；If KRECs and reference gene TRAC detect normal ((the present embodiment
Middle finger TRAC copy number >=5000copies/ μ l blood, and KRECs copy number >=10copies/ μ l blood), then determine
XLA is negative；If reference gene TRAC is extremely low or be not detected (the present embodiment middle finger TRAC copy number be less than 5000copies/ μ l
Blood), then determine that this time detection is unqualified, need to examine again.
Fig. 6 is that TRECs the and TRAC gene ddPCR of sample 1 detects two-dimension analysis figure, and horizontal axis is that TRECs gene-correlation is glimmering
Optical channel intensity, the longitudinal axis are TRAC gene-correlation fluorescence channel intensity.Respective channel delimited according to blank control product acquired results
Threshold line, obtain 4 quadrants.Point in the quadrant of the upper left corner indicates TRAC positive droplet, and the point in bottom right quadrant indicates
TRECs positive droplet carries after software calculated through instrument, and quantitative result is that TRECs gene copy number is 252copies/ μ
L blood, TRAC gene copy number is 22800copies/ μ l blood, is SCID negative sample.
Fig. 7 is that KRECs the and TRAC gene ddPCR of sample 1 detects two-dimension analysis figure, and horizontal axis is that KRECs gene-correlation is glimmering
Optical channel intensity, the longitudinal axis are TRAC gene-correlation fluorescence channel intensity.Respective channel delimited according to blank control product acquired results
Threshold line, obtain 4 quadrants.Point in the quadrant of the upper left corner indicates TRAC positive droplet, and the point in bottom right quadrant indicates
KRECs positive droplet carries after software calculated through instrument, and quantitative result is that KRECs gene copy number is 272copies/ μ
L blood, TRAC gene copy number is 21680copies/ μ l blood, is XLA negative sample.
Fig. 8 is that TRECs the and TRAC gene ddPCR of sample 2 detects two-dimension analysis figure, and horizontal axis is that TRECs gene-correlation is glimmering
Optical channel intensity, the longitudinal axis are TRAC gene-correlation fluorescence channel intensity.Respective channel delimited according to blank control product acquired results
Threshold line, obtain 4 quadrants.Point in the quadrant of the upper left corner indicates TRAC positive droplet, and the point in bottom right quadrant indicates
TRECs positive droplet carries after software calculated through instrument, and quantitative result is that TRECs gene copy number is 0copies/ μ l
Blood, TRAC gene copy number are 19640copies/ μ l blood, are SCID positive sample.
Fig. 9 is that KRECs the and TRAC gene ddPCR of sample 2 detects two-dimension analysis figure, and horizontal axis is that KRECs gene-correlation is glimmering
Optical channel intensity, the longitudinal axis are TRAC gene-correlation fluorescence channel intensity.Respective channel delimited according to blank control product acquired results
Threshold line, obtain 4 quadrants.Point in the quadrant of the upper left corner indicates TRAC positive droplet, and the point in bottom right quadrant indicates
KRECs positive droplet carries after software calculated through instrument, and quantitative result is that KRECs gene copy number is 76copies/ μ l
Blood, TRAC gene copy number are 21240copies/ μ l blood, are XLA negative sample.
Figure 10 is that TRECs the and TRAC gene ddPCR of sample 3 detects two-dimension analysis figure, and horizontal axis is that TRECs gene-correlation is glimmering
Optical channel intensity, the longitudinal axis are TRAC gene-correlation fluorescence channel intensity.Respective channel delimited according to blank control product acquired results
Threshold line, obtain 4 quadrants.Point in the quadrant of the upper left corner indicates TRAC positive droplet, and the point in bottom right quadrant indicates
TRECs positive droplet carries after software calculated through instrument, and quantitative result is that TRECs gene copy number is 200copies/ μ
L blood, TRAC gene copy number is 19920copies/ μ l blood, is SCID negative sample.
Figure 11 is that KRECs the and TRAC gene ddPCR of sample 3 detects two-dimension analysis figure, and horizontal axis is that KRECs gene-correlation is glimmering
Optical channel intensity, the longitudinal axis are TRAC gene-correlation fluorescence channel intensity.Respective channel delimited according to blank control product acquired results
Threshold line, obtain 4 quadrants.Point in the quadrant of the upper left corner indicates TRAC positive droplet, and the point in bottom right quadrant indicates
KRECs positive droplet carries after software calculated through instrument, and quantitative result is that KRECs gene copy number is 0copies/ μ l
Blood, TRAC gene copy number are 20440copies/ μ l blood, are XLA positive sample.
The clinical sample testing result of kit of the present invention and the sample use the T in flow cytomery sample blood
Cell, B cell quantity, gene sequencing result and clinical symptoms are consistent, illustrate that the method result is reliable, high sensitivity, repeat
Property is good, provides effective diagnostic means for Clinical screening newborn SCID and XLA.
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 LISTING
<110>Zhu Zhi；Chen Jun
<120>It is a kind of to utilize newborn's TRECs and KRECs gene copy number detection kit of digital pcr technology and its application
<130> 20181041cn
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>It is artificial synthesized（Artificially synthesized）
<400> 1
ccatgctgac acctctggtt 20
<210> 2
<211> 21
<212> DNA
<213>It is artificial synthesized（Artificially synthesized）
<400> 2
tcgtgagaac ggtgaatgaa g 21
<210> 3
<211> 23
<212> DNA
<213>It is artificial synthesized（Artificially synthesized）
<400> 3
cacggtgatg cataggcacc tgc 23
<210> 4
<211> 27
<212> DNA
<213>It is artificial synthesized（Artificially synthesized）
<400> 4
tcccttagtg gcattatttg tatcact 27
<210> 5
<211> 24
<212> DNA
<213>It is artificial synthesized（Artificially synthesized）
<400> 5
aggagccagc tcttacccta gagt 24
<210> 6
<211> 21
<212> DNA
<213>It is artificial synthesized（Artificially synthesized）
<400> 6
tctgcacggg cagcaggttg g 21
<210> 7
<211> 21
<212> DNA
<213>It is artificial synthesized（Artificially synthesized）
<400> 7
tggcctaacc ctgatcctct t 21
<210> 8
<211> 27
<212> DNA
<213>It is artificial synthesized（Artificially synthesized）
<400> 8
ggatttagag tctctcagct ggtacac 27
<210> 9
<211> 27
<212> DNA
<213>It is artificial synthesized（Artificially synthesized）
<400> 9
tcccacagat atccagaacc ctgaccc 27
Claims (11)
1. a kind of newborn's TRECs and KRECs gene copy number detection kit using digital pcr technology, which is characterized in that
The kit includes：For detecting the upstream primer, downstream primer and TRECs genetic test probe of TRECs gene, for examining
Upstream primer, downstream primer and the KRECs genetic test probe of KRECs gene are surveyed, and for detecting reference gene TRAC's
Swim primer, downstream primer and reference gene TRAC detection probe；The content of the upstream primer and downstream primer is respectively 400~
1000nM；The TRECs genetic test probe, KRECs genetic test probe, reference gene TRAC detection probe content difference
For 200~400nM.
2. newborn TRECs as described in claim 1 and KRECs gene copy number detection kit, which is characterized in that described
The content of upstream primer and downstream primer is respectively 900nM；The TRECs genetic test probe, KRECs genetic test probe,
The content of reference gene TRAC detection probe is respectively 250nM.
3. newborn TRECs as described in claim 1 and KRECs gene copy number detection kit, which is characterized in that described
For detecting the sequence such as SEQ ID NO of the upstream primer of TRECs gene:Shown in 1, the sequence of primer such as SEQ ID downstream
NO:Shown in 2, the nucleotide sequence of the TRECs genetic test probe such as SEQ ID NO:Shown in 3；It is described to be used to detect KRECs
The sequence of the upstream primer of gene such as SEQ ID NO:Shown in 4, the sequence of primer such as SEQ ID NO downstream:It is described shown in 5
The nucleotide sequence of KRECs genetic test probe such as SEQ ID NO:Shown in 6；The upstream for being used to detect reference gene TRAC
The sequence of primer such as SEQ ID NO:Shown in 7, the sequence of primer such as SEQ ID NO downstream:Shown in 8, the reference gene
The nucleotide sequence of TRAC detection probe such as SEQ ID NO:Shown in 9.
4. newborn TRECs as claimed in claim 2 and KRECs gene copy number detection kit, which is characterized in that described
TRECs genetic test probe, KRECs genetic test probe, TRAC genetic test probe are Taqman probe, the TRECs
5 ' ends of genetic test probe label are HEX fluorophor, and 3 ' ends are BHQ1 quenching group；The KRECs genetic test probe
5 ' ends of label are HEX fluorophor, and 3 ' ends are BHQ1 quenching group；5 ' ends of the TRAC genetic test probe label are
FAM fluorophor, 3 ' ends are BHQ1 quenching group.
5. newborn TRECs as described in claim 1 and KRECs gene copy number detection kit, which is characterized in that described
Also containing universal 2X ddPCR amplification premixed liquid and operation instructions in kit.
6. the described in any item kits of claim 1-5 are in severe combined immunodeficiency and agammaglobulinemia screening
In application.
7. application as claimed in claim 6, which is characterized in that include the following steps：
1) DNA of dried blood spot sample to be checked is extracted；
2) DNA obtained using step 1) is template, and the DNA profiling to be measured, the upstream and downstream for being used to detect TRECs gene are drawn
Object, TRECs genetic test probe, the upstream and downstream primer for detecting TRAC gene, TRAC genetic test probe and 2X
DdPCR expands premixed liquid mixing, prepares ddPCR mixed liquor I；The DNA obtained using step 1) is template, by the DNA mould to be measured
Plate, KRECs genetic test probe, draws for detecting the upstream and downstream of TRAC gene at the upstream and downstream primer for detecting KRECs gene
Object, TRAC genetic test probe and 2X ddPCR amplification premixed liquid mixing, prepare ddPCR mixed liquor I I；
3) ddPCR mixed liquor I and ddPCR mixed liquor I I are used, the micro- reaction drop of ddPCR is made on drop generators；
4) it after preparing the micro- reaction drop of ddPCR, is transferred in 96 hole PCR plates, and pcr amplification reaction is carried out in PCR instrument；
5) product after pcr amplification reaction reads instrument using droplet and carries out signal collection, to be measured according to fluorescence signal type interpretation
Copy number containing TRAC gene, TRECs gene or KRECs gene in sample, and equivalent is to copies/ μ l blood.
8. the use as claimed in claim 7, which is characterized in that DNA mould in the ddPCR mixed liquor I and ddPCR mixed liquor I I
The volume of plate is 2~8 μ l；Preferably, the volume of DNA profiling is 5 μ l in the ddPCR mixed liquor I and ddPCR mixed liquor I I.
9. the use as claimed in claim 7, which is characterized in that be used in the ddPCR mixed liquor I and ddPCR mixed liquor I I
It detects TRAC gene, TRECs gene or the upstream primer of KRECs gene and the content of downstream primer is 400~1000nM,
The content of TRAC genetic test probe, TRECs genetic test probe or KRECs genetic test probe is 200~400nM；
Preferably, it is used to detect TRAC gene, TRECs gene or KRECs in the ddPCR mixed liquor I and ddPCR mixed liquor I I
The upstream primer of gene and the content of downstream primer are 900nM, TRAC genetic test probe, TRECs genetic test probe or
The content of KRECs genetic test probe is 250nM.
10. the use as claimed in claim 7, which is characterized in that in step 3), use the ddPCR mixed liquor I and ddPCR
Mixed liquor I I makes the micro- method for reacting drop of ddPCR：Droplet is added in ddPCR mixed liquor I and ddPCR mixed liquor I I
Device generates 10000-20000 micro- reaction drops.
11. the use as claimed in claim 7, which is characterized in that in step 3), the pcr amplification reaction condition is：93-97
DEG C initial denaturation 3-15 minutes；93-95 DEG C denaturation 5-50 seconds, 55-60 DEG C of annealing/extension 30-70 seconds, altogether carry out 35-45 recycle；
95-98 DEG C of enzyme inactivates 10 minutes；2-12 DEG C of termination reaction；
Preferably, in step 3), the pcr amplification reaction condition is：95 DEG C initial denaturation 10 minutes；94 DEG C are denaturalized 30 seconds, 60 DEG C
Annealing/extension 1 minute, 40 circulations were carried out altogether；98 DEG C of enzymes inactivate 10 minutes；12 DEG C of termination reactions.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408713A (en) * | 2019-05-16 | 2019-11-05 | 重庆医科大学附属儿童医院 | A kind of kit and method detecting newborn TREC and KREC |
| CN111206092A (en) * | 2020-03-31 | 2020-05-29 | 上海捷易生物科技有限公司 | Disease screening primer probe combination and application thereof, kit and detection method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173533A (en) * | 2012-11-20 | 2013-06-26 | 无锡联合利康临床检验所有限公司 | Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs and KRECs genes and application thereof |
| CN105274230A (en) * | 2015-11-02 | 2016-01-27 | 上海领检科技有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of TRECs and KRECs genes and its application |
| WO2017023194A1 (en) * | 2015-08-06 | 2017-02-09 | Общество С Ограниченной Ответственностью "Абв -Тест" | Immunodiagnostic method and set for the analysis of dna molecules of trec and krec and of the number of dna genome equivalents |
-
2018
- 2018-07-31 CN CN201810857811.1A patent/CN108913770A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173533A (en) * | 2012-11-20 | 2013-06-26 | 无锡联合利康临床检验所有限公司 | Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs and KRECs genes and application thereof |
| WO2017023194A1 (en) * | 2015-08-06 | 2017-02-09 | Общество С Ограниченной Ответственностью "Абв -Тест" | Immunodiagnostic method and set for the analysis of dna molecules of trec and krec and of the number of dna genome equivalents |
| CN105274230A (en) * | 2015-11-02 | 2016-01-27 | 上海领检科技有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of TRECs and KRECs genes and its application |
Non-Patent Citations (4)
| Title |
|---|
| ALESSANDRA SOTTINI等: "Simultaneous quantification of recent thymic T-cell and bone marrow B-cell emigrants in patients with primary immunodeficiency undergone to stem cell transplantation", 《CLINICAL IMMUNOLOGY》 * |
| MARION VAGLIO TESSITORE等: "Detection of newly produced T and B lymphocytes by digital PCR in blood stored dry on nylon flcked swabs", 《J TRANSL MED》 * |
| 欧雪玲: "实时荧光定量PCR检测正常人外周血sjTREC含量的研究", 《中华临床医师杂志》 * |
| 顾鸣敏主编: "《医学遗传学》", 31 August 2013, 上海科学技术文献 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408713A (en) * | 2019-05-16 | 2019-11-05 | 重庆医科大学附属儿童医院 | A kind of kit and method detecting newborn TREC and KREC |
| CN111206092A (en) * | 2020-03-31 | 2020-05-29 | 上海捷易生物科技有限公司 | Disease screening primer probe combination and application thereof, kit and detection method |
| CN111206092B (en) * | 2020-03-31 | 2022-11-15 | 上海捷易生物科技有限公司 | Disease screening primer probe combination and application thereof, kit and detection method |
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Effective date of registration: 20190923 Address after: Block B, Level 2, Lane 2, 1238, Zhangjiang Road, Shanghai Free Trade Experimental Zone, 201210 Applicant after: Shanghai Jieyi Biotechnology Co.,Ltd. Address before: 201210 2nd Floor, Building 3, Hengyue International Building, 1238 Lane, Zhangjiang Road, Pudong New Area, Shanghai Applicant before: Zhu Zhi Applicant before: Chen Jun |
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