CN106381344A - High-sensitivity EBV DNA quantitative detection kit based on ddPCR and using method thereof - Google Patents
High-sensitivity EBV DNA quantitative detection kit based on ddPCR and using method thereof Download PDFInfo
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- CN106381344A CN106381344A CN201610807133.9A CN201610807133A CN106381344A CN 106381344 A CN106381344 A CN 106381344A CN 201610807133 A CN201610807133 A CN 201610807133A CN 106381344 A CN106381344 A CN 106381344A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention provides a high-sensitivity EBV DNA quantitative detection kit based on ddPCR and a using method thereof. The kit comprises DNA extraction buffer solution, PCR primers and probes, a PCR reaction solution and EBV positive and negative control. When the kit provided by the invention is used for detecting ddPCR, the influences of PCR equipment, standard substances and the like can be effectively eliminated, and further high-sensitivity absolute quantification is further realized. The EBV DNA quantitative detection kit provided by the invention is capable of performing high-sensitivity accurate quantification on EBV in serum and plasma through a ddPCR technology, and providing a reference basis for diagnosis and curative effect monitoring of EBV related diseases. According to the kit, a function relationship between the international unit and common unit of EBV DNA international standard substances is determined to be 1IU/ml=2.5Copy/ml, the international traceability of EBV DNA is realized, and the international comparability between the detection results and quantification unit is realized.
Description
Technical field
The present invention relates to DNA detection field is and in particular to a kind of tried based on the highly sensitive EBV DNA detection by quantitative of ddPCR
Agent box and using method.
Background technology
Epstein-Barr virus (Epstein-Barr virus, EBV) are also known as nerpes vinrus hominises VI type (Human herpesvirus
Type4), belong to Gammaherpesvirinae Lymphocryptovirus to belong to, in the carrying rate up to more than 90% of crowd, and EBV is once
Infection is carried all the life.EBV main infection human nasopharynx's epithelial cell and bone-marrow-derived lymphocyte, research just shows after EBV primary infection
Settle down all the life in human B lymphocyte.EBV relevant disease scope is wider, is roughly divided into blood system and non-blood system two is big
Class.EBV associated blood systemic disease mainly include infectious monocytosis (Infectious mononucleosis),
Burkitt lymphoma, Primary Lymphoma, t cell lymphoma etc.；EBV correlation non-blood systemic disease mainly includes gastric cancer, nose
The detection of pharyngeal cancer, pulmonary carcinoma, leiomyosarcoma etc., therefore EBV is great to the diagnosis of EBV relevant disease and therapeutic potential.
Current academic viewpoint thinks that the existence form of EBV DNA has three kinds：1. EBV DNA is present in complete Epstein-Barr virus grain
In the viral genome of son；2. the ring-type dissociated or wire EBV DNA；3. dissociate EBV DNA.Presence shape due to EBV DNA
Formula has three kinds, therefore, extracts EBV DNA and has important impact for the detection of EBV.At present, the conventional side of EBV DNA extraction
Method is boiling lysis, phenol-chloroform method, paramagnetic particle method and affinity chromatography.Boiling lysis needs repeatedly to heat, and is susceptible to quick-fried
Manage and produce pollution；Phenol-chloroform method complex operation, has high demands to equipment and human users, the specimen recall rate of low virus load
Low, and agents useful for same has certain toxicity；Paramagnetic particle method for clip size 82-181bp small molecule segment extraction effect
Bad；Affinity chromatography complex operation, needs frequently to change centrifuge tube, but extraction efficiency is high.And for three kinds of existence forms
EBV DNA, existing EBV detection kit completely by DNA extraction out, and then can not affect the detection of EBV.
The method of diagnosis EBV is usually immunological method and molecular biology, and wherein, immunological method mainly includes adopting
Specificity EBV in ELISA (enzyme linked immunosorbent assay, elisa) detection serum
Antibody；Molecular biology method mainly passes through RTFQ PCR (realtime fluorescence quantitative
Polymerase Chain Reaction, quantitative fluorescent PCR) technology qualitatively or quantitatively examined to EBV DNA carrying capacity in sample
Survey.The reagent cost of ELISA is low, needs test serum consumption few, as clinically main screening method；But ELISA deposits
In the shortcoming that diagnostic sensitivity is low, specificity is poor, thus the examination of EBV relevant disease can not be played with early warning or early stage
The purpose of diagnosis, meanwhile, nor the observation of curative effect for disease treatment and prognosis prediction.RTFQ PCR being capable of detection by quantitative
During the DNA of EBV, RTFQ PCR detection by quantitative, the result influence factor of detection by quantitative includes DNA extracting, genes of interest is chosen, drawn
Thing and probe design, PCR equipment, premix enzymatic reagent composition, period and standard substance selection etc., therefore, the quantitation of RTFQ PCR
Testing result influence factor is numerous, is difficult standardization, thus affecting quantitative result from many aspects.
Content of the invention
The present invention provides a kind of highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR and using method, using this
The test kit that invention provides can solve the problem that the existing problem that detection kit Detection results are poor, sensitivity is low；Simultaneously to EBV
The method for extracting of DNA and PCR amplification procedure are standardized, and realize testing result and the international comparability of quantitative unit.
The present invention provides a kind of highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR, and described test kit includes
DNA extraction buffer, PCR primer and probe, PCR reactant liquor and the comparison of EBV positive and negative, wherein,
Described DNA extraction buffer includes proteinase K buffer, buffer1, buffer2, buffer3 and buffer4；
Described buffer1 includes Tris-HCl, EDTA and guanidine hydrochloride；Described buffer2 include isopropanol, Tris-HCl and
EDTA；Described buffer3 and buffer4 includes all different dehydrated alcohol of concentration, Tris-HCl and EDTA respectively；
Described PCR reactant liquor includes 5 × PCR reaction buffer, 0.8-1.2mmol/L dideoxyribonucleotide triphosphate, 1-
7U/ μ l H-Taq archaeal dna polymerase；
Described PCR primer and probe include 700nmol/L-1.0 μm of ol/L and draw for the upstream and downstream expanding target polynucleotide
Thing and 0.2mol/L-0.5 μm of ol/L are used for detecting the probe of target polynucleotide；The described probe for detecting target polynucleotide
Base-pair sequence is：
5’FAM-CACACACTACACACACCCACCCGTCTC-BHQ1 3’.
Preferably, the base sequence of the described upstream and downstream primer for expanding target polynucleotide is：
Forward primer：5’-CCCAACACTCCACCACACC-3’；
Downstream primer：5’-TCTTAGGAGCTGTCCGAGGG-3’.
Preferably, described proteinase K buffer includes the described Tris-HCl that concentration is 10-20mmol/L, and concentration is
The described EDTA of 1.0-3.0mmol/L and concentration are the described E.C. 3.4.21.64 of 10ug/ml, wherein, described Tris-HCl and described
The pH value of EDTA is 7.5.
Preferably, described buffer1 includes the described Tris-HCl that concentration is 40-70mmol/L, and concentration is 2.0-
The described EDTA of 4.0mmol/L and concentration are the described guanidine hydrochloride of 4.0-6.0mol/L, wherein, described Tris-HCl and described
The pH value of EDTA is 7.5.
Preferably, described buffer2 includes the described isopropanol that concentration is 30%-60%, and concentration is 10-20mmol/L's
Described Tris-HCl and concentration are the described EDTA of 1.0-3.0mmol/L, wherein, the pH value of described Tris-HCl and described EDTA
It is 7.5.
Preferably, described buffer3 includes the described dehydrated alcohol that concentration is 50%-60%, and concentration is 10-20mmol/L
Described Tris-HCl and concentration be 1.0-3.0mmol/L described EDTA, wherein, the pH of described Tris-HCl and described EDTA
Value is 7.5.
Preferably, described buffer4 includes the described dehydrated alcohol that concentration is 60%-70%, and concentration is 8-20mmol/L
Described Tris-HCl and concentration be the described EDTA of 0.5-2.0mmol/L, the pH value of described Tris-HCl and described EDTA is equal
For 8.0.
Preferably, described EBV negative control is negative artificial serum, and described EBV positive control is in B958 cell culture
Clearly.
A kind of using method of the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR, described using method bag
Include：
Add the proteinase K buffer of 10-30 μ l, mix homogeneously in 100-300 μ l sample to be tested, form mixed liquor a；
Add 80-240 μ l buffer1 in described mixed liquor a, overturn and mix rear 60 DEG C of heating in water bath 20-40min, shape
Become mixed liquor b, the globule that described mixed liquor b centrifugation is covered removes；
Add 240-480 μ l buffer2 in described mixed liquor b, concussion mixes ice bath insulation 4-6min after 10-20s,
Form mixed liquor c, the globule that described mixed liquor c centrifugation is covered removes；
Add described mixed liquor c to being placed in the adsorption column in collecting pipe, in the bar for 10000-15000rpm for the rotating speed
It is centrifuged 40-80s, described adsorption column is left and taken standby under part；
Described adsorption column is put in described collecting pipe, adds 80-150 μ l buffer3 in described adsorption column, turning
Speed for being centrifuged 40-80s under conditions of 6000-12000rpm, leave and take standby by described adsorption column；
Described adsorption column is put in described collecting pipe, adds 100-200 μ l buffer4 in described adsorption column, turning
Speed for being centrifuged 2-4min under conditions of 10000-15000rpm, leave and take standby by described adsorption column；
Described adsorption column is put in new collecting pipe, adds 120-200 μ l dehydrated alcohol in described adsorption column, turning
Speed for being centrifuged 2-4min under conditions of 10000-15000rpm, leave and take standby by described adsorption column；
Described adsorption column is put in new collecting pipe, is centrifuged 1- under conditions of rotating speed is for 10000-15000rpm
2min, described adsorption column is left and taken standby；
Described adsorption column is put in EP pipe, room temperature stands 3-8min；
After standing terminates, to the adsorbed film middle part hanging Deca 40-100 μ l ultra-pure water of described adsorption column, room temperature is quiet
Put 2-5min；
After standing terminates, it is centrifuged 1-2min under conditions of rotating speed is for 8000-15000rpm, leave and take mixed in described EP pipe
Close liquid；
PCR mixed liquor is configured according to described sample to be tested quantity, wherein, described PCR mixed liquor includes 18 μ l PCR reactions
Liquid and sample to be tested described in 2 μ l, described PCR mixed liquor covers sealer；
Described PCR mixed liquor is carried out ddPCR detection.
Preferably, described ddPCR detection includes：Described PCR mixed liquor is carried out microdroplet process, by the system after processing
Transfer in PCR plate, sealer laggard performing PCR amplified reaction.
The technical scheme that embodiments of the invention provide can include following beneficial effect：
The present invention provides a kind of highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR, and described test kit includes
DNA extraction buffer, PCR primer and probe, PCR reactant liquor and EBV negative control, wherein, described buffer includes E.C. 3.4.21.64
Buffer, buffer1, buffer2, buffer3 and buffer4；Described buffer1 includes Tris-HCl, EDTA and guanidine hydrochloride；
Described buffer2 includes isopropanol, Tris-HCl and EDTA；It is all different that described buffer3 and buffer4 includes concentration respectively
Dehydrated alcohol, Tris-HCl and EDTA；Described PCR reactant liquor includes 5 × PCR reaction buffer, 0.8-1.2mmol/L deoxidation core
Riboside triphosphoric acid, 1-7U/ μ l H-Taq archaeal dna polymerase；PCR primer and probe, 700nmol/L-1.0 μm of ol/L are used for expanding
Increase the upstream and downstream primer of target polynucleotide and 0.2mol/L-0.5 μm of ol/L is used for detecting the probe of target polynucleotide；Described for
The base-pair sequence of probe of detection target polynucleotide is：5’FAM-CACACACTACACACACCCACCCGTCTC-BHQ1 3’.
Meanwhile, present invention also offers the using method of highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR.The present invention carries
For the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR pass through proteinase K buffer by sample protein drop
Solution, by gentle chemical cracking liquid buffer1, DNA and RNA in sample is cracked out completely, and then by buffer2,
Buffer3 and buffer4 step chromatography goes out DNA and RNA that whole forms exist, and improves the purity of EBV DNA, and then improves
The sensitivity of EBV DNA immue quantitative detection reagent box；Choose EBV quantitative amplification region (the BamHI-W area that international organization is recommended simultaneously
Domain) upstream and downstream primer and probe, by PCR reactant liquor, selected upstream and downstream primer and probe are expanded, Jin Erti
The specificity of high EBV DNA immue quantitative detection reagent box.The highly sensitive EBV DNA detection by quantitative based on ddPCR that the present invention provides
Test kit, when using, the sample to be tested preparing is carried out ddPCR detection, due to ddPCR only extraction and the purpose base with DNA
The selection of cause is relevant, therefore, using ddPCR, the EBV DNA being extracted is expanded, can effectively exclude PCR equipment, amplification
The impact of efficiency, standard substance and interfering material, premix enzymatic reagent composition etc., and then realize absolute quantitation, make result to be measured more
Plus accurately.The highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR that the present invention provides can be right by ddPCR technology
EBV in serum, plasma sample carries out accurate quantification, and the diagnosis for EBV relevant disease and curative effect monitoring provide reference frame.This
Invention establishes the letter between iu IU of EBVDNA international standard thing and conventional unit Copy/ml also by ddPCR technology
Number relation, realizes the quantitative international comparability of EBV DNA.The present invention is extracted by DNA, genes of interest selects, ddPCR is definitely fixed
Amount and EBV DNA international standard substance etc. realize the standardization of EBV DNA detection by quantitative and trace to the source in the world.
It should be appreciated that above general description and detailed description hereinafter are only exemplary and explanatory, not
The present invention can be limited.
Brief description
Fig. 1 is the use of the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR providing in the embodiment of the present invention
Method flow diagram；
Fig. 2 is the testing result figure to EBV DNA international reference materials for the ddPCR providing in the embodiment of the present invention.
Specific embodiment
Here will in detail exemplary embodiment be illustrated, its example is illustrated in the accompanying drawings.Explained below is related to
During accompanying drawing, unless otherwise indicated, the same numbers in different accompanying drawings represent same or analogous key element.Following exemplary embodiment
Described in embodiment do not represent all embodiments consistent with the present invention.On the contrary, they be only with such as appended
The example of the consistent device of some aspects being described in detail in claims, the present invention.
Each embodiment in this specification is all described by the way of going forward one by one, identical similar portion between each embodiment
Divide mutually referring to what each embodiment stressed is the difference with other embodiments.
Embodiment one
Provided in an embodiment of the present invention highly sensitive based on ddPCR (Droplet digital PCR, microdroplet digital pcr)
EBV DNA immue quantitative detection reagent box includes DNA extraction buffer, PCR primer and probe, PCR reactant liquor and EBV positive and negative pair
According to.
Wherein, DNA extraction buffer include proteinase K buffer, buffer1 (buffer), buffer2, buffer3 and
buffer4；PCR reactant liquor includes 5 × PCR reaction buffer, and concentration is dezyribonucleoside three phosphorus of 0.8-1.2mmol/L
Acid, concentration is the H-Taq archaeal dna polymerase (Thermusaquaticus, H- hot resistant DNA polymerase) of 1-7U/ μ l；PCR primer and
Probe includes the upstream and downstream primer for expanding target polynucleotide for 700nmol/L-1.0 μm of ol/L for the concentration and concentration is
The probe for detecting target polynucleotide of 0.2mol/L-0.5 μm of ol/L；EBV negative control is negative artificial serum, EBV sun
Property compares as B958 cells and supernatant.
Provided in an embodiment of the present invention based in the highly sensitive EBV DNA immue quantitative detection reagent box of ddPCR, in EBV base
Because choosing base-pair sequence and the amplification target multinuclear of the probe for detecting target polynucleotide in BamHI-W in the conservative region of group
The base sequence of the upstream and downstream primer of thuja acid, and then improve the specificity of EBV DNA immue quantitative detection reagent box.Implement in the present invention
In the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR that example provides, BamHI-W in the conservative region of EBV genome
Gene order be：CCCAACACTCCACCACACCCAGGCACACACTACACACACCCACCCGTCTCAGGGTCCCCTCGGA
CAGCTCCTAAGAAGGCACCGGTCGCCCAGTCCTACCAGAGGGGGCCAAGAACCCAGACGAGTCCGTAGAAGGGTCCT
C；The base-pair sequence of the selected probe for detecting target polynucleotide is：5’FAM-CACACACTACACACACCCACCC
GTCTC-BHQ1 3’；The base sequence of the upstream and downstream primer for expanding target polynucleotide is：
Forward primer：5’-CCCAACACTCCACCACACC-3’；
Downstream primer：5’-TCTTAGGAGCTGTCCGAGGG-3’.
Further, proteinase K buffer includes the Tris-HCl (Tris that concentration is 10-20mmol/L
(hydroxymethyl) aminomethane, three (methylol) aminomethane hydrochloride), concentration is 1.0-3.0mmol/L's
EDTA (Ethylene Diamine Tetraacetic Acid, ethylenediaminetetraacetic acid) and concentration are the E.C. 3.4.21.64 of 10ug/ml,
Wherein, the pH value of Tris-HCl and EDTA is 7.5.
Buffer1 includes Tris-HCl, EDTA and guanidine hydrochloride, and wherein, the concentration of Tris-HCl is 40-70mmol/L,
The concentration of EDTA is 2.0-4.0mmol/L, and the concentration of guanidine hydrochloride is 4.0-6.0mol/L, and the pH value of Tris-HCl and EDTA is
7.5.
Buffer2 includes isopropanol, Tris-HCl and EDTA, and wherein, the concentration of isopropanol is 30%-60%, Tris-
The concentration of HCl is the concentration of 10-20mmol/L and EDTA is 1.0-3.0mmol/L, and wherein, the pH value of Tris-HCl and EDTA is equal
For 7.5.
The composition of buffer3 and buffer4 is dehydrated alcohol, Tris-HCl and EDTA, and wherein, buffer3 includes dense
Spend the dehydrated alcohol for 50%-60%, concentration is the Tris-HCl of 10-20mmol/L and concentration is 1.0-3.0mmol/L's
EDTA, wherein, the pH value of Tris-HCl and EDTA is 7.5；Buffer4 includes the dehydrated alcohol that concentration is 60%-70%, dense
Spending the Tris-HCl for 8-20mmol/L and concentration is the EDTA of 0.5-2.0mmol/L, and the pH value of Tris-HCl and EDTA is
8.0.
Highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR provided in an embodiment of the present invention passes through in buffer
Proteinase K buffer the protein in sample to be tested is degraded, will be to be measured by gentle chemical cracking liquid buffer1
DNA and RNA in sample cracks out completely, and then is gone out whole by buffer2, buffer3 and buffer4 step chromatography
DNA and RNA of various existence forms, improves the purity of EBV DNA, and then improves the sensitive of EBV DNA immue quantitative detection reagent box
Degree.Pass through to choose the upstream and downstream primer for expanding target polynucleotide in EBV conservative region and be used for detecting target multinuclear simultaneously
The probe of thuja acid, is expanded to selected upstream and downstream primer and probe by PCR reactant liquor, and then it is fixed to improve EBV DNA
The specificity of amount detection kit.
Embodiment two
Provided in an embodiment of the present invention based on the highly sensitive EBV DNA immue quantitative detection reagent box of ddPCR can be applied to examine
The content of EBV DNA in survey blood plasma, serum sample, the operation of detection comprises the following steps that：
First, sample to be tested prepares
1st, plasma sample prepares
Under conditions of rotating speed is for 2000r/min, by EDTA anticoagulated whole blood in 2h centrifugation 3min, take separate after
Supernatant, supernatant is centrifuged 10min to remove broken cell debriss under conditions of rotating speed is for 20000g/min, collects supernatant
Liquid obtains plasma sample, if plasma sample does not temporarily use, by plasma sample under the conditions of -20 DEG C freezen protective, standby.
2nd, serum sample prepares
By centrifugation under conditions of rotating speed is for 20000g/min after fresh EDTA anticoagulated whole blood room temperature standing 30min
3min, take separate after supernatant, this supernatant be serum sample, if serum sample does not temporarily use, by serum sample in-
Freezen protective under the conditions of 20 DEG C, standby.
2nd, test sample sample process
S01：To be added in 100-300 μ l sample to be tested in EP (Epoxy resin, epoxy resin) pipe, in EP pipe
Add 10-30 μ l proteinase K buffer, after mix homogeneously, form mixed liquor a；
S02：Add 80-240 μ l buffer1 in mixed liquor a, after fully reverse mixing, heat 20- in 60 DEG C of water-baths
40min forms mixed liquor b, and the globule that centrifugation is covered after terminating by heating removes；
S03：Add 240-480 μ l buffer2 in mixed liquor b, fully shaking mixes ice bath insulation 4- after 10-20s
6min, forms mixed liquor c, and the globule that centrifugation is covered after terminating by ice bath insulation removes；
S04：After adsorption column is nested in collecting pipe, mixed liquor c is added in adsorption column, is 10000- in rotating speed
It is centrifuged 40-80s, centrifugation terminates the waste liquid in rear reject collecting pipe, and adsorption column is left and taken standby under conditions of 15000rpm；
S05：Adsorption column is put in collecting pipe, adds 80-150 μ l buffer3 in adsorption column, be 6000- in rotating speed
It is centrifuged 40-80s, centrifugation terminates the waste liquid in rear reject collecting pipe, and adsorption column is left and taken standby under conditions of 12000rpm；
S06：Adsorption column is put in collecting pipe, adds 100-200 μ l buffer4 in adsorption column, in rotating speed be
It is centrifuged 2-4min, centrifugation terminates the waste liquid in rear reject collecting pipe, and adsorption column is left and taken standby under conditions of 10000-15000rpm；
S07：Adsorption column is put in new collecting pipe, adds 120-200 μ l dehydrated alcohol in adsorption column, in rotating speed be
It is centrifuged 2-4min, centrifugation terminates the waste liquid in rear reject collecting pipe, and adsorption column is left and taken standby under conditions of 10000-15000rpm；
S08：Adsorption column is put in new collecting pipe, is centrifuged 1- under conditions of rotating speed is for 10000-15000rpm
2min, centrifugation terminates the waste liquid in rear reject collecting pipe, and adsorption column is left and taken standby；
S09：Adsorption column is put in clean EP pipe, room temperature stands 3-8min；
S10：After standing terminates, to the adsorbed film middle part hanging Deca 40-100 μ l ultra-pure water of adsorption column, room temperature is quiet
Put 2-5min；
S11：After standing terminates, it is centrifuged 1-2min under conditions of rotating speed is for 8000-15000rpm, leave and take mixed in EP pipe
Close liquid, if mixed liquor temporarily without, by mixed liquor under the conditions of -20 DEG C freezen protective, standby；
S12：PCR mixed liquor is configured according to sample to be tested quantity, wherein, PCR mixed liquor includes 18 μ l PCR reactant liquors and 2
μ l sample to be tested, also needs in negative control add 2 μ lDDW (deuterium depleted water, depleted water), will configure
Good PCR mixed liquor covers sealer；
S13：PCR mixed liquor after sealer is carried out microdroplet process, and the system after microdropletization process is transferred to 96
In the PCR plate of hole, after 96 hole PCR plate sealers, carry out ddPCR amplified reaction.
3rd, ddPCR reaction
1st, PCR reaction tube is put into amplification instrument sample cell, testing sample title and positive and negative pair are set by corresponding order
According to.
2nd, fluorescence detection channel selects：Select FAM (Reportere:FAM,Quencher:None) sense channel.
3rd, ddPCR reaction condition refers to table 1：
Table 1：DdPCR reaction condition
4th, interpretation of result
After reaction terminates, carry software using detecting instrument and automatically analyzed, record out the absolute quantitation of sample to be tested
Number, absolute quantitation number unit is Copy/ μ l.
At present, international standard thing (the NIBSC code of existing EBV DNA in the world:09/260), the embodiment of the present invention carries
For determination test EBV DNA international standard thing being detected using ddPCR technology, can be obtained according to determination test
Functional relationship between the international standard substance value of EBV DNA and absolute quantitation number, acquired results refer to accompanying drawing 2.
Can learn from accompanying drawing 2, using ddPCR technology provided in an embodiment of the present invention to EBV DNA international standard thing
The testing result being detected is 1.25 × 104Copy/ul, and the international standard substance value of EBV DNA international standard thing is 5.0
×103IU/ul, therefore, between the international standard substance value of EBV DNA and absolute quantitation number exist functional relationship be 5.0 ×
103IU/ul=1.25 × 104Copy/ul, i.e. 1IU/ul=2.5Copy/ul.Provided in an embodiment of the present invention based on ddPCR
The use of highly sensitive EBV DNA immue quantitative detection reagent box in, be applied to the quantitation of ddPCR by EBV DNA international standard substance
In detection, it is capable of high sensitivity, standardized EBV DNA detection by quantitative, realize the quantitative international comparability of EBV DNA.
The embodiment of the present invention also detects to the non-keratinization type plasma of patients with nasopharyngeal carcinoma made a definite diagnosis but without treatment,
Testing result refer to table 2.
Table 2：Plasma of patients with nasopharyngeal carcinoma testing result
| Packet | Number of cases | Median (IU/ml) | Recall rate (%) |
| Nasopharyngeal carcinoma group | 67 | 1250 | 100 |
| Matched group | 75 | 0 | 0 |
It can be seen that provided in an embodiment of the present invention tried based on the highly sensitive EBV DNA detection by quantitative of ddPCR from table 2
Agent box can detect to non-keratinization type nasopharyngeal carcinoma, and positive rate is 100%.
Meanwhile, the embodiment of the present invention also to normal person's plasma sample, serum sample, HAV, HBV, HCV, HEV, HIV-1 and
HCMV infection sample is detected, testing result is feminine gender, and negative match-rate is 100%.In conjunction with to non-keratinization type nasopharynx
The testing result of cancer patients blood plasma understands, the highly sensitive EBV DNA detection by quantitative examination based on ddPCR provided in an embodiment of the present invention
Agent box has good specificity.
The above is only the specific embodiment of the present invention, makes skilled artisans appreciate that or realizing this
Bright.Multiple modifications to these embodiments will be apparent to one skilled in the art, as defined herein
General Principle can be realized without departing from the spirit or scope of the present invention in other embodiments.Therefore, the present invention
It is not intended to be limited to the embodiments shown herein, and be to fit to and principles disclosed herein and features of novelty phase one
The scope the widest causing.
Claims (10)
1. a kind of highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR is it is characterised in that described test kit includes DNA
Extraction buffer, PCR primer and probe, PCR reactant liquor and the comparison of EBV positive and negative, wherein,
Described DNA extraction buffer includes proteinase K buffer, buffer1, buffer2, buffer3 and buffer4；
Described buffer1 includes Tris-HCl, EDTA and guanidine hydrochloride；Described buffer2 include isopropanol, Tris-HCl and
EDTA；Described buffer3 and buffer4 includes all different dehydrated alcohol of concentration, Tris-HCl and EDTA respectively；
Described PCR reactant liquor includes 5 × PCR reaction buffer, 0.8-1.2mmol/L dideoxyribonucleotide triphosphate, 1-7U/ μ l
H-Taq archaeal dna polymerase；
Described PCR primer and probe include 700nmol/L-1.0 μm of ol/L for expand target polynucleotide upstream and downstream primer and
0.2mol/L-0.5 μm of ol/L is used for detecting the probe of target polynucleotide；
The base-pair sequence of the described probe for detecting target polynucleotide is：
5’FAM-CACACACTACACACACCCACCCGTCTC-BHQ1 3’.
2. the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR according to claim 1 is it is characterised in that institute
The base sequence stating the upstream and downstream primer for expanding target polynucleotide is：
Forward primer：5’-CCCAACACTCCACCACACC-3’；
Downstream primer：5’-TCTTAGGAGCTGTCCGAGGG-3’.
3. the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR according to claim 1 is it is characterised in that institute
State proteinase K buffer and include the described Tris-HCl that concentration is 10-20mmol/L, concentration is the described of 1.0-3.0mmol/L
EDTA and concentration are the described E.C. 3.4.21.64 of 10ug/ml, and wherein, the pH value of described Tris-HCl and described EDTA is 7.5.
4. the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR according to claim 1 is it is characterised in that institute
State buffer1 include concentration be 40-70mmol/L described Tris-HCl, concentration be 2.0-4.0mmol/L described EDTA and
Concentration is the described guanidine hydrochloride of 4.0-6.0mol/L, and wherein, the pH value of described Tris-HCl and described EDTA is 7.5.
5. the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR according to claim 1 is it is characterised in that institute
State buffer2 and include the described isopropanol that concentration is 30%-60%, concentration is the described Tris-HCl of 10-20mmol/L and dense
Spend the described EDTA for 1.0-3.0mmol/L, wherein, the pH value of described Tris-HCl and described EDTA is 7.5.
6. the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR according to claim 1 is it is characterised in that institute
State buffer3 include concentration be 50%-60% described dehydrated alcohol, concentration be 10-20mmol/L described Tris-HCl and
Concentration is the described EDTA of 1.0-3.0mmol/L, and wherein, the pH value of described Tris-HCl and described EDTA is 7.5.
7. the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR according to claim 1 is it is characterised in that institute
State buffer4 and include the described dehydrated alcohol that concentration is 60%-70%, concentration is the described Tris-HCl of 8-20mmol/L and dense
Spend the described EDTA for 0.5-2.0mmol/L, the pH value of described Tris-HCl and described EDTA is 8.0.
8. the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR according to claim 1 is it is characterised in that institute
Stating EBV negative control is negative artificial serum, and described EBV positive control is B958 cells and supernatant.
9. a kind of highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR as described in any one in claim 1-8
Using method it is characterised in that described using method includes：
Add the proteinase K buffer of 10-30 μ l, mix homogeneously in 100-300 μ l sample to be tested, form mixed liquor a；
Add 80-240 μ l buffer1 in described mixed liquor a, overturn and mix rear 60 DEG C of heating in water bath 20-40min, formed mixed
Close liquid b, the globule that described mixed liquor b centrifugation is covered removes；
Add 240-480 μ l buffer2 in described mixed liquor b, concussion mixes ice bath insulation 4-6min after 10-20s, is formed
Mixed liquor c, the globule that described mixed liquor c centrifugation is covered removes；
Add described mixed liquor c to being placed in the adsorption column in collecting pipe, under conditions of rotating speed is for 10000-15000rpm
Centrifugation 40-80s, described adsorption column is left and taken standby；
Described adsorption column is put in described collecting pipe, adds 80-150 μ l buffer3 in described adsorption column, in rotating speed be
It is centrifuged 40-80s, described adsorption column is left and taken standby under conditions of 6000-12000rpm；
Described adsorption column is put in described collecting pipe, adds 100-200 μ l buffer4 in described adsorption column, in rotating speed be
It is centrifuged 2-4min, described adsorption column is left and taken standby under conditions of 10000-15000rpm；
Described adsorption column is put in new collecting pipe, adds 120-200 μ l dehydrated alcohol in described adsorption column, in rotating speed be
It is centrifuged 2-4min, described adsorption column is left and taken standby under conditions of 10000-15000rpm；
Described adsorption column is put in new collecting pipe, is centrifuged 1-2min, institute under conditions of rotating speed is for 10000-15000rpm
State adsorption column leave and take standby；
Described adsorption column is put in EP pipe, room temperature stands 3-8min；
After standing terminates, to the adsorbed film middle part hanging Deca 40-100 μ l ultra-pure water of described adsorption column, room temperature stands 2-
5min；
After standing terminates, it is centrifuged 1-2min under conditions of rotating speed is for 8000-15000rpm, leaves and takes the mixing in described EP pipe
Liquid；
PCR mixed liquor is configured according to described sample to be tested quantity, wherein, described PCR mixed liquor includes 18 μ l PCR reactant liquors and 2
Sample to be tested described in μ l, described PCR mixed liquor covers sealer；
Described PCR mixed liquor is carried out ddPCR detection.
10. the using method of the highly sensitive EBV DNA immue quantitative detection reagent box based on ddPCR according to claim 9, its
It is characterised by, described ddPCR detection includes：
Described PCR mixed liquor is carried out microdroplet process, the system after processing is transferred in PCR plate, the laggard performing PCR of sealer expands
Increase reaction.
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| CN111020063A (en) * | 2020-01-10 | 2020-04-17 | 上海润达榕嘉生物科技有限公司 | Detection primer of EB virus and kit thereof |
| CN111088401A (en) * | 2020-01-10 | 2020-05-01 | 上海润达榕嘉生物科技有限公司 | Multi-virus detection primer and kit thereof |
| CN111041131A (en) * | 2020-03-16 | 2020-04-21 | 广东永诺医疗科技有限公司 | EB virus detection kit based on droplet type digital PCR |
| CN111041131B (en) * | 2020-03-16 | 2020-07-10 | 广东永诺医疗科技有限公司 | EB virus detection kit based on droplet type digital PCR |
| CN111235318A (en) * | 2020-03-30 | 2020-06-05 | 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） | Primer, probe and kit for detecting EB virus in nasopharyngeal carcinoma |
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