CN110684863A

CN110684863A - Digital PCR kit for quantitatively detecting nucleic acid of EB virus and detection method

Info

Publication number: CN110684863A
Application number: CN201810737842.3A
Authority: CN
Inventors: 严晓锋; 刘凤麟; 熊慧
Original assignee: WUHAN BIOTECH GENE ENGINEERING Co Ltd; Suzhou Yuntai Biological Pharmaceutical Co Ltd
Current assignee: WUHAN BIOTECH GENE ENGINEERING Co Ltd; Suzhou Yuntai Biological Pharmaceutical Co Ltd
Priority date: 2018-07-06
Filing date: 2018-07-06
Publication date: 2020-01-14

Abstract

The invention belongs to the field of virus nucleic acid detection, and particularly relates to a digital PCR kit and a detection method for quantitatively detecting EB virus nucleic acid. The kit contains a digital PCR reaction solution and a quality control product; the digital PCR reaction solution contains primers and probes for detecting EB virus nucleic acid, and comprises a forward primer shown by SEQ ID NO. 1, a reverse primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3; the two ends of the probe are marked with fluorescent groups. The kit and the detection method can realize absolute quantification of nucleic acid molecules, thereby avoiding deviation caused by PCR amplification efficiency difference, having higher accuracy, sensitivity and repeatability, and being easy to standardize.

Description

Digital PCR kit for quantitatively detecting nucleic acid of EB virus and detection method

Technical Field

The invention belongs to the field of virus nucleic acid detection, and particularly relates to a digital PCR kit and a detection method for quantitatively detecting EB virus nucleic acid.

Background

Humans are the only natural host for Epstein-Barr virus (EBV), which has a worldwide human infection rate of over 90%. Primary infections usually occur in childhood and are asymptomatic, appearing as latent infections; however, if the infection occurs in adolescents or adults, infectious mononucleosis may result. More than 95% of people in China are reported to be infected with EBV at the age of 3-5 years. After primary infection, the virus can persist for a long time, in a latent state of infection, and in most cases asymptomatic. The EB virus is mainly transmitted through saliva, but is also reported to be transmitted through blood and sexual activity. There are two forms of EB virus infected cells: latent infections and proliferative infections. In latent infections, the virus exists in episomal and/or integrative form. Under certain conditions, such as low immune function or the action of certain physicochemical factors (ultraviolet rays, butyric acid, croton oil, uracil and the like), the EB virus can change from a latent state to a proliferative state.

China is a high-incidence country of nasopharyngeal carcinoma, and the incidence rate of the nasopharyngeal carcinoma accounts for the first place of head and neck tumors. A large number of experimental studies show that EB virus is closely related to nasopharyngeal carcinoma, plasma EBVDNA detection before radiotherapy has important clinical significance for clinical staging and prognosis judgment of NPC, plasma EBV DNA detection after the radiotherapy is finished has important significance for judging residual focus and total survival, and has monitoring effect on tumor metastasis and recurrence after the radiotherapy. Traditional detection of epstein barr virus-associated antibodies is the most common method for diagnosing and monitoring patient disease, but it is less sensitive and specific. The existing fluorescent quantitative PCR can only carry out relative quantification on the original nucleic acid, the nucleic acid quantity is determined by depending on a standard curve or a reference gene, and uncertain detection results exist in the detection of whether the virus is positive, so that the detection is carried out for multiple times or the detection result is inaccurate. The introduction of ddPCR technology provides a new non-invasive means for directly and truly reflecting the virus replication level in patients, and the diagnosis, treatment and prognosis of tumors.

The digital PCR technique is a third-generation absolute quantitative PCR technique which has been recently developed, and is independent of the threshold value (C) of the amplification curve as compared with the real-time quantitative PCR technique_T) The quantification is carried out without the influence of amplification efficiency and the adoption of a standard curve, has good accuracy and reproducibility, and can realize absolute quantitative analysis. In the current stage of low abundance factor detection, the real-time fluorescence quantitative PCR technology is interfered by background DNA or impurities, so that the detection sensitivity and accuracy can not meet the requirement of accurate quantification, and the digital PCR detection system separates rare detection fragments from a large amount of complex backgrounds through microdroplet treatment, so that the detection sensitivity and accuracy are improved.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a digital PCR kit and a detection method for quantitatively detecting EB virus nucleic acid.

The invention also aims to provide a primer and a probe for detecting EB virus nucleic acid by adopting digital PCR.

In order to achieve the purpose of the invention, the technical scheme is as follows:

the invention relates to a digital PCR kit for quantitatively detecting nucleic acid of EB virus, which contains a digital PCR reaction solution and a quality control product; the digital PCR reaction solution contains a primer and a probe for detecting EB virus nucleic acid, and the primer and the probe for detecting EB virus nucleic acid comprise a forward primer shown by SEQ ID NO. 1, a reverse primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3; and fluorescent groups are marked at two ends of the probe.

Optionally, the 5 'end of the nucleotide sequence shown in SEQ ID NO. 3 is labeled with FAM, and the 3' end is labeled with BHQ.

Optionally, the digital PCR reaction solution further contains a digital PCR buffer solution.

Optionally, the digital PCR reaction solution contains 1-1.5 muL of DEPC treated water, 8-12 muL of digital PCR buffer solution, 0.01-0.02 nmol of forward primer shown in SEQ ID NO. 1, 0.01-0.02 nmol of reverse primer shown in SEQ ID NO. 2, and 0.005-0.01 nmol of probe shown in SEQ ID NO. 3;

preferably, the digital PCR reaction solution contains 1.2. mu.L of DEPC treated water, 10. mu.L of digital PCR buffer solution, 0.015nmol of forward primer shown by SEQ ID NO. 1, 0.015nmol of reverse primer shown by SEQ ID NO. 2, and 0.008nmol of probe shown by SEQ ID NO. 3.

Optionally, the quality control product comprises a negative quality control product and a positive quality control product;

the negative quality control product is sterile normal saline, and the concentration of the positive quality control product is 5.25 multiplied by 10⁵The nucleotide sequence of the EB virus standard plasmid is shown as SEQ ID NO. 4.

Optionally, the sample for which the digital PCR kit is applied is selected from serum or plasma.

The invention also relates to a method for quantitatively detecting EB virus nucleic acid by using the digital PCR kit, which at least comprises the following steps:

(1) collecting a sample to obtain a sample collection liquid;

(2) sample treatment: extracting the sample collection liquid by adopting a commercial extraction kit to obtain a sample treatment liquid;

(3) preparing a digital PCR reaction mixed solution: mixing the digital PCR reaction solution with the sample treatment solution to obtain a digital PCR reaction mixed solution; preferably, the volume ratio of the digital PCR reaction solution to the sample treatment solution is 3: 1;

(4) adding the digital PCR reaction mixed solution and the droplet generation oil into a droplet generation card, placing the droplet generation card into a droplet generation instrument to generate droplets, and then carrying out digital PCR amplification reaction;

(5) reading the fluorescence signal: placing the amplified 96-well plate in a microdroplet reader, and directly reading and analyzing the result by using software; and automatically calculating according to a poisson distribution principle to obtain the copy number of the EB virus DNA in the ddPCR reaction system.

Optionally, the conditions of the digital PCR amplification reaction are: firstly, preserving heat for 9-11 min at the temperature of 95 ℃; then, preserving heat at 94 ℃ for 13-16 seconds and preserving heat at 58 ℃ for 58-60 seconds, and performing 39-41 cycles in total; finally, preserving the heat for 10min at the temperature of 98 ℃, and cooling to 4 ℃ to stop the reaction;

preferably, the temperature is firstly preserved for 10min at the temperature of 95 ℃; then preserving heat for 15 seconds at 94 ℃ and 60 seconds at 58 ℃ for 40 cycles; finally, preserving the heat for 10min at the temperature of 98 ℃, and cooling to 4 ℃ to stop the reaction;

more preferably, the temperature rising and falling speed is less than or equal to 2 ℃/second.

The invention also relates to a primer and a probe for detecting EB virus nucleic acid by adopting digital PCR, wherein the sequences of the primer and the probe comprise a forward primer shown by SEQ ID NO. 1, a reverse primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3; and fluorescent groups are marked at two ends of the probe.

The technical scheme of the invention at least has the following beneficial effects:

the kit and the detection method can realize absolute quantification of nucleic acid molecules, thereby avoiding deviation caused by PCR amplification efficiency difference.

The kit disclosed by the invention does not need to depend on certified standard substances or other standard substances, has higher accuracy, sensitivity and repeatability, and is easy to standardize.

The kit and the detection method are more suitable for detecting nucleic acid with low copy number, and the minimum detection amount is 5.25 copies/mu L of EB virus DNA.

Because the digital PCR adopted by the invention is end-point detection and has higher tolerance to the inhibitor, the kit and the detection method can reduce the detection deviation caused by the type of the matrix.

Drawings

FIG. 1 is a graph of the results of the experiment provided in example 1 of the present invention; wherein the abscissa represents the cycle number (cyclember) and the ordinate represents the fluorescence value (Delta Rn).

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Detailed Description

The embodiment of the invention relates to a digital PCR kit and a detection method for quantitatively detecting EB virus nucleic acid, and has the technical advantages of simple operation and accurate result. The kit is suitable for use in a digital PCR (ddPPCR) system comprising a droplet generator and droplet analyser and their associated consumables. The droplet generator can partition the DNA to be tested into 20000 uniform nanoliter droplets. The droplets were transferred to a 96-well PCR plate and PCR amplification of DNA was performed using a thermal cycler, with PCR reactions performed independently in each droplet. After amplification, each droplet in the sample is analyzed individually using a droplet analyzer for fluorescence readings. After the droplets are inhaled, the tube breaks up the emulsified droplets and passes them through a dichroic optical detection system in sequence. Droplets with fluorescence signal were positive and droplets without fluorescence signal were negative, and the number of positive and negative droplets and the proportion of positive droplets were calculated. Finally, according to the Poisson distribution principle and the proportion of the positive microdroplets, the analysis software can calculate the concentration or copy number of the target molecules to be detected. In the embodiment of the invention, the EB virus specific conserved sequence is selected as a target detection fragment, the EB virus specific conserved sequence is obtained through the national center for Biotechnology information (NCBI, http:// www.ncbi.nlm.nih.gov), and the design of specific primers and probes suitable for digital PCR is carried out by using professional design software Beacon Designer 7.0. And primer sequences with strong specificity and high sensitivity are obtained through primer screening.

The kit of the embodiment of the invention contains a digital PCR reaction solution and a quality control product; the digital PCR reaction solution contains a primer and a probe for detecting EB virus nucleic acid, and the primer and the probe for detecting EB virus nucleic acid comprise a forward primer shown by SEQ ID NO. 1, a reverse primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3; two ends of the probe are marked with fluorescent groups; preferably, the nucleotide sequence shown in SEQ ID NO. 3 is labeled with FAM at the 5 'end and BHQ at the 3' end.

Specifically, the sequences of the primers and probes are shown in table 1:

TABLE 1

Synthesized by Bao bioengineering (Dalian) Co., Ltd, according to the design results in the above table; optionally, the digital PCR reaction solution further contains a digital PCR buffer. Specifically, the digital PCR buffer solution may be 2 XQX 200ddPCRSupermix For Probes from BioRad.

Optionally, the digital PCR reaction solution contains 1-1.5 muL of DEPC treatment water, 8-12 muL of digital PCR buffer solution, 0.01-0.02 nmol of forward primer shown in SEQ ID NO. 1, 0.01-0.02 nmol of reverse primer shown in SEQ ID NO. 2, and 0.005-0.01 nmol of probe shown in SEQ ID NO. 3;

preferably, the digital PCR reaction solution contains 1.2. mu.L of DEPC treated water, 10. mu.L of digital PCR buffer solution, 0.015nmol of forward primer shown in SEQ ID NO. 1, 0.015nmol of reverse primer shown in SEQ ID NO. 2, and 0.008nmol of probe shown in SEQ ID NO. 3.

Optionally, the quality control product comprises a negative quality control product and a positive quality control product; wherein the negative quality control product is sterile physiological saline, and the positive quality control product has a concentration of 5.25 × 10⁵copies/. mu.L EB virus standard plasmid solution;

the nucleotide sequence of the EB virus standard plasmid is specifically shown in a table 2:

TABLE 2

The preparation method of the positive quality control product comprises the following steps: b95-8 cells were used as template with primers SEQ ID NO:1 to SEQ ID NO: amplifying a specific fragment of about 93bp, wherein the specific fragment comprises a ddPCR amplified target gene; constructing recombinant plasmid, sequencing by BAO bioengineering (Dalian) Co., Ltd, and performing homology analysis on the sequencing result and EB virus specific conserved sequence, wherein the homology reaches 100%. The recombinant plasmid is transformed into Escherichia coli DH5 alpha, proliferated, extracted by alkali cracking method, purified by DNA purification kit, quantified by A260 spectrophotometer, converted according to formula and diluted to 5.25 × 10⁵copies/. mu.L. Stored at the temperature of minus 20 ℃ and used as a positive quality control product of the kit.

Optionally, the sample for which the digital PCR kit is adapted is selected from serum or plasma.

Optionally, the microdroplet digital PCR kit further comprises a microdroplet generation oil, a microdroplet generation card, a 96-well plate and an aluminum foil heat-sealing film.

The embodiment of the invention also relates to a method for quantitatively detecting EB virus nucleic acid by using the digital PCR kit, which at least comprises the following steps:

(1) collecting a sample to obtain a sample collection liquid;

the specific collection method can be as follows:

a. using a sterile syringe to extract 1ml venous blood of a detected person, injecting the venous blood into a sterile 1.5ml centrifuge tube, standing the venous blood for 2 hours at room temperature, standing the venous blood for 1 hour at 4 ℃, centrifuging the venous blood for 5 minutes at 8000rpm, sucking 200 mu L of supernatant (paying attention to not sucking red blood cells), and transferring the supernatant into another sterile 1.5ml centrifuge tube to obtain a serum specimen.

b. Specimen preservation and transportation: if the specimen is not tested immediately, it should be stored at-20 deg.C to avoid repeated freezing and thawing. The long-distance transportation of the specimen should adopt a 0 ℃ curling stone.

(2) Sample treatment: and extracting the sample collection solution by adopting a commercial extraction kit to obtain a sample treatment solution, namely a ddPCR amplification DNA template.

(3) Preparing a digital PCR reaction mixed solution: mixing the digital PCR reaction solution with the sample treatment solution to obtain a digital PCR reaction mixed solution;

preferably, the volume ratio of the digital PCR reaction solution to the sample treatment solution is 3: 1; specifically, as shown in table 3;

TABLE 3

Components	Working fluid concentration	Sample addition amount of mu L
			QX200Bio-rad ddPCR Supermix For Probes	2×	10
SEQ ID NO:1	10μmol/L	1.5
			SEQ ID NO:2	10μmol/L	1.5
SEQ ID NO:3	10μmol/L	0.8
			DEPC treated Water	/	1.2
Form panel	50-100ng/μL	5

(4) Adding 20 μ L of the digital PCR reaction system and 70 μ L of the droplet generation oil into 8-well droplet generation cards, respectively, and placing the cards in a droplet generation instrument (QX200, Bio-Rad, Pleasanton, CA) to generate droplets;

(5) the resulting water-in-oil droplets (40. mu.L) were slowly transferred to a 96-well plate, sealed and then subjected to an amplification reaction on a PCR instrument (Veriti thermal cycler, Applied BioSystems, Foster City, Calif.) under the digital PCR amplification reaction conditions shown in Table 4:

TABLE 4

Wherein the temperature rising and falling speed is less than or equal to 2 ℃/second.

(5) Reading the fluorescence signal: the amplified 96-well plate was placed in a microdroplet reader (QX200, Bio-Rad, Pleasanton, CA) and direct results reading and analysis was performed using QuantaSoft software; the positive microdroplet containing the amplification product and the negative microdroplet without the amplification product show the difference of fluorescence signal intensity, and a threshold line is set by taking the highest point of the fluorescence amplitude of the negative microdroplet cluster as a boundary; and automatically calculating according to a poisson distribution principle to obtain the copy number of the EB virus DNA in the ddPCR reaction system.

Wherein the determination of invalid result: the total number of droplets in each reaction tube is more than or equal to 8000, and if the total number of droplets is less than 8000, the droplets in the reaction holes are not generated ideally, and the droplets are generated again.

The embodiment of the invention also relates to a primer and a probe for detecting the nucleic acid of the EB virus by adopting the digital PCR, and the primer and the probe have the technical advantages of strong specificity and high sensitivity. The sequences of the primers and the probes comprise a forward primer shown by SEQ ID NO. 1, a reverse primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3; the two ends of the probe are marked with fluorescent groups.

Preferably, the nucleotide sequence shown in SEQ ID NO. 3 is labeled with FAM at the 5 'end and BHQ at the 3' end.

Example 1: primer screening

1. Designing a primer and a probe: referring to relevant documents at home and abroad, an EBV specific conserved sequence is selected as a target detection fragment, an EB virus specific conserved sequence is obtained through the national center for Biotechnology information (NCBI, http:// www.ncbi.nlm.nih.gov), and the design of specific primers and probes suitable for ddPCR is carried out by using professional design software Beacon Designer 7.0.

This example shows the process of screening the best primers, selecting several pairs of candidate primers designed by software for screening, the candidate primers are shown in Table 5.

TABLE 5

2. Screening of primers

2.1 the primers are matched with six pairs: F1R1, F1R2, F2R1, F2R2, F3R1, F3R 2;

specific probe P: the 5 'end fluorescence reporter group is FAM, and the 3' end quenching group is BHQ.

2.2 then carrying out fluorescence quantitative PCR, wherein the formula of a PCR system comprises the following steps: takara Premix Ex Taq (Probe qPCR) (2X) 25. mu.L, forward primer 0.015nmol, reverse primer 0.015nmol, Probe 0.008nmol, DEPC water 16.2. mu.L, template 5. mu.L.

The amplification condition of the fluorescence PCR is 50 ℃ and 2 min; at 95 ℃ for 3 min; fluorescence was collected at 95 ℃, 15sec, 60 ℃, 60sec for 40 cycles; the reaction was terminated.

2.3 the results of the experiment are shown in FIG. 1.

Based on the analysis of the results in FIG. 1, the primer pair F1R1 with the best amplification effect is selected.

Example 2: kit composition

The specific composition of the kit is shown in table 6:

TABLE 6

Example 3: specificity test

1. Materials: serum samples of hepatitis B virus, human cytomegalovirus, and EB virus infected patients, and serum samples of healthy persons.

2. The method comprises the following steps: the serum samples of hepatitis B virus, human cytomegalovirus and EB virus infected patients and the serum samples of healthy people are respectively extracted by using a commercial extraction kit to obtain DNA templates, the kit provided by the embodiment of the invention is used for ddPCR amplification detection, and the kit is observed to see whether non-specific reaction occurs.

3. As a result: after the DNA of the serum samples of patients infected by hepatitis B virus, human cytomegalovirus and EB virus and the serum samples of healthy people are amplified by a PCR instrument, a microdroplet reader is adopted for detection, and QuantaSOFT software is utilized for automatically reading and analyzing results. The results of the specific experiments are shown in table 7.

TABLE 7

Name (R)	The result of the detection
		Hepatitis B virus	0copies/μL
Human cytomegalovirus	0copies/μL
		EB virus	3.56×10⁴copies/μL
Healthy person	0copies/μL

Experimental results show that only EB virus infected patients have positive microdroplets, and the rest are negative microdroplets, so that the kit and the detection method provided by the embodiment of the invention are proved to have good specificity.

Example 4: sensitivity test

1. Materials: EB virus positive quality control product (5.25X 10)⁵copies/. mu.L) were diluted to 5.25X 10, respectively⁴copies/μL、5.25×10³copies/μL、5.25×10²The specific compositions of copies/. mu.L and 5.25 copies/. mu.L, as sensitivity reference substances, are shown in Table 8.

TABLE 8

Numbering	Theoretical concentration
		YL1	5.25×10⁵copies/μL
YL2	5.25×10⁴copies/μL
		YL3	5.25×10³copies/μL
YL4	5.25×10²copies/μL
		YL5	5.25copies/μL

2. The detection method comprises the following steps: mu.L of each dilution gradient was taken and ddPCR detection was performed using the kit of the invention, each sample was repeated 3 times, and the lowest limit detectable by the assay kit was determined. The experimental results obtained are shown in table 9:

TABLE 9

Numbering	Average copy number of test results
		YL1	5.38×10⁵copies/μL
YL2	4.98×10⁴copies/μL
		YL3	5.47×10³copies/μL
YL4	5.19×10²copies/μL
		YL5	5.38copies/μL

Experimental results show that the kit and the detection method provided by the embodiment of the invention can detect 5.25 copies/mu L EB virus DNA, greatly improve the sensitivity of the EB-related detection method, do not depend on a standard product, can realize absolute quantification, and have intuitive and reliable results.

Example 5: accuracy test

Accuracy (tress) refers to the degree of agreement between the average of a set of test results and an acceptable reference value. According to the European Union and Codex standards, the accuracy criterion is that the measured value should be within + -25% of the reference value within the whole dynamic range of detection.

1. Materials: EB virus positive quality control product (5.25X 10)⁵copies/. mu.L) were diluted to 5.25X 10, respectively⁴copies/μL、5.25×10³copies/μL。

2. The method comprises the following steps: the ddPCR detection is carried out on each sample by using the kit of the invention, each sample is provided with 10 replicates, and 1 negative control (a negative quality control is used for replacing template DNA) is arranged at the same time.

The results of the experiment are shown in table 10:

watch 10

Theoretical concentration	Average copy number of test results	Relative standard deviation (CV)
			5.25×10⁵copies/μL	5.40×10⁵copies/μL	10.66％
5.25×10⁴copies/μL	5.13×10⁴copies/μL	10.31％
			5.25×10³copies/μL	5.56×10³copies/μL	12.09％

The experimental result shows that the measured values of the ddPCR of the EB virus are close to the expected values, and the standard deviation is less than 15 percent and less than 25 percent, which indicates that the kit and the detection method have very good accuracy.

Example 6: repeatability test

EB virus positive control prepared in example 2 (5.25X 10)⁵copies/. mu.L) to 5.25X 10²The results of experiments performed by using copies/. mu.L and the kit of the present invention to perform ddPCR detection with 10 replicates each and 1 blank control (negative quality control was used instead of template DNA) are shown in Table 11:

TABLE 11

Theoretical concentration	Average copy number of test results	Relative standard deviation (CV)
			5.25×10⁵copies/μL	5.30×10⁵copies/μL	8.02％
5.25×10²copies/μL	5.44×10²copies/μL	9.76％

As can be seen from table 11, the kit and the detection method of the embodiment of the present invention have a coefficient of variation CV of less than 25% when used for quantitative detection of EB virus, which proves that the kit and the detection method of the embodiment of the present invention have good repeatability when used for quantitative detection of EB virus.

Although the present invention has been described with respect to the preferred embodiments, it is not intended to be limited to the embodiments disclosed, and many modifications and variations are possible to those skilled in the art without departing from the spirit of the invention.

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Claims

1. A digital PCR kit for quantitatively detecting nucleic acid of EB virus is characterized in that the kit contains a digital PCR reaction solution and a quality control product;

the digital PCR reaction solution contains a primer and a probe for detecting EB virus nucleic acid, and the primer and the probe for detecting EB virus nucleic acid comprise a forward primer shown by SEQ ID NO. 1, a reverse primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3; and fluorescent groups are marked at two ends of the probe.

2. The digital PCR kit according to claim 1, wherein the nucleotide sequence of SEQ ID NO. 3 is labeled with FAM at the 5 'end and BHQ at the 3' end.

3. The digital PCR kit according to claim 1, wherein the digital PCR reaction solution further contains a digital PCR buffer.

4. The digital PCR kit according to claim 3, wherein the digital PCR reaction solution contains: 1-1.5 muL of DEPC treated water, 8-12 muL of digital PCR buffer solution, 0.01-0.02 nmol of forward primer shown in SEQ ID NO. 1, 0.01-0.02 nmol of reverse primer shown in SEQ ID NO. 2, and 0.005-0.01 nmol of probe shown in SEQ ID NO. 3;

5. The digital PCR kit of claim 1, wherein the quality control substances comprise negative quality control substances and positive quality control substances;

6. The digital PCR kit of claim 1, wherein the sample for which the digital PCR kit is applied is selected from serum or plasma.

7. A method for quantitatively detecting EB virus nucleic acid by using the digital PCR kit of any one of claims 1 to 6, which is characterized by comprising at least the following steps:

(1) collecting a sample to obtain a sample collection liquid;

(5) reading the fluorescence signal: placing the amplified 96-well plate in a microdroplet reader, and directly reading and analyzing the result by using software; and (4) automatically obtaining the copy number of the EB virus DNA in the ddPCR reaction system according to the calculation of a poisson distribution principle.

8. The method of claim 7, wherein the digital PCR amplification reaction is performed under the following conditions: firstly, preserving heat for 9-11 min at the temperature of 95 ℃; then, preserving heat at 94 ℃ for 13-16 seconds and preserving heat at 58 ℃ for 58-60 seconds, and performing 39-41 cycles in total; finally, preserving the heat for 10min at the temperature of 98 ℃, and cooling to 4 ℃ to stop the reaction;

9. A primer and a probe for detecting EB virus nucleic acid by adopting digital PCR are characterized in that the sequences of the primer and the probe comprise a forward primer shown by SEQ ID NO. 1, a reverse primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3; and fluorescent groups are marked at two ends of the probe.

10. The primers and probes according to claim 9, wherein the nucleotide sequence of SEQ ID NO. 3 is labeled with FAM at the 5 'end and BHQ at the 3' end.