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

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

Info

Publication number
CN110684863A
CN110684863A CN201810737842.3A CN201810737842A CN110684863A CN 110684863 A CN110684863 A CN 110684863A CN 201810737842 A CN201810737842 A CN 201810737842A CN 110684863 A CN110684863 A CN 110684863A
Authority
CN
China
Prior art keywords
digital pcr
seq
probe
virus
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810737842.3A
Other languages
Chinese (zh)
Inventor
严晓锋
刘凤麟
熊慧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
WUHAN BIOTECH GENE ENGINEERING Co Ltd
Suzhou Yuntai Biological Pharmaceutical Co Ltd
Original Assignee
WUHAN BIOTECH GENE ENGINEERING Co Ltd
Suzhou Yuntai Biological Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by WUHAN BIOTECH GENE ENGINEERING Co Ltd, Suzhou Yuntai Biological Pharmaceutical Co Ltd filed Critical WUHAN BIOTECH GENE ENGINEERING Co Ltd
Priority to CN201810737842.3A priority Critical patent/CN110684863A/en
Publication of CN110684863A publication Critical patent/CN110684863A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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 techniqueT) 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.
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.
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
Figure BDA0001722483680000051
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
Figure BDA0001722483680000061
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
Figure BDA0001722483680000071
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
Figure BDA0001722483680000081
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
Figure BDA0001722483680000091
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.
Sequence listing
<110> Suzhou Yuntai biomedical science and technology Co., Ltd
Wuhanbaitai Gene engineering Co Ltd
<120> digital PCR kit for quantitatively detecting nucleic acid of EB virus and detection method
<160>7
<170>SIPOSequenceListing 1.0
<210>1
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
tcaccaggcc tgccaaa 17
<210>2
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
ggtccaaatc actaccagag attacc 26
<210>3
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
cagccccaaa gcgggtgcag taac 24
<210>4
<211>93
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
tcaccaggcc tgccaaagag ccagatctaa ggccgggaga ggcagcccca aagcgggtgc 60
agtaacaggt aatctctggt agtgatttgg acc 93
<210>5
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
tcaccaggcc tgccaaag 18
<210>6
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
gggtccaaat cactaccaga gatta 25
<210>7
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
caccaggcct gccaaaga 18

Claims (10)

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;
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.
5. The digital PCR kit of claim 1, wherein the quality control substances comprise negative quality control substances and positive quality control substances;
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.
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;
(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 (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;
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.
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.
CN201810737842.3A 2018-07-06 2018-07-06 Digital PCR kit for quantitatively detecting nucleic acid of EB virus and detection method Pending CN110684863A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810737842.3A CN110684863A (en) 2018-07-06 2018-07-06 Digital PCR kit for quantitatively detecting nucleic acid of EB virus and detection method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810737842.3A CN110684863A (en) 2018-07-06 2018-07-06 Digital PCR kit for quantitatively detecting nucleic acid of EB virus and detection method

Publications (1)

Publication Number Publication Date
CN110684863A true CN110684863A (en) 2020-01-14

Family

ID=69107468

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810737842.3A Pending CN110684863A (en) 2018-07-06 2018-07-06 Digital PCR kit for quantitatively detecting nucleic acid of EB virus and detection method

Country Status (1)

Country Link
CN (1) CN110684863A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
CN116790812A (en) * 2023-05-19 2023-09-22 华中科技大学同济医学院附属同济医院 Kit for detecting EB virus nucleic acid copy number and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101855363A (en) * 2007-08-03 2010-10-06 香港中文大学 Analysis of nucleic acids by digital pcr
CN106381344A (en) * 2016-09-07 2017-02-08 林勤 High-sensitivity EBV DNA quantitative detection kit based on ddPCR and using method thereof
US20190085417A1 (en) * 2015-12-18 2019-03-21 Lucence Diagnostics Pte Ltd Detection and Quantification of Target Nucleic Acid Sequence of a Microorganism

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101855363A (en) * 2007-08-03 2010-10-06 香港中文大学 Analysis of nucleic acids by digital pcr
US20190085417A1 (en) * 2015-12-18 2019-03-21 Lucence Diagnostics Pte Ltd Detection and Quantification of Target Nucleic Acid Sequence of a Microorganism
CN106381344A (en) * 2016-09-07 2017-02-08 林勤 High-sensitivity EBV DNA quantitative detection kit based on ddPCR and using method thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHENG-TE MAJOR LIN ET AL.,: ""Human herpesvirus multiplex ddPCR detection in brain tissue from low-and high-grade astrocytoma cases and controls"", 《INFECTIOUS AGENTS AND CANCER》 *
ELINA KUUSELA ET AL.,: ""Serum Epstein-Barr virus DNA, detected by droplet digital PCR, correlates with disease activity in patients with rheumatoid arthritis"", 《CLIN EXP RHEUMATOL》 *
JESS HONGANH VO ET AL.,: ""Comparison of Circulating Tumour Cells and Circulating Cell-Free Epstein-Barr Virus DNA in Patients with Nasopharyngeal Carcinoma Undergoing Radiotherapy"", 《SCIENTIFIC REPORTS》 *
L. TANG ET AL.,: ""Quantitative Assessment of Commutability for Clinical Viral Load Testing Using a Digital PCR-Based Reference Standard"", 《JOURNAL OF CLINICAL MICROBIOLOGY》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
CN116790812A (en) * 2023-05-19 2023-09-22 华中科技大学同济医学院附属同济医院 Kit for detecting EB virus nucleic acid copy number and application thereof
CN116790812B (en) * 2023-05-19 2024-03-12 华中科技大学同济医学院附属同济医院 Kit for detecting EB virus nucleic acid copy number and application thereof

Similar Documents

Publication Publication Date Title
CN106957927B (en) African swine fever fluorescent PCR detection reagent, African swine fever fluorescent PCR detection kit and application thereof
WO2022095723A1 (en) Kit and method for detecting sars-cov-2
Gnanamony et al. Detection and quantitation of HPV 16 and 18 in plasma of Indian women with cervical cancer
CN110684863A (en) Digital PCR kit for quantitatively detecting nucleic acid of EB virus and detection method
CN111286559B (en) Primer, probe and kit for detecting African swine fever virus
Pow et al. Salivary Epstein-Barr virus DNA level in patients with nasopharyngeal carcinoma following radiotherapy
CN105886663B (en) Locked nucleic acid sensitization detection kit for fluorescent quantitative PCR of wild strain of porcine pseudorabies virus
CN110684864A (en) Microdroplet digital PCR kit for quantitatively detecting human cytomegalovirus and detection method
US20220195542A1 (en) Use of primer probe combination and kit thereof in hbv detection
CN115873993B (en) Kit for detecting 9 genotypes of hepatitis B virus and application thereof
WO2023093387A1 (en) Raa primer and kit for detecting hepatitis c virus, and application of raa primer
Yi et al. Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of goose astrovirus genotypes 1 and 2
CN113122655A (en) TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for African swine fever virus EP402R gene
CN114350848A (en) Dual fluorescent probe primer combination and kit for identifying African swine fever type I strain and type II strain and application of dual fluorescent probe primer combination and kit
Kowalczyk et al. Diagnostics and genotyping of Canine parvovirus type 2 (CPV-2) from disease cases in south-eastern Poland
CN110938712A (en) Primer, probe, kit and method for detecting human papilloma virus based on real-time fluorescent quantitative PCR technology
CN103131797B (en) A kind of bocavirus real-time fluorescence PCR detection reagent kit and application thereof
CN103882149A (en) Dual real-time fluorescence PCR detection method of type I and type II of porcine torque teno virus
CN114182046A (en) Primer probe combination and kit for detecting pathogen nucleic acid of human herpesvirus and application of primer probe combination and kit
EP3363915B1 (en) Nucleic acid sequences for typing detection of cutaneous human papillomaviruses and use thereof
CN110684862B (en) Microdroplet digital PCR kit for quantitatively detecting hepatitis B virus and detection method
CN105567838B (en) MiR-331-5p is as the application in acute HIV infection detection marker
CN114990261B (en) Multiplex qPCR detection reagent for detecting respiratory tract infectious disease pathogens
RU2731716C1 (en) Kit for differentiating cattle pestiviruses and method for differentiating cattle pestiviruses
Supchokpul et al. E4 expression of human papillomavirus in cervical samples with different cytology categories

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20200114