CN103866046A

CN103866046A - Detection kit for human herpes viruses EBV and VZV

Info

Publication number: CN103866046A
Application number: CN201410028810.8A
Authority: CN
Inventors: 陶然; 尚世强; 舒强; 杜立中; 李伟
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2014-01-21
Filing date: 2014-01-21
Publication date: 2014-06-18

Abstract

The invention provides a detection kit for human herpes viruses EBV and VZV. The detection kit comprises quantitative PCR reaction solution, an EBV standard, a VZV standard, an EBV positive control, a VZV positive control, a negative control, instructions and the kit body, the quantitative PCR reaction solution comprises PCR buffer, MgCl2, dNTPs, heat-resistant DNA polymerase, an EBV forward amplification primer, an EBV reverse amplification primer, a VZV forward amplification primer, a VZV reverse amplification primer, a EBV fluorescent probe and a VZV fluorescence probe. The human herpes viruses EBV and VZV can be detected in one step by using real-time quantitative PCR and two-color fluorescent probes according to the detection kit, synchronous diagnosis of EBV and VZV infections is realized, positive virus can be accurately quantified in real time, urgent need for accurate diagnosis of EBV and VZV infections in the early clinical diagnosis is met and a basis is provided for timely definitive therapy of EBV and VZV infections.

Description

Herpes virus hominis EBV and VZV detection kit

Technical field

The invention belongs to biological technical field, relate to fluorescent quantificationally PCR detecting kit, be specifically related to a kind of two probe for real-time fluorescence quantitative PCR and detect Epstein-Ma Er virus (Epstein-Barr virus in blood samples of patients, urine, cerebrospinal fluid equal samples simultaneously, and the diagnostic kit of varicella zoster virus (varicella-zoster virus, VZV) EBV).

Background technology

EBV and VZV are the common herpes virus hominises who causes pediatric viral encephalitis.EBV invades after central nervous system, can cause cerebral edema, hemorrhage, immune complex deposit, the change of cerebral tissue demyelination sample etc., clinically shows as Acute onset or chronic active infringement.Acute EBV encephalitis mostly is virus and directly invades neural system, as the neural axis at meninx, cerebral tissue and spinal cord, the multiple positions of peripheral nerve; Chronic EBV encephalitis is relevant with autoimmune response, and the cerebral tissue demyelination sample causing for the rear Inflammatory response of deposition, infection of immunocomplex changes.VZV primary infection causes varicella, mainly betides children; After varicella recovery from illness, virus lays dormant is in ganglion cell, and reactivation causes zoster.VZV encephalitis often betided after eruption in 1 week, and its pathogenesis may be that virus is directly invaded central nervous system, also may be for due to immune response damages.In addition, infect in addition the Case report of EBV and VZV secondary viral encephalitis simultaneously, between two-strain, may have the situation of mutually promoting and activating.

The early diagnosis that EBV and VZV infect and in time antiviral therapy, to can obviously improving prognosis, reduce case fatality rate.Because of the clinical manifestation that EBV and VZV infect various, and compared with other simplexviruss clinical symptom and sign many without specificity, therefore early diagnosis and treatment in time need to rely on laboratory and detect.Traditional EBV and VZV detection method mainly comprise that viral separation and Culture and serology detect, the former was once the diagnosis " gold standard " of simplexvirus, but had defects such as taking long (3-30 days), susceptibility low (especially after antiviral treatment), somatotype difficulty; The latter is divided into again antigen direct Detection Method and antibody indirect detection method, antigen direct Detection Method causes susceptibility low because some virus does not produce detectable antigen, antibody indirect detection method needs just to produce the antibody of effective concentration in about 1 week and cannot carry out early diagnosis after due to herpesvirus infection, and between different simplexviruss, have cross reaction, antibodies specific is not high.

Along with developing rapidly of Protocols in Molecular Biology, polymerase chain reaction (polymerase chain reaction, PCR) technology, the detection that the Real-Time Fluorescent Quantitative PCR Technique particularly rising is in recent years pathogenic micro-organism provides new direction.The ultimate principle of Real-Time Fluorescent Quantitative PCR Technique is to utilize 5 '-3 ' excision enzyme nucleic acid activity of Taq enzyme, designs fluorescence double-tagging probe on regular-PCR basis, and two ends are mark fluorescent reporter group (R) and quenching group (Q) respectively; When probe keeps complete, the fluorescent signal of R group is suppressed by Q group, once probe is cut off, the restraining effect of Q group disappears, and the fluorescent signal of R group just can be detected.This technology is by realizing the Real-Time Monitoring to product amount in PCR process to the detection of fluorescent signal, and can accurate calculation original template amount, except having quantitatively accurately, detect the advantage such as quick, maximum advantage is to adopt complete stopped pipe to detect, save the aftertreatment to PCR product, avoided crossed contamination.

Current Real-Time Fluorescent Quantitative PCR Technique is along with material and instrument further develops, by different fluorescence dye (FAM, HEX, ROX etc.) and the hyperchannel quantitative real time PCR Instruments of 5 ' end mark at fluorescent probe, can realize the researchs such as gene type, detection in Gene Mutation, snp analysis.The present invention is based on above-mentioned technical background, design is for the specific probe of EBV and VZV, exploitation energy single stage method detects the PCR kit for fluorescence quantitative with quantitative EBV and VZV simultaneously, be intended to meet clinical EBV and VZV and infect demand early stage, quick diagnosis, for the clinical immunotherapy targeted autoantibody that carries out in time provides foundation.

Summary of the invention

The invention provides a kind of herpes virus hominis EBV and VZV detection kit, it is a kind of real-time fluorescence quantitative PCR detection kit, be made up of quantitative PCR reaction solution, EBV standard substance, VZV standard substance, EBV positive reference substance, VZV positive reference substance, negative control product, specification sheets and box body, wherein quantitative PCR reaction solution contains PCR damping fluid, MgCl ₂, dNTPs, hot resistant DNA polymerase, EBV upstream amplimer, EBV downstream amplimer, VZV upstream amplimer, VZV downstream amplimer, EBV fluorescent probe and VZV fluorescent probe.

Pcr amplification primer sequence is:

EBV upstream amplimer sequence (SEQ ID No:1): 5 '-TCATCTACGGGGACACGGAC-3 ';

EBV downstream amplimer sequence (SEQ ID No:2): 5 '-GAGCTCCACCCCCTTCATC-3 ';

VZV upstream amplimer sequence (SEQ ID No:3): 5 '-TAGGCGGATGGTGGACGACT-3 ';

VZV downstream amplimer sequence (SEQ ID No:4): 5 '-GCCCGCAAACTTGTAGAACTG-3 '.

EBV fluorescent probe sequence (SEQ ID No:5): 5 ' FAM-GCACTCGATAAACAGCGAG-BHQ-1-3 ';

VZV fluorescent probe sequence (SEQ ID No:6): 5 ' HEX-TTGTTATGACGGCCGAGCT-BHQ-1-3 ';

EBV standard substance sequence (SEQ ID No:7): GAGCTCCACC CCCTTCATCA GGGTCTTGCC GTCCGTCAGC ACCCCCACAT ATCTCTTCTT TGTAATCAGC ATCAGGCAGG AGAAGGTCTT CTCGGCCTCC AGGGAGATGG GGGCCACAAA CAGGCTCCGG GTGGTGTGGG CGGCCAGGGC CTCGGCAAAG CGCAGGGTCT CGCTCTCTGA AAACCCCCGG CACTCGATAA ACAGCGAGTC CGTGTCCCCG TAGATGA;

VZV standard substance sequence (SEQ ID No:8): TAGGCGGATG GTGGACGACT AAGCTCGGCC GTCATAACAA ACTTATTAAT ATCCAATTTG GGTGATGTAA TCTGGCGATG TGCATCTGCA ATTATGCGTC CAAACCCGGCCATCCCAGAC GGCATGGCCC GTCTATTCCA TTCAGCAATG GAAACACACG ACGCCTCCGC CGCAGCACGC GAGACGGTGT CGTCATATAA CAACAGTTCT ACAAGTTTGC GGGC.

EBV positive control and VZV positive control are respectively EBV and VZV inactivation of viruses strain sample, and negative control is sterile water for injection sample.

Test kit of the present invention should be stored in-20 ℃, reduces multigelation as far as possible.

Test kit using method of the present invention:

Each detection all should be set up positive control and negative control, and standard substance are 1 × 10 with aseptic deionized water dilution ⁴-1 × 10 ⁷copy.

The extraction of viral DNA: in strict accordance with commercial QIAamp DNA extraction agent box operation, extract viral DNA from clinical samples or viral separation and Culture thing.

Pcr amplification detects: on double-colored (or more than) quantitative real time PCR Instrument, carry out, each PCR reaction system cumulative volume is 25 , comprising 20

pCR reaction solution and 5

template (viral DNA of extraction, standard substance, positive or negative contrast).Probe in detecting pattern is: FAM, HEX passage are respectively used to detect EBV, VZV.PCR reaction conditions: 94 ℃ of 5 minutes denaturations, 94 ℃ 20 seconds → 60 ℃ 60 seconds, totally 40 circulations.After setting completes, preserve file, working procedure.

Fluorescent quantitation report the test: 1. detect the amplification curve of sample without increased logarithmic phase or two probe CT(threshold cycle) value is all >=40 negative; 2. detecting sample a certain probe CT value≤38 and amplification curve has obvious increased logarithmic phase, and the virus that this probe is corresponding is positive; In same reaction system, two probe CT value≤38 and amplification curve have obvious increased logarithmic phase, are that EBV and VZV mix positive; 3. detect a certain probe 38 < CT value < 40 of sample, need re-start DNA extraction and PCR detection to sample.

The present invention adopts Real-Time Fluorescent Quantitative PCR Technique and Two Colour Fluorescence probe, has developed the test kit for EBV and VZV detection by quantitative.This invention test kit energy single stage method detects herpes virus hominis EBV and VZV, not only realize the synchronous diagnosis that EBV and VZV infect, and can carry out real-time accurate quantitative analysis to positive-virus, can meet that clinical early stage, Accurate Diagnosis EBV and VZV infect in the urgent need to, the timely immunotherapy targeted autoantibody infecting for EBV and VZV provides foundation.

Accompanying drawing explanation

Fig. 1 is the structural representation of test kit of the present invention.

Fig. 2 is the typical curve of EBV and VZV concentration gradient standard substance.

Embodiment

The present invention is described further with accompanying drawing in conjunction with the embodiments.Should be appreciated that, these embodiment, only for illustration purpose, limit the scope of the invention and be not used in.

embodiment 1

Referring to Fig. 1, herpes virus hominis EBV provided by the invention and VZV detection kit, be made up of quantitative PCR reaction solution 1, EBV standard substance 2, VZV standard substance 3, EBV positive reference substance 4, VZV positive reference substance 5, negative control product 6, specification sheets 7 and box body 8.

Wherein quantitative PCR reaction solution contains PCR damping fluid, MgCl ₂, dNTPs, hot resistant DNA polymerase, EBV upstream amplimer, EBV downstream amplimer, VZV upstream amplimer, VZV downstream amplimer, EBV fluorescent probe and VZV fluorescent probe.

Pcr amplification primer sequence is:

EBV upstream amplimer sequence (SEQ ID No:1): 5 '-TCATCTACGGGGACACGGAC-3 ';

EBV downstream amplimer sequence (SEQ ID No:2): 5 '-GAGCTCCACCCCCTTCATC-3 ';

VZV upstream amplimer sequence (SEQ ID No:3): 5 '-TAGGCGGATGGTGGACGACT-3 ';

VZV downstream amplimer sequence (SEQ ID No:4): 5 '-GCCCGCAAACTTGTAGAACTG-3 '.

EBV positive control 1 and VZV positive control 2 are respectively EBV and VZV inactivation of viruses strain sample, and negative control is sterile water for injection sample.

embodiment 2the susceptibility of herpes virus hominis EBV and VZV detection kit and specific test

(1) material:

Choosing pathogenic micro-organism comprises: experimental group: EBV(B95-8 strain) VZV(EF strain is provided by Beijing institute of viruses) provided by microorganism teaching and research group of Medical University Of Anhui; Control group: streptococcus aureus, escherichia coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome are provided by attached children's hospital of Zhejiang University Bacteriology Room, Viral Laboratory and gene amplification chamber.

(2) design of primer and probe is with synthetic:

Conserved regions sequence to EBV and VZV is carried out bioinformatic analysis, designs and screen pcr amplification primer and specificity fluorescent probe, entrusts Shanghai Sheng Gong biotech firm synthetic.

EBV upstream amplimer sequence (SEQ ID No:1): 5 '-TCATCTACGGGGACACGGAC-3 ';

EBV downstream amplimer sequence (SEQ ID No:2): 5 '-GAGCTCCACCCCCTTCATC-3 ';

VZV upstream amplimer sequence (SEQ ID No:3): 5 '-TAGGCGGATGGTGGACGACT-3 ';

VZV downstream amplimer sequence (SEQ ID No:4): 5 '-GCCCGCAAACTTGTAGAACTG-3 '.

VZV fluorescent probe sequence (SEQ ID No:6): 5 ' HEX-TTGTTATGACGGCCGAGCT-BHQ-1-3 '.(3) preparation of examination criteria product:

By EBV upstream amplimer and EBV downstream amplimer amplification EBV virus strain, by VZV upstream amplimer and VZV downstream amplimer amplification VZV virus strain, after each amplified fragments purifying, insert pGEM-T-Easy cloning vector construction recombination plasmid, each plasmid is carried out quantitatively with spectrophotometer, and carry out quantitative fluorescent PCR sensitivity test with the each plasmid of 10 doubling dilution with aseptic deionized water.

(4) susceptibility of test kit and specific test:

Adopt herpes virus hominis EBV and VZV detection kit to detect above-mentioned encountered pathogenic microorganism, experimental group two-strain detected result all positive and somatotype meets, the detected results such as control group streptococcus aureus, escherichia coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome are all negative, and specificity is 100%.After the recombinant plasmid standard substance doubling dilution building, detection sensitivity can reach 10 copies; With 10 ⁴, 10 ⁵, 10 ⁶, 10 ⁷four quantitative concentration gradient standard substance are made typical curve, have good linear relationship (Fig. 2) between its CT value and plasmid copy number.

The present invention is described in conjunction with most preferred embodiment, but is reading after foregoing of the present invention, and those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the scope of the application's appended claims institute limit equally.

<110> Zhejiang University

<120> herpes virus hominis EBV and VZV detection kit

<160> 8

<210> 1

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> detects herpes virus hominis EBV upstream primer sequence

<400> 1

TCATCTACGGGGACACGGAC 20

<210> 2

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> detects herpes virus hominis EBV downstream primer sequence

<400> 2

GAGCTCCACCCCCTTCATC 19

<210>3

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> detects herpes virus hominis VZV upstream primer sequence

<400> 3

TAGGCGGATGGTGGACGACT 20

<210> 4

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> detects herpes virus hominis VZV downstream primer sequence

<400> 4

GCCCGCAAACTTGTAGAACTG 21

<210> 5

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> herpes virus hominis EBV fluorescent probe sequence

<400> 5

GCACTCGATAAACAGCGAG 19

<210> 6

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> herpes virus hominis VZV fluorescent probe sequence

<400> 6

TTGTTATGACGGCCGAGCT 19

<210> 7

<211> 227

<212> DNA

<213> artificial sequence

<220>

<223> is according to the EBV fluorescent quantitation examination criteria product sequence of herpes virus hominis EBV BALF5 gene order design

<400> 7

GAGCTCCACC CCCTTCATCA GGGTCTTGCC GTCCGTCAGC ACCCCCACAT ATCTCTTCTT 60

TGTAATCAGC ATCAGGCAGG AGAAGGTCTT CTCGGCCTCC AGGGAGATGG GGGCCACAAA 120

CAGGCTCCGG GTGGTGTGGG CGGCCAGGGC CTCGGCAAAG CGCAGGGTCT CGCTCTCTGA 180

AAACCCCCGG CACTCGATAA ACAGCGAGTC CGTGTCCCCG TAGATGA 227

<210> 8

<211> 224

<212> DNA

<213> artificial sequence

<220>

<223> is according to the VZV fluorescent quantitation examination criteria product sequence of herpes virus hominis VZV ORF28 gene order design

<400> 8

TAGGCGGATG GTGGACGACT AAGCTCGGCC GTCATAACAA ACTTATTAAT ATCCAATTTG 60

GGTGATGTAA TCTGGCGATG TGCATCTGCA ATTATGCGTC CAAACCCGGC CATCCCAGAC 120

GGCATGGCCC GTCTATTCCA TTCAGCAATG GAAACACACG ACGCCTCCGC CGCAGCACGC 180

GAGACGGTGT CGTCATATAA CAACAGTTCT ACAAGTTTGC GGGC 224

Claims

1. a herpes virus hominis EBV and VZV detection kit, it is characterized in that, formed by quantitative PCR reaction solution (1), EBV standard substance (2), VZV standard substance (3), EBV positive reference substance (4), VZV positive reference substance (5), negative control product (6), specification sheets (7) and box body (8), wherein: PCR reaction solution (1) contains PCR damping fluid, MgCl ₂, dNTPs, hot resistant DNA polymerase, EBV upstream amplimer, EBV downstream amplimer, VZV upstream amplimer, VZV downstream amplimer, EBV fluorescent probe and VZV fluorescent probe;

EBV upstream amplimer sequence: 5 '-TCATCTACGGGGACACGGAC-3 ';

EBV downstream amplimer sequence: 5 '-GAGCTCCACCCCCTTCATC-3 ';

VZV upstream amplimer sequence: 5 '-TAGGCGGATGGTGGACGACT-3 ';

VZV downstream amplimer sequence: 5 '-GCCCGCAAACTTGTAGAACTG-3 ';

EBV fluorescent probe sequence: 5 ' FAM-GCACTCGATAAACAGCGAG-BHQ-1-3 ';

VZV fluorescent probe sequence: 5 ' HEX-TTGTTATGACGGCCGAGCT-BHQ-1-3 ';

EBV standard substance sequence: GAGCTCCACC CCCTTCATCA GGGTCTTGCC GTCCGTCAGC ACCCCCACAT ATCTCTTCTT TGTAATCAGC ATCAGGCAGG AGAAGGTCTT CTCGGCCTCC AGGGAGATGG GGGCCACAAA CAGGCTCCGG GTGGTGTGGG CGGCCAGGGC CTCGGCAAAG CGCAGGGTCT CGCTCTCTGA AAACCCCCGG CACTCGATAA ACAGCGAGTC CGTGTCCCCG TAGATGA;

VZV standard substance sequence: TAGGCGGATG GTGGACGACT AAGCTCGGCC GTCATAACAA ACTTATTAAT ATCCAATTTG GGTGATGTAA TCTGGCGATG TGCATCTGCA ATTATGCGTC CAAACCCGGCCATCCCAGAC GGCATGGCCC GTCTATTCCA TTCAGCAATG GAAACACACG ACGCCTCCGC CGCAGCACGC GAGACGGTGT CGTCATATAA CAACAGTTCT ACAAGTTTGC GGGC.

2. a kind of herpes virus hominis EBV according to claim 1 and VZV detection kit, is characterized in that, EBV positive control and VZV positive control are respectively EBV and VZV inactivation of viruses strain sample, and negative control is sterile water for injection sample.

3. a kind of herpes virus hominis EBV according to claim 1 and VZV detection kit, is characterized in that, test kit of the present invention is stored in-20 ℃, reduces multigelation.