CN105132584B - For the kit and its production method of parting detection varicellazoster virus and application - Google Patents
For the kit and its production method of parting detection varicellazoster virus and application Download PDFInfo
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Abstract
The present invention provides a kind of kits for parting detection varicellazoster virus, it is characterised in that it includes with the nucleotide sequence as shown in specification table 3 and the corresponding specific primer of 5 type varicellazoster virus of the clade1 of chemical constitution and specific probe.The kit has the function of the various varicellazoster virus DNA of detection, detection sensitivity 102Copy/reaction, and with human genome, the type of herpes simplex virus type 1/2, cytomegalovirus, Epstein-Barr virus no cross reaction, it is diagnosed suitable for the viral gene of clinical varicellazoster virus the infected, it may also be used for the epidemiological survey of different Clade types varicellazoster virus.
Description
Technical field
The invention belongs to microbial gene diagnostic techniques fields, and in particular to one kind detects varicella zoster for parting
The kit and its production method of virus and application.
Background technology
Varicella virus (Varicella-zoster virus, VZV) is herpetoviridae Varicellavirus
A kind of virus is clinical varicella and the common pathogen of herpes zoster.Varicella symptom can occur as patient primary infection VZV,
And generate with lifelong latent infection, the virus hidden when immunity of organisms declines after the several years may activate again cause it is band-like
Bleb.Varicella such as diagnoses and treatment not in time, by secondary varicella pneumonia, encephalitis, myocarditis etc. or even causes death, and band-like
Bleb can bring patient and have an intense pain for a long time, and many inconvenience are brought to minimal invasive treatment, and burden is brought to Public Health Expenditure.
There is ascendant trend in varicella, herpes zoster incidence in recent years, and the Case report that the whole world has severe varicella lethal successively has text
The domestic and international some areas adult varicella incidence of report is offered significantly to rise, and explosive stream is be easy to cause in gathering of people region
Row.
It looks back the passing clinical diagnostic method to VZV infection of people and finds that clinician, which relies primarily on, at present typically faces
Bed symptom and skin lesion carry out VZV Infect And Diagnoses, but other viral (such as Coxsackie virus, herpes simplex virus) infection are equally
Varicelliform eruption can be caused to bring difficulty to antidiastole.And existing laboratory diagnostic method have Virus culture, directly it is glimmering
Photoactivated antibody analysis, PCR, Serum Antibody Detection, all in the presence of needing, sample size is big, complicated for operation, speed is relatively slow, cannot batch for they
It detects, be unable to the shortcomings such as Multiple detection, sensibility is low, these all annoying clinical diagnosis and treatment, prevention.Wherein Virus culture is VZV
The goldstandard of Infect And Diagnose, but it needs special experimental laboratory condition, it is complicated for operation, time a couple of days is needed, and positive rate is low；Directly
Fluorescence antibody analysis operation is cumbersome, needs professional skilled engineer, and can only once detect a sample, it is impossible to high pass
Amount detection；Standard PCR detection cannot carry out batch, Multiple detection, and parting needs to carry out multitube detection, cumbersome and time-consuming；Blood
Clear antibody test is clinically used viral diagnosis method, but VZV antibody cannot function as the diagnosis index of its infection, because of its shadow
The factor of sound is more and related with immunization campaign state.In addition, as flow of personnel is frequent, the wild strain of various regions VZV viruses it is new
The variation of the epidemics such as hair and gene mutation also proposes new test to VZV preventions, it is desirable that laboratory is quickly to people
In group while popular VZV viral diagnosis, moreover it is possible to carry out accurate parting, hold various VZV fashions trend, but existing reality
Parting detection cannot be carried out by testing room detection method.In short, research and develop novel varicellazoster virus diagnostic kit still
It is one of challenge that people face.
Streaming liquid-phase chip technology is also known as streaming fluorescent technique or suspension array streaming fluorescent technique, and principle is by fluorescence
Unicellular (or particle) suspension after label enters flow chamber with sheath fluid, and the drop that pressure forces sheath fluid to wrap up includes single cell
Or particle is perpendicular through detection zone.The cell or particle of fluorescent marker send out fluorescence under laser excitation, are marked according to itself
Fluorescence and excitation after the fluorescence comprehensive analysis that sends out be detected.Its small sample, high throughput, it is easy to operate, quick and precisely,
The aspects such as susceptibility, specificity height are better than traditional analysis method, and its all reaction carries out in the liquid phase, keeps natural
Conformation, is conducive to probe and determinand fully reacts, and is widely used in all kinds of nucleic acid and Protein Detection at present, as Genotyping,
Antigen/antibody detection, infectious diseases diagnosis etc., wherein HPV detection and parting kit, Respirovirus detection kit,
Cytokine detection kits, tumor markers detection kit etc. are used in the U.S. and China through FDA and SFDA approvals
Clinical detection.VZV has genome most conservative in herpesviral, is divided VZV for 5 type (clade according to international conference in 2008
1-5).Conservative genome is conducive to design it primer and probe of his opposite sex, carries out multiplex PCR and hybridization.
Invention content
It can quick, high-throughput, Multiple detection varicella the object of the present invention is to provide a set of streaming liquid-phase chip platform that is based on
Herpes zoster virus infection patient's bleb liquid varicellazoster virus DNA and the microbial gene that Clade partings are carried out to it
Diagnostic kit, suitable for quick, the high-throughput diagnosis to clinical varicellazoster virus the infected, it may also be used for different
The epidemiological survey of Clade type varicellazoster virus.
Technical scheme is as follows：
For the kit of parting detection varicellazoster virus, which is characterized in that including having following nucleotides sequence
Row specific primer corresponding with the clade1-5 type varicellazoster virus of chemical constitution and specific probe：
Clade1- sense primers：5’-Biotin-CACGGAACATGAATGTAGGA-3’
Clade1- downstream primers：5’-Biotin-ATCCTTTTCGTCTGGCTTA-3’
Clade1- probes：5’-NH2-TTTTTTTTTTCGTACAAAATGCGGTGGGT-3’
Clade2- sense primers：5’-Biotin-CGTCTACCCGACCTTCATAC-3’
Clade2- downstream primers：5’-Biotin-CTTGTGGCATACGTTCGT-3’
Clade2- probes：5’-NH2-TTTTTTTTTTGAAAAGCGTTCTTGTCTCC-3’
Clade3- sense primers：5’-Biotin-CACTGTCAGACACACTTGGGA-3’
Clade3- downstream primers：5’-Biotin-TATAAGGGGACGTTGGAACT-3’
Clade3- probes：5’-NH2-TTTTTTTTTTTTCATCGGTGCTATTGGG-3’
Clade4- sense primers：5’-Biotin-CGTTATAGCAGGGGTCTCC-3’
Clade4- downstream primers：5’-Biotin-TTTATTGGCACGTTGTCG-3’
Clade4- probes：5’-NH2-TTTTTTTTTTCCGTGACAGAAACATCAACC-3’
Clade5- sense primers：5’-Biotin-TTCGGTCCACTAGCGTTG-3’
Clade5- downstream primers：5’-Biotin-GCTTGATGGTTATGGCAGTA-3’
Clade5- probes：5’-NH2-TTTTTTTTTTGCCCTACAAAGACATTCCG-3’.
The nucleotide sequence of above-mentioned primer/probe is from top to bottom successively such as sequence table Seq ID No.1-Seq ID No.15
It is shown；Listed in sequence table is only nucleotide sequence, and the 5` ends of each pair of upstream and downstream primer primer are repaiied with biotin (biotin)
Decorations, the 5` ends of every probe are by NH2 base group modifications.
The kit is further included for carrying out the reagent of PCR, fluorescent microsphere, for by probe and fluorescent microsphere progress
Coupling prepares the reagent of microballoon hybridization solution, and/or the reagent for PCR product to be hybridized with microballoon hybridization solution.
It is described to include for carrying out the reagent of PCR：PCR buffer solutions, dNTPs, Taq enzyme, distilled water；
The fluorescent microsphere is Luminex carboxyl fluorescent microspheres；
It is described be used to probe and fluorescent microsphere carrying out coupling preparing the reagent of microballoon hybridization solution include：MES buffer solutions (PH
4.5), EDC solution, Tween 20, SDS, TE buffer solution, TMAC hybridization buffers；
It is described to be used to include PCR product with the reagent that microballoon hybridization solution hybridizes：SA-PE；
The TMAC hybridization buffers include：5mol/L TMAC, 1mol/L Tris-HCl (pH 8.0), 20%
Sarkosyl、0.5mol/L EDTA。
A kind of preparation method of kit for parting detection varicellazoster virus, which is characterized in that assembling examination
The reagent of agent box includes the corresponding specific primer of the clade1-5 types varicellazoster virus and specific probe.
It assembles and is further included in the reagent of kit for carrying out the reagent of PCR, fluorescent microsphere, for by probe and fluorescence
Microballoon carries out coupling and prepares the reagent of microballoon hybridization solution, and/or the examination for PCR product to be hybridized with microballoon hybridization solution
Agent.
For the method for parting detection varicellazoster virus, which is characterized in that carried out such as using the kit
Lower step：
(1) using the corresponding specific primer of the clade1-5 types varicellazoster virus to the gene of sample to be tested
Group DNA carries out multiplexed PCR amplification；
(2) it is other glimmering with not jack per line respectively with the corresponding specific probe of the clade1-5 types varicellazoster virus
Light microballoon carries out coupling and microballoon hybridization solution is prepared；
(3) PCR product is mixed to the hybrid product for carrying out obtaining after hybridization reaction to be splined on the microballoon hybridization solution
Luminex200 streaming fluorescence analysers detect the characteristic fluorescence signal that the hybrid product is sent out, record and this feature fluorescence
The corresponding fluorescent microsphere number of signal is not so as to know corresponding varicellazoster virus type；
The corresponding specific primer of the clade1-5 types varicellazoster virus and specific probe refer to：
Clade1- sense primers：5’-Biotin-CACGGAACATGAATGTAGGA-3’
Clade1- downstream primers：5’-Biotin-ATCCTTTTCGTCTGGCTTA-3’
Clade1- probes：5’-NH2-TTTTTTTTTTCGTACAAAATGCGGTGGGT-3’
Clade2- sense primers：5’-Biotin-CGTCTACCCGACCTTCATAC-3’
Clade2- downstream primers：5’-Biotin-CTTGTGGCATACGTTCGT-3’
Clade2- probes：5’-NH2-TTTTTTTTTTGAAAAGCGTTCTTGTCTCC-3’
Clade3- sense primers：5’-Biotin-CACTGTCAGACACACTTGGGA-3’
Clade3- downstream primers：5’-Biotin-TATAAGGGGACGTTGGAACT-3’
Clade3- probes：5’-NH2-TTTTTTTTTTTTCATCGGTGCTATTGGG-3’
Clade4- sense primers：5’-Biotin-CGTTATAGCAGGGGTCTCC-3’
Clade4- downstream primers：5’-Biotin-TTTATTGGCACGTTGTCG-3’
Clade4- probes：5’-NH2-TTTTTTTTTTCCGTGACAGAAACATCAACC-3’
Clade5- sense primers：5’-Biotin-TTCGGTCCACTAGCGTTG-3’
Clade5- downstream primers：5’-Biotin-GCTTGATGGTTATGGCAGTA-3’
Clade5- probes：5’-NH2-TTTTTTTTTTGCCCTACAAAGACATTCCG-3’.
The nucleotide sequence of above-mentioned primer/probe is from top to bottom successively such as sequence table Seq ID No.1-Seq ID No.15
It is shown；Listed in sequence table is only nucleotide sequence, and the 5` ends of each pair of upstream and downstream primer primer are repaiied with biotin (biotin)
Decorations, the 5` ends of every probe are by NH2 base group modifications.
The reaction system of the PCR amplification is：4 × PCR buffer solutions：5μl；2.5mol/L dNTPs：2μl；Taq enzyme：0.5
μ l, Clade 1/2/4 type sense primer：Each 0.2 μ l；Downstream primer：Each 0.4 μ l；3/5 type sense primers of Clade：Each 0.4 μ
l；Downstream primer：Each 0.2 μ l；Genomic DNA：2μl；Distilled water：7.5μl.
The response procedures of the PCR amplification are：95 DEG C of pre-degeneration 10min；Using 95 DEG C of 15s, 57 DEG C of 30s, 72 DEG C of 30s as 1
A cycle, totally 35 recycle, last 72 DEG C of extensions 5min.
The coupling refers to：Luminex carboxyl fluorescent microsphere vortex mixing 30s are taken, 400ul 12000rpm is taken to centrifuge 2 points
Clock abandons supernatant, and microballoon is resuspended in 0.1mol/l MES buffer solution (PH 4.5) the whirlpools mixing for adding in 45 μ l, adds 2 μ l
Probe described in 0.1mol/l, mixing；The EDC solution of 2.5 μ l 10g/l twice is added in, reacts 30 under the conditions of room temperature is protected from light every time
Minute；The 20 whirlpool mixings of Tween of 1.0ml 0.02% are added in, 12000rpm is centrifuged 2 minutes, abandons supernatant；Use 1.0ml
0.1% SDS washed once, and 12000rpm is centrifuged 2 minutes, abandons supernatant, and microballoon finally is resuspended with 80 μ l TE buffer solutions, is protected from light 4
DEG C preserve；Above-mentioned 1 μ l of resuspension microballoon is taken to add in 45 μ l TE buffer solutions and 170 μ l TMAC hybridization buffers, microballoon hybridization is made
Liquid.
The hybridization refers to：3 μ l of PCR product is taken to be mixed with microballoon hybridization solution described in 22 μ l, 95 DEG C of denaturation 5min, 48 DEG C of hybridization
After 30min, SA-PE 75 μ l, 48 DEG C of incubation 15min are added in.
The present invention provides a kind of varicellazoster virus combined using streaming liquid-phase chip technology and round pcr
(Varicella-zoster virus, VZV) detection and genotyping kit, technical principle are as follows：Design is for each hypotypes of VZV
The specific primer and specific probe of clade1-5, the 5` ends biotin modification of the primer, is gone out by multiplexed PCR amplification
Specific fragment PCR product；It is other glimmering with not jack per line respectively using the corresponding specific probes of each hypotype clade1-5 of VZV simultaneously
Light microballoon carries out coupling and 5 kinds of numbers not different hybridization microballoons that can send out respective characteristic fluorescence signal respectively is prepared,
It is mixed as hybridization solution；Again PCR product is hybridized to obtain hybrid product with the microballoon hybridization solution；With streaming fluorescence point
Analyzer detects the hybrid product, records the characteristic fluorescence signal of generation, finds out corresponding number other hybridization microballoon, and then know that this is miscellaneous
Liquid is handed over to correspond to the specific probe of coupling, so as to know the specific hypotype of VZV.
Kit of the present invention for parting detection varicellazoster virus, which is characterized in that including strictly according to the facts
Apply the corresponding specific primers of clade1-5 types VZV and specific probe shown in table 3 in example 1.Inventor utilizes existing
Ncbi database downloads to the genome sequence of each Strain of VZV, carries out Multiple sequence alignments, and it is special to find out various VZV Strain
Different in nature SNP site, while in view of in multiplexed PCR amplification, the annealing temperature of each primer pair need to approach can just make PCR smoothly into
Row, inventor go out 5 pairs of primers that are clade1-5 types VZV specificity and meeting multiplex PCR condition for above-mentioned every point design,
Each pair of primer 5` ends are modified with biotin (biotin) simultaneously, biotin modification is advantageous in that：It is with phycoerythrin fluorescence mark
The strong bonded and multistage enlarge-effect of high affinity, can effectively promote probe mark between note Streptavidin (SA-PE)
Note and related tracer analysis become sensitiveer；The corresponding specific primers of clade1-5 types VZV are expanding sample to be tested base
Because group DNA obtains the specific PCR products segment of a certain type VZV；Design specific probe be for not jack per line is other sends out
The fluorescent microsphere coupling for going out characteristic fluorescence signal obtains microballoon hybridization solution；Product after PCR product is hybridized with microballoon hybridization solution
It is placed in detection acquisition characteristic fluorescence signal on streaming fluorescence analyser and would know which kind of type sample to be tested genomic DNA belongs to
VZV。
Further, the kit is further included for carrying out the reagent of PCR, fluorescent microsphere, for by probe and fluorescence
Microballoon carries out coupling and prepares the reagent of microballoon hybridization solution, and/or the examination for PCR product to be hybridized with microballoon hybridization solution
Agent.Specifically, it is described to include for carrying out the reagent of PCR：PCR buffer solutions, dNTPs, Taq enzyme, distilled water；PCR reagent is used for
Complete reaction system is formed with the specific primer and reacts amplification so as to carry out PCR, and above-mentioned is that this field is used for PCR amplification
Conventional reagent, those skilled in the art can also use the various existing products for PCR reactions, such as common commercially available
PCR kit, PCR reactions Mix etc..The fluorescent microsphere is specially Luminex carboxyl fluorescent microspheres, which marks
Two kinds of fluorescent dyes, the concentration of each dyestuff have 10 gradients, and 100 kind (1 is can obtain by adjusting the ratio of both dyestuffs
Numbers-No. 100) it can respectively send out the microballoon of different characteristic fluorescence signals；It, can be by different specific probes based on this feature
Be coupled with the other microballoon of not jack per line, the microballoon after coupling with specific fragment PCR product hybridized after production
Object detects specific fluorescence signal through streaming fluorescence analyser, so as to know corresponding sample to be tested type.It is described to be used to visit
Needle and fluorescent microsphere carry out coupling and prepare the reagent of microballoon hybridization solution including：MES buffer solutions (PH 4.5), EDC solution, Tween
20th, SDS, TE buffer solution, TMAC hybridization buffers (5mol/L TMAC, 1mol/L Tris-HCl (pH 8.0), 20%
Sarkosyl、0.5mol/L EDTA)；Mentioned reagent is the common agents being coupled with Luminex carboxyls fluorescent microsphere, this field
Technical staff can need to adjust concentration and dosage according to experiment or even do related improvement to each solution formula, but as long as being with this
Specific primer described in the kit of invention and probe are technological core, adjustment or improvement for the purpose of parting detection VZV,
It belongs to the scope of protection of the present invention.It is described to be used to include PCR product with the reagent that microballoon hybridization solution hybridizes：SA-PE, it
Can be strong bonded with biotin, so as to improve the sensitivity entirely reacted.
The preparation method of the kit is also claimed in the present invention, and any scale is sold or produced based on commercial object
The behavior of the kit belongs to the claimed range of the present invention, wherein the behavior for producing the kit refers to assembling reagent
The reagent of box includes the corresponding specific primers of clade1-5 types VZV as shown in Table 1 and specific probe.
Application of the kit in terms of parting detects varicellazoster virus is also claimed in the present invention.Experience
Card, the varicella band in varicellazoster virus the infected's bleb liquid, serum can be detected using kit of the present invention
Shape herpes virus DNA simultaneously carries out Clade partings to it, and detection sensitivity can reach 102Copy/reaction, with human genome, merely
The type of 1 type of herpesviral/2, cytomegalovirus, Epstein-Barr virus no cross reaction, also no cross reaction between various VZV, reach to facing
The purpose of bed varicellazoster virus the infected's diagnosis, it may also be used for the stream of different Clade types varicellazoster virus
Row disease learns investigation.Also, when kit using the present invention is detected, using 96 orifice plates, whole operation process can be in 4h
Within complete provide as a result, for clinical varicellazoster virus the infected diagnosis, especially suitable for reply burst it is big
Area eruption and prevalence is detected simultaneously parting, hence it is evident that better than traditional VZV laboratory diagnostic methods in the short time.
In conclusion the present invention is that the micro sample of streaming liquid-phase chip platform, fast speed, high throughput, Multiple detection is special
Point hybridizes combination with specific PCR amplification and probe, and the kit is finally made to have had both high specificity, sensitivity and use
Micro sample detect simultaneously multiple samples, multiple indexs (parting) function, can be used not only for clinical varicellazoster virus
The infected's bleb liquid viral gene detects, and varicellazoster virus infection is diagnosed and Clade partings, it may also be used for no
With the epidemiological survey of type varicellazoster virus.
Description of the drawings
1 type parting sites of Fig. 1 sequence alignments Clade
2 type parting sites of Fig. 2 sequence alignments Clade
3 type parting sites of Fig. 3 sequence alignments Clade
4 type parting sites of Fig. 4 sequence alignments Clade
5 type parting sites of Fig. 5 sequence alignments Clade
Fig. 6 is various standard items amplified production electrophoresis pattern, wherein Type1, Type3, Type4, Type5 Clade1/
3/4/5 type positive criteria product, pOka plants of vaccine are 2 type positive criteria products of Clade, and Type2 is Clade2 types negative control, HGD
It is distilled water for human genome, NC.
Fig. 7 VZV streaming liquid-phase chip detection parameters and result interface
Fig. 8 VZV streaming liquid-phase chips detectability is analyzed
Specific embodiment
The present invention is described in further detail With reference to embodiment, but is not intended to limit the model of the present invention
It encloses.Unless otherwise specified, the operation used in following embodiments is conventional method, and used reagent commercially available can obtain
.
Reagent and consumptive material
Tween 20, lauryl sodium sulfate (SDS), PCR buffer solutions, dNTPs, Taq enzyme, 2-morpholine ethane sulfonic acid (MES),
Water-soluble carbodiimide (EDC), tetramethyl sal-ammoniac (TMAC), trishydroxymethylaminomethane (Tris), flesh aminoacyl
(Sarkosyl), ethylenediamine tetra-acetic acid (EDTA) phycoerythrin fluorescent marker Streptavidin (SA-PE) is commercially available；
Luminex carboxyls fluorescent microsphere is purchased from Luminex companies；
The formula of TMAC hybridization buffers is as shown in table 1 below：
Table 1
MES buffer solutions (PH 4.5), EDC solution, TE buffer solutions, Tris-HCl solution (pH 8.0) can be according to《Molecule gram
Grand experiment guide》The step of described in one book, is prepared.
Clade1-5 type VZV standard items, virus genom DNA extracts kit are commercially available.
The source of biomaterial and record source
It is as shown in table 2 below with recording source for the source of each Strain of parting in embodiment 1.
Table 2
The sample to be tested bleb liquid used in 3 clinical verification of embodiment is always cured collected from Chengdu Military Area Command of the Chinese People's Liberation Army
The varicella and Patients with Herpes Zoster that institute accepts for medical treatment.
The preparation of embodiment 1, kit of the present invention
First, varicellazoster virus detection and the selection in parting site
Using NCBI nucleic acid databases (http://www.ncbi.nlm.nih.gov/nuccore/) search and download VZV
Each Strain genome sequence, and parting is carried out to each Strain according to Clade classifying methods.Using DNAman softwares to VZV
Each Strain, herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), cytomegalovirus (CMV), Epstein-Barr virus
(EBV) strain, human gene group DNA's sequence carry out Multiple sequence alignments, exclude homologous sequence, it is special to find out various VZV Strain
Property SNP site.
Find out 21 plants of varicellazoster virus altogether, wherein Dumas, 32passage 72,32passage 22,
32passage 5,49,36, kel, SD, MSP, BC belong to 1 types of Clade, Varivax, VarilRix, pOka, vOka belong to
2 types of Clade, 03-500,22,11, HJ0 belong to 3 types of Clade, 8, DR belongs to 4 types of Clade, CA123 belongs to 5 types of Clade,
Table 2 is referred in the source and record source of each Strain.36802T/C、51936A/C、83724A/G、70927C/A、50860C/A
5 SNP sites can effectively distinguish that Clade is various, and sequence alignment result is shown in Fig. 1-Fig. 5.
2nd, the screening and verification of VZV DNA cloning primer
Using primer 5 and 7 softwares of oligo corresponding spy is designed for the respective above-mentioned sites of clade1-5 types VZV
Specific primer and specific probe, primer are shown in Table 3, and grope reaction system and condition with probe sequence, are shown in Table 4, wherein,
Template is the genomic DNA of sample to be tested, and the template in the present embodiment is clade1-5 type VZV standard items；4X
Buffer, dNTPs (2.5mM) and Taq Polymerase are mixed into premix taq DNA polymerase reaction liquid in advance.It will
Clade1-5 type VZV standard items are diluted to 105、104、103Copy/reaction is expanded, and amplified production draws 2 μ l and carries out 3%
Agarose gel electrophoresis, scanning result are shown in attached drawing 6, various 103It more than copy/reaction density can effectively expand, and have no bright
Aobvious nonspecific reaction, with human genome no cross reaction.
The corresponding specific primers of table 3clade1-5 types VZV and specific probe
The nucleotide sequence of above-mentioned primer/probe is from top to bottom successively such as sequence table Seq ID No.1-Seq ID No.15
It is shown；Listed in sequence table is only nucleotide sequence, and the 5` ends of each pair of upstream and downstream primer primer are repaiied with biotin (biotin)
Decorations, the 5` ends of every probe are by NH2 base group modifications.
4 varicellazoster virus DNA PCR reaction systems of table and reaction condition
3rd, kit is assembled
Based on above-mentioned screening verification as a result, assembling kit of the present invention.The reagent of assembling kit includes：Table
The corresponding specific primers of clade1-5 types VZV and specific probe shown in 3；
It assembles and is still further comprised in the reagent of kit：
For carrying out the reagent of PCR：PCR buffer solutions, dNTPs, Taq enzyme, distilled water；
Luminex carboxyl fluorescent microspheres；
For probe and fluorescent microsphere to be carried out the reagent that coupling prepares microballoon hybridization solution：MES buffer solutions (PH4.5), EDC
Solution, Tween 20, SDS, TE buffer solution, TMAC hybridization buffers；
For the reagent for hybridizing PCR product with microballoon hybridization solution：SA-PE；
The TMAC hybridization buffers include：5mol/L TMAC, 1mol/L Tris-HCl (pH 8.0), 20%
Sarkosyl、0.5mol/L EDTA。
Embodiment 2, kit sensitivity of the present invention and the verification of specificity
By the use of clade1-5 type VZV standard items as sample to be tested, with human genome, herpes simplex virus 1, herpe simplex disease
Poison 2, cytomegalovirus, epstein barr virus dna are control sample, using globulin (globin) probe as internal standard, are tested in accordance with the following steps
Demonstrate,prove the sensitivity and specificity of kit of the present invention：
(1) multiplex PCR expansion is carried out to the genomic DNA of sample to be tested using the specific primer shown in 1 table 3 of embodiment
Increase；
(2) coupling is carried out with the other fluorescent microsphere of not jack per line respectively with the specific probe shown in 1 table 3 of embodiment to be prepared into
To mixing microballoon hybridization solution；
(3) PCR product is mixed to the hybrid product for carrying out obtaining after hybridization reaction to be splined on the microballoon hybridization solution
Luminex200 streaming fluorescence analysers detect the characteristic fluorescence signal that the hybrid product is sent out, record and this feature fluorescence
The corresponding fluorescent microsphere number of signal is not so as to know corresponding varicellazoster virus type；
The specific probe is coupled with fluorescent microsphere to be as follows：Take 1.25X107Luminex carboxyls
Fluorescent microsphere (MicroPlex microballoons) mixing 30s on eddy blending machine takes 400ul12000rpm to centrifuge 2 minutes, abandons supernatant,
It takes 0.1mol/l MES buffer solution (PH 4.5) the whirlpools mixing for adding in 45ul that microballoon is resuspended, adds 2ul 0.1mol/l implementations
Probe described in example 1, mixing；Dichloroethanes (EDC) solution of the 10g/l of 2.5 microlitres of fresh configurations twice is added in, room temperature is protected from light instead
It answers each 30 minutes, then adds in the 20 whirlpool mixings of Tween of 1.0ml0.02%, 12000rpm is centrifuged 2 minutes, abandons supernatant, is used
The SDS of 1.0ml 0.1% washed once, and 12000rpm is centrifuged 2 minutes, abandon supernatant, and microballoon is resuspended in last 80ul TE buffer solutions,
It is protected from light 4 DEG C of preservations.
1 type probe and No. 19 fluorescent microspheres are coupled with reference to above method, 2 type probes and No. 45 fluorescent microspheres couple, 3 types
Probe is coupled with No. 21 fluorescent microspheres, 4 type probes are coupled with No. 29 fluorescent microspheres, 5 type probes couple with No. 26 fluorescent microspheres, is interior
Mark globulin (globin) probe is coupled with No. 28 fluorescent microspheres, and each 1 μ l of the various microballoons coupled is taken to add in 45 μ l TE bufferings
Liquid and 170 μ l tetramethyls sal-ammoniac (TMAC) hybridization buffers are prepared as microballoon hybridization solution (configuration method is shown in Table 4).
The multiplexed PCR amplification the specific steps are：Various VZV standard items are diluted to 105、104、103、102Copy/
Reaction is expanded, and with 5 pairs of primers shown in 1 table 3 of embodiment as the primer of multiplex PCR, reaction system and response procedures are such as
Shown in 1 table 4 of embodiment.3 μ l of PCR product is taken to be mixed with 22 μ l microballoon hybridization solutions, after 95 DEG C of denaturation 5min, 48 DEG C of hybridization 30min,
Add in SA-PE 75 μ l, 48 DEG C of incubation 15min.Hybrid product, setting are detected using Luminex200 streamings fluorescence analyser
Event is more than 500, according to Instrument measuring peak-settings door threshold value, record average fluorescent strength (mean fluorescence
Intensity, MFI), operating parameter and result interface are shown in Fig. 7, wherein, clade1 type VZV samples correspond to product send out No. 19 it is micro-
The characteristic fluorescence signal of ball；Clade2 type VZV samples correspond to the characteristic fluorescence signal that product sends out No. 45 microballoons；Clade3 types
VZV samples correspond to the characteristic fluorescence signal that product sends out No. 21 microballoons；Clade4 type VZV samples correspond to product and send out No. 29 microballoons
Characteristic fluorescence signal；Clade5 type VZV samples correspond to the characteristic fluorescence signal that product sends out No. 26 microballoons.Same method expands
Increase and detect human genome, herpes simplex virus 1, herpes simplex virus 2, cytomegalovirus, epstein barr virus dna.As a result such as attached drawing 8
And shown in table 5, it is strong positive that Globin internal standards, which detect all samples, illustrates that Globin can be expanded by stablizing, reaction system has
Effect.Clade 1-5 type standard items are diluted to 105、104、103、102Copy number/reaction has stronger MFI, illustrates various VZV
Minimum detectability can reach 102Copy；MFI is all higher than 300 during each fluorescent microsphere detection target type, and to other types and
MFI is relatively low during 1ng/ μ L HGD Samples detections, and respectively less than 50, there are not nonspecific signals when this reaction system being prompted to detect.
Table 5VZV streaming liquid-phase chips detect the analysis of other herpetoviridae virus results
The clinical verification of embodiment 3, kit of the present invention
It is varicella and Patients with Herpes Zoster to acquire 44 clinical diagnosises that in January, 2013 in December, 2014, our hospital accepted for medical treatment
Bleb liquid, wherein varicella 19, herpes zoster 25.Kit using the present invention and the common detection methods of this field simultaneously
Parallel testing and parting are carried out to these samples to be tested.
The VZV detection methods of this field routine are：Direct fluorescence antibody detection.
Same a collection of sample to be tested is detected using kit described in embodiment 1, bleb liquid passes through virus genom DNA
Extracts kit extracts viral DNA, is expanded, be coupled, hybridized successively according to step described in embodiment 2 and by hybrid product
Upper 200 streaming fluorescence analysers of Luminex detection.
Two methods testing result is as shown in table 6 below：
Table 6
Using kit of the present invention detection VZV viruses and parting it can be seen from above-mentioned testing result, with this field
Conventional method is compared, and result difference is not notable, and consistency is good, so as to confirm kit of the present invention in parting detection VZV viruses
The reliability and accuracy of aspect.
Claims (5)
1. for the kit of parting detection varicellazoster virus, which is characterized in that including having following nucleotide sequence
Specific primer corresponding with the clade1-5 type varicellazoster virus of chemical constitution and specific probe：
Clade1- sense primers：5’-Biotin-CACGGAACATGAATGTAGGA-3’
Clade1- downstream primers：5’-Biotin-ATCCTTTTCGTCTGGCTTA-3’
Clade1- probes：5’-NH2-TTTTTTTTTTCGTACAAAATGCGGTGGGT-3’
Clade2- sense primers：5’-Biotin-CGTCTACCCGACCTTCATAC-3’
Clade2- downstream primers：5’-Biotin-CTTGTGGCATACGTTCGT-3’
Clade2- probes：5’-NH2-TTTTTTTTTTGAAAAGCGTTCTTGTCTCC-3’
Clade3- sense primers：5’-Biotin-CACTGTCAGACACACTTGGGA-3’
Clade3- downstream primers：5’-Biotin-TATAAGGGGACGTTGGAACT-3’
Clade3- probes：5’-NH2-TTTTTTTTTTTTCATCGGTGCTATTGGG-3’
Clade4- sense primers：5’-Biotin-CGTTATAGCAGGGGTCTCC-3’
Clade4- downstream primers：5’-Biotin-TTTATTGGCACGTTGTCG-3’
Clade4- probes：5’-NH2-TTTTTTTTTTCCGTGACAGAAACATCAACC-3’
Clade5- sense primers：5’-Biotin-TTCGGTCCACTAGCGTTG-3’
Clade5- downstream primers：5’-Biotin-GCTTGATGGTTATGGCAGTA-3’
Clade5- probes：5’-NH2-TTTTTTTTTTGCCCTACAAAGACATTCCG-3’.
2. kit according to claim 1, which is characterized in that further include for carry out the reagent of PCR, fluorescent microsphere,
The reagent of microballoon hybridization solution is prepared for probe to be carried out coupling with fluorescent microsphere, and/or for PCR product to be hybridized with microballoon
The reagent that liquid is hybridized.
3. kit according to claim 2, which is characterized in that described to include for carrying out the reagent of PCR：PCR is buffered
Liquid, dNTPs, Taq enzyme, distilled water；
The fluorescent microsphere is Luminex carboxyl fluorescent microspheres；
It is described be used to probe and fluorescent microsphere carrying out coupling preparing the reagent of microballoon hybridization solution include：MES buffer solutions, EDC are molten
The pH values of liquid, Tween 20, SDS, TE buffer solution, TMAC hybridization buffers, wherein MES buffer solutions are 4.5；
It is described to be used to include PCR product with the reagent that microballoon hybridization solution hybridizes：SA-PE；
The TMAC hybridization buffers include：5mol/L TMAC, 1mol/L Tris-HCl, 20%Sarkosyl, 0.5mol/L
The pH values of EDTA, wherein Tris-HCl are 8.0.
A kind of 4. preparation method of kit for parting detection varicellazoster virus, which is characterized in that assembling reagent
The reagent of box includes the corresponding specific primer of clade1-5 types varicellazoster virus described in claim 1 and special
Property probe.
5. preparation method according to claim 4, which is characterized in that assemble and further include to carry out in the reagent of kit
The reagent of PCR, fluorescent microsphere prepare the reagent of microballoon hybridization solution, and/or are used for for probe and fluorescent microsphere to be carried out coupling
The reagent that PCR product is hybridized with microballoon hybridization solution.
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| CN106119425B (en) * | 2016-09-22 | 2020-02-18 | 武汉百格资产管理有限公司 | Primer, probe and kit for synchronously detecting types 6 and 7 of human herpesviruses |
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| FR3051482B1 (en) * | 2016-05-19 | 2021-01-29 | Life & Soft | PROCESS FOR DETERMINING THE PRESENCE AND QUANTIFICATION OF AT LEAST ONE MICRO-ORGANISM IN A BIOLOGICAL SAMPLE |
| CN106119424B (en) * | 2016-09-22 | 2020-02-18 | 武汉百格资产管理有限公司 | Primer, probe and kit for synchronously detecting types 6, 7 and 8 of human herpesviruses |
| CN106244731A (en) * | 2016-09-22 | 2016-12-21 | 武汉百格资产管理有限公司 | Synchronous detecting human herpes virus 6, the primer of 8 types, probe and test kit |
| CN114790494B (en) * | 2021-11-04 | 2024-03-22 | 江汉大学 | MNP (varicella-zoster virus) marking site, primer composition, kit and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880733A (en) * | 2010-08-05 | 2010-11-10 | 中国检验检疫科学研究院 | Avian influenza virus typing detection method based on flow microsphere technique |
| CN103146841A (en) * | 2013-01-25 | 2013-06-12 | 海尔施生物医药股份有限公司 | Kit capable of synchronously detecting eighteen kinds of fever with eruption pathogens and detection method thereof |
| CN103866046A (en) * | 2014-01-21 | 2014-06-18 | 浙江大学 | Detection kit for human herpes viruses EBV and VZV |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880733A (en) * | 2010-08-05 | 2010-11-10 | 中国检验检疫科学研究院 | Avian influenza virus typing detection method based on flow microsphere technique |
| CN103146841A (en) * | 2013-01-25 | 2013-06-12 | 海尔施生物医药股份有限公司 | Kit capable of synchronously detecting eighteen kinds of fever with eruption pathogens and detection method thereof |
| CN103866046A (en) * | 2014-01-21 | 2014-06-18 | 浙江大学 | Detection kit for human herpes viruses EBV and VZV |
Non-Patent Citations (2)
| Title |
|---|
| A real-time PCR assay for the detection of varicellla-zoster virus DNA and differentiation of vaccine, wild-type and control strains;Graham A. Tipples等;《Journal of Virological Methods》;20030228;第113卷(第2期);113 * |
| Genotyping of Varicella-zoster Virus and the Discrimination of Oka Vaccine strains by Taqman Real-time PCR;S.P. Parker等;《Journal of Clinical Microbiology》;20061130;第44卷(第11期);第3912页实验结果部分第2段，第3913页图2，第3914页讨论部分第1-2段 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119425B (en) * | 2016-09-22 | 2020-02-18 | 武汉百格资产管理有限公司 | Primer, probe and kit for synchronously detecting types 6 and 7 of human herpesviruses |
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