CN108220484A - One-step method detects EBV, CMV, the kit and detection method of HSV-6 herpesvirals simultaneously - Google Patents

One-step method detects EBV, CMV, the kit and detection method of HSV-6 herpesvirals simultaneously Download PDF

Info

Publication number
CN108220484A
CN108220484A CN201810172845.7A CN201810172845A CN108220484A CN 108220484 A CN108220484 A CN 108220484A CN 201810172845 A CN201810172845 A CN 201810172845A CN 108220484 A CN108220484 A CN 108220484A
Authority
CN
China
Prior art keywords
sample
magnetic bead
nucleic acid
kit
hsv
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.)
Granted
Application number
CN201810172845.7A
Other languages
Chinese (zh)
Other versions
CN108220484B (en
Inventor
杨尚鑫
王芳
王一芳
刘杰
褚伟峰
潘玲玲
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.)
Shaoxing Xun Xun Kang Biological Technology Co Ltd
Original Assignee
Shaoxing Xun Xun Kang Biological Technology 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 Shaoxing Xun Xun Kang Biological Technology Co Ltd filed Critical Shaoxing Xun Xun Kang Biological Technology Co Ltd
Publication of CN108220484A publication Critical patent/CN108220484A/en
Application granted granted Critical
Publication of CN108220484B publication Critical patent/CN108220484B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

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)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a kind of herpes virus hominis EBV for detecting β, γ hypotype common in sample, CMV, the kit of HSV 6, the kit includes nucleic acid cleavage solution, wherein, nucleic acid cleavage solution includes magnetic bead, and the nucleic acid cleavage solution includes 1M NaCl, 0.01%Triton X and 0.001M KCl, and magnetic bead is the hydroxyl magnetic bead of 0.5 microlitre of 10mg/mL.It is that kit can extract nucleic acid without additional step with one-step method using this, the augmentation detection of progress nucleic acid that can be quickly and easily can realize full-automatic operation.

Description

One-step method detects EBV, CMV, the kit of HSV-6 herpesvirals and detection simultaneously Method
Technical field
The invention belongs to molecular diagnostics biological technical fields, are related to one-step method nucleic acid extraction and fluorescence quantitative PCR detection Kit, and in particular to the real-time fluorescence quantitative PCR reagent containing three probes, detection blood samples of patients, urine, cerebrospinal fluid equal samples Middle EBV, CMV, the novel agent box of common β, γ hypotype herpes virus hominis of tri- kinds of HSV-6.
Background technology
The herpesviral related with the mankind is clinically known as nerpes vinrus hominis in VD hospital.Herpesviral is mainly invaded Violate the tissue of ectodermal origin, including skin, mucous membrane and nerve fiber.Infection site and caused disease are varied, and have The trend of latent infection threatens human health.
Herpesviral (herpesviruses, HPV) is the medium sized double-stranded DNA virus of a group, have 100 or more into Member, is divided into tri- subfamilies of α, β, γ according to its physicochemical property.α herpesvirals (such as herpes simplex virus, varicella-zoster disease Poison) growth rate is fast, cytopathy can be caused.β herpesvirals (such as cytomegalovirus), growth cycle is long, and infection cell is formed Giant cell.Gamma herpes viruses (such as Epstein-Barr virus), the target cell of infection is lymphoid cell, can cause lymphocytic hyperplasia.Herpesviral The host range of infection is extensive, can infect the mankind and other vertebrates.The herpesviral of human diseases is caused to be shown in Table 1.
The type and its caused principal disease of 1 nerpes vinrus hominis of table
Metainfective common presentation is：Neuromere body of gland, kidney lymphoid tissue, lymphoid tissue herpes febrilis；Lip, eye, brain sense Dye；Genital herpes varicella；Herpes zoster monocytosis,mononucleosis, eye, kidney, brain and congenital infection infectious mononucleosis Disease, Burkitt lymthomas, nasopharyngeal carcinoma, baby's urgency rash and some other such as unknown abdominal pain illness.
BlebVirusExtremely widespread, mostly subclinical infection is infected, minority is apparent infection.Different blebs in crowd are investigated VirusSerum antibodyIt was found that 10 years old children in the poverty-stricken area in the world, have about 90% to infect herpes simplex virus I-form (HSV- 1) almost all of, being grown up infected HSV-1.In Temperate Region in China, 90% 14 years old children infected varicella-zoster Virus.In West Europe, Britain and the U.S., about 20~80% teenagers infectedCytomegalovirus(CMV).In third world countries In, nearly all children had infected CMV.Herpesviral easily causes fetal congenital to infect in addition to Epstein-Barr virus, causes to flow Production, premature labor, fetal congenital deformity and persistent postnatal infection.Since CMV and HSV easily cause pregnant woman'sCervixInfection, tire The incidence of youngster's congenital infection is also higher.Infants with congenital cmv infection incidence is 0.5~1.5%.Fetal congenital infects Approach be that the virus being contaminted in pregnant woman's body is broadcast to fetus or transcervical uplink by placenta and infects fetus.Herpesviral Though not completely certainly, many researchs have shown that herpesvirus infection is related with the generation of certain carcinomas to carcinogenicity.Such asEpstein-Barr virusWithBurkitt's lymphomaIt is closely related with nasopharyngeal carcinoma, HSV-1 and lip cancer, HSV-2 and cervix cancer.It is believed that this viroid Gene can be partly or entirely integrated in the gene of host cell, cause the canceration of host cell.Herpesviral is that one kind has The DNA virus of coating, it has now been found that 60 kinds or more.Mammality, birds, amphibian animal and the infection of fish can be caused.State in 1978 Herpesviral is divided into tri- groups of α, β and γ by the border viral nomenclature committee.Can infect the mankind herpes simplex virus I-form and II type and Varicella virus divides α groups into, and cytomegalovirus is β groups, and Epstein-Barr virus is γ groups.The herpesviral of the infection mankind has altogether Same form and structure.Herpesviral is spherical in shape, and a diameter of 150~200nm, intact virus is made of core, nucleocapsid and coating. The core of virus is distrand DNA, and has a small amount of zymoprotein.Nucleocapsid forms 20 face bodies by 162 hollow tubular shell particles.It is outermost One layer is coating, is made of fat and sugar albumen, antigenic structure is present in glycoprotein.The herpesviral of the mankind is infected except EB diseases It is malicious outer, it can be grown in diploid cell tissue cultures, and cause apparent cytopathy.
After herpesvirus infection, can behave as primary infection,Latent infectionOr recurrent infection.Bleb is infected for the first time Virus is primary infection without immunity person.After primary infection herpesviral, virus is not proliferated in human body cell, is not also broken Bad cell, but in latence, for patient without clinical symptoms, this is known as latent infection at this time, once due to environmental stimuli, such as by Cool, wound, infection decline using resistance of human body such as immunosuppressor, and the virus of latence just replicates proliferation again, destroys Cell, causes a series of clinical symptoms, this is known as recurrent infection.Herpes simplex virus andVaricellaHerpes zoster virus is being felt Feel latent infection in neuromere and gasserian ganglion, sacral ganglia.Epstein-Barr virus latent infection in lymphocyte.Herpesviral sense The immunity generated after dye is incomplete, the killed vaccine of attenuated live vaccine or the conventional method inactivation of some herpesvirals (such as HSV) Under study for action, but some herpesvirals (such as Epstein-Barr virus) have oncogenic potential for immunization campaign.The above-mentioned dangerous property of vaccine, someone's research The membranous antigen that extraction Epstein-Barr virus determines from people's lymph matricyte system cell of production Epstein-Barr virus manufactures vaccine.
The diseases such as fever, jaundice, encephalitis or meningitis caused by herpesvirus infection, because sings and symptoms are without special Property, early diagnose the diagnosis in the laboratory that needs to rely on treatment in time.Herpesviral relatively common at present is 1-6 types, And the herpesviral case study for 1-6 types is also relatively more, and symptom description is clearer and more definite.
Examining the common detection methods of virus has：1. virus purification：Peripheral blood mononuclear cells culture of isolated virus is inspection Survey the goldstandard of herpesviral.It was drawer peripheral blood mononuclear cells in the past.With fresh normal circumference blood lymphocyte Or cord blood lymphocytes cell holds high also middle mixed culture, but generally require 5-21 days general.At present, scholar replaces training with culture plate Bottle is supported, is more convenient for obtaining cell film flying；Progress immunohistochemistry monitoring on slide is fixed in, can obviously reduce reagent use Amount not only reduces cost, and shortens viral detection time (taking only 1 day or so), while significantly reduces culture pollution machine Meeting, it is reproducible.2. the more commonly used diagnostic method in Serologic detection antibody assay room is the measure of serum antibody, specific to detect Method has and can be divided into antigen direct Detection Method and antibody indirect detection method.Antigen direct Detection Method due to certain viruses do not generate can The antigen of detection and cause sensitivity low, antibody indirect detection method is effective due to needing just to generate for 1 week or so after herpesvirus infection The antibody of concentration and can not be early diagnosed, and there are cross reaction between different herpesviral, antibody specificity is not high.
With the rapid development of Protocols in Molecular Biology, polymerase chain reaction (polymerase chain reaction, PCR) technology, providing property of detection direction of the Real-Time Fluorescent Quantitative PCR Technique particularly risen in recent years for pathogenic microorganism. It has merged round pcr and nucleic acid efficient amplification, the high specific of probe technique, the hypersensitivity of spectral technique and high-precision determines The advantages that amount, the variation of fluorescence signal is to obtain quantitative result during direct detection PCR.Such as Chinese patent application CN103866046A discloses a kind of herpes virus hominis EBV and VZV detection kits, by quantitative PCR reaction solution, EBV standard items, VZV standard items, EBV positive reference substances, VZV positive reference substances, negative controls, specification and box body composition.The invention reagent Box uses Real-Time Fluorescent Quantitative PCR Technique and Two Colour Fluorescence probe, and energy one-step method detection herpes virus hominis EBV and VZV are realized The synchronous diagnosis that EBV and VZV infect, to positive-virus can real-time accurate quantitative analysis, can meet clinical early stage, Accurate Diagnosis EBV and VZV infection there is an urgent need to, for EBV and VZV infection timely immunotherapy targeted autoantibody foundation is provided.With round pcr detection EB diseases Malicious DNA mainly includes the extraction of Epstein-Barr virus nucleic acid and the PCR amplification of nucleic acid, but the kit is not directed to Epstein-Barr virus nucleic acid and carries Take aspect, and in practice, except PCR in itself in addition to, nucleic acid extraction efficiency and antijamming capability accurately detect PCR viral core Acid content has a significant impact.
At present, it is domestic clinically mainly using direct boiling method, phenol-chloroform extraction process or nucleic acid extraction kit to blood Herpesviral nucleic acid in slurry or serum sample extracts.Direct boiling method extraction process is more complicated, and when handling sample By multiple steps such as boiling lysis, high speed centrifugation enrichment DNAs, there is loss in the DNA in sample, while snead process is extracted It is nucleic acid-templated in containing more impurity, these existing impurity can Interference Detections progress, such as protein, heparin, institute To generally require nucleic acid extraction liquid being further purified, purification procedures are tediously long and often fall flat.
Invention content
In view of the above-mentioned problems, other than three kit for detecting nucleic acid to α herpesvirals, to common β, γ hypotype Herpes virus hominis EBV, CMV, HSV-6 invented three kit for detecting nucleic acid, the invention provides a kind of paramagnetic particle methods The one-step method kit of absorption is cracked to the progress Rapid nucleic acid in sample and keeps preferable detection sensitivity, to supplement bleb The diagnostic area of type.
Directly sample is extracted using magnetic bead, without the purifying and washing of sample, directly uses magnetic bead and amplification Reagent contact carry out nucleic acid amplification, reduce operating procedure, meanwhile, avoid interference of the impurity to detection.
One aspect of the present invention provides common tri- joint inspection of β, γ hypotype herpes virus hominis EBV, CMV, HSV-6 of detection and surveys Kit, the kit include lysate and bead suspension, wherein, the ingredient of lysate includes 1M NaCl, 0.01% Triton-X and 0.001M KCl, bead suspension include the magnetic bead of hydroxyl modified, polydispersity coefficient ＜ 0.2；Magnetic bead is diameter Less than or equal to 500nM, wherein, the content of magnetic bead is 10mg/mL.
In some preferred modes, when lysate and the mixing of magnetic bead solution, the volume ratio of the two is 200:1. Alternatively, providing a kind of mixture, which is solution mixture, which includes 1M NaCl, 0.01%Triton-X With the hydroxyl magnetic bead of 0.001M KCl and 0.005 microlitre of 10mg/mL.Alternatively, magnetic bead lysate is provided in the way of table 1, The magnetic bead lysate is by lysate and magnetic bead liquid according to volume 200：1 mode mixes.
In some preferred modes, which further includes the necessary ingredient of PCR reaction amplifications, the amplification Reagent includes the Mg2+ of 1~10mmol/L, the Tris-HCl buffer solutions of 10~50mmol/L；The K ions of concentration 25mmol/L, The enzyme stability reagent of 100 μ g/ml, such as BSA etc..Preferably, glycerine is further included in amplifing reagent.In some preferred sides In formula, special probe sequence is further included, the sequence is SEQ.1.1-1.3；SEQ.2.1-2.3 and SEQ.3.1-3.3.
On the other hand, the object of the present invention is to provide the herpes virus hominis of a kind of one-step method nucleic acid extraction and β, γ hypotype Tri- link detection reagent kit of EBV, CMV, HSV-6, wherein including magnetic bead lysate, quantitative PCR detection liquid to kit.Preferably, The kit further includes：Negative control, EBV, CMV, HSV-6 positive controls, specification and box body composition.Specific component is seen below Table.
Table 1：The component and volume ratio of magnetic bead lysate.
Table 2:The composition of amplifing reagent and specific ingredient
Preferably, quantitative PCR detection liquid contains PCR buffer solutions, hot resistant DNA polymerase, three kinds of primer and probes.Described Primer is specifically, being table 3.
Table 3：Fluorescent quantitative PCR primed probe
In some modes, negative control is further included as TE buffer；Positive control is EBV, and CMV, HSV-6 inactivation are sick Strain mixing sample.Kit of the present invention should be stored in -20 DEG C, within multigelation 8 times.
On the one hand, the present invention provides a kind of tri- joint inspection of herpes virus hominis EBV, CMV, HSV-6 for detecting β, γ hypotype and surveys Method, this method includes providing the reagent of table 1-3, the extraction step of nucleic acid and the amplification step of nucleic acid, wherein, nucleic acid carries Take including：
First, nucleic acid DNA extracts：
1. magnetic bead lysate is taken out, oscillation shakes up rear of short duration centrifugation；
2. taking in suitable centrifuge tube or PCR pipe, often pipe adds in the magnetic bead lysate of 80 μ L；
3. in the centrifuge tube of step 2 or PCR pipe, often pipe adds in 20 μ L samples；
4. test every time should at least set a hole negative control (TE buffer) and a hole positive control (positive control is EBV, CMV, HSV-6 inactivation of viruses strain mixing sample), the same sample of loading methods, negative method for extracting nucleic acid and inspection with the positive The method of test sample sheet is consistent.
5. covering pipe lid, centrifuge tube or PCR pipe are placed in 80 DEG C of constant temperature and are incubated 5min by of short duration centrifugation, and then room temperature is put Put 5min.If so, eight unions can be put into PCR instrument, 80 DEG C × 5min is set, 20 DEG C × 5min.
6. step 5 centrifuge tube or eight unions are placed on magnetic frame and stand 1-2min；Liquid is removed, retains magnetic bead, simultaneously Any further washed, washing or processing are not done to magnetic bead.If being accidentally drawn onto magnetic bead, then liquid is returned in pipe, weight It is inhaled again after new standing 1min.Liquid is abandoned in suction to take out liquid completely or using automated process, retain magnetic bead in PCR pipe.
Two：Fluorescent quantitative PCR：
7. with liquid-transfering gun draw 50 μ L kits 2 in quantitative PCR detection liquid, be added separately to step 6 centrifuge tube or In eight unions.
8. it is inhaled to beat to magnetic bead to be completely dispersed repeatedly with liquid-transfering gun and (please notes that part pipette tips may adsorb magnetic bead, it will influence Testing result, mixing here can not also use liquid-transfering gun, but the method for using vibrations mixes automatically).Such as 1.5mL from Heart pipe must be transferred in PCR pipe or PCR disks.Close the lid or seal up glued membrane.
9. being immediately placed in PCR instrument carries out augmentation detection.If do not detected temporarily, PCR pipe or PCR disks must be kept in dark place in 2- It is no more than 2 hours in 8 DEG C of refrigerators.
10. following loop parameter is set in PCR instrument：
95℃×10min；It is recycled 40 times by 95 DEG C × 15s, 60 DEG C × 45s again；
Fluoroscopic examination is at 60 DEG C；Reaction system is 50 μ L
11. fluorescence channel detection selection (EBV collection fluorescence-FAM, CMV collection fluorescence-NED, HSV-6 collections fluorescence- CY5)；If with the PCR instrument of ABI series, Quencher Dye selections None.Save file runs program.
12. experiment terminates, suitable threshold line is set, obtains Ct values, judgement sample feminine gender positive findings.
Sample in all modes can be liquid type sample：Serum, urine, mouthwash, hydrothorax, ascites, cerebrospinal fluid, room Water is used directly for nucleic acid extraction；Or swab sample, such as Nasopharyngeal swabs, sputum class sample, swab or sputum sample It is dissolved in 1-5mL physiological saline.
Advantageous effect：
1. pair nucleic acid carries out easy rapid extraction：With magnetic bead adsorption of DNA nucleic acid, without washing elution, to be directly used in PCR anti- It should；2. the sample size of requirement is few：Nucleic acid extraction sample in general existing traditional technology is 1mL, and to sample in the present invention It is required that being 20ul or can less realize；3. β, γ hypotype herpesviral are used for quickly detecting by quantitative fluorescent PCR, There are good specificity, sensitivity and Classification Identification.
Description of the drawings
Fig. 1 is the experimental result picture of the detection positive or negative sample in a specific embodiment of the invention, wherein, Figure 1A is the test signal figure for detecting negative sample and positive sample；Figure 1B is the EBV positives, and the detection of CMV, HSV-6 feminine gender As a result new signal figure；Fig. 1 C are the CMV positives, and the testing result new signal figure of EBV, HSV-6 feminine gender；Fig. 1 D are the HSV-6 positives, And the testing result new signal figure of EBV, CMV feminine gender.
Fig. 2 is the signal graph of specific detection experiment.
Fig. 3 is to positive sample sensitivity technique signal graph.
Specific embodiment
The present invention is described in further detail below in conjunction with the drawings and specific embodiments, the scheme that embodiment provides is Preferred embodiment is how to be put into practice, but not as right as those of ordinary skill in the art marrow according to the invention to crack The restriction of the application, scope of the present application embody in the claims.
Explanation：It is Bio-RAD CFX96 and ABI 7500 in PCR amplification instrument used in following embodiment.
Embodiment 1：The EBV for confirming positive or negative sample, CMV, HSV-6 samples are passed through in the detection of magnetic bead one-step method
The reagent of offer such as following table：
Table 1：The component and volume ratio of magnetic bead lysate.
Table 2:The composition of amplifing reagent and specific ingredient
Table 3：Fluorescent quantitative PCR primed probe
Specific method is as follows：
First, nucleic acid DNA extracts：
1. the magnetic bead lysate in such as table 1 is taken out, oscillation shakes up rear of short duration centrifugation.
2. take the magnetic bead lysate that 80 μ L are respectively added in one or eight connecting leg, five pipe, 1 is used for negative sample nucleic acid extraction, 4 For different positive sample nucleic acid extractions
3. in the PCR pipe of step 2, it is separately added into below 20ul samples：
4. covering pipe lid, eight unions are placed in 80 DEG C of constant temperature and are incubated 5min, are then placed at room temperature for 5min by of short duration centrifugation.
5. step 4 centrifuge tube or eight unions are placed on magnetic frame and stand 1-2min.Liquid is carefully sucked with pipettor. If being accidentally drawn onto magnetic bead, then liquid is returned in pipe, inhaled again after standing 1min again, it is complete that liquid is abandoned in suction.
6. adding in the above-mentioned PCR detections liquid of 50ul, of short duration centrifugation after mixing is vibrated.
PCR temperature controls：45℃ 10min；
95℃ 10min；
95 DEG C of 15s, 60 DEG C of 45s；40 cycles；
Fluorescence selects tri- channels of FAM, NED, CY5
As a result referring to Fig. 1, from figure one as can be seen that Fig. 1 (is EBV, CMV, HSV-6 sun of the method based on invention in Fig. 1 Property sample and negative sample empirical curve；Wherein, figure a is a negative sample and EBV, CMV, HSV-6 mixing sample；Figure B is single EBV positive samples；It is single CMV positive samples to scheme c；It is single HSV-6 positive samples to scheme d.)
Interpretation of result
With the present invention magnetic bead one-step method QPCR methods can detect accurately distinguish it is negative and single or mix EBV, CMV, Tri- kinds of herpesviral positives of HSV-6, positive curve have preferable exponential increase signal.
Meanwhile (health is century universal pillar genome extraction examination using existing commercially available nucleic acid extraction kit Agent box CW2298S) and carried out according to the method for commercial reagents box, the PCR detection liquid of the extraction product present invention is expanded, and is tied Fruit finds that its Ct value steps back 1-2 cycle cycle, and illustrating small system sample, there are larger losses with traditional extracting mode.
Likewise, confirm that positive sample carries out single and three kinds of mixing saliva sample, swab sample is adopted for clinical Carry out the extraction of nucleic acid with the method for the present embodiment 1, then similarly expanded, it is similary obtain it is positive as a result, showing The positive can be obtained, and reliable result still can be obtained for clinical sample not only for plasmid sample.
Examples of implementation 2：Specific test
According to examples of implementation 1 magnetic bead method for extracting nucleic acid to following sample carry out nucleic acid extraction, PCR amplification system and Amplification condition is identical with examples of implementation 1, and it is respectively HSV-1, HSV-2, VZV, rotavirus, enteron aisle to take 7 parts of specific reference materials Virus, streptococcus pneumonia (ATCC 49618), Escherichia coli (reference culture ATCC 35150) positive sample carry out fluorescent PCR The specific test of kit.
As a result it shows (such as Fig. 2), shows that only EBV, CMV, HSV-6 positive plasmid amplification curve are shown as positive, and HSV1, HSV2, VZV, rotavirus, enterovirus, streptococcus pneumonia, Escherichia coli positive sample do not occur specific amplification song Line illustrates that the combination specificity is very high.
Meanwhile using existing business diagnostic products (up to peace gene Epstein-Barr virus nucleic acid amplification (PCR) fluorogenic quantitative detection reagent Box, human cytomegalovirus nucleic acid quantitative determination reagent kit (PCR- fluorescence methods), human herpes virus type 6 kit for detecting nucleic acid (PCR- fluorescence probe methods)) carry out herpesviral positive sample of the interfering effects of drug test discovery commercial product to water melon frost interfering effects of drug Originally false negative is occurred, and the method for the present invention test result has correct amplification curve, illustrates same sample, present invention inspection Test agent can be obtained more accurately with stable testing result.
Examples of implementation 3：Sensitivity tests
To being diluted (dilution 10 with tri- kinds of plasmid mixing samples of EBV, CMV, HSV-61/102/103/104/105/106), Then PCR amplification is carried out according to the kit mode of operation that the method and reagent of examples of implementation 1 refer to respectively to detect three times.
As a result it is as follows；
Show：Fluorescence quantifying PCR method minimum detection limit in combination is about 10copies/ul, there is preferable sensitivity Resolution ratio.This can not only be detected well, and can be very high sensitivity, be also for detecting medication effect Can with, this may have relationship with the quality of extraction nucleic acid of the present invention.
Equally, for existing commercial product (up to peace gene Epstein-Barr virus nucleic acid amplification (PCR) fluorogenic quantitative detection reagent Box, human cytomegalovirus nucleic acid quantitative determination reagent kit (PCR- fluorescence methods), human herpes virus type 6 kit for detecting nucleic acid (PCR- fluorescence probe methods)) it extracts nucleic acid according to the reagent of existing product and is expanded, it is only able to detect 1 × 103Threshold values, And for 1 × 102With 1 × 101Sample, it is impossible to detect positive findings (summary of specific experiment data).
The all patents and publications mentioned in description of the invention all represents that these are the public technologies of this field, this hair It is bright to use.All patents referred to herein and publication are all equally listed in bibliography, with each publication It is specific to be individually referenced equally.The present invention described here can lack any element or multiple element, and one It is realized in the case of kind limitation or a variety of limitations, this limitation here is not particularly illustrated.Such as art in each example here Language "comprising", " essence by ... form " and " by ... form " can be replaced with remaining 2 term of one of both.Here it adopts Describing mode carried out by terms and expressions mode, and be not limited except as, also indicate that this book describes without any intention here These terms and explain and eliminate any equivalent feature, but it is recognised that can be in the model of the present invention and claim Any suitable be altered or modified is done in enclosing.Preferably implement it is appreciated that examples of implementation described in the invention are all some Example and feature, any those of ordinary skill in the art can be changed and become according to some are done under the marrow of the invention that describe Change, these are changed and variation is recognized as belonging to the scope of the present invention and independent claims and appended claims are limited Range.

Claims (9)

1. a kind of kit for the herpes virus hominis EBV, CMV, HSV-6 for detecting β, γ hypotype common in sample, the kit Including nucleic acid cleavage solution, wherein, nucleic acid cleavage solution includes magnetic bead, the nucleic acid cleavage solution include 1M NaCl, 0.01%Triton-X and 0.001M KCl, magnetic bead are the hydroxyl magnetic bead of 0.5 microlitre of 10mg/mL.
2. a kind of tri- link detection reagent kit of herpes virus hominis EBV, CMV, HSV-6 for detecting common β, γ hypotype, the kit Including lysate and bead suspension, wherein, the ingredient of lysate includes 1M NaCl, 0.01%Triton-X and 0.001M KCl, bead suspension include the magnetic bead of hydroxyl modified, polydispersity coefficient ＜ 0.2；Magnetic bead is less than or equal to 500nM for diameter, In, the content of magnetic bead is 10mg/mL.
3. according to the kit described in one of claim 1-2, wherein, after lysate and the mixing of magnetic bead solution, the body of the two Product is than being 200:1.
4. according to the kit described in one of claim 1-3, wherein, which further includes a kind of mixture, which is Solution mixture, the mixture include cracking ingredient and magnetic bead, are divided into 1M NaCl, 0.01%Triton-X wherein being cracked into With 0.001M KCl, magnetic bead is the hydroxyl magnetic bead of 0.005mg.
5. according to the kit described in one of claim 1-4, wherein, the institute which further includes PCR reaction amplifications is necessary Ingredient, the amplifing reagent includes the Mg2+ of 1~10mmol/L, the Tris-HCl buffer solutions of 10~50mmol/L；Concentration The K ions of 25mmol/L, the enzyme stability reagent of 100 μ g/ml, such as BSA etc.；Glycerine and such as SEQ.1.1-1.3；SEQ.2.1- Primer sequence and probe sequence shown in 2.3 and SEQ.3.1-3.3.
6. according to the kit described in one of claim 1-5, wherein the kit further includes：Negative control, EBV, CMV, HSV-6 positive controls, specification and box body composition.
7. according to the kit described in one of claim 1-6, wherein, the sample is serum, urine, mouthwash, hydrothorax, Ascites, cerebrospinal fluid or aqueous humor equal samples, the sample are used directly for nucleic acid extraction；Alternatively, swab sample, such as nasopharyngeal swab Son, sputum class sample, wherein swab or sputum sample are dissolved in 1-5mL physiological saline.
8. a kind of method for detecting β, γ hypotype herpes virus hominis EBV, CMV, HSV-6 common in sample, this method include：
Kit as described in one of claim 1-7 is provided,
The extraction and amplification of sample of nucleic acid, wherein, the method for the nucleic acid extraction is as follows：
(1), sample is allowed to be contacted in PCR pipe with magnetic bead lysate, wherein the volume ratio of magnetic bead lysate and sample is 4:1, In, sample is 20 microlitres；
(2), 60-100 DEG C is heated to the PCR pipe of step (1), 10min is placed at room temperature for after 5min；
(3), the liquid in PCR pipe is removed, only retains magnetic bead, while any subsequent processing is not done to magnetic bead；
(4), the necessary reagent of nucleic acid is added into PCR pipe, such reagent includes such as SEQ.1.1-1.3；SEQ.2.1-2.3 and Primer sequence and probe sequence shown in SEQ.3.1-3.3；
(5), the amplification of at least one cycles of PCR is carried out.
9. according to the method described in claim 8, the sample for serum, urine, mouthwash, hydrothorax, ascites, cerebrospinal fluid or Person's aqueous humor equal samples, the sample are used directly for nucleic acid extraction；Alternatively, swab sample, such as Nasopharyngeal swabs, sputum class sample This, wherein swab or sputum sample is dissolved in 1-5mL physiological saline.
CN201810172845.7A 2017-03-03 2018-03-01 Kit for simultaneously detecting EBV, CMV and HSV-6 herpes viruses by one-step method and detection method Active CN108220484B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2017101221522 2017-03-03
CN201710122152.2A CN106636446A (en) 2017-03-03 2017-03-03 Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample

Publications (2)

Publication Number Publication Date
CN108220484A true CN108220484A (en) 2018-06-29
CN108220484B CN108220484B (en) 2020-10-16

Family

ID=58846931

Family Applications (6)

Application Number Title Priority Date Filing Date
CN201710122152.2A Pending CN106636446A (en) 2017-03-03 2017-03-03 Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample
CN201810172545.9A Pending CN108315325A (en) 2017-03-03 2018-03-01 A kind of method and reagent for extracting nucleic acid substances using magnetic bead
CN201810172845.7A Active CN108220484B (en) 2017-03-03 2018-03-01 Kit for simultaneously detecting EBV, CMV and HSV-6 herpes viruses by one-step method and detection method
CN201810172541.0A Active CN108441580B (en) 2017-03-03 2018-03-01 Kit for simultaneously detecting HSV-1, HSV-2, VZV herpes viruses by one-step method and detection method
CN201810172491.6A Pending CN108486259A (en) 2017-03-03 2018-03-01 One-step method detects the kit and detection method of pertussis nucleic acid
CN201810172492.0A Active CN108411036B (en) 2017-03-03 2018-03-01 Nucleic acid detection kit and method for rapidly detecting influenza A and influenza B viruses

Family Applications Before (2)

Application Number Title Priority Date Filing Date
CN201710122152.2A Pending CN106636446A (en) 2017-03-03 2017-03-03 Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample
CN201810172545.9A Pending CN108315325A (en) 2017-03-03 2018-03-01 A kind of method and reagent for extracting nucleic acid substances using magnetic bead

Family Applications After (3)

Application Number Title Priority Date Filing Date
CN201810172541.0A Active CN108441580B (en) 2017-03-03 2018-03-01 Kit for simultaneously detecting HSV-1, HSV-2, VZV herpes viruses by one-step method and detection method
CN201810172491.6A Pending CN108486259A (en) 2017-03-03 2018-03-01 One-step method detects the kit and detection method of pertussis nucleic acid
CN201810172492.0A Active CN108411036B (en) 2017-03-03 2018-03-01 Nucleic acid detection kit and method for rapidly detecting influenza A and influenza B viruses

Country Status (2)

Country Link
CN (6) CN106636446A (en)
WO (1) WO2018157844A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113373267A (en) * 2021-07-16 2021-09-10 浙江大学 Multiplex fluorescence quantitative RT-PCR kit for detecting blood-borne infectious viruses
CN114591945A (en) * 2022-04-02 2022-06-07 予果生物科技（北京）有限公司 DNA virus nucleic acid extraction detection reagent, kit, method and application thereof

Families Citing this family (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636446A (en) * 2017-03-03 2017-05-10 绍兴迅敏康生物科技有限公司 Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample
CN107619775B (en) * 2017-09-20 2021-03-26 宝瑞源生物技术(北京)有限公司 Portable nucleic acid detection platform suitable for PCR chromatography
CN108374059A (en) * 2018-03-01 2018-08-07 绍兴迅敏康生物科技有限公司 Respiratory Syncytial Virus(RSV) kit for detecting nucleic acid and its detection method
CN109055490A (en) * 2018-08-30 2018-12-21 郑州安图生物工程股份有限公司 A kind of reverse transcription PCR reaction solution and method for extracting nucleic acid with Nucleic Acid Elution function
CN108866049A (en) * 2018-09-05 2018-11-23 河南东格生物技术有限公司 A kind of kit and its method extracting genomic DNA from the sample of oral cavity
CN109337898A (en) * 2018-10-10 2019-02-15 南通中科基因医学检验所有限公司 A kind of Oral Mucosal Cells genome DNA rapid extraction method
CN109487013B (en) * 2019-01-09 2022-08-19 中生方政生物技术股份有限公司 Herpes simplex virus type I and type II detection marker, primer probe pair, kit and detection method
CN111560444A (en) * 2019-02-13 2020-08-21 湖南宏雅基因技术有限公司 Bordetella pertussis nucleic acid detection kit
CN110317902A (en) * 2019-06-20 2019-10-11 上海伯杰医疗科技有限公司 I/II/III/V type nucleic acid parting detecting reagent of herpes virus hominis and detection method
CN112280838A (en) * 2019-07-22 2021-01-29 苏州态和生物科技有限公司 Method for removing PCR (polymerase chain reaction) pollution by using magnetic bead-nuclease filtration chromatographic column
CN111334502A (en) * 2020-03-17 2020-06-26 广州奕昕生物科技有限公司 Method for rapidly extracting group B streptococcus nucleic acid
CN111518872A (en) * 2020-04-28 2020-08-11 上海勇求实业发展有限公司 Method for instant detection of nucleic acid
CN113736914A (en) * 2020-05-29 2021-12-03 绍兴迅敏康生物科技有限公司 Novel coronavirus one-step magnetic bead nucleic acid detection kit and use method thereof
CN111662901A (en) * 2020-06-09 2020-09-15 佛山市博朋生物科技有限公司 Method for extracting virus nucleic acid from animal low nucleic acid content sample
CN112176109A (en) * 2020-10-29 2021-01-05 上海伯杰医疗科技有限公司 Influenza A and B virus nucleic acid detection kit and use method thereof
CN112430327B (en) * 2020-11-25 2022-09-09 南开大学 Reticular magnetic molecular imprinting covalent organic framework material and preparation method and application thereof
CN112501162A (en) * 2020-12-28 2021-03-16 上海纳米技术及应用国家工程研究中心有限公司 Kit for extracting new coronavirus RNA by using nano magnetic beads and extraction method
CN113151397B (en) * 2021-02-03 2023-06-23 广东粤港澳大湾区国家纳米科技创新研究院 Nucleic acid extraction kit for extracting virus sample based on magnetic bead method
CN113122534A (en) * 2021-03-15 2021-07-16 济凡生物科技（北京）有限公司 Kit for extracting dry blood spot genome DNA by paramagnetic particle method and extraction method
CN112795623A (en) * 2021-03-22 2021-05-14 武汉纳磁生物科技有限公司 Virus nucleic acid extraction lysate, kit and method
CN113234589B (en) * 2021-05-10 2024-03-26 宁波康程德诺生物医药有限公司 Four-pipe device, kit and extraction method for rapid extraction of nucleic acid
CN113270142B (en) * 2021-05-19 2024-09-24 东南大学 Space transcriptome sequencing decoding method based on transient coding
CN113583803B (en) * 2021-07-26 2024-07-09 上海思路迪生物医学科技有限公司 Nucleic acid extraction and amplification POCT consumable and method applied to molecular diagnosis
CN113913494A (en) * 2021-09-14 2022-01-11 苏州锐讯生物科技有限公司 Kit for quickly and conveniently extracting virus DNA in body fluid and extraction method
CN116218835A (en) * 2021-12-02 2023-06-06 圣湘生物科技股份有限公司 Composition, kit, method and application for washing-free extraction of nucleic acid
CN114277026B (en) * 2021-12-21 2023-07-14 深圳市易瑞生物技术股份有限公司 Magnetic bead for nucleic acid extraction, preparation method thereof and kit for nucleic acid extraction by magnetic bead method
CN114250323B (en) * 2021-12-27 2023-09-29 武汉百泰基因工程有限公司 HSV1/HSV2/VZV virus triple fluorescence PCR detection kit and using method thereof
CN114371065A (en) * 2021-12-28 2022-04-19 上海固容生物科技有限公司 Method for processing liquid biopsy sample of biological sample (magnetic bead separation method) and application thereof
CN114438074A (en) * 2022-02-11 2022-05-06 欧蒙医学诊断（中国）有限公司 Method for increasing extraction amount of nucleic acid from liquid sample
CN114908080B (en) * 2022-03-17 2023-10-31 合肥中科易康达生物医学有限公司 Functionalized magnetic particle for nucleic acid extraction, preparation method and application thereof
CN115044712B (en) * 2022-04-22 2024-05-24 江苏先声医学诊断有限公司 Primer set suitable for ONT sequencing platform and used for detecting herpesviruses in infection samples and application thereof
CN115261183A (en) * 2022-05-31 2022-11-01 李治国 Instant gene detection kit
CN118240982A (en) * 2024-05-22 2024-06-25 深圳市明鉴检测专业技术有限公司 Herpesvirus detection kit and detection method

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040048304A1 (en) * 1998-03-19 2004-03-11 Human Genome Sciences, Inc. 95 human secreted proteins
CN105420403A (en) * 2016-01-14 2016-03-23 北京纳捷诊断试剂有限公司 Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube
CN106636446A (en) * 2017-03-03 2017-05-10 绍兴迅敏康生物科技有限公司 Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample
CN106676198A (en) * 2016-12-30 2017-05-17 上海星耀医学科技发展有限公司 High-sensitivity quantitative detection kit for herpes virus 4 and herpes virus 5
CN106755584A (en) * 2017-01-12 2017-05-31 广州华弘生物科技有限公司 A kind of ebb virus's detection kit and detection method

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101812444A (en) * 2010-04-23 2010-08-25 北京博迈世纪生物技术有限公司 Blood genome magnetic bead small-amount extraction reagent kit and extraction method thereof
CN102634596B (en) * 2012-05-07 2016-08-03 江苏和创生物科技有限公司 The specific detection of Bordetella pertussis and Bordetella parapertussis primed probe combination and test kit
CN104032034A (en) * 2014-03-07 2014-09-10 王全意 Method for simultaneously detecting influenza A virus, influenza B virus and influenza C virus and kit
CN104745689A (en) * 2015-01-30 2015-07-01 湖北永邦医疗科技有限公司 Primers, probe and kit used for detecting bordetella pertussis
EP3390671B1 (en) * 2016-02-11 2019-10-09 HTG Molecular Diagnostics, Inc. Method of direct target sequencing using nuclease protection
CN106497915A (en) * 2016-09-23 2017-03-15 广东国盛医学科技股份有限公司 A kind of paramagnetic particle method extracts the lysate of saliva nucleic acid
CN106676098B (en) * 2016-11-25 2018-02-27 广州奇辉生物科技有限公司 A kind of magnetic bead composition for improving nucleic acid extraction rate and its application
CN106591297A (en) * 2017-02-28 2017-04-26 解码（上海）生物医药科技有限公司 Magnetic bead nucleic acid extraction method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040048304A1 (en) * 1998-03-19 2004-03-11 Human Genome Sciences, Inc. 95 human secreted proteins
CN105420403A (en) * 2016-01-14 2016-03-23 北京纳捷诊断试剂有限公司 Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube
CN106676198A (en) * 2016-12-30 2017-05-17 上海星耀医学科技发展有限公司 High-sensitivity quantitative detection kit for herpes virus 4 and herpes virus 5
CN106755584A (en) * 2017-01-12 2017-05-31 广州华弘生物科技有限公司 A kind of ebb virus's detection kit and detection method
CN106636446A (en) * 2017-03-03 2017-05-10 绍兴迅敏康生物科技有限公司 Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
李明月 等: "巢式聚合酶链反应检测人疱疹病毒在人群中的感染情况", 《国际检验医学杂志》 *
杨洪艳 等: "《病理生理学实验指导》", 31 August 2003, 郑州大学出版社 *
谢欣 等: "基于多功能纳米磁珠的DNA制备与基因分型", 《科学通报》 *
郑之北 等: "多重PCR-基因芯片技术检测多种人疱疹病毒", 《浙江省科学技术协会会议论文集》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113373267A (en) * 2021-07-16 2021-09-10 浙江大学 Multiplex fluorescence quantitative RT-PCR kit for detecting blood-borne infectious viruses
CN113373267B (en) * 2021-07-16 2022-04-29 浙江大学 Multiplex fluorescence quantitative RT-PCR kit for detecting blood-borne infectious viruses
CN114591945A (en) * 2022-04-02 2022-06-07 予果生物科技（北京）有限公司 DNA virus nucleic acid extraction detection reagent, kit, method and application thereof

Also Published As

Publication number Publication date
CN108486259A (en) 2018-09-04
CN108220484B (en) 2020-10-16
WO2018157844A1 (en) 2018-09-07
CN108441580B (en) 2021-09-28
CN108411036B (en) 2021-09-28
CN108411036A (en) 2018-08-17
CN108441580A (en) 2018-08-24
CN108315325A (en) 2018-07-24
CN106636446A (en) 2017-05-10

Similar Documents

Publication Publication Date Title
CN108220484A (en) One-step method detects EBV, CMV, the kit and detection method of HSV-6 herpesvirals simultaneously
Peng et al. Human papillomavirus types 16 and 33, herpes simplex virus type 2 and other risk factors for cervical cancer in Sichuan Province, China
Linde et al. Specific diagnostic methods for herpesvirus infections of the central nervous system: a consensus review by the European Union Concerted Action on Virus Meningitis and Encephalitis
CN109487013B (en) Herpes simplex virus type I and type II detection marker, primer probe pair, kit and detection method
Nishi et al. Polymerase chain reaction for the detection of the varicella-zoster genome in ocular samples from patients with acute retinal necrosis
WO2020224395A1 (en) Probe, primer pair, fluorescent quantitative pcr kit and method for detecting replicative lentivirus
Sandmeyer et al. Comparison of polymerase chain reaction tests for diagnosis of feline herpesvirus, Chlamydophila felis, and Mycoplasma spp. infection in cats with ocular disease in Canada
CN103966358A (en) Fluorescent quantitative PCR detection kit for infectious spleen and kidney necrosis virus and Fluorescent quantitative PCR detection method of infectious spleen and kidney necrosis virus
CN105755169A (en) Reagent kit for detecting and typing high-risk type human papilloma viruses and application of reagent kit
CN104195266A (en) Quadruple fluorogenic quantitative PCR kit for detecting three pathogens of infantile pneumonia
CN101760560A (en) Fluorescent PCR detection method for human cytomegalovirus (HCMV)
CN109207641A (en) A kind of multiple RT-PCR detection kit and application
CN108034767A (en) Differentiate serum I type Marek's disease virus gene deletion of vaccine, primer, probe and the kit of Attenuation vaccine and wild poison
CN107475389B (en) Primer group, kit and method for detecting mycoplasma pneumoniae
CN105132584B (en) For the kit and its production method of parting detection varicellazoster virus and application
CN106048081A (en) HPV (human papilloma virus) typing detection primers as well as detection method and application thereof
CN105779644A (en) Realtime fluorescent nucleic acid constant temperature amplification detection kit of human cytomegalovirus (HCMV)
CN105349661A (en) Chlamydia trachomatis and gonococcus nucleic acid detection kit
CN103352088A (en) Primer pair, probe, reagent kit and method for detecting avian influenza virus H7 subtype
Liu et al. Detection of congenital cytomegalovirus in newborns using nucleic acid amplification techniques and its public health implications
McCaughtry et al. Inapparent genital herpes simplex virus infection in college women
CN103866049B (en) A kind of LAMP primer group for the I type herpes simplex virus that increases and test kit and application
CN102618665B (en) Kit for fluorescence PCR detection of herpes simplex virus I
CN106755590A (en) FISH detection probe for detecting EB virus of leaflet patient
CN102146468B (en) Special primer for assisted identification of Streptococcus suis type 2 and Streptococcus suis type 7 and application thereof

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
GR01 Patent grant
GR01 Patent grant