CN106676198A - High-sensitivity quantitative detection kit for herpes virus 4 and herpes virus 5 - Google Patents

High-sensitivity quantitative detection kit for herpes virus 4 and herpes virus 5 Download PDF

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Publication number
CN106676198A
CN106676198A CN201611261968.5A CN201611261968A CN106676198A CN 106676198 A CN106676198 A CN 106676198A CN 201611261968 A CN201611261968 A CN 201611261968A CN 106676198 A CN106676198 A CN 106676198A
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seq
dna
fluorescent probe
herpesviruss
internal reference
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CN201611261968.5A
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Inventor
吴大治
夏懿
吴梅
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Shanghai Xingyao Medical Technology Development Co Ltd
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Shanghai Xingyao Medical Technology Development Co Ltd
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Abstract

The invention relates to a kit for high-sensitivity quantitative detection of nucleic acids of a herpes virus 4 and a herpes virus 5 (HHV4 and HHV5) by using a fluorescent PCR technology. Specific nucleic acid sequences of the HHV4 and the HHV5 are amplified by using two pairs of primers separately, and an HHV4 DNA and an HHV5 DNA are quantitatively detected at different wavelengths through corresponding fluorescent probes; and meanwhile, an internal reference DNA is detected by using an endogenous gene specific primer and the fluorescent probes. Existence of the HHV4 DNA, the HHV5 DNA and the internal reference DNA is detected at the same time through a single-tube three-wavelength fluorescent PCR technology; the HHV4 DNA and the HHV5 DNA in a whole-blood sample and a plasma sample can be quantitatively detected; whether template loss caused by a PCR inhibitor or detection misoperation exists in each sample or not is judged through the detection result of an internal reference nucleic acid; and false negative leaking detection is reduced. The kit is simple and fast in operation, the quantitative result is sensitive and accurate, and the kit can be widely applied to quantitative detection of clinical infection of the herpes virus 4 and the herpes virus 5.

Description

Herpesviruss 4, the highly sensitive immue quantitative detection reagent box of 5 types
Technical field
The present invention relates to a kind of herpesviruss 4, the highly sensitive fluorescent quantificationally PCR detecting kit of 5 types, belong to biotechnology neck Domain.
Background technology
Herpesviruss（Herpesviruses）It is a group median size, tunicary double-stranded DNA viruses, it is known to more than 120 Kind, according to its physicochemical property point α, β, γ, four subfamilies of unfiled herpesviruss.They can infect the mankind and other vertebras are moved Thing, the main tissue for invading ectodermal origin, including skin, mucosa and nervous tissue, its infection site and the disease for causing are more Plant various, and have the trend of latent infection.Now know infection the mankind common herpesviruss have 1 type of nerpes vinrus hominises, 2 types, 3 Type, 4 types, 5 types, 6 types, 7 types, 8 types（HHV 1~8）This eight kinds, they cause the various diseases of the mankind, can also hide for a long time In tissue, when body's immunity is low, particularly to leukemia, immunosuppressive patient, the herpesviruss hidden are lived Change forms recurrent infection, causes transplant organ necrosis and endangers patients ' lives, threaten human health when serious.
Substantial amounts of clinical sample research discovery, ebb virus（HHV4, also known as Epstein-Barr virus, belongs to gamma herpes viruses Subfamily）With 5 types（HHV5, also known as human cytomegalic inclusion disease virus, belongs to Betaherperesvirinae）Most common, the target cell of HHV4 infection is Lymphoid cell, can cause lymphocytic hyperplasia；HHV5 is maximum animal viruss, and its growth cycle is long, and infection cell forms big and small Born of the same parents.Traditional herpesviruss detection typically adopts goldstandard virus purification culture, serology and nucleic acid detection method, and respectively has Pluses and minuses.As HHV4 is difficult to separation and Culture, HHV5 growth cycles are long（30 days）, virus purification culture method is not suitable for clinical experiment Room uses.Serologic detection detects viral associated proteins using antigen antibody reaction, quick, simple, easily operated with detecting Advantage, but detection sensitivity is not high, and as after infection, antibody produces " window phase ", detection also has certain hysteresis quality.Core In sour detection technique, particularly fluorescence PCR method have specificity and sensitivity it is high, can quantitative, easy to operate, cost it is more low Advantage, has been disclosed for many technical schemes in herpesviruss detection（CN201110373390、CN201410026783、 CN201410027036、CN201410028810、CN201610682298、CN201610841711、CN201610848377）. It will be appreciated, however, that above-mentioned technical proposal much simply carries out qualitative detection of nucleic acids to herpesviruss, it is impossible to report that nucleic acid is fixed Amount result, particularly scheme in fluorescent PCR detection all use internal reference, when clinical sample extract nucleic acid in contain PCR During mortifier, or nucleic acid is lost in experimental implementation error, will cause testing result " false negative ", and cause missing inspection and mistaken diagnosis, and be delayed Patient treats and gets well.
The present invention is based on above-mentioned technical background, it is therefore an objective to which the common herpesviruss 4 of design screening, 5 types and internal reference are special Property primed probe, exploitation single-tube quantitative typing detection herpesviruss 4, the fluorescent PCR kit of 5 type nucleic acid meet clinic to bleb Exanthema virus 4, the infection of 5 types be quick, accurately, the demand of detection by quantitative, for clinical herpesvirus infection immunotherapy targeted autoantibody provide according to According to.
The content of the invention
The present invention provides a kind of highly sensitive quantitative typing detection herpesviruss 4, the fluorescent PCR kit of 5 type nucleic acid, and which is special Levy and be, the test kit simultaneously adopt a pair of herpesvirus 4 primers and fluorescent probe, a pair of 5 type primers of herpesviruss and Fluorescent probe, a pair of internal reference specific primers and fluorescent probe, herpesvirus 4 upstream and downstream primer have SEQ ID NO：1 With SEQ ID NO：2 sequence, fluorescent probe have SEQ ID NO：3 sequences and its 5 ' end flag F AM fluorophor, blister sore Malicious 5 type upstream and downstream primers have SEQ ID NO：4 and SEQ ID NO：5 sequence, fluorescent probe have SEQ ID NO：6 sequences And its 5 ' end labelling ROX fluorophor, internal reference upstream and downstream primer has SEQ ID NO：7 and SEQ ID NO：It is 8 sequence, glimmering Light probe has SEQ ID NO：9 sequences and its 5 ' end labelling Cy5 fluorophor.Present invention introduces internal reference can be used for prison There is bleb from nucleic acid extraction to the maloperation of augmentation detection overall process in possible response inhabitation thing and monitoring in control PCR amplifications The double-negative sample of exanthema virus, internal reference can pass through the template for diluting extraction or improve sample processing method reinspection to obtain correct knot Really, so as to avoid detect missing inspection；Test kit determines that sample, with the presence or absence of herpesviruss 4,5 type nucleic acid accurate carrying capacity, is adapted to face Bed is promoted the use of.
Technical scheme
By to herpesviruss in GenBank 4,5 types and human endogenous house keeper（House-keeping）Gene order is inquired about, and is used The softwares such as Vector NTI, Oligo are compared to the gene order in various sources design, design according to TaqMan fluorescent PCRs Principle, preferred a pair of herpesvirus 4 specific primers and fluorescent probe amplification gene group camber is conservative, multicopy weight Multiple genes（BamHI-W）On 115bp fragments, in a pair of 5 type-special primers of herpesviruss and fluorescent probe amplification gene group Highly conserved can early gene（IE2）The 91bp fragments in middle UL122 regions, a pair of people GAPDH（Glyceraldehyde- 3-phosphate dehydrogenase）Gene-specific primer and fluorescent probe expand its conservative region 144bp fragments, 9 Primer and probe nucleotide sequence（5’ -> 3’）It is as follows：
HHV4 forward primer：CCCATAgACTCCCATgTAAgC, sequence are designated as SEQ ID NO：1.
HHV4 downstream primers：CCCTggACATCTggACAAAg, sequence are designated as SEQ ID NO：2.
HHV4 fluorescent probes：CgAgTAggTgCCTCCAgAgCC, sequence are designated as SEQ ID NO：3, its middle probe 5 ' is held Flag F AM（6-carboxy-fluorescein）Fluorophor, 3 ' end labelling BHQ（Black hole quencher）Base is quenched Group.
HHV5 forward primer：ACCCTgTTCTTCCTCgCTAT, sequence are designated as SEQ ID NO：4.
HHV5 downstream primers：GCCCgAgCCCgACTTTA, sequence are designated as SEQ ID NO：5.
HHV5 fluorescent probes：GATACAgCCggCggTATCgA, sequence are designated as SEQ ID NO：6, its end of middle probe 5 ' mark Note ROX（5-Carboxy-X-rhodamine）Fluorophor, 3 ' end labelling BHQ quenching groups.
Internal reference forward primer：TgCTTTTAACTCTggTAAAgTgg, sequence are designated as SEQ ID NO：7.
Internal reference downstream primer：ATgACAAgCTTCCCgTTCTC, sequence are designated as SEQ ID NO：8.
Internal reference fluorescent probe：TTgTTgCCATCAATgACCCCTTCA, sequence are designated as SEQ ID NO：9, its middle probe 5 ' end labelling Cy5（Cyanine 5）Fluorophor, 3 ' end labelling BHQ quenching groups.
Present invention design herpesviruss 4,5 types and internal control primer probe have Tm values closely, and between sequence Without obvious primer dimer or hairpin structure, it is ensured that three target sequence amplifications have close amplification efficiency, are conducive to Improve test kit detection sensitivity；Above-mentioned primer probe sequence can also be the sequence with above-mentioned sequence homology more than more than 85% Row.
The amplification reaction system is respectively as follows into being grouped into：10mM Tris-HCl（pH8.3）, 50mM KCl, 0.2~ 0.5mM dNTPs, 1.5~5 mM MgCl2, each 0.1~0.5 μM of HHV4, HHV5 and internal reference primer and fluorescent probe（SEQ ID NO：1~9）, 1~5 U Taq archaeal dna polymerases and 0.01~1 U UNG enzymes, the 10 μ l of template of extraction, total reaction volume 30 μl.More specifically, each composition of amplification reaction solution is final concentration of：Tris-HCl（pH8.3）10 mM、KCl 50 mM、dATP、 DGTP, dCTP, dUTP each 0.2 mM, MgCl23.5 mM, HHV4 and HHV5 primers and fluorescent probe（SEQ ID NO：1~6） Each 0.3 μM, internal reference primer and fluorescent probe（SEQ ID NO：7~9）Each 0.15 μM, 2 U, UNG enzyme of Taq archaeal dna polymerases 0.5 U。
The herpesviruss 4,5 type PCR kit for fluorescence quantitative are as follows using amplification program：50℃ 2min、94℃ According to 94 DEG C of 10sec, 60 DEG C of 45sec cyclic amplifications 45 times after 5min, FAM, ROX and Cy5 in circulation step, when 60 DEG C, are gathered Wavelength fluorescent signal.
The herpesviruss 4,5 type PCR kit for fluorescence quantitative include 1 negative control, 1 positive control and 4 The viral nucleic acid quantitative calibration product of linear gradient concentration, 4 calibration object HHV4, HHV5 nucleic acid concentrations are respectively 5.0x107 copies/ml、5.0x106 copies/ml、5.0x105 copies/ml、5.0x104Copies/ml, calibration object be containing The SEQ ID NO of HHV4：1~2, the SEQ ID NO of HHV5：TA clone's matter of 4~5 primer pair amplifies target gene tandem sequences Grain.Positive control is the escherichia coli containing the cloned plasmids.
The herpesviruss 4,5 type PCR kit for fluorescence quantitative, are adopted and are carried based on the viral DNA of paramagnetic particle method nucleic acid gradient Reagent is taken, realizes that sample nucleic acid automatization extracts on instrument for extracting nucleic acid.
By preferred a pair of the herpesviruss 4 for obtaining of above-mentioned design, 5 type primers and fluorescent probe, a pair of internal reference primers And fluorescent probe, the invention provides the test kit of real-time fluorescence quantitative PCR detection, optimizes PCR reaction systems and amplification journey Sequence.Certainly this research field technical staff can adjust PCR reaction systems and program according to the general requirement of fluorescent PCR, also together Sample can realize the purpose for detecting.
Beneficial effect
The present invention is vesiculated by optimizing reaction system and amplification condition development according to the primed probe and technical scheme of above-mentioned design Exanthema virus 4,5 type nucleic acid fluorescent PCR immue quantitative detection reagent boxes, its main feature are as follows：
（1）The herpesviruss 4 of design, 5 type primed probe Tm values are close to and without secondary structure is intersected, primer expands target gene length It is close to, it is ensured that two-strain has close amplification efficiency, test kit inspection can not only be also improved to the accurate typing of herpesviruss Survey herpesviruss 4, the sensitivity of 5 types.
（2）The internal reference being introduced into can be monitored in reaction system with the presence or absence of the false negative caused because of PCR mortifiers etc., can be with Avoid the inaccurate mistaken diagnosis for causing of testing result.
（3）Test kit has used noncompetitive internal reference, and herpesviruss 4,5 types and internal reference gene order are special, fluorescence is visited Pin will not be interfered between fluorescence signal using different fluorophor labellings, it is ensured that detection specificity.
（4）DUTP-UNG enzyme systems in amplification system further avoid result caused by gene-amplification product pollution False positive, and play " double Quality Control effects " after internal reference is used in combination, improve the accuracy of test kit testing result.
（5）Test kit extracts sample nucleic acid, single tube using paramagnetic particle method automatization and detects herpesviruss 4,5 types and internal reference simultaneously According to, it is to avoid the repeated work that two-strain need to be detected at twice during same sample, and it is easy to operate quick, can at 2 hours Interior report testing result, improves work efficiency.
The These characteristics of test kit, are herpesviruss 4 using specific designs, 5 types and internal reference primed probe simultaneously Directly cause with Fluorescence PCR assay combination application.From the principle, test kit of the present invention is in herpesviruss 4,5 type pathogen By the analysis to three channel fluorescence signals of real-time fluorescent PCR amplification in clinical sample detection, herpesviruss 4,5 type cores are judged The presence of sour specific fragment and virus load, technical scheme are reasonable in design, can be widely applied to clinical herpess Virus-4, the detection by quantitative of 5 types infection.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limit the scope of the present invention.In addition, it is to be understood that after the content for having read instruction of the present invention, people in the art Member can make various technical changes or modification by technology general knowledge to the present invention, and these equivalent form of values equally fall within the application institute Attached claims limited range.
Embodiment 1：The design of herpesviruss 4,5 type nucleic acid fluorescent PCR immue quantitative detection reagent box primed probes
According to HHV4, HHV5, GAPDH gene order inquired about in NCBI GenBank data bases, using Vector NTI, The primer-design softwares such as Oligo, as shown in table 1, the amplification of HHV4 primers is the virus to the preferred primer probe sequence for obtaining What on BamHI-W genes, 115bp fragments, HHV5 primers were expanded is 91bp fragments on the viral IE2 genes, the amplification of internal reference primer Be 144bp fragments on people's GAPDH genes.
The test kit primed probe of the design of table 1
The synthesis of above primed probe commission Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Embodiment 2：Herpesviruss 4,5 type nucleic acid fluorescent PCR immue quantitative detection reagent boxes are prepared
Test kit reaction buffer prepares 32 person-portion test kits reaction buffering by 2 each component concentration of table and volume voluntarily to prepare Liquid, the reaction buffer of preparation each reaction subpackage consumption is 20 μ l, and after adding 10 μ l of template, total reaction volume is 30 μ l.
2 test kit reaction buffer of table prepares each group partial volume
Test kit negative control adopts normal person's negative plasma；Quantitative calibration product are the clone containing HHV4, HHV5 amplified fragments Plasmid, is diluted to 4 concentration as kit calibration object with TE buffer：5.0x107 copies/ml、5.0x106 copies/ml、5.0x105 copies/ml、5.0x104copies/ml；Positive control is the large intestine containing above-mentioned cloned plasmids Bacillus, employment negative plasma are diluted to concentration for 5.0x103 copies/ml。
The paramagnetic particle method virus that test kit viral DNA extracts reagent is developed using Shanghai Xingyao Medical Technology Development Co., Ltd. DNA extraction kit, it includes magnetic bead, lysate, E.C. 3.4.21.64, cleaning buffer solution, 5 components of elution buffer, tries during use Agent box negative control, positive control, specimen extract nucleic acid using the test kit.
Test kit amplification program is：Expand according to 94 DEG C of 10sec, 60 DEG C of 45sec circulations after 50 DEG C of 2min, 94 DEG C of 5min Increase 45 times, in circulation step, when 60 DEG C, gather FAM, ROX and Cy5 channel fluorescence signal.
Included by the herpesviruss 4 of above-mentioned preparation, 5 type nucleic acid fluorescent PCR immue quantitative detection reagent box components：Reaction buffering Liquid, negative control, positive control and 4 quantitative calibration product, it is desirable to -20 DEG C of storage and transport.
Embodiment 3：The measure of test kit detection sensitivity
（1）Herpes virus DNA is extracted
The positive blood plasma of herpesviruss 4 known to clinical identification, concentration, 5 types of learning from else's experience is dense by two-strain with normal person's negative plasma Degree is adjusted to the mixing of identical and equal-volume, and in mixing sample, herpesviruss 4,5 type concentration are 5x104copies/ml）.Then Using normal person's negative plasma by 10 times of continuous gradient dilutions of the mixing sample to 5x103 copies/ml、5x102 copies/ ml、5x101Copies/ml, draws above-mentioned viral dilution liquid, test kit negative control, each 400 μ l of positive control, is used together Paramagnetic particle method viral DNA extracts reagent in embodiment 2 carries out viral DNA extraction on instrument for extracting nucleic acid, and last each sample is obtained The each 50 μ l of nucleic acid for obtaining.
（2）Fluorescent PCR is detected
Test kit reaction buffer in embodiment 2 is taken out into freeze thawing, mixing, of short duration centrifugation from -20 DEG C of refrigerators, by 20 μ l/ person-portions Reaction buffer is dispensed in PCR reaction tubes, be then respectively adding the test kit negative control of said extracted, positive control, Template and each 10 μ l of quantitative calibration product without the need for nucleic acid extraction that viral dilution liquid is extracted.Each reaction pipe volume is 30 μ L, above-mentioned reaction tube is put on ABI7500 fluorescent PCR instrument, sets each reaction tube sample type and to arrange quantitative calibration product dense Angle value, reacts 2 minutes according to 50 DEG C, and then 94 DEG C are incubated 5 minutes, then circulate 45 times within 45 seconds by 94 DEG C 10 seconds → 60 DEG C（ The signal of 60 DEG C of collection FAM, ROX, Cy5 fluorescence channels）Response procedures operation.Expand after terminating according to instrument software and reagent Box description analyzes judgment experiment result.
（3）Interpretation of result
Test kit is the positive to all samples detection internal reference；Negative control FAM and ROX Air conduct measurement result is herpesviruss 4 types, 5 types are negative, and positive control testing result is herpesvirus 4, the 5 types positive, is set by concentration value respectively in FAM, ROX passage Determine herpesvirus 4,5 type quantitative calibration product values, calibration object detection linearly dependent coefficient is all higher than 0.98, Pass Test Quality Control Require.It was observed that 5x103 copies/ml、5x102 copies/ml、5x101The biased sample gradient of copies/ml concentration point Detection is not repeated 5 times, herpesvirus 4,5 type results are the positive, thus may determine that test kit is to herpesvirus 4,5 types Detection sensitivity can reach 5x101Copies/ml, test kit can correct typing and highly sensitive quantitative inspections to two-strain Survey.
SEQUENCE LISTING
<110>Shanghai Xingyao Medical Technology Development Co., Ltd.
<120>Herpesviruss 4, the highly sensitive immue quantitative detection reagent box of 5 types
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artificial
<223>HHV4 forward primer
<400> 1
cccatagact cccatgtaag c 21
<210> 2
<211> 20
<212> DNA
<213> Artificial
<223>HHV4 downstream primers
<400> 2
ccctggacat ctggacaaag 20
<210> 3
<211> 23
<212> DNA
<213> Artificial
<223>HHV4 fluorescent probes
<221> 3
<222> (1)..(23)
<223> b=FAM; d=BHQ
<400> 3
bcgagtaggt gcctccagag ccd 23
<210> 4
<211> 20
<212> DNA
<213> Artificial
<223>HHV5 forward primer
<400> 4
accctgttct tcctcgctat 20
<210> 5
<211> 17
<212> DNA
<213> Artificial
<223>HHV5 downstream primers
<400> 5
gcccgagccc gacttta 17
<210> 6
<211> 22
<212> DNA
<213> Artificial
<223>HHV5 fluorescent probes
<221> 6
<222> (1)..(22)
<223> b=ROX; d=BHQ
<400> 6
bgatacagcc ggcggtatcg ad 22
<210> 7
<211> 23
<212> DNA
<213> Artificial
<223>Internal reference forward primer
<400> 7
tgcttttaac tctggtaaag tgg 23
<210> 8
<211> 20
<212> DNA
<213> Artificial
<223>Internal reference downstream primer
<400> 8
atgacaagct tcccgttctc 20
<210> 9
<211> 26
<212> DNA
<213> Artificial
<223>Internal reference fluorescent probe
<221> 9
<222> (1)..(26)
<223> b=Cy5; d=BHQ
<400> 9
bttgttgcca tcaatgaccc cttcad 26

Claims (5)

1. a kind of single-tube quantitative typing detects herpesviruss 4, the fluorescent PCR kit of 5 type nucleic acid, it is characterised in that the examination Agent box simultaneously adopt a pair of herpesvirus 4 primers and fluorescent probe, a pair of 5 type primers of herpesviruss and fluorescent probe, a pair Internal reference specific primer and fluorescent probe, herpesvirus 4 upstream and downstream primer have SEQ ID NO：1 and SEQ ID NO：2 Sequence, fluorescent probe there is SEQ ID NO：3 sequences and its 5 ' end flag F AM fluorophor, 5 type upstream and downstream of herpesviruss draw Thing has SEQ ID NO：4 and SEQ ID NO：5 sequence, fluorescent probe have SEQ ID NO：6 sequences and its 5 ' end labelling ROX fluorophors, internal reference upstream and downstream primer have SEQ ID NO：7 and SEQ ID NO：8 sequence, fluorescent probe have SEQ ID NO：9 sequences and its 5 ' end labelling Cy5 fluorophor.
2. test kit as claimed in claim 1, the amplification reaction system are respectively as follows into being grouped into：10mM pH8.3's Tris-HCl, 50mM KCl, 0.2~0.5mM dNTPs, 1.5~5 mM MgCl2, each 0.1~0.5 μM of SEQ ID NO：1~ 9 primers and fluorescent probe, 1~5 U Taq archaeal dna polymerases and 0.01~1 U UNG enzymes, the 10 μ l of template of extraction, overall reaction body 30 μ l of product.
3. test kit as claimed in claim 1, each composition of the amplification reaction solution are final concentration of：The Tris-HCl 10 of pH8.3 50 mM of mM, KCl, dATP, dGTP, dCTP, dUTP each 0.2 mM, MgCl23.5 mM, herpesviruss SEQ ID NO：1~6 Primer and fluorescent probe are each 0.3 μM, internal reference SEQ ID NO：7~9 primers and fluorescent probe are each 0.15 μM, and Taq DNA gather 2 U of synthase, 0.5 U of UNG enzymes.
4. test kit as claimed in claim 1, the amplification program for using are as follows：According to 94 after 50 DEG C of 2min, 94 DEG C of 5min DEG C 10sec, 60 DEG C of 45sec cyclic amplifications 45 times, gather FAM, ROX and Cy5 wavelength fluorescent signal in 60 DEG C of each circulation.
5. test kit as claimed in claim 1, including negative control, positive control and viral nucleic acid quantitative calibration product.
CN201611261968.5A 2016-12-30 2016-12-30 High-sensitivity quantitative detection kit for herpes virus 4 and herpes virus 5 Withdrawn CN106676198A (en)

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CN110923362A (en) * 2019-12-19 2020-03-27 武汉中帜生物科技股份有限公司 Colloidal gold chromatography kit for simultaneously detecting herpes simplex virus I/II and application thereof

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CN102115794A (en) * 2009-12-30 2011-07-06 上海复星医学科技发展有限公司 Internal-reference-containing HCMV fluorescence quantitative PCR detection kit
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