CN106916907A - The fluorescence PCR method and kit of a kind of specific detection herpes simplex virus I, II type nucleic acid - Google Patents
The fluorescence PCR method and kit of a kind of specific detection herpes simplex virus I, II type nucleic acid Download PDFInfo
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- CN106916907A CN106916907A CN201710308453.4A CN201710308453A CN106916907A CN 106916907 A CN106916907 A CN 106916907A CN 201710308453 A CN201710308453 A CN 201710308453A CN 106916907 A CN106916907 A CN 106916907A
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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
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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
- C12Q1/68—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 present invention relates to a kind of specific detection herpes simplex virus I, the fluorescence PCR method and kit of II type nucleic acid, the specific primer pair and fluorescence probe that the kit is included are as follows：Herpes simplex virus I-form：Forward primer：5’‑CCCCGCTGGAACTACTATGACA‑3’；Reverse primer：5’‑TCCGTCCAGTCGTTTATCTTCA‑3’；Fluorescence probe：5’‑ACGCCCCCGCGTTTGAGACC‑3’；Herpes simplex virus type II：Forward primer：5’‑AAGCTCCCGCTAAGGACATG‑3’；Reverse primer：5’‑GGTGCTGATGATAAAGAGGATATCTAGA‑3’；Fluorescence probe：5’‑AACACATCCCCCTGTTCTGGTTCCTAACG‑3’.Kit of the present invention has high sensitivity, high specific, efficient feature, it is possible to achieve to herpes simplex virus I, the qualitative detection of II type, can turn into effective auxiliary detection instrument.
Description
Technical field
The invention belongs to microorganism beyond body nucleic acid detection field, and in particular to a kind of specific detection herpes simplex virus I,
The fluorescence PCR method and kit of II type nucleic acid, can carry out qualitative detection to the DNA of herpes simplex virus I-form and II type, can
As effective detection instrument.
Background technology
Herpes simplex virus (Herpes Simplex Viruses, HSV) belongs to herpetoviridae, and virion diameter is about
180 nanometers, the mankind are the unique natural reservoir (of bird flu viruses) of herpes simplex virus.Meanwhile, herpes simplex virus mainly divides amphitypy, i.e., simple blister
The type of exanthema virus I (HSV1) and herpes simplex virus type II (HSV2).HSV1 mainly causes oral cavity beyond genital tract, skin, viscous
Film or other organ infections, and HSV2 mainly causes RTI.
First infection of the pregnant woman in phase morning, noon and afternoon of pregnant journey has a strong impact on to fetus, can cause to miscarry, hypoevolutism,
Congenital abnormality, dysnoesia, retinochoroiditis etc..Most of infections of newborn are because vaginal delivery is contaminted, although
Appearance can be normal during birth, but engenders symptom in 3 weeks postpartum：One is blister occur at skin, oral cavity, eye combination film etc.
Rash；Two is central nervous system exception, increased intracranial pressure, twitch etc.；Three is general diffusion-type, shows as jaundice, hepatitis, lung
Inflammation etc..Herpes simplex viral infection in newborns without timely treatment, loses after having very maximum probability to cause death or having nervous system
Disease.
At present, clinical labororatory's detection uses serological test, such as ELISA, RIA and IFA, and detection specificity is anti-
Body.Serum herpes simplex virus-IgM the positives prove recent infection, and oneself continues 2~4 months.Serum herpes simplex virus-IgG sun
Property then points out previous infection.Because herpes simplex virus antigens source is difficult, the still difficult popularization of serological test, and some crowds
Antibody response is very weak after infection virus or does not produce antibody, therefore only can not accurately judge herpe simplex disease with antibody test result
Malicious infection conditions, need to be aided with antigen or detection of nucleic acids result as diagnosis and the foundation for the treatment of.The nucleic acid inspection of current comparative maturity
Survey method is to use fluorescence PCR method, the method overcome Standard PCR method poor specificity, easily pollution, interpretation of result operating procedure many
The shortcomings of, using TaqMan methods or double mark sonde methods (fluorescent quenching probe or FQ probes), i.e., at its 5 ' end and 3 ' ends
One fluorescent reporter group of mark and a quenching group respectively.The specific probe of target sequence and specific PCR primer are same
When use, the probe of design is annealed in the range of upstream and downstream PCR primer, and purpose base is incorporated into jointly by three oligonucleotides
Because of fragment, greatly enhance its specificity.In the extension stage of PCR, the exonuclease activity of Taq archaeal dna polymerases 5 ' -3 '
Fluorescent reporter group is cut down from probe.With the increase of PCR cycle number, the quantity of free reporter group constantly increases
Plus, the fluorescence signal that real-time detection discharges from free fluorophor, so as to carry out qualitative analysis to target sequence.The PCR reacts
Carry out in closed system, analysis result without Kaifeng, therefore, it is to avoid the generation of PCR primer pollution.
At present, also have fluorescence PCR method for detecting the report of herpes simplex virus, but develop it is more effectively, it is quick,
Accurately, sensitivity is high, primer and probe of high specificity fluorescence PCR method, and this primer and probe are used to detect try
Agent box is also the target that this area is pursued.
The content of the invention
The technical problems to be solved by the invention are to overcome the deficiencies in the prior art, there is provided a kind of simple blister of specific detection
The fluorescent PCR kit of the type of exanthema virus I and II type nucleic acid, the kit has sensitive, special and efficient feature, so that real
The nucleic acid of herpes simplex virus in sample is now detected, qualitatively purpose is realized.
To solve above technical problem, the present invention is adopted the following technical scheme that：
The fluorescent PCR kit of a kind of specific detection herpes simplex virus I, II type nucleic acid, including herpes simplex virus I
Type-special primer pair and fluorescence probe, herpes simplex virus type II specific primer pair and fluorescence probe,
Described herpes simplex virus I-form specific primer pair and fluorescence probe are as follows：
Forward primer：5’-CCCCGCTGGAACTACTATGACA-3’
Reverse primer：5’-TCCGTCCAGTCGTTTATCTTCA-3’
Fluorescence probe：5’-ACGCCCCCGCGTTTGAGACC-3’；
Described herpes simplex virus type II specific primer pair and fluorescence probe are as follows：
Forward primer：5’-AAGCTCCCGCTAAGGACATG-3’
Reverse primer：5’-GGTGCTGATGATAAAGAGGATATCTAGA-3’
Fluorescence probe：5’-AACACATCCCCCTGTTCTGGTTCCTAACG-3’.
Described herpes simplex virus I-form fluorescence probe, the two ends of herpes simplex virus type II fluorescence probe mark respectively
Reporter fluorescence element or fluorescent dye, quenching group.The reporter fluorescence element or fluorescent dye of mark be FAM or other any can make
Be probe mark fluorescein or fluorescent dye, such as HEX, TET, JOE, Yakima Yellow, Cy3, Cy3.5, Cy5, NED,
VIC、PET、ROX、LIZ、RED、Texas Red；Quenching group be TAMRA or other it is any can be as the chemistry of quenching group
Material, such as Dabcyl, BHQ1, BHQ2.
Further, described kit also includes PCR reaction solutions, herpes simplex virus I-form positive quality control product, simple blister
The type positive quality control product of exanthema virus II and negative quality-control product.
Preferably, described herpes simplex virus I-form positive quality control product contains artificial synthesized sequence, the artificial synthesized sequence
For it is artificial synthesized containing the herpes simplex virus I-form specific primer to the sequence of amplified production fragment.The artificial synthesized sequence
Content is：
CGCGGCAAATATGCCTTGGTGGATGCCTCTCTCAAGATGGCCGACCCCAATCGCTTTCGCGGCAAAGACCTTCCGGT
CCTGGACCAGCTGACCGACCCTCCGGGGGTCCGGCGCGTGTACCACATCCAGGCGGGCCTACCGGACCCGTTCCAGC
CCCCCAGCCTCCCGATCACGGTTTACTACGCCGTGTTGGAGCGCGCCTGCCGCAGCGTGCTCCTAAACGCACCGTCG
GAGGCCCCCCAGATTGTCCGCGGGGCCTCCGAAGACGTCCGGAAACAACCCTACAACCTGACCATCGCTTGGTTTCG
GATGGGAGGCAACTGTGCTATCCCCATCACGGTCATGGAGTACACCGAATGCTCCTACAACAAGTCTCTGGGGGCCT
GTCCCATCCGAACGCAGCCCCGCTGGAACTACTATGACAGCTTCAGCGCCGTCAGCGAGGATAACCTGGGGTTCCTG
ATGCACGCCCCCGCGTTTGAGACCGCCGGCACGTACCTGCGGCTCGTGAAGATAAACGACTGGACGGAGATTACACA
GTTTATCCTGGAGCACCGAGCCAAGGGCTCCTGTAAGTACGCCCTCCCGCTGCGCATCCCCCCGTCAGCCTGCCTCT
CCCCCCAGGCCTACCAGCAGGGGGTGACGGTGGACAGCATCGGGATGCTGCCCCGCTTCATCCCCGAGAACCAGCGC
ACCGTCGCCGTATACAGCTTGAAGATCG。
Preferably, described herpes simplex virus I-form positive quality control product is to insert described by pUC57 plasmid vectors
The artificial synthesized sequence restructuring containing the herpes simplex virus I-form specific primer to amplified production fragment is obtained.
A specific aspect of the invention, the restructuring contained by described herpes simplex virus I-form positive quality control product
PUC57 vector plasmids concentration is 1ng/ μ L.
Preferably, described herpes simplex virus type II positive quality control product contains artificial synthesized sequence, the artificial synthesized sequence
Be classified as it is artificial synthesized containing the herpes simplex virus type II specific primer to the sequence of amplified production fragment.The artificial synthesized sequence
Row content is：
CCACGACTCCGGGGCCCCAAACAACCCCTCCCGGACCCGCAACCCCGGGTCCGGTGGGCGCCTCCGCCGCGCCCACG
GCCGATTCCCCCCTCACCGCCTCGCCCCCCGCTACCGCGCCGGGGCCCTCGGCCGCCAACGTTTCGGTCGCCGCGAC
CACCGCCACGCCCGGAACCCGGGGCACCGCCCGTACCCCCCCAACGGACCCAAAGACGCACCCACACGGACCCGCGG
ACGCTCCCCCCGGCTCGCCAGCCCCCCCACCCCCCGAACATCGCGGCGGACCCGAGGAGTTTGAGGGCGCCGGGGAC
GGCGAACCCCCCGAGGACGACGACAGCGCCACCGGCCTCGCCTTCCGAACTCCGAACCCCAACAAACCACCCCCCGC
GCGCCCCGGGCCCATCCGCCCCACGCTCCCGCCAGGAATTCTTGGGCCGCTCGCCCCCAACACGCCTCGCCCCCCCG
CCCAAGCTCCCGCTAAGGACATGCCCTCGGGCCCCACACCCCAACACATCCCCCTGTTCTGGTTCCTAACGGCCTCC
CCTGCTCTAGATATCCTCTTTATCATCAGCACCACCATCCACACGGCGGCGTTCGTTTGTCTGGTCGCCTTGGCAGC
ACAACTTTGGCGCGGCCGGGCGGGGCGCAGGCGATACGCGCACCCGAGCGTGCGTTACGTATGTCTGCCACCCGAGC
GGGATTAGGGGGTGGGGGTGGGGGGCGAGAAACGATGAAGGACGGGAAAGGGAACAGCGACCAAATGTCACGATAAG
AACAATAAA。
Preferably, described herpes simplex virus type II positive quality control product is by inserting institute in pUC57 plasmid vectors
The artificial synthesized sequence restructuring containing the herpes simplex virus type II specific primer to amplified production fragment is stated to be obtained.
A specific aspect of the invention, the restructuring contained by described herpes simplex virus type II positive quality control product
PUC57 vector plasmids concentration is 1ng/ μ L.
Preferably, sequence of the described negative quality-control product containing artificial synthesized mankind's house-keeping gene GAPDH Partial Fragments.
Primer pair for expanding described artificial synthesized mankind's house-keeping gene GAPDH Partial Fragments is as follows：
Forward primer：5’-CCGGGAAGGAAATGAATG-3’
Reverse primer：5’-CTTCTCTAAGTCCCTCCTAC-3’.
It is highly preferred that the sequence of described artificial synthesized mankind's house-keeping gene GAPDH Partial Fragments is：
TTCTTTCCTTTCGCGCTCTGCGGGGTCACGTGTCGCAGAGGAGCCCCTCCCCCACGGCCTCCGGCACCGCAGGCCCC
GGGATGCTAGTGCGCAGCGGGTGCATCCCTGTCCGGATGCTGCGCCTGCGGTAGAGCGGCCGCCATGTTGCAACCGG
GAAGGAAATGAATGGGCAGCCGTTAGGAAAGCCTGCCGGTGACTAACCCTGCGCTCCTGCCTCGATGGGTGGAGTCG
CGTGTGGCGGGGAAGTCAGGTGGAGCGAGGCTAGCTGGCCCGATTTCTCCTCCGGGTGATGCTTTTCCTAGATTATT
CTCTGGTAAATCAAAGAAGTGGGTTTATGGAGGTCCTCTTGTGTCCCCTCCCCGCAGAGGTGTGGTGGCTGTGGCAT
GGTGCCAAGCCGGGAGAAGCTGAGTCATGGGTAGTTGGAAAAGGACATTTCCACCGCAAAATGGCCCCTCTGGTGGT
GGCCCCTTCCTGCAGCGCCGGCTCACCTCACGGCCCCGCCCTTCCCCTGCCAGCCTAGCGTTGACCCGACCCCAAAG
GCCAGGCTGTAAATGTCACCGGGAGGATTGGGTGTCTGGGCGCCTCGGGGAACCTGCCCTTCTCCCCATTCCGTCTT
CCGGAAACCAGATCTCCCACCGCACCCTGGTCTGAGGTTAAATATAGCTGCTGACCTTTCTGTAGCTGGGGGCCTGG
GCTGGGGCTCTCTCCCATCCCTTCTCCCCACACACATGCACTTACCTGTGCTCCCACTCCTGATTTCTGGAAAAGAG
CTAGGAAGGACAGGCAACTTGGCAAATC。
It is highly preferred that the negative quality-control product is by inserting the artificial synthesized mankind in pTG19-T plasmid vectors
The sequence restructuring of house-keeping gene GAPDH Partial Fragments is obtained.
Preferably, the PCR reaction solutions composition such as including Taq enzyme, dNTP, buffer solution.
According to the present invention, the reaction system of described kit include the reaction system of detection herpes simplex virus I-form and
Detect the reaction system of herpes simplex virus type II.
According to the present invention, the reaction system of the detection herpes simplex virus I-form is 20 μ L-50 μ L, when the detection is single
The reaction system of pure chimpanzee agent is 20 μ L systems, then include：The μ L of 10 μ L, HSV1 forward primer of PCR reaction solutions (2x) 0.5
(10 μM of ol/mL), the μ L of HSV1 reverse primers 0.5 (10 μM of ol/mL), the μ L of HSV1 fluorescence probes 0.5 (10 μM of ol/mL) are detected
Thing cell, tissue, the μ L of DNA extracts 4 of body fluid (secretion, sputum, urine etc.), sterilizing ultra-pure water add to 20 μ L；Work as institute
The reaction system for stating detection herpes simplex virus I-form is 50 μ L systems, then include：μ L, the HSV1 forward directions of PCR reaction solutions (2x) 25 are drawn
The μ L of thing 1.25 (10 μM of ol/mL), the μ L of HSV1 reverse primers 1.25 (10 μM of ol/mL), the μ L of HSV1 fluorescence probes 1.25 (10 μM of ol/
ML), the DNA extract 4-10 μ L of detected material cell, tissue, body fluid (secretion, sputum, urine etc.), sterilize ultra-pure water
Add to 50 μ L.
According to the present invention, the reaction system of the detection herpes simplex virus type II is 20 μ L-50 μ L, when the detection is single
The reaction system of pure herpesvirusⅡtype is 20 μ L systems, then include：The μ L of 10 μ L, HSV2 forward primer of PCR reaction solutions (2x) 0.5
(10 μM of ol/mL), the μ L of HSV2 reverse primers 0.5 (10 μM of ol/mL), the μ L of HSV2 fluorescence probes 0.5 (10 μM of ol/mL) are detected
Thing cell, tissue, the μ L of DNA extracts 4 of body fluid (secretion, sputum, urine etc.), sterilizing ultra-pure water add to 20 μ L；Work as institute
The reaction system for stating detection herpes simplex virus type II is 50 μ L systems, then include：PCR reaction solutions (2x) 25 μ L, HSV2 are positive
The μ L of primer 1.25 (10 μM of ol/mL), the μ L of HSV2 reverse primers 1.25 (10 μM of ol/mL), μ L (10 μ of HSV2 fluorescence probes 1.25
Mol/mL), the DNA extract 4-10 μ L of detected material cell, tissue, body fluid (secretion, sputum, urine etc.), sterilizing is super
Pure water adds to 50 μ L.
According to the present invention, the reaction time of the fluorescent PCR amplification of described kit and temperature are：50 DEG C, 2min 1
Circulation (is omitted) if PCR reaction solutions are free of UNG enzymes；95 DEG C, 2-10min (depending on the PCR reaction solutions of separate sources) 1
Individual circulation；95 DEG C, 15s, 60 DEG C, 1min, 40 circulate and collect fluorescence signal.
The present invention use another technical scheme be：A kind of specific detection herpes simplex virus I, II type nucleic acid it is glimmering
Light PCR method, is detected using mentioned reagent box.
Due to the implementation of above-mentioned technical proposal, the present invention has the following advantages that compared with prior art：
Kit of the present invention detects by a large amount of clinical samples, can effective detection go out herpes simplex virus I, II
Type, the detection of a large amount of clinical samples is provided and ensured to the validity of kit, large-scale use.
Kit of the present invention, the reaction efficiency of herpes simplex virus I-form fluorescent PCR detection is up to 89.8%, herpe simplex
The reaction efficiency of virus type II fluorescent PCR detection is up to 85.1%, with reaction efficiency higher.
Brief description of the drawings
Fig. 1 is herpes simplex virus I, the quality inspection electrophoretogram of II type fluorescence PCR primer pair；
In figure, 1：HSV1 Fluorescence PCR forward primers；2：HSV1 Fluorescence PCR reverse primers；3：HSV2 fluorescence
PCR reaction forward primers；4：HSV2 Fluorescence PCR reverse primers；M：Electrophoresis Marker, length is followed successively by 17 from top to bottom,
24,34 (units：Base).
Fig. 2 schemes for the quality inspection HPLC of HSV1 Fluorescence PCR probes.
Fig. 3 schemes for the quality inspection HPLC of HSV2 Fluorescence PCR probes.
Fig. 4 constitutes schematic diagram for the pUC57 recombinant plasmids of herpes simplex virus I-form.
Fig. 5 constitutes schematic diagram for the pUC57 recombinant plasmids of herpes simplex virus type II.
Fig. 6 is that pTG19-T plasmid vectors constitute schematic diagram.
Fig. 7 is the amplified reaction figure of herpes simplex virus I-form positive quality control product fluorescent PCR；
Curve in figure from left to right corresponds to the concentration of herpes simplex virus I-form positive quality control product from high to low respectively.
Fig. 8 is the corresponding canonical plottings of Fig. 7.
Fig. 9 is the amplified reaction figure of herpes simplex virus type II positive quality control product fluorescent PCR；
Curve in figure from left to right corresponds to the concentration of herpes simplex virus type II positive quality control product from high to low respectively.
Figure 10 is the corresponding canonical plottings of Fig. 9.
Specific embodiment
With reference to specific embodiment, the present invention will be further described in detail, should illustrate that some embodiment is only used
In illustrating, of the invention rather than limitation is of the invention.
Embodiment 1：The preparation of kit
1st, the design and synthesis of primer and probe
Using the softwares of Primer Express 3.0, the gpG genes of gpD genes, II type to herpes simplex virus I-form
DNA sequence dna has separately designed specific primer pair and its specific probe, and primer and probe are limited by Shanghai JaRa biology
Company synthesizes, and double fluorescence and quenching group mark have been carried out on the probe of synthesis.Wherein, primer is PAGE purifying, and probe is
HPLC is purified.The primer pair and probe designed are respectively：
Herpes simplex virus I-form：Forward primer：5’-CCCCGCTGGAACTACTATGACA-3’(SEQ ID NO.1)
Reverse primer：5’-TCCGTCCAGTCGTTTATCTTCA-3’(SEQ ID NO.2)
Fluorescence probe：5’-ACGCCCCCGCGTTTGAGACC-3’(SEQ ID NO.3)
Herpes simplex virus type II：Forward primer：5’-AAGCTCCCGCTAAGGACATG-3’(SEQ ID NO.4)
Reverse primer：5’-GGTGCTGATGATAAAGAGGATATCTAGA-3’(SEQ ID NO.5)
Fluorescence probe：5’-AACACATCCCCCTGTTCTGGTTCCTAACG-3’(SEQ ID NO.6).
Lyophilized powder is prepared into after primer and probe synthesis, 100 μM of concentration is then diluted to 1 × TE buffer
As mother liquor, -20 DEG C of preservations.Working solution is that mother liquor is obtained for 10 times by sterilized water dilution, is used for conventional.
Quality inspection analysis is carried out to specific primer pair and fluorescence probe, as a result as shown in Figure 1,2 and 3.
2nd, the preparation of positive quality control product and negative quality-control product
1) preparation of I type positive quality control product of herpe simplex poison：Specific primer containing herpes simplex virus I-form is to amplified production
Specific fragment by Nanjing Jin Sirui biology Co., Ltd synthesize, then manually loaded with recombinant dna gene engineering method
In pUC57 plasmid vectors, then recombinant plasmid dna is obtained by converting the steps such as Escherichia coli, plasmid amplification and plasmid extraction, this
DNA is exactly the herpe simplex I type positive quality control product of poison used in kit of the present invention, and the herpe simplex I type of poison is positive
Artificial synthesized sequence contained by quality-control product is as shown in SEQ ID NO.7.The pUC57 recombinant plasmids of herpes simplex virus I-form are constituted
Schematic diagram is as shown in Figure 4.
2) preparation of II type positive quality control product of herpe simplex poison：Specific primer containing herpes simplex virus type II is produced to amplification
The specific fragment of thing is synthesized by Nanjing Jin Sirui biologies Co., Ltd, is then manually loaded with recombinant dna gene engineering method
In pUC57 plasmid vectors, then recombinant plasmid dna is obtained by converting the steps such as Escherichia coli, plasmid amplification and plasmid extraction, this
DNA is exactly the herpe simplex II type positive quality control product of poison used in kit of the present invention, and the herpe simplex I type of poison is positive
Artificial synthesized sequence contained by quality-control product is as shown in SEQ ID NO.8.The pUC57 recombinant plasmids of herpes simplex virus type II are constituted
Schematic diagram is as shown in Figure 5.
3) preparation of negative quality-control product：With mankind's house-keeping gene GAPDH genes as template, Standard PCR primer, primer are designed
Sequence is entered performing PCR and is expanded as shown in SEQ ID NO.9 and SEQ ID NO.10 in the genomic DNA extracted from human tissue cells
Increase, obtain amplified production, then carried amplified production manual assembly to pTG19-T plasmids with the method for recombinant dna gene engineering
In body, then recombinant plasmid dna is obtained by converting the steps such as Escherichia coli, plasmid amplification and plasmid extraction, this DNA is exactly
Negative quality-control product used in kit of the present invention, the artificial synthesized mankind's house-keeping gene contained by the negative quality-control product
The sequence of GAPDH Partial Fragments is as shown in SEQ ID NO.11.It is as shown in Figure 6 that pTG19-T plasmid vectors constitute schematic diagram.
3rd, PCR reaction solutions
The PCR reaction solutions composition such as including Taq enzyme, dNTP, buffer solution, it is possible to use purchased from the Fluorescence PCR of ABI companies
Mixed liquor, title isUniversal Master Mix II,with UNG。
Embodiment 2：The extraction of sample nucleic acid
Use the blood/tissue/cellular genome extracts kit of TIANGEN Biotech (Beijing) Co., Ltd.
(DP304), according to kit specification the step of, sample nucleic acid is extracted.After the completion of extraction, product puts -20 DEG C of preservations.
Embodiment 3：The making of standard curve
1) herpes simplex virus I-form
Herpes simplex virus I-form positive quality control product is chosen as reference material, diluted concentration is then proceeded to 10 to 1 μ g/mL
Times five concentration of gradient dilution, come to 6 concentration gradients, i.e. 1 μ g/mL, 10-1μg/mL、10-2μg/mL、10-3μg/mL、10-4
μg/mL、10-5μg/mL.Then fluorescent PCR amplified reaction is carried out, with the Software Create standard curve supporting with ABI7500 instruments,
As shown in Figure 7 and Figure 8, and the system of instrument shows result, and reaction efficiency is up to 89.8%.
2) herpes simplex virus type II
Choose herpes simplex virus type II positive quality control product as reference material, diluted concentration to 1 μ g/mL, then proceed to
10 times of gradient dilutions, five concentration, come to 6 concentration gradients, i.e. 1 μ g/mL, 10-1μg/mL、10-2μg/mL、10-3μg/mL、
10-4μg/mL、10-5μg/mL.Then fluorescent PCR amplified reaction is carried out, with the Software Create standard supporting with ABI7500 instruments
Curve, as a result as shown in Figure 9 and Figure 10, and the system of instrument shows, reaction efficiency is up to 85.1%.
Embodiment 4：Fluorescent PCR is expanded and interpretation of result
1st, use reaction system as shown in Tables 1 and 2.After good each system of configuration in special eight union of fluorescent PCR, put
Enter in ABI7500 fluorescent PCR instruments (or other types of open quantitative fluorescent PCR instrument), be ready for reaction.It is real every time
Test and be required for configuring 1 reaction tube of positive quality control product as positive control and a reaction tube for negative quality-control product as feminine gender
Control.
Table 1
| HSV1 syllabus and contents | Volume (μ L) |
| PCR reaction solutions | 10 |
| HSV1 forward primers | 0.5 |
| HSV1 reverse primers | 0.5 |
| HSV1 fluorescence probes | 0.5 |
| Sterilized water | 4.5 |
| Sample to be tested | 4 |
| Amount to | 20 |
Table 2
| HSV2 syllabus and contents | Volume (μ L) |
| PCR reaction solutions | 10 |
| HSV2 forward primers | 0.5 |
| HSV2 reverse primers | 0.5 |
| HSV2 fluorescence probes | 0.5 |
| Sterilized water | 4.5 |
| Sample to be tested | 4 |
| Amount to | 20 |
Sample to be tested in table 1 and 2 is the sample to be tested prepared by way of embodiment 2.
2nd, reaction condition sets as shown in table 3：
Table 3
3rd, interpretation of result
1) after reaction terminates, first, the selection of baseline is set to " automatic ".Threshold value (threshold) setting principle be
Threshold line is just above the peak of the amplification curve of negative quality-control product, makes its Ct value=40 or " Undetermined ".
2) after setting good threshold, whether the amplification curve for observing positive quality control product is normal S types Increasing Curve of Logarithm, and
Ct value≤37.Meet above-mentioned condition and then illustrate that this experiment reaction is normal, otherwise, it is necessary to reform.
3) result judgement：Ct value≤37, represent that the sample results are the positive, Ct values>37 or " Undetermined ", represent
The sample results are feminine gender.
Application Example
124 tissue samples being collected into are detected using kit of the present invention.124 samples are collected for clinical
Fetal tissue, be divided to two groups：Normal induced labor group (control group) 42, spontaneous abortion group (ill group) 82.According to above-mentioned reality
Apply example 2：The extraction of sample nucleic acid.Then according to above-described embodiment 4：Fluorescent PCR is expanded and interpretation of result, is tried using the present invention
Agent box carries out fluorescent PCR detection in ABI7500 fluorescent PCRs instrument, as a result as shown in table 4 below.
Table 4
Can be drawn from the result in table, this kit can successfully detect herpes simplex virus I, II type, and from just
Often from the point of view of the positive rate result of group and ill group, this kit can well distinguish Normal group and ill group.Simple blister
The type testing result of exanthema virus I, the no positive result of control group, and ill group is 4.76%；Herpes simplex virus type II testing result,
Control group is 1 to 2.55 with the ratio of ill group positive rate, and that is the infection rate of ill group herpes simplex virus type II is
2.55 times of Normal group, as a result show that this kit has good sensitivity and specificity, can be simple as detection
Herpesviral I, the effective tool of II type.Validity, large-scale use offer of the detection of a large amount of clinical samples to kit
Ensure.
Kit of the present invention, the reaction efficiency of herpes simplex virus I-form is up to 89.8%, herpes simplex virus type II it is anti-
Efficiency high up to 85.1% is answered, with reaction efficiency higher.
The present invention is described in detail above, its object is to allow the personage for being familiar with this art to will appreciate that this
The content of invention is simultaneously carried out, and it is not intended to limit the scope of the present invention, and the invention is not restricted to above-mentioned implementation
Example, the equivalent change or modification that all Spirit Essences of the invention are made should all be included within the scope of the present invention.
Sequence table
<110>Zhangjiagang indigo plant is revived thing Engineering Co., Ltd
<120>The fluorescence PCR method and kit of a kind of specific detection herpes simplex virus I, II type nucleic acid
<130>
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>Detect the forward primer of herpes simplex virus I-form
<400> 1
ccccgctgga actactatga ca 22
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>Detect the reverse primer of herpes simplex virus I-form
<400> 2
tccgtccagt cgtttatctt ca 22
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Detect the fluorescence probe of herpes simplex virus I-form
<400> 3
acgcccccgc gtttgagacc 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Detect the forward primer of herpes simplex virus type II
<400> 4
aagctcccgc taaggacatg 20
<210> 5
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>Detect the reverse primer of herpes simplex virus type II
<400> 5
ggtgctgatg ataaagagga tatctaga 28
<210> 6
<211> 29
<212> DNA
<213>Artificial sequence
<220>
<223>Detect the fluorescence probe of herpes simplex virus type II
<400> 6
aacacatccc cctgttctgg ttcctaacg 29
<210> 7
<211> 721
<212> DNA
<213>Artificial sequence
<220>
<223>Artificial synthesized sequence contained by kit herpe simplex I type positive quality control product of poison
<400> 7
cgcggcaaat atgccttggt ggatgcctct ctcaagatgg ccgaccccaa tcgctttcgc 60
ggcaaagacc ttccggtcct ggaccagctg accgaccctc cgggggtccg gcgcgtgtac 120
cacatccagg cgggcctacc ggacccgttc cagcccccca gcctcccgat cacggtttac 180
tacgccgtgt tggagcgcgc ctgccgcagc gtgctcctaa acgcaccgtc ggaggccccc 240
cagattgtcc gcggggcctc cgaagacgtc cggaaacaac cctacaacct gaccatcgct 300
tggtttcgga tgggaggcaa ctgtgctatc cccatcacgg tcatggagta caccgaatgc 360
tcctacaaca agtctctggg ggcctgtccc atccgaacgc agccccgctg gaactactat 420
gacagcttca gcgccgtcag cgaggataac ctggggttcc tgatgcacgc ccccgcgttt 480
gagaccgccg gcacgtacct gcggctcgtg aagataaacg actggacgga gattacacag 540
tttatcctgg agcaccgagc caagggctcc tgtaagtacg ccctcccgct gcgcatcccc 600
ccgtcagcct gcctctcccc ccaggcctac cagcaggggg tgacggtgga cagcatcggg 660
atgctgcccc gcttcatccc cgagaaccag cgcaccgtcg ccgtatacag cttgaagatc 720
g 721
<210> 8
<211> 779
<212> DNA
<213>Artificial sequence
<220>
<223>Artificial synthesized sequence contained by kit herpe simplex II type positive quality control product of poison
<400> 8
ccacgactcc ggggccccaa acaacccctc ccggacccgc aaccccgggt ccggtgggcg 60
cctccgccgc gcccacggcc gattcccccc tcaccgcctc gccccccgct accgcgccgg 120
ggccctcggc cgccaacgtt tcggtcgccg cgaccaccgc cacgcccgga acccggggca 180
ccgcccgtac ccccccaacg gacccaaaga cgcacccaca cggacccgcg gacgctcccc 240
ccggctcgcc agccccccca ccccccgaac atcgcggcgg acccgaggag tttgagggcg 300
ccggggacgg cgaacccccc gaggacgacg acagcgccac cggcctcgcc ttccgaactc 360
cgaaccccaa caaaccaccc cccgcgcgcc ccgggcccat ccgccccacg ctcccgccag 420
gaattcttgg gccgctcgcc cccaacacgc ctcgcccccc cgcccaagct cccgctaagg 480
acatgccctc gggccccaca ccccaacaca tccccctgtt ctggttccta acggcctccc 540
ctgctctaga tatcctcttt atcatcagca ccaccatcca cacggcggcg ttcgtttgtc 600
tggtcgcctt ggcagcacaa ctttggcgcg gccgggcggg gcgcaggcga tacgcgcacc 660
cgagcgtgcg ttacgtatgt ctgccacccg agcgggatta gggggtgggg gtggggggcg 720
agaaacgatg aaggacggga aagggaacag cgaccaaatg tcacgataag aacaataaa 779
<210> 9
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>The forward primer of the amplification artificial synthesized fragment of negative quality-control product
<400> 9
ccgggaagga aatgaatg 18
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>The reverse primer of the amplification artificial synthesized fragment of negative quality-control product
<400> 10
cttctctaag tccctcctac 20
<210> 11
<211> 798
<212> DNA
<213>Artificial sequence
<220>
<223>Artificial synthesized sequence contained by kit feminine gender quality-control product
<400> 11
ttctttcctt tcgcgctctg cggggtcacg tgtcgcagag gagcccctcc cccacggcct 60
ccggcaccgc aggccccggg atgctagtgc gcagcgggtg catccctgtc cggatgctgc 120
gcctgcggta gagcggccgc catgttgcaa ccgggaagga aatgaatggg cagccgttag 180
gaaagcctgc cggtgactaa ccctgcgctc ctgcctcgat gggtggagtc gcgtgtggcg 240
gggaagtcag gtggagcgag gctagctggc ccgatttctc ctccgggtga tgcttttcct 300
agattattct ctggtaaatc aaagaagtgg gtttatggag gtcctcttgt gtcccctccc 360
cgcagaggtg tggtggctgt ggcatggtgc caagccggga gaagctgagt catgggtagt 420
tggaaaagga catttccacc gcaaaatggc ccctctggtg gtggcccctt cctgcagcgc 480
cggctcacct cacggccccg cccttcccct gccagcctag cgttgacccg accccaaagg 540
ccaggctgta aatgtcaccg ggaggattgg gtgtctgggc gcctcgggga acctgccctt 600
ctccccattc cgtcttccgg aaaccagatc tcccaccgca ccctggtctg aggttaaata 660
tagctgctga cctttctgta gctgggggcc tgggctgggg ctctctccca tcccttctcc 720
ccacacacat gcacttacct gtgctcccac tcctgatttc tggaaaagag ctaggaagga 780
caggcaactt ggcaaatc 798
Claims (11)
1. a kind of specific detection herpes simplex virus I, the fluorescent PCR kit of II type nucleic acid, including herpes simplex virus I-form
Specific primer pair and fluorescence probe, herpes simplex virus type II specific primer pair and fluorescence probe, it is characterised in that：
Described herpes simplex virus I-form specific primer pair and fluorescence probe are as follows：
Forward primer：5’-CCCCGCTGGAACTACTATGACA-3’
Reverse primer：5’-TCCGTCCAGTCGTTTATCTTCA-3’
Fluorescence probe：5’-ACGCCCCCGCGTTTGAGACC-3’；
Described herpes simplex virus type II specific primer pair and fluorescence probe are as follows：
Forward primer：5’-AAGCTCCCGCTAAGGACATG-3’
Reverse primer：5’-GGTGCTGATGATAAAGAGGATATCTAGA-3’
Fluorescence probe：5’-AACACATCCCCCTGTTCTGGTTCCTAACG-3’.
2. specific detection herpes simplex virus I according to claim 1, the fluorescent PCR kit of II type nucleic acid, it is special
Levy and be：Described herpes simplex virus I-form fluorescence probe, the two ends of herpes simplex virus type II fluorescence probe mark report respectively
Accuse fluorescein or fluorescent dye, quenching group.
3. specific detection herpes simplex virus I according to claim 2, the fluorescent PCR kit of II type nucleic acid, it is special
Levy and be：Described reporter fluorescence element or fluorescent dye are to include FAM or other any fluoresceins that can be marked as probe
Or fluorescent dye；Described quenching group be include TAMRA or other it is any can be as the chemical substance of quenching group.
4. specific detection herpes simplex virus I according to claim 1, the fluorescent PCR kit of II type nucleic acid, it is special
Levy and be：Described kit also includes PCR reaction solutions, herpes simplex virus I-form positive quality control product, herpes simplex virus type II
Positive quality control product and negative quality-control product.
5. specific detection herpes simplex virus I according to claim 4, the fluorescent PCR kit of II type nucleic acid, it is special
Levy and be：Described herpes simplex virus I-form positive quality control product contains artificial synthesized sequence, and described artificial synthesized sequence is artificial
Synthesis contains sequence of the herpes simplex virus I-form specific primer to amplified production fragment.
6. specific detection herpes simplex virus I according to claim 5, the fluorescent PCR kit of II type nucleic acid, it is special
Levy and be, the artificial synthesized sequence contained by described herpes simplex virus I-form positive quality control product is：
CGCGGCAAATATGCCTTGGTGGATGCCTCTCTCAAGATGGCCGACCCCAATCGCTTTCGC
GGCAAAGACCTTCCGGTCCTGGACCAGCTGACCGACCCTCCGGGGGTCCGGCGCGTGTAC
CACATCCAGGCGGGCCTACCGGACCCGTTCCAGCCCCCCAGCCTCCCGATCACGGTTTAC
TACGCCGTGTTGGAGCGCGCCTGCCGCAGCGTGCTCCTAAACGCACCGTCGGAGGCCCCC
CAGATTGTCCGCGGGGCCTCCGAAGACGTCCGGAAACAACCCTACAACCTGACCATCGCT
TGGTTTCGGATGGGAGGCAACTGTGCTATCCCCATCACGGTCATGGAGTACACCGAATGC
TCCTACAACAAGTCTCTGGGGGCCTGTCCCATCCGAACGCAGCCCCGCTGGAACTACTAT
GACAGCTTCAGCGCCGTCAGCGAGGATAACCTGGGGTTCCTGATGCACGCCCCCGCGTTT
GAGACCGCCGGCACGTACCTGCGGCTCGTGAAGATAAACGACTGGACGGAGATTACACAG
TTTATCCTGGAGCACCGAGCCAAGGGCTCCTGTAAGTACGCCCTCCCGCTGCGCATCCCC
CCGTCAGCCTGCCTCTCCCCCCAGGCCTACCAGCAGGGGGTGACGGTGGACAGCATCGGG
ATGCTGCCCCGCTTCATCCCCGAGAACCAGCGCACCGTCGCCGTATACAGCTTGAAGATC
G。
7. specific detection herpes simplex virus I according to claim 4, the fluorescent PCR kit of II type nucleic acid, it is special
Levy and be：Described herpes simplex virus type II positive quality control product contains artificial synthesized sequence, and the artificial synthesized sequence is artificial
Synthesis contains sequence of the herpes simplex virus type II specific primer to amplified production fragment.
8. described specific detection herpes simplex virus I, the fluorescent PCR reagent of II type nucleic acid according to claim 7
Box, it is characterised in that the artificial synthesized sequence contained by described herpes simplex virus type II positive quality control product is：
CCACGACTCCGGGGCCCCAAACAACCCCTCCCGGACCCGCAACCCCGGGTCCGGTGGGCG
CCTCCGCCGCGCCCACGGCCGATTCCCCCCTCACCGCCTCGCCCCCCGCTACCGCGCCGG
GGCCCTCGGCCGCCAACGTTTCGGTCGCCGCGACCACCGCCACGCCCGGAACCCGGGGCA
CCGCCCGTACCCCCCCAACGGACCCAAAGACGCACCCACACGGACCCGCGGACGCTCCCC
CCGGCTCGCCAGCCCCCCCACCCCCCGAACATCGCGGCGGACCCGAGGAGTTTGAGGGCG
CCGGGGACGGCGAACCCCCCGAGGACGACGACAGCGCCACCGGCCTCGCCTTCCGAACTC
CGAACCCCAACAAACCACCCCCCGCGCGCCCCGGGCCCATCCGCCCCACGCTCCCGCCAG
GAATTCTTGGGCCGCTCGCCCCCAACACGCCTCGCCCCCCCGCCCAAGCTCCCGCTAAGG
ACATGCCCTCGGGCCCCACACCCCAACACATCCCCCTGTTCTGGTTCCTAACGGCCTCCC
CTGCTCTAGATATCCTCTTTATCATCAGCACCACCATCCACACGGCGGCGTTCGTTTGTC
TGGTCGCCTTGGCAGCACAACTTTGGCGCGGCCGGGCGGGGCGCAGGCGATACGCGCACC
CGAGCGTGCGTTACGTATGTCTGCCACCCGAGCGGGATTAGGGGGTGGGGGTGGGGGGCG
AGAAACGATGAAGGACGGGAAAGGGAACAGCGACCAAATGTCACGATAAGAACAATAAA。
9. specific detection herpes simplex virus I according to claim 4, the fluorescent PCR kit of II type nucleic acid, it is special
Levy and be：Sequence of the described negative quality-control product containing artificial synthesized mankind's house-keeping gene GAPDH Partial Fragments, it is described for expanding
Artificial synthesized mankind's house-keeping gene GAPDH Partial Fragments primer pair it is as follows：
Forward primer：5’-CCGGGAAGGAAATGAATG-3’
Reverse primer：5’-CTTCTCTAAGTCCCTCCTAC-3’.
10. specific detection herpes simplex virus I according to claim 9, the fluorescent PCR kit of II type nucleic acid, its
It is characterised by：The sequence of described artificial synthesized mankind's house-keeping gene GAPDH Partial Fragments is：
TTCTTTCCTTTCGCGCTCTGCGGGGTCACGTGTCGCAGAGGAGCCCCTCC
CCCACGGCCTCCGGCACCGCAGGCCCCGGGATGCTAGTGCGCAGCGGGTG
CATCCCTGTCCGGATGCTGCGCCTGCGGTAGAGCGGCCGCCATGTTGCAA
CCGGGAAGGAAATGAATGGGCAGCCGTTAGGAAAGCCTGCCGGTGACTAA
CCCTGCGCTCCTGCCTCGATGGGTGGAGTCGCGTGTGGCGGGGAAGTCAG
GTGGAGCGAGGCTAGCTGGCCCGATTTCTCCTCCGGGTGATGCTTTTCCT
AGATTATTCTCTGGTAAATCAAAGAAGTGGGTTTATGGAGGTCCTCTTGT
GTCCCCTCCCCGCAGAGGTGTGGTGGCTGTGGCATGGTGCCAAGCCGGGA
GAAGCTGAGTCATGGGTAGTTGGAAAAGGACATTTCCACCGCAAAATGGC
CCCTCTGGTGGTGGCCCCTTCCTGCAGCGCCGGCTCACCTCACGGCCCCG
CCCTTCCCCTGCCAGCCTAGCGTTGACCCGACCCCAAAGGCCAGGCTGTA
AATGTCACCGGGAGGATTGGGTGTCTGGGCGCCTCGGGGAACCTGCCCTT
CTCCCCATTCCGTCTTCCGGAAACCAGATCTCCCACCGCACCCTGGTCTG
AGGTTAAATATAGCTGCTGACCTTTCTGTAGCTGGGGGCCTGGGCTGGGG
CTCTCTCCCATCCCTTCTCCCCACACACATGCACTTACCTGTGCTCCCAC
TCCTGATTTCTGGAAAAGAGCTAGGAAGGACAGGCAACTTGGCAAATC。
A kind of 11. specific detection herpes simplex virus Is, the fluorescence PCR method of II type nucleic acid, using in claim 1~10
Kit described in any one claim is detected.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109487013A (en) * | 2019-01-09 | 2019-03-19 | 中生方政生物技术股份有限公司 | Herpes simplex virus I-type and II type detection marker, primed probe are to, kit and detection method |
| CN109609699A (en) * | 2019-01-30 | 2019-04-12 | 浙江省人民医院 | A kind of kit for HSV-2 detection of nucleic acids |
| 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 |
| CN111172323A (en) * | 2020-01-17 | 2020-05-19 | 郑州安图生物工程股份有限公司 | Herpes simplex virus 1 type and 2 type typing nucleic acid detection kit |
| CN112063749A (en) * | 2019-10-28 | 2020-12-11 | 北京康美天鸿生物科技有限公司 | Primer probe combination for identifying herpes simplex virus and fluorescent quantitative PCR kit |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979667A (en) * | 2010-11-05 | 2011-02-23 | 武汉百泰基因工程有限公司 | Fluorescence quantitative PCR kit for synchronously detecting herpes simplex virus I and II |
| CN102102131A (en) * | 2009-12-17 | 2011-06-22 | 上海裕隆临床检验中心有限公司 | Fluorescent PCR kit for detecting herpes simplex virus type II |
| CN102373304A (en) * | 2011-12-12 | 2012-03-14 | 北京利德曼生化股份有限公司 | Identification method for detecting type I herpes simplex virus and type II herpes simplex virus at the same time |
| CN103773898A (en) * | 2014-01-21 | 2014-05-07 | 浙江大学 | Triple detection kit for human herpes viruses HSV-1, HSV-2 and HCMV |
| WO2014145975A2 (en) * | 2013-03-15 | 2014-09-18 | Whitehead Institute For Biomedical Research | Cellular discovery platform for neurodegenerative diseases |
-
2017
- 2017-05-04 CN CN201710308453.4A patent/CN106916907A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102102131A (en) * | 2009-12-17 | 2011-06-22 | 上海裕隆临床检验中心有限公司 | Fluorescent PCR kit for detecting herpes simplex virus type II |
| CN101979667A (en) * | 2010-11-05 | 2011-02-23 | 武汉百泰基因工程有限公司 | Fluorescence quantitative PCR kit for synchronously detecting herpes simplex virus I and II |
| CN102373304A (en) * | 2011-12-12 | 2012-03-14 | 北京利德曼生化股份有限公司 | Identification method for detecting type I herpes simplex virus and type II herpes simplex virus at the same time |
| WO2014145975A2 (en) * | 2013-03-15 | 2014-09-18 | Whitehead Institute For Biomedical Research | Cellular discovery platform for neurodegenerative diseases |
| CN103773898A (en) * | 2014-01-21 | 2014-05-07 | 浙江大学 | Triple detection kit for human herpes viruses HSV-1, HSV-2 and HCMV |
Non-Patent Citations (1)
| Title |
|---|
| 张修发等: ""单纯疱疹病毒实时检测以及与巢式和病毒培养方法的比较"", 《深圳中西医结合杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109487013A (en) * | 2019-01-09 | 2019-03-19 | 中生方政生物技术股份有限公司 | Herpes simplex virus I-type and II type detection marker, primed probe are to, kit and detection 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 |
| CN109609699A (en) * | 2019-01-30 | 2019-04-12 | 浙江省人民医院 | A kind of kit for HSV-2 detection of nucleic acids |
| 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 |
| CN112063749A (en) * | 2019-10-28 | 2020-12-11 | 北京康美天鸿生物科技有限公司 | Primer probe combination for identifying herpes simplex virus and fluorescent quantitative PCR kit |
| CN111172323A (en) * | 2020-01-17 | 2020-05-19 | 郑州安图生物工程股份有限公司 | Herpes simplex virus 1 type and 2 type typing nucleic acid detection kit |
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