CN107299157A - A kind of primer combination for detecting encephalitis viruses and application, kit - Google Patents
A kind of primer combination for detecting encephalitis viruses and application, kit Download PDFInfo
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- CN107299157A CN107299157A CN201710759502.6A CN201710759502A CN107299157A CN 107299157 A CN107299157 A CN 107299157A CN 201710759502 A CN201710759502 A CN 201710759502A CN 107299157 A CN107299157 A CN 107299157A
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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
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- 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]
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- 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
Abstract
The invention provides a kind of primer combination for detecting encephalitis viruses and application, kit, belong to field of medical examination.The primer combination for the detection encephalitis viruses that the present invention is provided can exactly, specifically detect the species of encephalitis viruses；The combination of above-mentioned primer is applied to prepare detection kit, can easily and fast, efficiently be checked applied to detection；By the method for multiplex PCR, kit is applied to detection, the efficiency of detection is improved.
Description
Technical field
The present invention relates to field of medical examination, combine and apply in particular to a kind of primer for detecting encephalitis viruses,
Kit.
Background technology
Viral encephalitis (VE) has been increasingly becoming the most common cental system infectious diseases of modern age paediatrics, in global point
Cloth, is distributed throughout the year, is in a bad way, and has death, seriously endangers the health of children.Its case fatality rate and sequelae hair
Raw rate is higher.Cause the viral species of viral encephalitis (Viral Encephalitis, VE) a lot, due to viral encephalitis
The diversity of cause of disease, certain difficulty is caused to clinical diagnosis, treatment and prevention.About more than 100 kind viruses can cause
The acute infection of central nervous system, encephalitis B virus adenovirus, enterovirus herpesviral, mumps virus and adenopathy
Poison etc. is the main pathogens of viral encephalitis.
At present, for above-mentioned encephalitis Causative virus inspection more than detected using the method in laboratory；Traditional experiment
Room detection method needs the time of several weeks to obtain accurate result mostly, it is impossible to obtain timely result；Accordingly, it would be desirable to one
Plant the means of quick detection.
The content of the invention
The first object of the present invention is that providing a kind of primer for detecting encephalitis viruses combines, and primer combination, which has, to be directed to
Different pathogens realize indifference amplification simultaneously, and sensitivity is high, embodies good advantage.
The second object of the present invention is to provide above-mentioned primer combination answering in the kit for preparing detection encephalitis viruses
With.
The third object of the present invention is to provide a kind of kit for detecting encephalitis viruses.
The fourth object of the present invention is the kit for providing above-mentioned detection encephalitis viruses in detection encephalitis viruses RNA
Using.
In order to realize the above-mentioned purpose of the present invention, using following technical scheme：
A kind of primer combination for detecting encephalitis viruses, primer combination includes detecting the 1st primer pair of enterovirus, detection second
At least one of 2nd primer pair of encephalovirus, the 3rd primer pair for detecting herpes simplex virus and universal primer pair；1-3
The base sequence of primer pair is as shown in SEQ ID No.1-6；The base sequence of universal primer pair is as shown in SEQ ID No.7-8.
Every primer of 1-3 primer pairs is all made up of two parts；The sequence label of a universal primers identification, b pathogen bases
Because of specific primer recognition sequence.
Whole amplification procedure is made up of following 2 stages：(1) concentration stage:1-3 primer pairs than relatively low concentration to add
Enter reaction system, the gene-specific primer of low concentration is given time enough and goes down discovery simultaneously in higher annealing temperature
With reference to template, the specificity of amplification is improved.This stage typically carries out 10 circulations；(2) stage is expanded:Universal primer to than
Higher concentration adds reaction system, and the universal primer indifference of high concentration efficiently expands all target sequences.The technology
The inconsistent difficulty of different primers annealing temperature is overcome, the indifference opposite sex of amplification is realized.This stage typically carries out 30 and followed
Ring.
Application of the above-mentioned primer combination in the kit for preparing detection encephalitis viruses.
A kind of kit for detecting encephalitis viruses, the primer that kit includes above-mentioned detection encephalitis viruses is combined.
Application of the above-mentioned detection kit in detection viral RNA.
Compared with prior art, beneficial effects of the present invention are：The primer combination for the detection encephalitis viruses that the present invention is provided
It can exactly, specifically detect the species of virus；The combination of above-mentioned primer is applied to prepare detection kit, can facilitate,
Fast and efficiently check and be applied to detection；By the method for nest-type PRC, kit is applied to detection, the effect of detection is improved
Rate.
Brief description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be attached to what is used required in embodiment
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, therefore is not construed as pair
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is the sample electrophoresis testing result figure that experimental example 1 of the present invention is provided；
Fig. 2 is the sample electrophoresis testing result figure that experimental example 1 of the present invention is provided.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be
The conventional products that can be obtained by commercially available purchase.
A kind of primer of detection encephalitis viruses of the embodiment of the present invention is combined below and application, kit are carried out specifically
It is bright.
A kind of primer combination for detecting encephalitis viruses, primer combination includes detecting the 1st primer pair of enterovirus, detection second
At least one of 2nd primer pair of encephalovirus, the 3rd primer pair for detecting herpes simplex virus, and universal primer pair；1-
The base sequence of 3 primer pairs is as shown in SEQ ID No.1-6；The base sequence of universal primer pair is as shown in SEQ ID No.7-8.
Further, 1-3 primer pairs and universal primer are to including sense primer and anti-sense primer；1-3 primer pairs
Upstream primer sequence includes the upstream primer sequence of universal primer, and the downstream primer sequence of 1-3 primer pairs includes universal primer
Downstream primer sequence.
Detect that the upstream primer sequence of the primer pair of enterovirus the 1st is as follows：
SEQ ID No.1：5’-AGGTGACACTATAGAATAGATGAGTCACCGCATTCC-3’；
Detect that the downstream primer sequence of the primer pair of enterovirus the 1st is as follows：
SEQ ID No.2：5’-GTACGACTCACTATAGGGACACACCACGTCCGTATTAG-3’。
Detect that the upstream primer sequence of the primer pair of encephalitis B virus the 2nd is as follows：
SEQ ID No.3：5’-AGGTGACACTATAGAATAGAGAATGGCATAGTCTTGGA-3’；
Detect that the downstream primer sequence of the primer pair of encephalitis B virus the 2nd is as follows：
SEQ ID No.4：5’-GTACGACTCACTATAGGGACGCAGGAATGGTCAATCT-3’。
Detect that the upstream primer sequence of the primer pair of herpes simplex virus the 3rd is as follows：
SEQ ID No.5：5’-AGGTGACACTATAGAATATGCCTTCAACCGAATATGT-3’；
Detect that the downstream primer sequence of the primer pair of herpes simplex virus the 3rd is as follows：
SEQ ID No.6：5’-GTACGACTCACTATAGGGAGATGACTCAAGTCCTCCAA-3’。
The upstream primer sequence of universal primer pair is as follows：
SEQ ID No.7：5’-AGGTGACACTATAGAATA-3’；
The downstream primer sequence of universal primer pair is as follows：
SEQ ID No.8：5’-GTACGACTCACTATAGGGA-3’。
Application of the above-mentioned primer combination in the kit for preparing detection encephalitis viruses.
A kind of kit for detecting encephalitis viruses, kit includes above-mentioned primer and combined.
Further, in addition at least one in PCR reaction buffers, Taq archaeal dna polymerases, dNTPs and reverse transcriptase
Kind.
Further, PCR reaction buffers include TrisHCl, KCl, MgSO4(NH4)2SO4。
Further, in addition to viral RNA extracts reagent.
Further, RNA extracts reagents include DNase solution, absolute ethyl alcohol and RNase solution.
Application of the above-mentioned detection kit in detection viral RNA.
Further, including using sample to be checked as template, performing PCR reaction is entered；
PCR response procedures are：50-51 DEG C of reverse transcription 30min；95-96 DEG C, pre-degeneration 15min；95-96 DEG C of denaturation 30s,
62-68 DEG C of renaturation 45s, 72-73 DEG C of extension 30s, 10 circulations；95-96 DEG C, it is denatured 30s, 52-58 DEG C of renaturation 30s, 72-73 DEG C
Extend 30s, 30 circulations.
The feature and performance to the present invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of primer combination for detecting encephalitis viruses, and primer combination includes detecting the 1st of enterovirus
At least one of primer pair, the 2nd primer pair for detecting encephalitis B virus, the 3rd primer pair for detecting herpes simplex virus, Yi Jitong
Use primer pair；The base sequence of 1-3 primer pairs is as shown in SEQ ID No.1-6；The base sequence of universal primer pair such as SEQ
Shown in ID No.7-8.
1-3 primer pairs and the universal primer are to including sense primer and anti-sense primer；Draw the upstream of 1-3 primer pairs
Thing sequence includes the upstream primer sequence of universal primer, and the downstream primer sequence of 1-3 primer pairs includes the downstream of universal primer
Primer sequence.
Detect that the upstream primer sequence of the primer pair of enterovirus the 1st is as follows：
SEQ ID No.1：5’-AGGTGACACTATAGAATAGATGAGTCACCGCATTCC-3’；
Detect that the downstream primer sequence of the primer pair of enterovirus the 1st is as follows：
SEQ ID No.2：5’-GTACGACTCACTATAGGGACACACCACGTCCGTATTAG-3’。
The product length of 1st primer pair is 134bp.
Detect that the upstream primer sequence of the primer pair of encephalitis B virus the 2nd is as follows：
SEQ ID No.3：5’-AGGTGACACTATAGAATAGAGAATGGCATAGTCTTGGA-3’；
Detect that the downstream primer sequence of the primer pair of encephalitis B virus the 2nd is as follows：
SEQ ID No.4：5’-GTACGACTCACTATAGGGACGCAGGAATGGTCAATCT-3’。
The product length of 2nd primer pair is 394bp.
Detect that the upstream primer sequence of the primer pair of herpes simplex virus the 3rd is as follows：
SEQ ID No.5：5’-AGGTGACACTATAGAATATGCCTTCAACCGAATATGT-3’；
Detect that the downstream primer sequence of the primer pair of herpes simplex virus the 3rd is as follows：
SEQ ID No.6：5’-GTACGACTCACTATAGGGAGATGACTCAAGTCCTCCAA-3’。
The product length of 3rd primer pair is 512bp.
The upstream primer sequence of universal primer pair is as follows：
SEQ ID No.5：5’-AGGTGACACTATAGAATA-3’；
The downstream primer sequence of universal primer pair is as follows：
SEQ ID No.6：5’-GTACGACTCACTATAGGGA-3’。
Embodiment 2
The present embodiment provides a kind of kit for detecting encephalitis viruses, and kit contains the detection encephalitis of the offer of embodiment 1
The primer combination of virus.
Embodiment 3
The present embodiment provides a kind of detection kit for detecting encephalitis viruses, and kit contains the detection of the offer of embodiment 1
The primer combination of encephalitis viruses.
Also include at least one of PCR reaction buffers, Taq archaeal dna polymerases, dNTPs and reverse transcriptase.
PCR reaction buffers include TrisHCl, KCl, MgSO4(NH4)2SO4。
Embodiment 4
The present embodiment provides a kind of while detecting the detection kit of a variety of encephalitis viruses, and kit contains embodiment 1 and carried
The primer combination of the detection encephalitis viruses of confession.
Also include at least one of PCR reaction buffers, Taq archaeal dna polymerases, dNTPs and reverse transcriptase.
PCR reaction buffers include TrisHCl, KCl, MgSO4(NH4)2SO4。
Also include RNA extracts reagents, RNA extracts reagents include DNase solution, absolute ethyl alcohol and RNase solution.
The application method for the kit that the present embodiment is provided, it is specific as follows：
Sample to be tested RNA extraction (experiment agents useful for same and consumptive material are RNase-free), specific method is as follows：
1.1 take 1mL person under test's serum samples, and 5000rpm centrifugation 10min abandon supernatant, add 1mL Trizol solution weights
It is outstanding；
The concussion of 1.2 whirlpools is mixed, and room temperature places 5-10min；
1.3 at 4 DEG C, and 13000g centrifugation 15min take 900 μ L of supernatant into new centrifuge tube, add 500 μ L chloroform whirlpools
Mix；
1.4 at 4 DEG C, and 13000g centrifugation 15min take 500 μ L of supernatant into new centrifuge tube, add the shake of 500 μ L isopropanols
Mixing is swung, room temperature places 10min；
1.5 at 4 DEG C, and 13000g centrifugation 15min abandon supernatant, precipitation washed with 75% ethanol 3 times, abandon ethanol, room temperature is dried in the air
Dry 5min；
1.6 add 50 μ L RNase-free water dissolving precipitation, obtain sample rna solution.
The quality of the RNA solution of Detection and Extraction, determines OD260/OD280 values, obtains the RNA extracted concentration and purity,
The RNA of Detection and Extraction simultaneously integrality.If the RNA extracted has DNA pollution, sample can be handled by DNase.
Pcr amplification reaction
React total system：42μL；Add PCR reaction buffers 5 μ L, dNTP (2mM each) 5 μ L, base sequence such as SEQ
Primer segments (5 μM) each 1.25 μ L shown in ID No.1-6, upstream and downstream sequence (50 μM) 1.25 μ L, the Taq DNA of universal primer
The μ L of polymerase 0.5, the μ L of reverse transcriptase 1；Reaction system is supplied to 42 μ L with ddH2O.
PCR response procedures：
50 DEG C of reverse transcription 30min；95 DEG C, pre-degeneration 15min；95 DEG C of denaturation 30s, 62 DEG C of renaturation 45s, 72 DEG C of extension 30s,
10 circulations；95 DEG C of denaturation 30s；52 DEG C of renaturation 30s；72 DEG C of extension 30s, 30 circulations.
Embodiment 5
The present embodiment provides a kind of detection kit for detecting encephalitis viruses, and kit contains the detection of the offer of embodiment 1
The primer combination of encephalitis viruses.
Also include at least one of PCR reaction buffers, Taq archaeal dna polymerases, dNTPs and reverse transcriptase.
PCR reaction buffers include TrisHCl, KCl, MgSO4 and (NH4) 2SO4.
Also include RNA extracts reagents, RNA extracts reagents include DNase solution, absolute ethyl alcohol and RNase solution.
The application method reference implementation example 4 for the kit that the present embodiment is provided.
PCR response procedures are：51 DEG C of reverse transcription 30min；96 DEG C, pre-degeneration 15min；96 DEG C of denaturation 30s, 63 DEG C of renaturation
45s, 73 DEG C of extension 30s, 10 circulations；96 DEG C of denaturation 30s；53 DEG C of renaturation 30s；73 DEG C, extend 30s, 30 circulations.
Embodiment 6
The present embodiment provides a kind of detection kit for detecting encephalitis viruses, and kit contains the detection of the offer of embodiment 1
The primer combination of encephalitis viruses.
Also include at least one of PCR reaction buffers, Taq archaeal dna polymerases, dNTPs and reverse transcriptase.
PCR reaction buffers include TrisHCl, KCl, MgSO4 and (NH4) 2SO4.
Also include RNA extracts reagents, RNA extracts reagents include DNase solution, absolute ethyl alcohol and RNase solution.
The application method reference implementation example 4 for the kit that the present embodiment is provided.
PCR response procedures are：51 DEG C of reverse transcription 30min；96 DEG C, pre-degeneration 15min；96 DEG C of denaturation 30s, 64 DEG C of renaturation
45s, 73 DEG C of extension 30s, 10 circulations；96 DEG C of denaturation 30s；54 DEG C of renaturation 30s；73 DEG C, extend 30s, 30 circulations.
Embodiment 7
The present embodiment provides a kind of detection kit for detecting encephalitis viruses, and kit contains the detection of the offer of embodiment 1
The primer combination of encephalitis viruses.
Also include at least one of PCR reaction buffers, Taq archaeal dna polymerases, dNTPs and reverse transcriptase.
PCR reaction buffers include TrisHCl, KCl, MgSO4 and (NH4) 2SO4.
Also include RNA extracts reagents, RNA extracts reagents include DNase solution, absolute ethyl alcohol and RNase solution.
The application method reference implementation example 4 for the kit that the present embodiment is provided.
PCR response procedures are：51 DEG C of reverse transcription 30min；96 DEG C, pre-degeneration 15min；96 DEG C of denaturation 30s, 65 DEG C of renaturation
45s, 72 DEG C of extension 30s, 10 circulations；96 DEG C of denaturation 30s；55 DEG C of renaturation 30s；73 DEG C, extend 30s, 30 circulations.
Embodiment 8
The present embodiment provides a kind of detection kit for detecting encephalitis viruses, and kit contains the detection of the offer of embodiment 1
The primer combination of encephalitis viruses.
Also include at least one of PCR reaction buffers, Taq archaeal dna polymerases, dNTPs and reverse transcriptase.
PCR reaction buffers include TrisHCl, KCl, MgSO4 and (NH4) 2SO4.
Also include RNA extracts reagents, RNA extracts reagents include DNase solution, absolute ethyl alcohol and RNase solution.
The application method reference implementation example 4 for the kit that the present embodiment is provided.
PCR response procedures are：51 DEG C of reverse transcription 30min；96 DEG C, pre-degeneration 15min；96 DEG C of denaturation 30s, 66 DEG C of renaturation
45s, 72 DEG C of extension 30s, 10 circulations；96 DEG C of denaturation 30s；56 DEG C of renaturation 30s；72 DEG C, extend 30s, 30 circulations.
Embodiment 9
The present embodiment provides a kind of detection kit for detecting encephalitis viruses, and kit contains the detection of the offer of embodiment 1
The primer combination of encephalitis viruses.
Also include at least one of PCR reaction buffers, Taq archaeal dna polymerases, dNTPs and reverse transcriptase.
PCR reaction buffers include TrisHCl, KCl, MgSO4 and (NH4) 2SO4.
Also include RNA extracts reagents, RNA extracts reagents include DNase solution, absolute ethyl alcohol and RNase solution.
The application method reference implementation example 4 for the kit that the present embodiment is provided.
PCR response procedures are：51 DEG C of reverse transcription 30min；96 DEG C, pre-degeneration 15min；96 DEG C of denaturation 30s, 67 DEG C of renaturation
45s, 72 DEG C of extension 30s, 10 circulations；96 DEG C of denaturation 30s；57 DEG C of renaturation 30s；73 DEG C, extend 30s, 30 circulations.
Embodiment 10
The present embodiment provides a kind of detection kit for detecting encephalitis viruses, and kit contains the detection of the offer of embodiment 1
The primer combination of encephalitis viruses.
Also include at least one of PCR reaction buffers, Taq archaeal dna polymerases, dNTPs and reverse transcriptase.
PCR reaction buffers include TrisHCl, KCl, MgSO4 and (NH4) 2SO4.
Also include RNA extracts reagents, RNA extracts reagents include DNase solution, absolute ethyl alcohol and RNase solution.
The application method reference implementation example 4 for the kit that the present embodiment is provided.
PCR response procedures are：51 DEG C of reverse transcription 30min；96 DEG C, pre-degeneration 15min；96 DEG C of denaturation 30s, 68 DEG C of renaturation
45s, 73 DEG C of extension 30s, 10 circulations；96 DEG C of denaturation 30s；58 DEG C of renaturation 30s；72 DEG C of extension 30s, 30 circulations.
Experimental example 1
The specificity for the kit that this experimental example is provided embodiment 4 is detected.
Sample to be checked includes：
Positive strain：Japanese encephalitis virus (37010411333), mankind's enteric virus71 type (0311S035F), herpe simplex
Positive sample (20120403).
Negative strain：11 plants of negative control virus strain：The type HFRSV (Z10) of hemorrhagic fever with renal syndrome virus I, syndrome goes out
The type HFRSV (Z37) of fever virus II, yellow fever ((54) Tiantan Bio-pharmaceuticals 0239 in 2006), coxsackie virus A 16
(2511S034F), Flu-A (H1N1) (SD11716) virus, rotavirus (0411F008F), H1N1
(SDSWL92), Flu-A H3 (SDTQ1188) (BY_SDTQ1100), influenza (BV_SDZQ1171).
All virus titers are all higher than or equal to 10 in this test4TCID50mL-1。
Sample rna is extracted and PCR reaction systems and response procedures reference implementation example 4.
As shown in figure 1, swimming lane 1-3 is encephalitis B virus in figure, swimming lane 4-6 is mankind's enteric virus71, swimming lane 7-9 herpe simplexes
Virus, swimming lane 10 is Marker (100bp Ladder), and swimming lane 11-13 is encephalitis B virus, enterovirus and herpes simplex virus
RNA mixed liquors, swimming lane 14 is negative control.Swimming lane 15 is Hantaan virus, and swimming lane 16 is yellow fever virus, and swimming lane 17 is Flu-A
H1 viruses, swimming lane 18 is Flu-A H1N1 viruses, and swimming lane 19 is Flu-A H3 viruses, and swimming lane 20 is Type B influenza virus
(Yamagata), swimming lane 21 is Type B influenza virus (Victoria), and swimming lane 22 is rotavirus, swimming lane 23 be Hep2 cells,
Mdck cell and RD mixing with cells RNA solutions.Marker (100bp Ladder) is followed successively by from down to up：100bp、200bp、
300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 1000bp and 1500bp.
As a result show that the kit that embodiment 4 is provided can effectively identify positive strain japanese encephalitis virus
(37010411333), mankind's enteric virus71 type (0311S035F), herpe simplex positive sample (20120403), and 11 plants of feminine genders
Strain testing result is feminine gender.
It can therefore be seen that the kit that embodiment 4 is provided has preferable specificity.
Experimental example 2
This experimental example provides the sensitivity of the checking kit that embodiment 4 is provided.
It is 10 to choose concentration4TCID50mL-1Encephalitis B virus, enterovirus and herpes simplex virus strain, extract RNA
(extracting method reference implementation example 4), then adjusts concentration to 20ng/ μ L；Then diluted, diluted respectively with 10 times of gradient concentrations
102、103With 104Times.Enter performing PCR reaction (reaction system and response procedures reference implementation example 4)；After reaction terminates, take 5 μ L's anti-
Product is answered to enter row agarose gel electrophoresis.
As a result as shown in Fig. 2 in figure, swimming lane 1 represents negative control, and swimming lane 2 represents encephalitis B virus, enterovirus and simple
Herpesviral Sample Dilution 104Mixing sample again, swimming lane 3 represents encephalitis B virus, enterovirus and herpes simplex virus sample
Dilution 103Mixing sample again, swimming lane 4 represents encephalitis B virus, enterovirus and herpes simplex virus Sample Dilution 102Times it is mixed
Sample is closed, swimming lane 5 represents Marker, and swimming lane 6 represents herpes simplex virus Sample Dilution 104Times, swimming lane 7 represents herpe simplex
Virus Sample dilution 103Times, swimming lane 8 represents herpes simplex virus Sample Dilution 102Times, swimming lane 9 represents enterovirus sample
Dilution 104Times, swimming lane 10 represents enterovirus Sample Dilution 103Times, swimming lane 11 represents enterovirus Sample Dilution 102Times
, swimming lane 12 represents encephalitis B virus Sample Dilution 104Times, swimming lane 13 represents encephalitis B virus Sample Dilution 103Times, swimming lane 14
Represent encephalitis B virus Sample Dilution 102Times；Wherein Marker is with experimental example 1.
As can be seen that enterovirus, herpes simplex virus are in dilution 104After times；I.e. concentration reaches 2pg/ μ L, still can
Enough detect；And encephalitis B virus also can still be detected in 20pg/ μ L concentration.
As can be seen that the kit that the embodiment of the present invention 4 is provided has higher sensitivity.
Experimental example 3
1102 parts of actual samples that this experimental example is collected to 2011-2013 detect that testing result statistics is shown in Table 1.
The actual sample testing result of table 1
As it can be seen from table 1 total positive 236 parts of the detection of actual sample, positive rate 21.05%, cerebrospinal fluid herpe simplex
Virus and the double positives 18 of enterovirus.The sensitivity of this detection method is 92.5%, wherein the positive mark of 104 parts of encephalitis B virus
This testing result is 94 parts of positives, 8 parts of feminine genders；The testing result of 87 parts of samples of herpe simplex is 80 parts of positives, 7 parts of feminine genders；
42 parts of positive testing results of enterovirus are 40 parts of positives, 2 parts of feminine genders；22 parts of sun of herpe simplex and enterovirus concurrent infection
Property, testing result is 18 parts of positives, 4 parts of feminine genders.
In the test, the specificity of this detection method is 100%, and the testing result of 847 parts of ' negative ' specimens is all negative,
Non-false positive result.
Checkout and diagnosis accuracy rate is 98.27%.In the inspection of all 847 non-encephalitis, herpe simplex and enterovirus sample
Equal non-false positive amplification in survey, it was demonstrated that the high degree of specificity of this method.The negative predictive value (NPV) of this detection method is
97.8%, positive predictive value (PPV) is 100%.
In summary, the primer combination for the detection encephalitis viruses that the present invention is provided is with preferably specific and higher spirit
Sensitivity, the kit for being applied to detection encephalitis viruses is combined using the primer equally also with preferably specific and higher spirit
Sensitivity；The kit is convenient to be extracted Virus Sample and is identified, with higher practicality and higher application value.
Embodiments described above is a part of embodiment of the invention, rather than whole embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
SEQUENCE LISTING
<110>Jinan Disease Prevention and Control Centre
<120>A kind of primer combination for detecting encephalitis viruses and application, kit
<160> 8
<170> PatentIn version 3.5
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aggtgacact atagaataga tgagtcaccg cattcc 36
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gtacgactca ctatagggac acaccacgtc cgtattag 38
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aggtgacact atagaataga gaatggcata gtcttgga 38
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aggtgacact atagaatatg ccttcaaccg aatatgt 37
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Claims (10)
1. a kind of primer combination for detecting encephalitis viruses, it is characterised in that the primer combination includes detecting the 1st of enterovirus
Primer pair, the 2nd primer pair for detecting encephalitis B virus, at least one of the 3rd primer pair for detecting herpes simplex virus and logical
Use primer pair；The base sequence of the 1-3 primer pairs is as shown in SEQ ID No.1-6；The base sequence of the universal primer pair
Row are as shown in SEQ ID No.7-8.
2. it is according to claim 1 detection encephalitis viruses primer combination, it is characterised in that the 1-3 primer pairs and
The universal primer is to including sense primer and anti-sense primer；The upstream primer sequence of the 1-3 primer pairs includes described logical
With the upstream primer sequence of primer, the downstream primer sequence of the 1-3 primer pairs includes the anti-sense primer of the universal primer
Sequence.
3. application of the primer combination as claimed in claim 1 or 2 in the kit for preparing detection encephalitis viruses.
4. a kind of kit for detecting a variety of encephalitis viruses, it is characterised in that the kit is included such as the institute of claim 1 or 2
The primer combination stated.
5. kit according to claim 4, it is characterised in that also including PCR reaction buffers, Taq archaeal dna polymerases,
At least one of dNTPs and reverse transcriptase.
6. kit according to claim 5, it is characterised in that the PCR reaction buffers include TrisHCl,
KCl、MgSO4(NH4)2SO4。
7. kit according to claim 5, it is characterised in that also including viral RNA extracts reagent.
8. kit according to claim 7, it is characterised in that the RNA extracts reagents include DNase solution, anhydrous
Ethanol, isopropanol and chloroform.
9. application of the kit in detection viral RNA as described in claim any one of 4-8.
10. application according to claim 9, it is characterised in that including using sample to be checked as template, entering performing PCR reaction；
PCR response procedures are：50-51 DEG C of reverse transcription 30min；95-96 DEG C, pre-degeneration 15min；95-96 DEG C of denaturation 30s, 62-68
DEG C renaturation 45s, 72-73 DEG C of extension 30s, 10 circulations；95-96 DEG C, it is denatured 30s；52-58 DEG C of renaturation 30s；72-73 DEG C, extension
30s, 30 circulations.
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| CN106167833A (en) * | 2016-05-20 | 2016-11-30 | 南方医科大学 | A kind of detection RT PCR primer of 8 kinds of entomophila encephalitises simultaneously and probe combinations and test kit |
| CN106868218A (en) * | 2017-03-30 | 2017-06-20 | 苏州大学 | For the multiple PCR reagent kit of viral encephalitis quick diagnosis |
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| CN102352411A (en) * | 2011-09-29 | 2012-02-15 | 苏州大学 | Multiplex polymerase chain reaction (PCR) amplification method and reagent kit |
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| CN106167833A (en) * | 2016-05-20 | 2016-11-30 | 南方医科大学 | A kind of detection RT PCR primer of 8 kinds of entomophila encephalitises simultaneously and probe combinations and test kit |
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Application publication date: 20171027 |