CN101942526B - Method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and kit - Google Patents
Method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and kit Download PDFInfo
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Abstract
The invention discloses a method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and a kit. A primer and a TaqMan probe is designed in accordance with the conserved region of the epidemic encephalitis B virus E gene, which is used for quantitatively detecting nucleic acid copy number of the epidemic encephalitis B virus in samples. Particularly, the forward primer of the primer has a nucleotide sequence of SEQ ID NO: 1 in a sequence table, the reverse primer of the primer has a nucleotide sequence of SEQ ID NO: 2 in the sequence table, and TaqMan probe has a nucleotide sequence in SEQ ID NO: 3 in the sequence table. The invention has larger valuable significance in the field of controlling the quality of biological products related to the human and animal sources and inspecting and quarantining the imported and exported animals, ensures the quality of the related biological products and controls the spread of the epidemic encephalitis B and the medication safety of people. The detection method and the kit of the invention can be used for inspecting the epidemic encephalitis B virus on cerebrospinal fluid samples of the clinical patients, miniature pig brain tissue samples and the biological products, and have wide application prospect.
Description
Technical field
The present invention relates to the molecular biology for detection of virus in the biological technical field, particularly relate to real-time fluorescence quantitative PCR detection method and the test kit of encephalitis b virus.
Background technology
Epidemic encephalitis B virus at first (nineteen fifty-three) from patient's cerebral tissue, separate acquisition in Japan, therefore be called japanese encephalitis virus (Japanese encephalitis virus, JEV), associated diseases claims Japanese encephalitis (Japanese encephalitis, JBE) in Japan.Nineteen fifty, China has carried out a large amount of etiologies and epidemiological study to this disease, in order to distinguish with encephalitis A, to name and is epidemic encephalitis type B (Japaneseencephalitis), is called for short encephalitis.According to the relevant regulations of China's Law on the Prevention and Control of Infectious Diseases, encephalitis belongs to Category B notifiable disease in China.Encephalitis is a kind of acute infectious disease of central nervous system infection, also is a kind of disease of natural focus of Zoonosis, clinically take high heat, the disturbance of consciousness, tic, respiratory insufficiency and meningeal irritation sign as feature.Encephalitis is the modal a kind of viral encephalitis in Asia.According to estimates, encephalitis b virus causes 50,000 routine clinical cases every year at least, and wherein majority is children below 10 years old, causes approximately 10,000 people dead, and other leaves long-term nerve-spirituality sequela at 15, the 000 routine cases of having an appointment.In recent decades, encephalitis had occurred breaking out without endemoepidemic area before some.Mosquito is the main communication media of JEV.In animal, pig is most important intermediate host, and encephalitis b virus is had amplification effect, makes infection and encephalitis B virus infection be pig-mosquito-people's chain.Although without clinical symptom, pregnant sow can produce stillborn foetus, mummy tire and weak son to Adult Pig after infecting encephalitis b virus.Most people shows as inferior clinical symptom after infecting encephalitis b virus, but children can cause the lethality encephalitis, the pregnant woman then can cause miscarriage (Chaturvedi MC, Mather A, Chandra A, et al.Transplacental infection withJapanese encephalitis virus.J Infect Dis, 1980,141,712-714.Endy TP, Nisalak A.Japanese encephalitis virus:ecology and epidemiology.Curr TopMicrobiol Immunol, 2002,267:11-48.).According to " People's Republic of China's animal epidemic prevention method " relevant regulations, the Ministry of Agriculture has formulated " infectious diseases common to human beings and animals register " together with sanitation Ministry tissue, and implements on January 19th, 2009.Pig japanese b encephalitis (https://www.ivdc.gov.cn/gg/200902/t20090209_31549.htm[2009-02-04]) is namely arranged in " infectious diseases common to human beings and animals register ".
Encephalitis b virus belongs to flaviviridae (Flaviviridae) Flavivirus (Flavivir μ s) member in classification.The genome of encephalitis b virus is the sub-thread positive chain RNA, and total length is 11kb approximately, and gene structure is that 5 ' end has the non-coding region of 95 Nucleotide to follow the coding region of one section 10 296 Nucleotide, is the non-coding region of 3 of 585 Nucleotide ' end subsequently.Genome only has an opening code-reading frame, the polyprotein precursor that its coding is comprised of 3 432 amino acid, this polyprotein precursor is in host cell, in the proteolytic enzyme (NS3) of virus self coding and born of the same parents under other enzyme effect, form 3 structural protein (C, pre M/M, E) and 7 Nonstructural Protein (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5), C wherein, pre M/M and the E gene capsid/core protein (capsid protein) of encoding respectively, albumen cyst membrane precursor protein/envelope protein (premembrane/membraneprotein) and E membrane glycoprotein (envelope glycoprotein).C albumen and geneome RNA consist of the nucleocapsid of virus, and virus is had certain provide protection.Membranin M is relevant with assembling, the maturation of virus.E albumen is the most important composition in virion surface; the surface antigen determinant that is formed by it has hemagglutination activity and neutralization activity; can stimulate body to produce neutralizing antibody; the protection body is avoided virus attack, participate in virus absorption, penetrate, cause a disease and closely related with the immunne response of inducing the host.E protein gene size is 1500bp, 500 amino-acid residues of encoding, and E albumen occupies critical positions in the encephalitis b virus virulence, and the simple point mutation of E gene can make encephalitis b virus lose its neurovirulence/invasiveness.The E district of SA14-14-2 attenuated strain strain SA14 maternal with it street strain of encephalitis b virus relatively has 8 amino-acid residue differences, particular location is at E107, E138, E176, E177, E264, E279, E315 and E439 position, key amino acid residue site (the Arroyo J of virulence attenuation of, Guirakhoo F, Fenner S, et al.Molecular basis for attenuation ofneurovirulence of a Yellow fever virus/Japanese encephalitis virus chimeravaccine (ChimeriVaxJE) .J Virol, 2001,934-942.Tsarev SA, Sanders ML, Va μ ghn DW, et al.Phylogenetic analysis suggests only one serotype ofJapanese encephalitis virus.Vaccine, 2000,18,36-43.Mchil PD, Satchidanandam V.Phylogenetic analysis of Japanese encephalitis virus:envelope gene based analysis reveals a fifth genotype, geographicclustering, and multiple introductions of the virus into the Indiansubcontinent.Am J Trop Med Hyg, 2001,65,242-251.).
The detection of encephalitis b virus mainly depends on ELISA (enzyme linked immunosorbent assay) test kit, therefore uses comparatively extensive.On the other hand, behind the infection and encephalitis B virus infection, viremia phase and IgM antibody occur also asynchronous in time, and the result who relies on the serology antibody test fully will certainly omit the sample of quite a few simple viral nucleic acid positive.
1985, U.S. Cetus company and University of California united and have created a kind of Progress of Nucleic Acid Amplification Technologies-polymerase chain reaction (polymerase chain reaction, PCR), and its principle is similar in the body of DNA and copies.Soon, PE-Cetus company has released first PC R thermal cycler, and the automatization of round pcr is become a reality.Because the popularity of the high susceptibility that has of round pcr, specificity and easy and simple to handle, applicable sample, so the tempo of it and correlation technique thereof is surprising, and be widely used in various fields.
The people such as Gao Zhengqin have set up the encephalitis b virus Nested RT-PCR Diagnostic Method, be used for the detection of animal derived sample and laboratory cell culture and virus liquid commonly used, demonstrate good specificity and susceptibility, to guaranteeing the significant (Gao Zhengqin of safety of associated biomolecule goods, He Zhengming, Xing Ruichang, etc. the foundation of encephalitis b virus-Nested RT-PCR Diagnostic Method and Preliminary Applications. Chinese Amphixenosis's magazine .2005,21 (4): 7-10.).
Adopt the method for nucleic acid amplification to detect viral RNA, present stage comparative maturity have following several: common reverse transcription PCR, nucleotide sequence rely on amplification (NASBA), reverse transcription real-time fluorescence PCR, reverse transcription loop-mediated isothermal amplification (LAMP) etc.Yet, the method that these are set up, quite a few is for cell culture and virus liquid, seldom directly method is applied to the lower clinical sample of virus load and detects.
The advantages such as in recent years, Real-Time Fluorescent Quantitative PCR Technique is highly sensitive with it, speed fast, high specificity are used widely at the aspects such as quantitative and qualitative analysis detection of gene expression dose analysis, sudden change and polymorphism research, pathogenic agent.Quantitative fluorescent PCR definition: refer in the PCR reaction system, add fluorophor, utilize the variation of fluorescent signal to detect in real time the variation of each cyclic amplification product amount in the pcr amplification reaction, pass through C
TValue and typical curve are realized the quantitative analysis to starting template.
Probe method real-time fluorescence quantitative PCR principle: in system, contain during pcr amplification on the basis of primer and add again a specific fluorescent probe, this probe is an oligonucleotide, and two ends are mark report fluorophor (R) and a cancellation fluorophor (Q) respectively.When probe was complete, the fluorescent energy that reporter group is launched was absorbed by quenching group, and instrument can't detect signal.Along with the carrying out of PCR, the Taq enzyme runs into the probe of being combined with template in the chain extension process, and its 5 ' → 3 ' exonuclease activity will cut off probe, and reporter group is away from quenching group, and its energy can not be absorbed, and namely produces fluorescent signal.Have not yet to see the detection by quantitative that the probe method Real-Time Fluorescent Quantitative PCR Technique is used for clinical patient and miniature pig sample encephalitis b virus.
Therefore, in order to realize the detection by quantitative of encephalitis b virus in the clinical sample, analyze the infection and encephalitis B virus infection situation of humans and animals, further improve the sensitivity that detects, set up the probe method real-time fluorescence quantitative PCR detection method of encephalitis b virus, and develop corresponding detection reagent and become very urgent.
Summary of the invention
The invention provides the primer and the TaqMan probe that detect for encephalitis b virus being carried out real-time fluorescence quantitative PCR.
Primer provided by the present invention and TaqMan probe are according to the design of the conservative region of encephalitis b virus E gene conservative region, in order to the nucleic acid copy number of encephalitis b virus in the detection by quantitative sample.
Specifically, the upstream primer of described primer has the nucleotide sequence of SEQ ID NO:1 in the sequence table, and downstream primer has the nucleotide sequence of SEQ ID NO:2 in the sequence table; Described TaqMan probe has the nucleotide sequence of SEQ ID NO:3 in the sequence table.
Described TaqMan probe is fluorescently-labeled for process, and its 5 ' end is marked with the report fluorophor, and 3 ' end is marked with the cancellation fluorophor.
Described report fluorophor is FAM, and the fluorescent quenching group is NFQ.
But second purpose of the present invention provides the real-time fluorescence quantitative PCR detection method of the encephalitis b virus of the higher and accurate quantitative analysis of a kind of sensitivity.
Detection method provided by the present invention is the real-time fluorescence quantitative PCR detection method that carries out with primer of the present invention and TaqMan probe.
Described 20ul real-time fluorescence quantitative PCR reaction system can comprise: TaqMan probe+primer 1ul (TaqMan concentration and probe concentration 250nM, the upstream and downstream primer concentration respectively is 900nM), sample cDNA 1ul (1 μ g/mL), without RNA enzyme water 8ul, Taqman Mix (TaqMan Gene Expression Master Mix 4369016 is available from U.S.'s application system (ABI) company) 10ul.
Described real-time fluorescence quantitative PCR reaction conditions can be: 50 ℃ of 2min of elder generation; Then 95 ℃ of 10min; Last 95 ℃ of 15s, 60 ℃ of 1min, totally 40 circulations are carried out fluorescent signal and are detected when the extension of each circulation finishes.
Described detection method also comprises the foundation of typical curve, and method is:, 1. its 10 times of serial dilutions are become: 8.86 * 10 as standard substance with the plasmid that contains the purpose fragment
9Copy/μ l; 2.: 8.86 * 10
8Copy/μ l; 3.: 8.86 * 10
7Copy/μ l; 4.: 8.86 * 10
6Copy/μ l; 5.: 8.86 * 10
5Copy/μ l; 6.: 8.86 * 10
4Copy/μ l; 7.: 8.86 * 10
3Copy/μ l; 8.: 8.86 * 10
2Copy/μ l; 9.: 8.86 * 10
1Copy/μ l.Plasmid is carried out real-time fluorescence quantitative PCR as template detect, obtain the typical curve that detects for encephalitis b virus.
The 20ul real-time fluorescence quantitative PCR reaction system of described typical curve comprises: TaqMan probe+primer 1ul (TaqMan concentration and probe concentration 250nM, the upstream and downstream primer concentration respectively is 900nM), plasmid DNA 1ul (0.0003219mg/mL), without RNA enzyme water 8ul, Taqman Mix (TaqMan Gene Expression Master Mix 4369016 is available from U.S.'s application system (ABI) company) 10ul; The real-time fluorescence quantitative PCR reaction conditions is same as described above.
The 3rd purpose of the present invention provides a kind of test kit for encephalitis b virus being carried out the real-time fluorescence quantitative PCR detection.
Test kit provided by the present invention comprises above-mentioned primer and TaqMan probe for encephalitis b virus being carried out the real-time fluorescence quantitative PCR detection.
The invention provides a kind of real-time fluorescence quantitative PCR detection method and primer special and TaqMan probe of encephalitis b virus.The method be encephalitis b virus nucleic acid in the sample to be checked as detected object, accuracy and precision are all better.The present invention has the following advantages:
1, set up first encephalitis b virus probe method real-time fluorescence quantitative PCR detection method, utilize this detection method both can the encephalitis b virus in clinical patient cerebrospinal fluid and the miniature pig cerebral tissue to be detected, also can the encephalitis b virus in the biological products be detected.
2, this detection method is compared with nested PCR with other conventional sense method of encephalitis b virus such as separation and Culture evaluation, the totivirus ELISA method of virus, sensitivity is higher, have sampling convenient, detection efficiency is greatly improved, and has effectively prevented pollution.
3, other conventional sense method of this detection method and encephalitis b virus, separation and Culture such as virus is identified, totivirus ELISA method is compared with nested PCR, has the advantages that schedule of operation is simple and easy to usefulness, and can further make detection kit, programming manipulation suits large area to popularize and uses.
4. this detection method not only can the appraiser and the infection and encephalitis B virus infection situation of pig, also provides foundation for the research of the association areas such as the epidemiology of encephalitis b virus, pathogenesis simultaneously.
5. this detection method also can be used for the pollution monitoring of exogenous encephalitis b virus in the cell culture commonly used of animal derived biological products and laboratory, provides effective tool for the security of biological products detects.
In sum, the present invention can detect the encephalitis b virus of people's (such as clinical patient CSF sample) and miniature pig (such as miniature pig cerebral tissue sample) quickly and accurately, and is significant to the quality and the human health that ensure Experimental Miniature Pig; The present invention also can be used for the pollution monitoring of exogenous encephalitis b virus in animal derived biological products and the laboratory cell culture commonly used, has larger practical significance to having in the quality of control humans and animals source property associated biomolecule goods and the Imported and exported animals inspection and quarantine field, can guarantee the quality of associated biomolecule goods, the control encephalitis b virus is propagated and people's drug safety.Detection method of the present invention and test kit can be used for the diagnosis of encephalitis and the encephalitis b virus calibrating of biological products, have a extensive future.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is that the encephalitis b virus probe method real-time fluorescence quantitative PCR of 10 times of serial dilution pMD-JEV-E plasmid standards detects amplification curve
Fig. 2 is the typical curve that the real-time fluorescence quantitative PCR of encephalitis b virus detects
Fig. 3 is that encephalitis b virus probe method real-time fluorescence quantitative PCR detects 23 this amplified fluorescence of increment curves
Fig. 4 is the specific amplified fluorescence curve of encephalitis b virus probe method Real-Time Optical quantitative PCR detection
Embodiment
Following embodiment only is not used in for explanation the present invention and limits the scope of the invention.The experimental technique of actual conditions is indicated at the end in the following example, usually compiles the condition described in " molecular cloning experiment guide " according to people such as normal condition such as sambrook, or the condition of advising according to manufacturer.The primer and TaqMan probe are synthetic by U.S.'s application system (ABI) company, and the work of all sequences mensuration is finished by precious biotechnology (Dalian) company limited.
The encephalitis b virus SA14 genom sequence (GeneBank:U14163) of including with reference to GenBank, to carry out respectively sequence alignment to very similar dengue virus and the west Nile virus of encephalitis b virus genome structure, select E gene conservative region, adopt ABI Primer Express 3.0 real-time fluorescence quantitative PCR primer-design softwares, the synthetic TaqMan probe of design and primer.The fluorescent mark of probe selects FAM (5 ' end) as the report luminophore, and NFQ (3 ' end) is quenching group.Sequence is as follows:
Upstream primer (JEV-1): 5 '-GAGAAACAGAGAACTCCTCATGGAA-3 ' (SEQ ID NO:1);
Downstream primer (JEV-2): 5 '-CTGTGACCCAAGAGCAACAAC-3 ' (SEQ ID NO:2 in the sequence table).
TaqMan probe: 5 ' FAM-CACGCCACAAAACAG-NFQ 3 ' (SEQ ID NO:3 in the sequence table).
The real-time fluorescence quantitative PCR of embodiment 2, encephalitis b virus detects
One, the foundation of typical curve
1, makes up the pMD-JEV-E recombinant plasmid
1.1 goal gene-encephalitis b virus E gene cloning
1.1.1 the extraction of encephalitis b virus RNA
Below operation must be carried out in P2 Lab Nagative pressure bio-safty cabinet in strict accordance with requiring.
(1) under the aseptic condition sample 50mg of encephalitis animal brain or encephalitis patient cerebrospinal fluid 500 μ L or encephalitis b virus liquid 300 μ L are inserted the clean centrifuge tube of 1.5mL, add 500 μ L Trizol Reagents (available from U.S. Promega company), fully vibration, room temperature leaves standstill 5min.
(2) add 100 μ L chloroforms, use forced oscillation 10sec, room temperature leaves standstill 5min, 4 ℃, the centrifugal 10min of 12000r/min.
(3) carefully that the upper water phase transition is secondary to another clean centrifuge tube, add the equal-volume Virahol, abundant mixing, the centrifugal 10min of 12000r/min.
(4) abandon supernatant, add 500 μ L, 75% ethanol (using without the fresh preparation of RNA enzyme water), abundant mixing, the centrifugal 10min of 12000r/min carefully draws most of ethanol.With centrifuge tube uncovered in air at room temperature dry 10min wait for that the ethanol volatilization is clean.
(5) adding 50 μ L precipitates without RNA enzyme water dissolution.
(6) measure gained RNA concentration (concentration is 1 μ g/mL) with the uv-spectrophotometric instrument, carry out immediately reverse transcription or place-70 ℃ of preservations.
Encephalitis b virus RNA also can obtain from other commercial channel, or extracts with other method that open source literature is introduced.
1.1.2 reverse transcription
The reaction system of reverse transcription is 20 μ L, adds successively following ingredients: 4 μ L MgCl
2(25mM), 2 μ LReverse Transcription, 10 * Buffer (Reverse Transcription System (reverse transcription system, available from U.S. Promega company), 2 μ L dNTP Mixture (10mM), 0.5 μ L RecombinantRNasin Ribonuclease inhibitor ((restructuring) ribonuclease inhibitor, available from U.S. Promega company) (40unit/ μ L), 0.5 μ L random primer (50pmol, Reverse Transcription System (reverse transcription system), available from U.S. Promega company), 0.1 μ L AMV ThermoScript II (15unit/ μ L, Reverse Transcription System (the reverse transcription system is available from U.S. Promega company), 6 μ L RNA, 4.9 μ l is without RNA enzyme water.
The reverse transcription reaction condition is: room temperature 10min, 42 ℃ of 15min, 95 ℃ of 5min, 4 ℃ of 5min.Gained cDNA namely can be used as the PCR reaction template.
1.1.3PCR amplification
The reaction system of PCR is 50 μ L, adds successively following ingredients: 5 μ L, 10 * EX Buffer (MgCl
2Plus, TaKaRa Ex Taq, available from precious biotechnology (Dalian) company limited), 4 μ L dNTP Mixture (10mM), 0.5 μ L forward primer: CGAAGTTGGCATTTTTGTGC (50pmol), the reverse primer of 0.5 μ L: GCTCGCAACGGAAACAATCG (50pmol), 0.25 μ L EX Taq (5unit/ μ L is available from precious biotechnology (Dalian) company limited), 5 μ L cDNA, 34.75 μ L are without RNA enzyme water.
The PCR reaction conditions is: 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 1min, totally 30 circulations.
1.1.4 agarose gel electrophoresis detects
Prepare 1% sepharose (containing 0.5 μ g/mL ethidium bromide).Get pcr amplification product 5 μ L, the 6 * load sample damping fluid mixing with 1 μ L is added in the gel pore lattice.Electrophoretic buffer is 1 * TAE damping fluid, and deposition condition is 110V constant voltage/30min, takes the record electrophoresis result under the gel imaging system.The result has obtained the purpose band of 622bp, reclaims and purifying with Agarose Gel DNA Purification Kit (sepharose reclaims test kit, available from precious biotechnology (Dalian) company limited).
1.2 the structure of encephalitis b virus E gene recombination plasmid
Encephalitis b virus E gene fragment is connected with pMD18-T carrier (available from precious biotechnology (Dalian) company limited), ligation system: 2 * Rapid Ligation Buffer, 5 μ L (precious biotechnology (Dalian) company limited test kit), pMD18-T carrier 0.5 μ L (concentration 50ng/ μ L), goal gene 4 μ L (45 μ g/mL), T4 DNA Ligase 0.5 μ L (5U/ μ L).Then will connect the product thermal transition to E.coliCompetent Cell JM109, coating LB is dull and stereotyped, 37 ℃ of incubated overnight.The selected single bacterium colony that contains positive colony is cultivated, extracted plasmid DNA, carry out enzyme with EcoR I and Hind III restriction enzyme and cut evaluation and order-checking.Enzyme is cut purpose band and the pMD18-T plasmid band that the result has obtained 622bp, conforms to expected results.Sequencing result shows and has obtained all correct recombinant plasmids that contains goal gene of sequence and on position, called after pMD-JEV-E.Measure plasmid DNA concentration, calculate copy number, copy number is 8.86 * 10 as a result
10Copy/μ L ,-40 ℃ of preservations.
2, standard substance detect
The pMD-JEV-E plasmid DNA is carried out encephalitis b virus probe method real-time fluorescence quantitative PCR as template detect the Criterion curve.Concrete operations are as follows: plasmid DNA is carried out 10 times of serial dilutions become 1.: 8.86 * 10
9Copy/μ L; 2.: 8.86 * 10
8Copy/μ L; 3.: 8.86 * 10
7Copy/μ L; 4.: 8.86 * 10
6Copy/μ L; 5.: 8.86 * 10
5Copy/μ L; 6.: 8.86 * 10
4Copy/μ L; 7.: 8.86 * 10
3Copy/μ L; 8.: 8.86 * 10
2Copy/μ L; 9.: 8.86 * 10
1Copy/μ L.Each extent of dilution repeats parallel test 3 times.
Standard substance detection reaction system is 20 μ l, add successively following ingredients: TaqMan probe+primer 1 μ L (TaqMan concentration and probe concentration 250nM, the upstream and downstream primer concentration respectively is 900nM)), plasmid DNA 1 μ L (0.0003219mg/mL), without RNA enzyme water 8 μ L, Taqman Mix (TaqMan Gene ExpressionMaster Mix 4369016 is available from U.S.'s application system (ABI) company) 10 μ L.
Standard substance detection reaction condition is: 50 ℃ of 2min of elder generation; Then 95 ℃ of 10min; Last 95 ℃ of 15s, 60 ℃ of 1min, totally 40 circulations are carried out fluorescent signal and are detected when the extension of each circulation finishes.
Standard substance probe method real-time fluorescence quantitative PCR amplification curve as shown in Figure 1 (1.: 8.86 * 10
9Copy/μ L; 2.: 8.86 * 10
8Copy/μ L; 3.: 8.86 * 10
7Copy/μ L; 4.: 8.86 * 10
6Copy/μ L; 5.: 8.86 * 10
5Copy/μ L; 6.: 8.86 * 10
4Copy/μ L; 7.: 8.86 * 10
3Copy/μ L; 8.: 8.86 * 10
2Copy/μ L; 9.: 8.86 * 10
1Copy/μ L.)。
3, the drafting of typical curve
According to gained C
TBe worth the logarithmic value drawing standard curve (Fig. 2) of the standard substance corresponding with it, the R square value of typical curve is 0.998, and the amplification efficiency of quantitative fluorescent PCR reaction is 99.022%.Typical curve shows: the encephalitis b virus probe method real-time fluorescence quantitative PCR detection method that the present invention sets up has the linear detection range of 9 orders of magnitude, further specifies this detection method and has very high sensitivity.
Two, encephalitis b virus probe method real-time fluorescence quantitative PCR detects
Carry out the detection of encephalitis b virus probe method real-time fluorescence quantitative PCR with the encephalitis patient cerebrospinal fluid cDNA of 1 part of clinical definite and the miniature pig cerebral tissue cDNA of 22 parts of encephalitis b virus positives as template.Every this repetition of increment parallel test 3 times.
The pattern detection system is 20 μ L, add successively following ingredients: (TaqMan concentration and probe concentration 250nM, the upstream and downstream primer concentration respectively is 900nM, sample cDNA 1 μ L (1 μ g/mL), without RNA enzyme water 8 μ L, TaqmanMix (TaqMan Gene Expression Master Mix 4369016 is available from U.S.'s application system (ABI) company) 10 μ L.
The detection reaction condition is: 50 ℃ of 2min of elder generation; Then 95 ℃ of 10min; Last 95 ℃ of 15s, 60 ℃ of 1min, totally 40 circulations are carried out fluorescent signal and are detected when the extension of each circulation finishes.
The probe method real-time fluorescence quantitative PCR detect to detect 23 these amplification curves of increment as shown in Figure 3, and the result shows: purpose amplified fluorescence curve all occurred take the miniature pig cerebral tissue cDNA of encephalitis patient cerebrospinal fluid cDNA, the encephalitis b virus positive as template.
The repeatability analysis of test one, encephalitis b virus probe method fluorescent quantitative PCR detection method
The repeated analytical procedure of encephalitis b virus probe method fluorescent quantitative PCR detection method is: after the DNA that will contain the recombinant plasmid pMD-JEV-E of encephalitis b virus E gene carries out respectively 10 times of serial dilutions, respectively get 1 μ L as template, adopting above-mentioned reaction system to carry out real-time fluorescence quantitative PCR detects, every this repetition of increment parallel test 3 times, divide two batches and carry out, test-results is got C
TMean value is by statistical computation C
TStandard deviation (SD), C
TThe variation coefficient (CV).The repeatability analytical results the results are shown in Table 1: its variation coefficient of replication CV value is less than 1.9% in batch, and replication CV value all illustrates having good stability of encephalitis b virus probe method fluorescent quantitative PCR detection method that the present invention sets up less than 0.8% between criticizing.
The replica test result of table 1 encephalitis b virus probe method fluorescent quantitative PCR detection method
The specificity of test two, encephalitis b virus probe method real-time fluorescence quantitative PCR detection method
Encephalitis b virus SA14-14-2 vaccine strain (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute) cDNA, encephalitis b virus Nakayama strain (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute) cDNA, BHK-21 cell (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute) cDNA are carried out the probe method real-time fluorescence quantitative PCR as template detect the specificity of evaluation response system.Reaction system and reaction conditions are identical with embodiment 2.Every this repetition of increment parallel test 2 times.
Probe method real-time fluorescence quantitative PCR detection specificity amplification curve as shown in Figure 4, the result shows: purpose amplified fluorescence curve occurred take encephalitis b virus-SA14-14-2 vaccine strain cDNA, encephalitis b virus-Nakayama strain cDNA as template, and purpose amplified fluorescence curve do not occurred take encephalomyelitis virus (GDVII) cDNA, BHK-21 cell cDNA as template.Presentation of results: the present invention is good for the specificity of the primer that encephalitis b virus is carried out the real-time fluorescence quantitative PCR detection and TaqMan probe, and quantitative reaction system specificity is good.
To detect JEV TaqMan assay mix 50 μ L (TaqMan concentration and probe concentration 250nM for encephalitis b virus being carried out real-time fluorescence quantitative PCR, the upstream and downstream primer concentration is respectively for 900nM), JEV TaqMan mix 500 μ L, JEV positive quality control product 50 μ L, negative quality control product 50 μ L and jointly pack without RNA enzyme water 500 μ L, obtain the real-time fluorescence quantitative PCR detection kit (50 secondary response) of encephalitis b virus.
Claims (2)
1. being used for encephalitis b virus is carried out primer and the TaqMan probe that real-time fluorescence quantitative PCR detects, is according to the conservative region design of encephalitis b virus E gene conservative region, in order to the nucleic acid copy number of encephalitis b virus in the detection by quantitative sample; The upstream primer of described primer is the Nucleotide shown in the SEQ ID NO:1 in the sequence table, and downstream primer is the Nucleotide shown in the SEQ ID NO.2 in the sequence table; Described TaqMan probe is the Nucleotide shown in the SEQ ID NO:3 in the sequence table, and its 5 ' end is marked with report fluorophor FAM, and 3 ' end is marked with cancellation fluorophor NFQ.
2. one kind is used for encephalitis b virus is carried out the test kit that real-time fluorescence quantitative PCR detects, and comprises described primer and the TaqMan probe for encephalitis b virus being carried out the real-time fluorescence quantitative PCR detection of claim 1.
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| CN107937615B (en) * | 2017-12-20 | 2021-06-25 | 广东省农业科学院动物卫生研究所 | Primers and probes for distinguishing wild strains and vaccine strains of swine Japanese encephalitis virus |
| CN110438123A (en) * | 2019-08-22 | 2019-11-12 | 昆明市儿童医院 | A kind of encephalitis B virus standard items plasmid construction primer and its application |
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| CN1786190A (en) * | 2004-12-10 | 2006-06-14 | 中国疾病预防控制中心病毒病预防控制所 | Method of detecting cerebritis B virus and kit |
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