CN106399586B - Multiplex PCR detects encephalitis meningitis correlated virus primer sets and kit - Google Patents
Multiplex PCR detects encephalitis meningitis correlated virus primer sets and kit Download PDFInfo
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Abstract
The present invention provides multiplex PCRs to detect encephalitis correlated virus primer sets, wherein the primer sets include primer shown in SEQ ID NO.1 and 3-22, and the present invention also provides the detection methods and kit of multiplex PCR detection encephalitis correlated virus.Through the above technical solution, the present invention provides complete technological means for quick, the accurate screening of encephalitis correlated virus, it can be realized morphology, impossible quick, comprehensive, sensitive, special, the automatic result judgement of immunology and substance real-time fluorescence detection institute, provide reliable foundation for rationally appropriate disposition epidemic situation.
Description
Technical field
The present invention relates to field of biotechnology, and in particular, to multiplex PCR detects encephalitis meningitis correlated virus primer sets
With kit and its detection method.
Background technique
Nervous system injury caused by virus infection oneself be increasingly becoming the clinically most common central nervous system infection
Property disease, a variety of virus infections such as arboviruse class, enterovirus can trigger different degrees of neurological symptom.
At present since the popularization of vaccine and section of infection intervening measure are capable, so that having regional, seasonal, popular
The multiple infectious diseases disease incidence such as encephalitis, tick-borne encephalitis meningitis be decreased obviously, and by enterovirus, herpesviral, respiratory tract
Central nervous system infection caused by virus etc. becomes common disease, frequently-occurring disease, seriously endangers children's health or even lethal, cause
It is residual.The cause of disease broad categories of central nervous system infection, it has been found that more than 130 viruses can cause viral encephalitis brain
Film is scorching, and the cause of disease distribution situation of various countries' viral encephalitis meningitis is different.Worldwide, the most common encephalitis meninx
Scorching cause of disease is encephalitis B meningitis disease (Japanese encephalitis virus, JEV), mumps virus (Mumps
Virus, MuV), enterovirus (Enterovirus, EV), herpes simplex virus (Herpes simplex virus, HSV), fiber crops
Exanthema virus (Measle virus, MEV), Respiratory Syncytial Virus(RSV) (Respiratory Syncytial Virus, RSV), western Buddhist nun
Sieve virus (West Nile Virus, WNV), tick-borne encephalitis meningitis virus (Tickborne Encephalitis Virus),
Influenza virus (Influenza viruses, IFV), adenovirus (Adenovirus, ADV) etc..
The early diagnosis of viral encephalitis meningitis can effectively mitigate immune caused brain tissue impairment, reduce sequelae hair
Raw rate and the death rate.Currently used detection means has cerebrospinal fluid virus purification, immunology detection, electroencephalogram and imageological examination
And the methods of PCR.
Traditional viral encephalitis meningitis pathogen detection technology includes cerebrospinal fluid virus purification, immunology detection etc., on
It states detection method and there is certain limitation in clinical application, although virus purification culture is diagnosis goldstandard, but to experiment
Room condition requires height, and required time is longer, larger workload, and contemporaneity can not handle great amount of samples.And in immunology detection,
It is viral easily to make a variation, it is possible to influence testing result there are factors such as cross reactivities between different virus.
PCR is easy to operate, quick because of sensibility and specificity with higher, is gradually applied to a variety of viral diseases
The clinical diagnosis of disease.It establishes and using round pcr traditional viral encephalitis meningitis pathogen is carried out fastly both at home and abroad at present
The technology of speed detection.But it is mostly detected using substance round pcr, the annealing temperature and concentration of multipair primer are not quite similar, and need
It to take turns PCR reaction to detect one by one, this method is cumbersome, expends time and manpower, and sensibility and specificity is poor, recall rate more
Low and coverage difference problem；Need to carry out the quality control of each reaction system, such as inner quality control respectively.
Summary of the invention
It is an object of the invention to solve present in existing meningitis correlated virus detection technique time-consuming, sensibility and
The problem that poor specificity, recall rate are low and coverage is poor provides a kind of new meningitis correlated virus detection primer and method.
To achieve the above objectives, the present invention provides a kind of multiplex PCRs to detect encephalitis correlated virus primer sets, wherein
The primer sets include primer shown in SEQ ID NO.1 and 3-22；The encephalitis correlated virus includes encephalitis B meningitis disease
Poison, mumps virus, enterovirus, herpes simplex virus, measles virus, Respiratory Syncytial Virus(RSV), west nile virus, tick pass brain
Scorching meningitis virus, influenza virus and adenovirus.
The present invention also provides a kind of detection method of multiplex PCR detection encephalitis correlated virus, this method includes following step
It is rapid:
(1) total nucleic acid of sample to be tested is extracted；
(2) reverse transcription is carried out to the total nucleic acid and obtains pcr template；With primer sets of the present invention to the PCR mould
Plate carries out multiplexed PCR amplification, the material after obtaining multiplexed PCR amplification；
(3) nucleic acid electrophoresis detection is carried out to the material after the multiplexed PCR amplification, obtains the result of nucleic acid electrophoresis detection；
If the amplification containing 581bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain encephalitis B meningitis virus in the sample to be tested；
If the amplification containing 189bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain mumps virus in the sample to be tested；
If the amplification containing 221bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain enterovirus in the sample to be tested；
If the amplification containing 378bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain herpes simplex virus in the sample to be tested；
If the amplification containing 330bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain measles virus in the sample to be tested；
If the amplification containing 123bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain Respiratory Syncytial Virus(RSV) in the sample to be tested；
If the amplification containing 277bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain west nile virus in the sample to be tested；
If the amplification containing 505bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain tick-borne encephalitis meningitis virus in the sample to be tested；
If the amplification containing 150bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain influenza virus in the sample to be tested；
If the amplification containing 402bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain adenovirus in the sample to be tested.
The present invention also provides the kits of detection encephalitis correlated virus, and the kit includes of the present invention multiple
PCR primer group, reverse transcriptase and archaeal dna polymerase；The encephalitis correlated virus includes encephalitis B meningitis virus, parotitis disease
Poison, enterovirus, herpes simplex virus, measles virus, Respiratory Syncytial Virus(RSV), west nile virus, tick-borne encephalitis meningitis disease
Poison, influenza virus and adenovirus.
On the other hand, the present invention also provides primer sets as described above in the kit for preparing detection encephalitis correlated virus
In purposes；Wherein, the encephalitis correlated virus include encephalitis B meningitis virus, it is mumps virus, enterovirus, simple
Herpesviral, measles virus, Respiratory Syncytial Virus(RSV), west nile virus, tick-borne encephalitis meningitis virus, influenza virus and adenopathy
Poison.The present invention establishes common encephalitis correlated virus multiple PCR detection primer group and detection method, energy through the above technical solution
Enough realize that morphology, immunology and the detection of substance real-time fluorescence are impossible quickly, comprehensive, sensitive, special, automatic
Result judgement is improved significantly to encephalitis correlated virus while the sensibility detected, specificity and simplicity.
Common ten kinds of encephalitis meningitis virus multi-PCR detection methods that the present invention establishes can be realized morphology, be immunized
It learns and substance real-time fluorescence detects impossible quick, comprehensive, sensitive, special, the automatic result judgement of institute, reach as follows
Detection effect:
(1) Multiple detection covering is comprehensive
Detection method established by the present invention can identify relevant ten kinds of diseases of encephalitis meningitis in a PCR reaction
Poison, i.e. encephalitis B meningitis virus, mumps virus, enterovirus, herpes simplex virus, measles virus, respiratory syncystial
Virus, west nile virus, tick-borne encephalitis meningitis virus, influenza virus, adenovirus, are quickly obtained testing result, save the time,
Cost of human and material resources.
(2) specificity is high
Detection method specificity established by the present invention is mainly reflected in the specificity of a whole set of primer, and full-automatic knot
The validity that fruit determines.All primers all pass through blast and compare analysis, conservative and specificity with height；Pass through simultaneously
Specificity experiments verifying, which can be good at distinguishing, belongs to similar virus, including metapneumovirus, coronavirus with detection target species
OC43, coronavirus N L63, Coronavirus HKU1, coronavirus 229E, rhinovirus, parainfluenza virus 1-4 type, echovirus,
Rotavirus, norovirus and letter such as virus etc., it was demonstrated that detection method has the specificity of height, can be quasi- by non-detection target
Really distinguish.
(3) high sensitivity
Detection while detection method established by the present invention can be realized 10 targets, it is each in a reaction system
The detection sensitivity of detection target can reach 10 copies.The main reason for sensitivity of the present invention improves is the present invention for all mesh
The specific primer LHP sequence specific to sequence design of gene is marked, the homologous universal primer in LHP sequence can be second
The amplification efficiency that multi-PRC reaction is improved in stage PCR, reduces the non-specific amplification and primer dimer of multi-PRC reaction
Generation, and 6 base catenation sequences in LHP can guarantee to have when homologous universal primer amplification different virulence gene it is identical
Amplification efficiency, the unbalance of different target gene yield during multiplexed PCR amplification is avoided, to improve on the whole multiple
The sensitivity of PCR detection.
(4) cost is relatively low
Multi-PCR detection method established by the present invention reduces human cost and time cost in operability, originally
Substance detection needs 10 artificial and 10 times of times, only needs 1 artificial and 1 reaction time using the method now；It should
Multiple detection method saves the reagent consumption that repetition detects the same sample simultaneously, and maximum can save 50% reagent cost.
(5) time is saved
The reaction of traditional two-step method needs to be first cDNA by the RNA template reverse transcription of virus, then carries out PCR reaction, consumes
When it is longer.The present invention uses the RT-PCR system of one-step method, and the whole experiment process including extracting only needs 3 hours, significantly
Time needed for shortening detection.
(6) interpretation of result and judgement are automated
The platform analyzed as a result using the full-automatic capillary electrophoresis analysis instrument of QIaxcel.Based on qiaxcel it is complete from
The intelligent interpretation of result program of dynamic operation and setting, realizes the automatic decision analysis of result.
(7) prevent false negative result
The positive internal reference added in reaction system of the present invention can be prompted effectively because of operation error, PCR mortifier
Etc. false negative testing result caused by reasons.
The present invention provides whole solution for encephalitis meningitis virus rapid screening, is able to achieve quick, sensitive spy
It is different, automatically detect ten kinds of common encephalitis meningitis virus.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Specific embodiment
Below in conjunction with attached drawing, detailed description of the preferred embodiments.It should be understood that this place is retouched
The specific embodiment stated is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The present invention provides a kind of multiplex PCRs to detect encephalitis correlated virus primer sets, wherein the primer sets include SEQ ID
Primer shown in NO.1 and 3-22；The encephalitis correlated virus includes encephalitis B meningitis virus (Japanese
Encephalitis virus, JEV), mumps virus (Mumps virus, MuV), enterovirus (Entero virus,
EV), herpes simplex virus (Herpes simplex virus, HSV), measles virus (Measle virus, MEV), respiratory tract
Syncytial virus (Respiratory Syncytial Virus, RSV), west nile virus (West Nile Virus, WNV), tick
Pass encephalitis meningitis virus (Tickborne Encephalitis Virus), influenza virus (Influenza viruses,
) and adenovirus (Adenovirus, ADV) IFV.
Wherein it is preferred to which the primer sets further include primer shown in SEQ ID NO.23-24.SEQ ID NO.23-24
Shown in primer be using pET-28a plasmid as the pair of primers of stencil design, as positive internal reference.
Specifically, meningitis correlated virus multiple PCR primer group is as shown in table 1.
1 multiple PCR primer group summary sheet of table
Wherein, SEQ ID NO.1 is homologous universal primer, which is connected with catenation sequence CATTAA, constitutes LHP sequence
(SEQ ID NO.2, homologous universal primer (the based on ligation-sequence homogenous based on catenation sequence
Primer, LHP)).
The present invention also provides the detection methods of encephalitis correlated virus, and this method comprises the following steps:
(1) total nucleic acid of sample to be tested is extracted；
(2) reverse transcription is carried out to the total nucleic acid and obtains pcr template；With primer sets of the present invention to the PCR mould
Plate carries out multiplexed PCR amplification, the material after obtaining multiplexed PCR amplification；
(3) nucleic acid electrophoresis detection is carried out to the material after the multiplexed PCR amplification, obtains the result of nucleic acid electrophoresis detection；
If the amplification containing 581bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain encephalitis B meningitis virus in the sample to be tested；
If the amplification containing 189bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain mumps virus in the sample to be tested；
If the amplification containing 221bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain enterovirus in the sample to be tested；
If the amplification containing 378bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain herpes simplex virus in the sample to be tested；
If the amplification containing 330bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain measles virus in the sample to be tested；
If the amplification containing 123bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain Respiratory Syncytial Virus(RSV) in the sample to be tested；
If the amplification containing 277bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain west nile virus in the sample to be tested；
If the amplification containing 505bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain tick-borne encephalitis meningitis virus in the sample to be tested；
If the amplification containing 150bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain influenza virus in the sample to be tested；
If the amplification containing 402bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain adenovirus in the sample to be tested.
Wherein it is preferred to the final of primer shown in SEQ ID NO.3-22 is respectively 0.1-0.8 μM using concentration, SEQ
The final use concentration of primer shown in ID NO.1 is 0.4-0.8 μM.
Wherein it is preferred to which the reaction condition of step (2) includes the program of following a-i:
A:49-51 DEG C, 28-32min；
B:94-96 DEG C, 13-18min；
C:94-96 DEG C, 28-32s,
D:54-56 DEG C, 28-32s,
E:71-73 DEG C, 58-62s, carry out the circulation of 10 c-e；
F:94-96 DEG C, 28-32s,
G:49-51 DEG C, 28-32s,
H:71-73 DEG C, 59-61s, carry out the circulation of 30 f-h；
I:71-73 DEG C, 5-10min.
Wherein, in above procedure, program a is to carry out the process that reverse transcription obtains pcr template to total nucleic acid, reverse transcription
It is cDNA that purpose, which is by the RNA reverse transcription in total nucleic acid, and the DNA in process of reverse-transcription in total nucleic acid is unaffected,
After process of reverse-transcription, the DNA in cDNA and total nucleic acid that reverse transcription obtains carries out the more of next step collectively as pcr template
Weight PCR amplification.
Wherein, the detection sequence of herpes simplex virus and adenovirus is DNA sequence dna.
In the method provided by the disclosure, the constituent of reaction system includes the total nucleic acid of sample to be tested, multiplex PCR
Primer sets, reverse transcriptase, archaeal dna polymerase, PCR reaction buffer and dNTP carry out reverse transcription mistake in the same reaction system
Journey and multiplexed PCR amplification reaction.
Wherein, the sample to be tested can include but is not limited in food, drug, excreta, vomitus and body fluid extremely
Few one kind.Preferably, the detection method of multiplex PCR of the invention detection encephalitis correlated virus is not used in diagnosis.In other words, encephalitis
The qualitative and quantitative result of correlated virus is not belonging to diagnostic result with whether disease occurs without one-to-one correlation, still
The testing result of the qualitative and quantitative of encephalitis correlated virus can be used as average information, for reference for clinicians.
Wherein it is preferred to which the nucleic acid electrophoresis detection includes nucleic acid gel electrophoresis and/or nucleic acid Capillary Electrophoresis.
In one embodiment of the invention, divided as a result using the full-automatic capillary electrophoresis analysis instrument of QIaxcel
The platform of analysis.The intelligent interpretation of result program of full automatic working and setting based on QIaxcel, to realize the automatic of result
Decision analysis.
The present invention also provides a kind of kit of multiplex PCR detection meningitis correlated virus, the kit includes this
Invention multiple PCR primer group, reverse transcriptase and the archaeal dna polymerase；The encephalitis correlated virus includes encephalitis B meninx
Scorching virus, mumps virus, enterovirus, herpes simplex virus, measles virus, Respiratory Syncytial Virus(RSV), west nile virus, tick
Pass encephalitis meningitis virus, influenza virus and adenovirus.
Wherein it is preferred to which the kit further includes PCR reaction buffer, reverse transcriptase and dNTP.
On the other hand, the present invention also provides primer sets as described above in the kit for preparing detection encephalitis correlated virus
In purposes；Wherein, the encephalitis correlated virus include encephalitis B meningitis virus, it is mumps virus, enterovirus, simple
Herpesviral, measles virus, Respiratory Syncytial Virus(RSV), west nile virus, tick-borne encephalitis meningitis virus, influenza virus and adenopathy
Poison.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is amplification of the multiple system of kit embodiment 1 of the present invention to substance target；
B12:100-1500bp Size Marker,
B1: encephalitis B meningitis virus amplified fragments 581bp,
B2: mumps virus amplified fragments 189bp,
B3: enterovirus amplified fragments 221bp,
B4: herpes simplex virus amplified fragments 378bp,
B5: measles virus amplified fragments 330bp,
B6: Respiratory Syncytial Virus(RSV) amplified fragments 123bp,
B7: west nile virus amplified fragments 277bp,
B8: tick-borne encephalitis meningitis virus amplified fragments 505bp,
B9: influenza viral amplification segment 150bp,
B10: adenovirus amplifies segment 402bp,
B11: blank control.
Fig. 2 is that kit of the present invention replaces the multiple system of primer to the amplification of substance target；
B12:100-1500bp Size Marker,
B1: encephalitis B meningitis virus amplified fragments 180bp,
B2: mumps virus amplified fragments 320bp,
B3: enterovirus amplified fragments 444bp,
B4: herpes simplex virus amplified fragments 372bp,
B5: measles virus amplified fragments 496bp,
B6: Respiratory Syncytial Virus(RSV) amplified fragments 560bp,
B7: west nile virus amplified fragments 378bp,
B8: tick-borne encephalitis meningitis virus amplified fragments 670bp,
B9: influenza viral amplification segment 229bp,
B10: adenovirus amplifies segment 280bp,
B11: blank control.
Embodiment 1
1, primer synthesizes:
According to sequence shown in table 1, the synthesis of the primer of SEQ ID NO.1 and 3-22 is carried out.
As unit of the amount of substance, the primer of SEQ ID NO.3-22 is respectively taken into 1 parts by weight, the SEQ ID with 4 parts by weight
The primer of NO.1 mixes, and constitutes the primer sets of the present embodiment.
2, the establishment of kit:
It is right that the kit contains 2 × RT PCR buffer, RT-PCR enzyme mix, 10 × primer mixed liquor, the positive
It is constituted according to, ultrapure water.
The reaction system of kit detection is 25 μ L, is configured as follows: 2 × RT PCR buffer, 12.5 μ L (mould containing IAC
Plate)；RT-PCR enzyme mix 1μL；10 × primer mixture, 2.5 μ L (2 μM each to concentration of the long primer including IAC,
Homologous 1 concentration of universal primer SEQ ID NO is 8 μM)；2 μ L of template, 7 μ L of ultrapure water.
3, the operation of kit and result judgement
(1) virus genomic extraction
Reagent is extracted using commercialization, disease is extracted from the cerebrospinal fluid for having made a definite diagnosis virus infection type or other clinical samples
DNA the and RNA nucleic acid of poison.
(2) configuration of reaction system
The PCR pipe of 200 μ L is taken to configure the reaction system of 25 μ L, configure as follows: 2 × RT PCR buffer, 12.5 μ L (contains
IAC template)；RT-PCR enzyme mix 1μL；10 × primer mixture, 2.5 μ L；2 μ L of template, 7 μ L of ultrapure water.
(3) PCR reacts
PCR pipe is put into Bio-Rad C1000 type PCR instrument, after opening heat lid, carries out PCR reaction according to the procedure below:
50 DEG C of 30min carry out reverse transcription；95℃15min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 60s, 10 circulations；95 DEG C of 30s, 50 DEG C
30s, 72 DEG C, 60s, 30 circulations；72℃5min.
(4) the full-automatic capillary electrophoresis analysis instrument of QIAxcel analyzes PCR product
PCR reaction tube is put into full-automatic capillary electrophoresis QIAxcel Advanced (Kai Jie company), uses high score
Resolution clip selects the size marker of the alignment marker, 100-1500bp of 15-2000bp, automatically analyzes target
The size of band.
(5) result judges
It is specific determine it is as follows: amplification the results show that all swimming lanes including blank control it is equal at least one
Band amplification, blank control has the amplification of positive internal reference (670bp), other detections have target stripe and (or) positive internal reference
Amplification shows that all PCR reactions are set up, excludes the result of false negative.Otherwise view experiment is invalid, needs to repeat.According to B-mode
Encephalitis meningitis virus 581bp, mumps virus 189bp, enterovirus 221bp, herpes simplex virus 378bp, measles virus
189bp, Respiratory Syncytial Virus(RSV) 123p, west nile virus 277bp, tick-borne encephalitis meningitis virus 505bp, influenza virus
150bp, adenovirus 402bp determine encephalitis meningitis virus type.
As a result as shown in Figure 1.
On the basis of IAC amplification, also there is the pcr amplification product of 1 581bp in swimming lane B1, is encephalitis B meningitis
The amplified band of virus；Also there is the pcr amplification product of 1 189189bp in swimming lane B2, is the amplified band of mumps virus；Swimming
Also there is the pcr amplification product of 1 221bp in road B3, is the amplified band of enterovirus；Swimming lane B4 also occurs 1 378bp's
Pcr amplification product is the amplified band of herpes simplex virus；Also there is the pcr amplification product of 1 330bp in swimming lane B5, is morbilli
The amplified band of virus；Also there is the pcr amplification product of 1 123bp in swimming lane B6, is the amplified band of Respiratory Syncytial Virus(RSV)；
Also there is the pcr amplification product of 1 277bp in swimming lane B7, is the amplified band of west nile virus；Swimming lane B8 also occurs 1 article
The pcr amplification product of 505bp is the amplified band of tick-borne encephalitis meningitis virus；The PCR that 1 150bp also occurs in swimming lane B8 expands
Increase production object, is the amplified band of influenza virus；Also there is the pcr amplification product of 1 402bp in swimming lane B10, is the amplification of adenovirus
Band.Swimming lane B11 is NTC, only IAC amplified band.
The specific test of 2 kit of embodiment
Select metapneumovirus (be originated from country CDC, number is IVDC 2.2445), Coronavirus OC43 (from country CDC,
Number is IVDC 2.2339), coronavirus N L63 (be originated from country CDC, number is IVDC 2.2338), Coronavirus HKU1
(being originated from country CDC, number IVDC2.2440), coronavirus 229E (being originated from country CDC, number is IVDC 2.2441), nose
Virus (being originated from country CDC, number is IVDC 2.2500), parainfluenza virus 1-4 type (are originated from country CDC, number IVDC
2.2610), echovirus (being originated from country CDC, number is IVDC 2.2333), rotavirus (are originated from country CDC, number is
IVDC 2.2450), norovirus (be originated from country CDC, number is IVDC 2.2461), letter such as virus (be originated from country CDC, volume
Number be IVDC 2.2462) etc. be used as specificity verification template.
These targets to be checked are detected using kit of the present invention, under conditions of positive control is set up, the equal nothing of target to be checked
Nonspecific miscellaneous band, show kit of the present invention can effective district sorting survey target and non-detection target, have preferable special
It is anisotropic.
The minimum detectability of 3 kit of embodiment is tested
Assessment detection sample: restriction enzyme SpeI and PvuII digestion encephalitis B meningitis virus, parotitis are used
Virus, enterovirus, measles virus, Respiratory Syncytial Virus(RSV), west nile virus, tick-borne encephalitis meningitis virus, influenza virus
Deng standard items plasmid, linearized and be transcribed in vitro；Choosing initial concentration is 106Copy/μ l transcription product gradient
Dilution, the template as minimum detectability assessment.Because herpes simplex virus and adenovirus are DNA virus, choosing initial concentration is
106Plasmid of the copy/μ l containing extension increasing sequence carries out gradient dilution, the template as minimum detectability assessment.
It is detected according to the method for embodiment 1, the minimum detectability of kit can reach 10 copies/anti-as the result is shown
It answers.
The kit of the invention of embodiment 4 is compared with literature method
Evaluate kit and document report (Development of a GeXP based multiplex RT- of the present invention
PCR assay for simultaneous detection of eight arboviruses related to
Encephalitis) effect compares, and is mainly compared in terms of susceptibility, specificity and coverage three.
Evaluation use positive sample: encephalitis B meningitis virus (be originated from country CDC, number respectively IVDC 2.24421,
2.24422,2.24423,2.24424,2.24425,2.24426,2.24427,2.24428,2.24429,2.24430), the parotid gland
Scorching virus (be originated from country CDC, number respectively IVDC2.25311,2.25312,2.25313,2.25314,2.25315,
2.25316,2.25317,2.25318,2.25319,2.25320), enterovirus (be originated from country CDC, number is
IVDC2.22332、2.22332、2.22333、2.22334、2.22335、2.22336、2.22337、2.22338、2.22339、
2.22340), herpes simplex virus (be originated from country CDC, number be IVDC 2.21343,2.21344,2.21345,2.21346,
2.21347,2.21348,2.21349,2.21350,2.21351,2.21352), measles virus (be originated from country CDC, number is
IVDC 2.21751、2.21752、2.21753、2.21754、2.21755、2.21756、2.21757、2.21758、2.21759、
2.21760), Respiratory Syncytial Virus(RSV) (be originated from country CDC, number be IVDC 2.27613,2.27614,2.27615,
2.27616,2.27617,2.27618,2.27619,2.27620,2.27621,2.27622), west nile virus (be originated from country
CDC, number be IVDC 2.27801,2.27802,2.27803,2.27804,2.27805,2.27806,2.27807,
2.27808,2.27809,2.27810), tick-borne encephalitis meningitis virus (be originated from country CDC, number be IVDC 2.27930,
2.27931,2.27932,2.27933,2.27934,2.27935,2.27936,2.27937,2.27938,2.27939), influenza
Virus (be originated from country CDC, number be IVDC 2.25331,2.25332,2.25333,2.25334,2.25335,2.25336,
2.25337,2.25338,2.25339,2.25340), adenovirus (be originated from country CDC, number be IVDC 2.29512,
2.29513,2.29514,2.29515,2.29516,2.29517,2.29518,2.29519,2.29520,2.29521) each 10
Strain.
Evaluation negative sample: metapneumovirus, Coronavirus OC43, coronavirus N L63, coronal disease in selection example 2
Malicious HKU1, coronavirus 229E, rhinovirus, parainfluenza virus 1-4 type, echovirus, rotavirus, norovirus, letter such as disease
The virus such as poison is used as specificity verification template.After nucleic acid is extracted in the above bacterium strain, equal proportion is mixed into a negative assessment mould
Plate.
Specificity assessment: specific evaluation operation is carried out according to the embodiment of the present invention 2.
Coverage assessment: 10 plants of evaluation positive samples are detected using kit of the present invention and literature method respectively.
Minimum detectability assessment: restriction enzyme SpeI and PvuII digestion encephalitis B meningitis virus, parotitis are used
Virus, enterovirus, measles virus, Respiratory Syncytial Virus(RSV), west nile virus, tick-borne encephalitis meningitis virus, influenza virus
Deng standard items plasmid, linearized and be transcribed in vitro；Choosing initial concentration is 106Copy/μ l transcription product gradient
Dilution, the template as minimum detectability assessment.Because herpes simplex virus and adenovirus are DNA virus, choosing initial concentration is
106Plasmid of the copy/μ l containing amplified fragments carries out gradient dilution, the template as minimum detectability assessment.It is tried using the present invention
Agent box and literature method detect the gradient dilution template of each encephalitis meningitis virus respectively.
Comparison result: kit and literature procedure detection negative sample result of the present invention is feminine gender, shows have
There is preferable specificity.
Kit of the present invention detects evaluation positive sample, acquisition positive findings, and literature procedure have 2 plants it is B-mode
The missing inspection of encephalitis meningitis virus and 2 plants of Respiratory Syncytial Virus(RSV) virus.
The minimum detectability comparison that kit and literature procedure of the present invention detect ten kinds of encephalitis meningitis virus is as follows
Table 2.
The minimum detectability comparing result of table 2 present invention and literature procedure
As seen from the above table, the minimum detectability that kit of the present invention detects 10 kinds of target genes can reach 10 copies/μ l
Level, and literature procedure only has enterovirus, measles virus, Respiratory Syncytial Virus(RSV), west nile virus that can reach
100 copies/μ l level, remaining can only achieve 1000 copies/μ l level.The minimum detectability of kit detection of the present invention is aobvious
It writes and is better than literature procedure.
Comparative example 1
Preparation comparison primer 2 5-44, is shown in Table 3.
Table 3 compares primer sequence table
It is detected according to the method for embodiment 1, difference is only that, by SEQ ID NO.3-4 in the primer sets of embodiment 1
Shown in primer replace with primer shown in SEQ ID NO.25-26 obtain comparison primer sets 1.
Primer shown in SEQ ID NO.5-6 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.27-28
Primer obtains comparison primer sets 2.
Primer shown in SEQ ID NO.7-8 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.29-30
Primer obtains comparison primer sets 3.
Primer shown in SEQ ID NO.9-10 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.31-32
Primer obtain comparison primer sets 4.
Primer shown in SEQ ID NO.11-12 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.33-34
Primer obtain comparison primer sets 5.
Primer shown in SEQ ID NO.13-14 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.35-36
Primer obtain comparison primer sets 6.
Primer shown in SEQ ID NO.15-16 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.37-38
Primer obtain comparison primer sets 7.
Primer shown in SEQ ID NO.17-18 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.39-40
Primer obtain comparison primer sets 8.
Primer shown in SEQ ID NO.19-20 replaces with shown in SEQ ID NO.41-42 in the primer sets of embodiment 1
Primer obtains comparison primer sets 9.
Primer shown in SEQ ID NO.21-22 replaces with shown in SEQ ID NO.43-44 in the primer sets of embodiment 1
Primer obtains comparison primer sets 10.
Wherein, encephalitis B meningitis virus standard items Plasmid samples are expanded with comparison primer sets 1；
Mumps virus standard items Plasmid samples are expanded with comparison primer sets 2；
Enterovirus standard items Plasmid samples are expanded with comparison primer sets 3；
Herpes simplex virus standard items Plasmid samples are expanded with comparison primer sets 4；
Measles virus standard items Plasmid samples are expanded with comparison primer sets 5；
Respiratory Syncytial Virus(RSV) standard items Plasmid samples are expanded with comparison primer sets 6；
West nile virus standard items Plasmid samples are expanded with comparison primer sets 7；
Tick-borne encephalitis meningitis virus standard items Plasmid samples are expanded with comparison primer sets 8；
Influenza virus standard items plasmid is expanded with comparison primer sets 9.
Adenovirus standard items plasmid is expanded with comparison primer sets 10.
Specificity is carried out according to the method for Examples 1 and 2 respectively and minimum detectability is tested, as the result is shown this comparative example
Specificity, minimum detectability and the coverage rate of primer 2 5-44 is worse than the primer (as shown in Figure 2) of embodiment 1-3.
The swimming lane of the 4th, 7 and 10 does not have amplified band other than Quality Control in the positive in Fig. 2, target amplification item occurs in remaining swimming lane
The display band color of band is shallower, therefore minimum detectability is lower than the 10 copies/μ L of this kit；In addition to this, comparative example side
The amplification of case has different degrees of primer dimer.
The preferred embodiment of the disclosure is described in detail in conjunction with attached drawing above, still, the disclosure is not limited to above-mentioned reality
The detail in mode is applied, in the range of the technology design of the disclosure, a variety of letters can be carried out to the technical solution of the disclosure
Monotropic type, these simple variants belong to the protection scope of the disclosure.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the disclosure to it is various can
No further explanation will be given for the combination of energy.
In addition, any combination can also be carried out between a variety of different embodiments of the disclosure, as long as it is without prejudice to originally
Disclosed thought equally should be considered as disclosure disclosure of that.
Claims (9)
1. a kind of multiplex PCR detects encephalitis correlated virus primer sets, wherein the primer sets include SEQ ID NO.1 and 3-22
Shown in primer；The encephalitis correlated virus includes encephalitis B meningitis virus, mumps virus, enterovirus, simple blister
Exanthema virus, measles virus, Respiratory Syncytial Virus(RSV), west nile virus, tick-borne encephalitis meningitis virus, influenza virus and adenopathy
Poison.
2. primer sets according to claim 1, wherein the primer sets further include drawing shown in SEQ ID NO.23-24
Object.
3. the detection method of the multiplex PCR detection encephalitis correlated virus for non-diagnostic purpose, which is characterized in that this method includes
Following steps:
(1) total nucleic acid of sample to be tested is extracted；
(2) reverse transcription is carried out to the total nucleic acid and obtains pcr template；With primer sets of any of claims 1 or 2 to the PCR
Template carries out multiplexed PCR amplification, the material after obtaining multiplexed PCR amplification；
(3) nucleic acid electrophoresis detection is carried out to the material after the multiplexed PCR amplification, obtains the result of nucleic acid electrophoresis detection；
If the amplified production containing 581bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain encephalitis B meningitis virus in the sample to be tested；
If the amplified production containing 189bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain mumps virus in the sample to be tested；
If the amplified production containing 221bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain enterovirus in the sample to be tested；
If the amplified production containing 378bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain herpes simplex virus in the sample to be tested；
If the amplified production containing 330bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain measles virus in the sample to be tested；
If the amplified production containing 123bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain Respiratory Syncytial Virus(RSV) in the sample to be tested；
If the amplified production containing 277bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain west nile virus in the sample to be tested；
If the amplified production containing 505bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain tick-borne encephalitis meningitis virus in the sample to be tested；
If the amplified production containing 150bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain influenza virus in the sample to be tested；
If the amplified production containing 402bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain adenovirus in the sample to be tested.
4. detection method according to claim 3, wherein the final of primer shown in SEQ ID NO.3-22 uses concentration
Respectively 0.1-0.8 μM, the final of primer shown in SEQ ID NO.1 using concentration is 0.4-0.8 μM.
5. detection method according to claim 3 or 4, wherein the reaction condition of step (2) includes the program of following a-i:
A:49-51 DEG C, 28-32min；
B:94-96 DEG C, 13-18min；
C:94-96 DEG C, 28-32s,
D:54-56 DEG C, 28-32s,
E:71-73 DEG C, 58-62s, carry out the circulation of 10 c-e；
F:94-96 DEG C, 28-32s,
G:49-51 DEG C, 28-32s,
H:71-73 DEG C, 59-61s, carry out the circulation of 30 f-h；
I:71-73 DEG C, 5-10min.
6. detection method according to claim 3 or 4, wherein nucleic acid electrophoresis detection include nucleic acid gel electrophoresis and/
Or nucleic acid Capillary Electrophoresis.
7. a kind of kit of multiplex PCR detection encephalitis correlated virus, which is characterized in that the kit includes claims 1 or 2
Multiple PCR primer group, reverse transcriptase and the archaeal dna polymerase；The encephalitis correlated virus includes encephalitis B meningitis disease
Poison, mumps virus, enterovirus, herpes simplex virus, measles virus, Respiratory Syncytial Virus(RSV), west nile virus, tick pass brain
Scorching meningitis virus, influenza virus and adenovirus.
8. kit according to claim 7, which is characterized in that the kit further include PCR reaction buffer and
dNTP。
9. purposes of the primer sets of any of claims 1 or 2 in the kit of preparation detection encephalitis correlated virus；Wherein, institute
Stating encephalitis correlated virus includes encephalitis B meningitis virus, mumps virus, enterovirus, herpes simplex virus, measles
Poison, Respiratory Syncytial Virus(RSV), west nile virus, tick-borne encephalitis meningitis virus, influenza virus and adenovirus.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796827A (en) * | 2011-05-24 | 2012-11-28 | 上海透景生命科技有限公司 | Method and kit for detecting multiple encephalitis related viruses |
| CN105385787A (en) * | 2015-12-04 | 2016-03-09 | 南京美宁康诚生物科技有限公司 | Multiplex PCR detection kit for 12 encephalitis virus nucleic acids and application thereof |
| CN105603128A (en) * | 2016-03-30 | 2016-05-25 | 江苏省疾病预防控制中心 | Encephalitis-related virus detection kit and application thereof |
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2016
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796827A (en) * | 2011-05-24 | 2012-11-28 | 上海透景生命科技有限公司 | Method and kit for detecting multiple encephalitis related viruses |
| CN105385787A (en) * | 2015-12-04 | 2016-03-09 | 南京美宁康诚生物科技有限公司 | Multiplex PCR detection kit for 12 encephalitis virus nucleic acids and application thereof |
| CN105603128A (en) * | 2016-03-30 | 2016-05-25 | 江苏省疾病预防控制中心 | Encephalitis-related virus detection kit and application thereof |
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