CN107400734A - Isothermal duplication zika virus detection kit based on ring mediation - Google Patents
Isothermal duplication zika virus detection kit based on ring mediation Download PDFInfo
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Abstract
The invention provides a kind of isothermal duplication (LAMP) primer based on ring mediation and related kit and the method using the primer detection zika virus (also referred to as zika virus, Zika virus, ZIKV).It is capable of the nucleic acid of specific ring mediated isothermal amplification zika virus using the primer.The inventive method specificity is good, high sensitivity, reproducible, simple, convenient quick, can be well applied to identify zika virus, suitable for clinical and test in laboratory.
Description
Technical field
The invention belongs to molecular biology and nucleic acid detection technique field.In particular it relates to it is based on ring
The isothermal duplication zika virus detection kit of mediation.
Background technology
Zika virus (Zika virus, ZIKV) is a kind of arboviruse propagated by mosquito, host
It is indefinite, main raw primate out of office and mosquito in the tree is inhabited, as circulated in aedes africanus.Should
Virus is most accidentally sent out earlier than nineteen forty-seven by yellow fever monitoring network in the rhesus macaque of stockaded village of Uganda card jungle
It is existing, found with after nineteen fifty-two in Uganda and Tanzania crowd.The viral activity is more hidden always
Hide, only the Africa of surrounding, America, the Asia-pacific region have zika virus to infect Sporadic cases under the line.
An earliest outbreak of epidemic is to occur on Western Pacific Mi Keluoni subgroup Dao Yapu islands for 2007, more greatly
Be once popular in -2014 years 2013 and occur in Oceanian French Polynesia, infected about
32000 people.Yellow-fever mosquito also propagates other three kinds of virus in flaviviridae, including dengue fever virus, datum hole are agreed
Refined virus and yellow fever virus are also mainly popular in subtropical and tropical zones.Decades ago, African research
Person notices that her mosquito-borne zika virus epidemic situation somehow or other follows yellow-fever mosquito to propagate chikungunya virus epidemic disease
After feelings.Similar rule starts from 2013, when chikungunya virus is propagated from west to east, stockaded village's card
Virus is closelyed follow.
In the prior art, the detection method of stockaded village's card mainly includes Virus culture separation, and enzyme linked immunosorbent detection is glimmering
Fluorescent Quantitative PCR detection technique.
The sample that Virus culture technology is collected co-cultures with cell, by observing cytopathy or other method
Viral amplification is detected to determine whether there is viral infection in sample.Although the method is virus infection once
Detect " golden standard ", but the virion with infection ability in sample can only be detected, sensitivity is not
Foot.And operation sequence is complicated, there are bio-safety class requirement, detection process length to experimental site and equipment
(at least wanting more than one week), it is impossible to be used in Site Detection, it is different to the testing result of different virus.
Enzyme mark detection based on antibody, directly with indirect fluorescent detection and collaurum quick detection using anti-
Specific bond between former and antibody, the antibody by being coupled colour developing group realize the visualization to pathogen
Detection.Antibody test is independent of large scale equipment, and testing result can obtain within 30 minutes.It is but anti-
Physical examination is surveyed due to the process of no signal amplification, and sensitivity is lower than Molecular Detection, and result easily occurs
False positive and false negative.Because the manufacturing cycle of new antibodies is longer, this method is difficult to new appearance virus
Detection, and it is also doubtful for the detectability of new virus subtype as caused by antigenic shift.The world
Health organization (WHO) research shows that ELISA is difficult to distinguish zika virus and dengue fever virus.
The nucleic acid molecules detection of cause of disease is expanded by polymerase to the nucleic acid molecules of cause of disease, then using glimmering
Light, monitoring is entered to course of reaction or product the methods of electrophoresis, whether carried out according to reaction or whether product occurs
To judge whether cause of disease occurs.But the method for PCR-based is required for the equipment such as thermal cycler, to detecting place
Also require, quantitative PCR also needs to specialized training, and these all limit PCR correlation techniques in quick detection
In application.
Therefore, there is an urgent need to develop fast and convenient effective zika virus detection method and reagent for this area.
The content of the invention
It is an object of the invention to provide the isothermal duplication zika virus detection method mediated based on ring and examination
Agent box.
In the first aspect of the present invention, there is provided a kind of primer collection for detecting zika virus, in the primer collection
Including primer can specifically bind to the NS4B genes of zika virus, and correspond to the base for expanding
The specific amplification products of cause.
In another preference, the primer collection includes the one or more primer pairs being selected from the group：
First primer pair：
F3 primers：Sequence such as SEQ ID NO:Shown in 1；
B3 primers：Sequence such as SEQ ID NO:Shown in 2；
Second primer pair：
FIP primers：Sequence such as SEQ ID NO:Shown in 3；
BIP primers：Sequence such as SEQ ID NO:Shown in 4；
Three-primer pair：
LF primers：Sequence such as SEQ ID NO:Shown in 5；
LB primers：Sequence such as SEQ ID NO:Shown in 6.
In another preference, described primer collection is used for the isothermal duplication that the ring of zika virus mediates
(LAMP)。
In the second aspect of the present invention, there is provided a kind of PCR amplification system, described system include：For
Primer collection described in the buffer system of amplification and first aspect present invention in the system.
In another preference, the first primer pair, the second primer pair and three-primer in described amplification system
To the ratio between mole be (0.8-1.2)：(6-10)：(4-6), preferably 1：(7-9)：(3-5), more preferably
Ground is 1:8:4.
In another preference, described amplification includes ring mediated isothermal amplification.
In another preference, the described buffer system for being used to expand includes：Buffer solution, amplification enzyme, dNTP,
And RNase inhibitor.
The dyestuff for detecting also is included in another preference, in the described buffer system for being used to expand.
In the third aspect of the present invention, there is provided a kind of kit for detecting zika virus, in the kit
Including (i) container, and (ii) is located at the primer collection as described in the first aspect of the invention in the container.
In another preference, described kit includes：
First container, and the first primer pair in first container；
Second container, and the second primer pair in the second container；And/or
3rd container, and the three-primer pair in the 3rd container；
Wherein, the first described container, second container, the 3rd container can be each independent or be same
Container (including two or three containers be same container).
In another preference, the first container, second container and the 3rd container are same containers；It is and described
First and second and three-primer to being mixed together, obtain a primer mixture.
In another preference, the single primer concentration of the first primer pair is in described primer mixture
0.1-0.3 μM, preferably 0.2 μM.
In another preference, the single primer concentration of the second primer pair is in described primer mixture
1.4-1.8 μM, preferably 1.5-1.6 μM.
In another preference, the single primer concentration of three-primer pair is in described primer mixture
0.6-1.0 μM, preferably 0.7-0.9 μM.
In another preference, described kit also includes the reagent for expanding, including amplification enzyme, slow
Fliud flushing, dNTP, RNase inhibitor etc.；RNA extracts reagents and the dyestuff for detection.
In another preference, described amplification enzyme includes Bst2.0DNA polymerases and thermal starting reverse transcription
Enzyme.
In another preference, described kit also includes the examination criteria product of zika virus.
Wherein, described buffer solution includes：Thermolpol RB(Isothermal Amplification
Buffer), magnesium sulfate and glycine betaine (Betaine).
In another preference, the described dyestuff for being used to detect is selected from the group：Hydroxynaphthol blue dyestuff
(Hydroxy naphthol blue, the HNB), (Green of SYTO 9 of green fluorescence nucleic acid dye SYTO 9
Fluorescent Nucleic Acid Stain), calcein dyestuff (Calcein) or its combination.
In another preference, the kit is also including the use of specification.
In the fourth aspect of the present invention, there is provided a kind of isothermal amplification method based on ring mediation, including step：
(a) using the primer collection described in first aspect present invention, the isothermal that ring mediation is carried out to detection sample expands
Increase.
In another preference, described method is nondiagnostic method.
In another preference, described method is in-vitro method.
In the fifth aspect of the present invention, there is provided a kind of method for detecting zika virus, including step：
(a) using the primer collection described in first aspect present invention, detection sample is expanded；
(b) amplification situation is detected, so as to judge the presence or absence of zika virus in the detection sample.
In another preference, described testing sample is nucleic acid extractive, preferably RNA extracts.
In another preference, in step (b), if specific amplification products produce, then explanation inspection
Zika virus be present in test sample product；If produced without specific amplification products, illustrate to detect in sample not
Zika virus be present.
In another preference, in step (a), described amplification is ring mediated isothermal amplification.
In another preference, the reaction temperature of described ring mediated isothermal amplification is 60-65 DEG C, preferably
For 61-63 DEG C.
In another preference, in step (a), the amplification system pair described in second aspect of the present invention is utilized
Detect sample and carry out ring mediated isothermal amplification, so as to obtain the reactant mixture comprising amplified production.
In another preference, in step (b), described detection is carried out by estimating, and step (a)
In the ring mediated isothermal amplification time be 30min-120min, preferably 50min-80min.
In another preference, in step (b), described detection is carried out by estimating, and is being estimated
HNB dyestuffs or Calcein dyestuffs are added in reactant mixture described in forward direction.
In another preference, HNB dyestuffs are added into described reactant mixture, and if reaction is mixed
Compound is changed into sky blue, then shows that amplification is positive, detect in sample and zika virus be present；If reaction mixing
Thing is changed into lilac, then shows that amplification is negative, detect in sample and zika virus is not present.
In another preference, Calcein dyestuffs are added into described reactant mixture, and it is if anti-
Answer mixture to be changed into green, then show that amplification is positive, detect in sample and zika virus be present；If reaction is mixed
Compound is changed into crocus, then shows that amplification is negative, detect in sample and zika virus is not present.
In another preference, in step (b), described detection is carried out by real-time PCR,
Also, the ring mediated isothermal amplification in step (a) includes 50-70 circulation, preferably 50-60 are followed
Ring.
In another preference, described circulation includes step：
(i) the 60-65 DEG C of insulation 20-40 second, preferably 30 seconds；
(ii) the 60-65 DEG C of collection fluorescence 20-40 second, preferably 30 seconds.
In another preference, described ring mediated isothermal amplification also includes 60-65 DEG C before being circulated
The step of isothermal reaction 1-3min, preferably 1-2min.
In another preference, described detection is carried out by real-time PCR, and the amplification body
Contain green fluorescence nucleic acid dye SYTO 9 in system.
In another preference, after loop-mediated isothermal amplification terminates, if amplification curve, then table
Bright amplification is positive, detects in sample and zika virus be present；If without amplification curve, show that amplification is negative,
Zika virus is not present in detection sample.
In another preference, described method is zika virus pre-detection method.
In another preference, described method is nondiagnostic method.
In another preference, described method is in-vitro method.
In the sixth aspect of the present invention, there is provided a kind of purposes of the primer collection described in first aspect present invention,
For preparing the kit of detection zika virus.
Present invention also offers a kind of method for identifying zika virus, methods described includes：
Using the RNA of testing sample as template, expanded with specificity amplification primer；If generation specificity expands
Increase, then show to include zika virus in testing sample；
Wherein, described specificity amplification primer includes：
Primer pair 1：SEQ ID NO:1 and SEQ ID NO:2；
Primer pair 2：SEQ ID NO:3 and SEQ ID NO:4；
Primer pair 3：SEQ ID NO:5 and SEQ ID NO:6；
In another preference, described amplification is ring mediated isothermal amplification.
In another preference, described ring mediated isothermal amplification condition is：
(1) 62 ± 2 DEG C of (preferably 62 ± 1 DEG C) 2 ± 1min of isothermal reaction (preferably 1 ± 1min)；
(2) 1. 62 ± 2 DEG C (preferably 62 ± 1 DEG C) 30 seconds, 2. 62 DEG C ± 2 DEG C (preferably 62 ± 1 DEG C) 30
Second collection fluorescence；Step 1. -2. repeat 60 ± 10 times (preferably 55 ± 5 times).
In another preference, the system of described ring mediated isothermal amplification includes：Primer pair 1-3 is mixed
Thing, loop-mediated isothermal amplification buffer solution, Bst2.0DNA polymerases (Bst2.0DNA polymerase,
Bst2.0), thermal starting reverse transcriptase (WarmStart RTx Reverse Transcriptase, WSRTx),
Hydroxynaphthol blue dyestuff (Hydroxy naphthol blue, HNB), green fluorescence nucleic acid dye SYTO 9
(SYTO 9Green Fluorescent Nucleic Acid Stain), calcein dyestuff (Calcein),
RNase inhibitor (Rnase inhibitor).
Wherein, described loop-mediated isothermal amplification buffer solution includes：DNTP, Thermolpol RB
(Isothermal Amplification Buffer), magnesium sulfate, glycine betaine (Betaine).
In another preference, (a) visual detection, described dyestuff is hydroxynaphthol blue dyestuff (Hydroxy
Naphthol blue, HNB)；After loop-mediated isothermal amplification terminates, day in amplified production be present
Blueness, then show that amplification is positive；Lilac in amplified production be present, then show that amplification is negative.(b) visually
Calcein dyestuff (Calcein) also may be selected in detection, described dyestuff；In loop-mediated isothermal amplification
After end, green in amplified production be present, then show that amplification is positive；Crocus in amplified production be present,
Then show that amplification is negative.(c) Real-time near real-time quantitative monitorings use green fluorescence nucleic acid dye SYTO 9
As dyestuff；After loop-mediated isothermal amplification terminates, there is amplification curve, then show that amplification is positive；Not yet
There is amplification curve, then show that amplification is negative.
In another preference, the method for the identification zika virus is non-methods for the diagnosis of diseases.
In another preference, described primer is used to expand as a mixture：
SEQ ID NO:1 or SEQ ID NO:2 in primer mixture final concentration be 0.2 μM；
SEQ ID NO:3 or SEQ ID NO:4 in primer mixture final concentration be 1.6 μM；
SEQ ID NO:5 or SEQ ID NO:6 in primer mixture final concentration be 0.8 μM；
Present invention also offers a kind of buffer solution for ring mediated isothermal amplification, the buffer solution includes：
DNTP, Thermolpol RB (Isothermal Amplification Buffer), magnesium sulfate, beet
Alkali.
In another preference, each reagent is respectively in the ultimate density of reaction system in the buffer solution：
DNTP (1.0-1.6mM), Thermolpol RB (1X), magnesium sulfate (6-10mM), glycine betaine (0-0.8M).
Present invention also offers a kind of kit for identifying zika virus, the kit includes：The examination
Agent box includes (i) container, and (ii) is located at the primer combination of the first aspect present invention in the container.
In another preference, also include in the kit：
Buffer solution or independent packing dNTP, Thermolpol RB and magnesium sulfate for ring mediated isothermal amplification；
Examination criteria product containing zika virus；RNA extracts reagents；Bst 2.0DNA polymerases；WSRTx is anti-
Transcriptase；RNase inhibitor；
Dyestuff：Visual detection uses HNB dyestuffs (ultimate density is 120 μM), or calcein dyestuff (calcium
Yellowish green element and manganese chloride are 1 by concentration ratio:100, ultimate density is respectively 500 μM：5mM)；real-time
Monitoring uses the dyestuffs of SYTO 9 (final concentration of 0.4 μM) in real time.
Illustrate the operation instructions for identifying the method for zika virus.
In another preference, the kit is used to identify zika virus from testing sample.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and below (such as embodiment)
It can be combined with each other between each technical characteristic of middle specific descriptions, so as to form new or preferable technical side
Case.As space is limited, no longer tire out one by one herein and state.
Brief description of the drawings
Fig. 1 shows the orthogonal experiments of Real-time real-time quantitative PCRs detection LAMP reaction conditions.
Fig. 2 shows Real-time real-time quantitative PCRs detection LAMP sensitivity.
Fig. 3 shows Real-time real-time quantitative PCRs detection LAMP specificity.
Fig. 4 shows the visual monitoring result figure of Zika LAMP course of reaction.Wherein, upper row is HNB
Dyeing, the positive is sky blue, and feminine gender is purple；Middle row and lower row are calcein dyeing, point
Do not show visible ray and the lower result of naked eyes, and calcein is positive for green, feminine gender for yellow (visually,
Visible ray), calcein is positive to be bright, and feminine gender is dark (ultraviolet).
Fig. 5 shows the real-time monitoring figure of Zika LAMP course of reaction.
Fig. 6 shows the Zika LAMP visual monitoring result figure to clinical sample HNB dyestuffs.
Fig. 7 shows the Zika LAMP real-time monitoring figure to simulating clinical sample.
Fig. 8 shows Zika LAMP clinical sample detection figures.
Fig. 9 shows RT-QPCR (electrophoresis) checking Zika LAMP result.
Figure 10 shows the real-time monitoring figure of different Zika LAMP primer compositions sensitivity.
Embodiment
The present inventor discloses a kind of zika virus (Zika first by in-depth study and experiment extensively
Virus, ZIKV) ring mediated isothermal amplification (loop-mediated isothermal amplification,
LAMP) detection method.By comparing screening, have found for the good primer of Zika virus-specifics, institute
State primer and be directed to and specific amplification does not occur containing the nucleic acid beyond Zika viruses.The method of the present invention can be good
Ground is applied to identification Zika virus compositions, and with good repeatability, sensitivity.
The present inventor have found in many Zika diseases by the analysis to a large amount of Zika virus genome sequences
Sequence section relatively conservative in virus gene group, based on the screening of this progress primer, obtaining one kind can specificity
The primer of Zika viruses is identified, specific amplification occurs for the RNA of Zika viruses in it, and to other diseases
Specific amplification does not occur for the nucleic acid of poison.
LAMP (Loop-mediated isothermal amplification, ring mediation isothermal duplication) be
Reported first in 2000 and behind by updating a kind of isothermal to be formed, it is continuously, quickly, high
Special and Visual retrieval nucleic acid amplification method.In view of the suitable, amplimer of a wider spectrum is difficult to find that,
And, it is difficult to realize and result judgement is carried out according to color change, at present both at home and abroad also without detection Zika diseases
The RT-LAMP detection products of poison occur.
Compared with PCR method, LAMP does not need thermal cycler (PCR instrument), and is produced in being reacted due to LAMP
Raw substantial amounts of accessory substance-white magnesium pyrophosphate precipitation, amplified production observes by the naked eye or nephelometer can be sentenced
Determine result；Therefore LAMP is suitable for scene, wartime field or the poor laboratory of condition and is used for quickly detecting.
LAMP technology is reflected with its advantages that quick, accurate and easy to operate in the scientific research of nucleic acid, pathogenic microorganism
The fields such as fixed and GM food detection have obtained increasingly extensive application.In general, LAMP methods are a kind of
Easy, quick, high special gene amplification method, it is not necessary to special reagent and instrument and equipment, using the party
Method can set up the cheap detection architecture of totle drilling cost.
The present invention uses ring mediated isothermal amplification (LAMP) Fast Detection Technique, and three pairs of design is directed to aim sequence
Eight sections of regions primer, utilize being combined with primer of being formed of strand displacement polymerase and amplified production itself
Neck ring structure realizes the cyclic amplification of purpose fragment, and final product is a series of aim sequence of different lengths
Series connection long-chain.The amplification efficiency of the amplification method is 100 times of PCR.The accessory substance pyrophosphoric acid of simultaneous reactions
Root can be accumulated largely, the amount for the white precipitate that can be formed by the magnesium ion in pyrophosphate and reaction system
To monitor the carry out degree of reaction and can be used to the positive and negative of result of determination.
Therefore, the present invention provides a kind of primer, and described primer is LAMP amplimers, including：Primer
To 1：SEQ ID NO:1 and SEQ ID NO:2；Primer pair 2：SEQ ID NO:3 and SEQ ID NO:
4；Primer pair 3：SEQ ID NO:5 and SEQ ID NO:6.
Using the primer of the present invention, LAMP reactions are carried out, by direct visual perception or turbidity can be passed through
Whether instrument detects, rapidly judge testing sample containing Zika viruses.Preferably, the present invention is anti-in LAMP
Answer in system with HNB as dyestuff, after loop-mediated isothermal amplification terminates, deposited in amplified production
In sky blue, then show that amplification is positive；Lilac in amplified production be present, then show that amplification is negative, naked eyes
Can substantially it observe.Preferably, the present invention in LAMP reaction systems with Calcein as dyestuff,
After loop-mediated isothermal amplification terminates, green in amplified production be present, then show that amplification is positive；Expand
Crocus be present in volume increase thing, then show that amplification is negative, visually can substantially observe.Preferably, Real-time
SYTO 9 can be used to be used as dyestuff near real-time quantitative monitoring, after loop-mediated isothermal amplification terminates, there is expansion
Increase curve, then show that amplification is positive；There is no amplification curve, then show that amplification is negative.
Present invention also offers a kind of method of identification Zika viruses, methods described includes：With testing sample
RNA be template, with specific amplification Zika virus primer expanded；If generation specific amplification,
Then show in testing sample comprising Zika viruses.It is highly preferred that it is applied to mirror based on provided by the present invention
Determine the specific primer of Zika viruses, methods described includes：Using the RNA of testing sample as template, to draw
Thing expands to the mix primer of 1~primer pair 3；If generation specific amplification, shows testing sample
In comprising Zika virus.
The method for obtaining the RNA of testing sample is technology well-known to those skilled in the art, such as can be taken
Traditional phenol/chloroform/isoamyl alcohol method, or the RNA extracts kits commercially available from some, this kind of reagent can be used
Box is well known to those skilled in the art.
The invention further relates to a kind of kit for being used to identify Zika viruses, contain in the kit and be directed to
The LAMP amplimers of Zika viruses.
In addition, reagent of the described kit also containing other identification Zika viruses, such as (but not limited to)：
Examination criteria product containing Zika viruses；Buffer solution for ring mediated isothermal amplification；Contain zika virus
Examination criteria product；RNA extracts reagents；Bs2.0 polymerases；WarmStart RTx reverse transcriptase；HNB；
Calcein and the dyestuffs of SYTO 9；Illustrate the operation instructions for identifying zika virus method.
In addition, the operation instructions also containing identification Zika viruses and standard operation in described kit
Program.
Kit of the present invention can realize quick detection, the purpose of batch detection Zika viruses.
Zika virus
As used herein, term " Zika viruses " and " zika virus " are used interchangeably.
Zika virus (Zika virus, ZIKV) category flaviviridae, Flavivirus, single strand plus RNA virus,
Diameter 20nm, it is a kind of arboviruse propagated by mosquito, host is indefinite, main creature out of office
Long class animal and mosquito in the tree is inhabited, as circulated in aedes africanus.
Zika virus, which is divided into, African 2 hypotypes of type and Asian type.Divided according to the source of infected host,
It can be divided into again：The zika virus in people source, the zika virus in mosquito source, the zika virus in monkey source (are namely felt
The zika virus of people is contaminated, infects the zika virus of mosquito, infects the zika virus of monkey).
The incubation period of zika virus disease is unclear (from time that symptom occur is touched), may be a couple of days.
In zika virus the infected, only about 20% can show light symptoms, and typical symptom includes Acute onset
Low-heat, maculopapule, arthralgia (mainly involving hand, sufficient Minor articulus), conjunctivitis, other symptoms include flesh
Bitterly, headache, eye socket are bitterly and powerless.Rare symptom includes stomachache, Nausea and vomiting, mucosa ulcer in addition
And pruitus.Symptom is generally relatively gentle, continue less than one week, it is necessary to the serious illness of hospitalization not
It is common.Had been reported that respectively during French Polynesia and Brazilian stockaded village's card epidemic situation within 2013 and 2015
Zika virus disease is claimed to be likely to result in nerve and self immune system complication.
Neonate (the new life of birth that many microcephaluses are found that in Brazilian stockaded village's card outbreak of epidemic in 2015
Youngster's head circumference has exceeded two standard deviations with the identical sex and the child in pregnant age ratio, subaverage matched).
Between in January, -2016 in May, 2015, report that 4000 pregnant woman childbirths for infecting zika virus are small altogether
Scaphocephaly, compared with the ratio of former years microcephalus, rise 20 times.35 microcephalus neonates
Head CT and head ultrasound prompting diffusivity brain tissue calcification be present, occur mainly in by telocoele, it is thin
By wall tissue and thalamus region, Basal ganglia region.Ventricles of the brain atrophy can also be shown in caused by atrophy under cortex and cortex
Arrive.There is the contracture of joint in fraction baby, prompts surrounding and central nervous system involvement.Stockaded village's card epidemic situation is opened
Exhibition investigation finds that increasing evidence, which shows to have between zika virus and microcephaly, to be associated.However,
Explain and still need to make more investigation before the relation between baby's microcephaly and zika virus.
Main advantages of the present invention are：
(1) a kind of primer that can be viral specificity identification Zika is disclosed first, and described primer specificity is good
It is good, it can realize specific amplification for Zika viruses.Also, the primer have good repeatability,
As a result it is reliable and stable.
(2) described primer or the detection kit containing the primer are utilized, can be examined quickly, in large quantity
Zika viruses are surveyed, Zika viruses are rapidly and accurately distinguished from testing sample, and required sample size is few,
It is simple to operate.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for
The bright present invention rather than limitation the scope of the present invention.The experiment side of unreceipted actual conditions in the following example
Method, generally write according to normal condition such as J. Pehanorm Brookers etc., Molecular Cloning:A Laboratory guide, the third edition,
Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.
Unless otherwise defined, all specialties used in text and scientific words and one skilled in the art institute are ripe
The meaning known is identical.In addition, any method similar or impartial to described content and material all can be applied to
In the present invention.Preferable implementation described in text only presents a demonstration with material to be used.
I. versatile material and method
Viral nucleic acid extracting method
The Zika viral RNAs fragment used in embodiment is inverted in vitro by Institut Pasteur of Shanghai of the Chinese Academy of Sciences
Record synthesis.Nucleic acid in other non-Zika Virus Samples uses QIAamp Viral RNA Mini Kit
(Qiagen) RNA extracts kits are extracted, and can be used as reaction mould with without RNase water elution, eluent
Plate uses.
LAMP reaction buffers
Take 1 μ L 100mM MgSO4, 3.5 μ L 10mM dNTPs, 2.5 μ L 10 × Thermolpol RB,
7 water of the μ L without RNase.
Wherein 10 × Thermolpol RB are the Bst 2.0DNA polymerase buffers of NEB companies, by 200
MM Tris-HCl, 100mM (NH4)2SO4, 500mM KCl, 20mM MgSO4, 1%Triton X-20
Composition, pH8.8 (25 DEG C).
Reaction enzyme
1 μ L 8U/ μ L Bst2.0 polymerases, 0.5 μ L 15U/ μ L thermal starting are added per secondary response
Reverse transcriptase (WarmStart RTx Reverse Transcriptase), 0.5 μ L 40U/ μ L RNA
Enzyme inhibitor (Rnase inhibitor).
Nucleic acid dye
Visual detection adds 1 μ L 3mM 500 μM of HNB's (Sigma companies) or 1 μ L
Calcein (Sigma companies), real-time Real_time quantitative detections use 10 μM of the 1 μ L (Life of SYTO 9
Company).
Zika virus
Except embodiment 6, zika virus used is the stockaded village of Pasteur institute cell injuring model in other embodiment
Card virus.
RT-LAMP reacts
RT-LAMP reacts primary structure such as table 1.
The Zika RT-LAMP of table 1 react primary structure
Reactions steps are as follows：
(a) visual detection：62 DEG C, 60min directly observes color change.
Dyestuff is HNB or Calcein.
(b) Real-time real time fluorescent quantitatives are detected as：
62 DEG C, 120s, 1 time circulations,
62 DEG C, 60s, 60 times circulations, gather fluorescence
Dyestuff is SYTO 9, and the path setting for gathering fluorescence is SYBR Green I passages.
II. embodiment
Embodiment 1
Design of primers and screening
The whole genome sequence of all Zika viruses of GenBank is obtained, carries out Multiple sequence alignments and sequence
Row analysis, find conservative region, Zika virus (by taking KU740184.1 as an example) sequences 7621bp-7813bp
The conservative of section is higher, is adapted as design of primers region.Above-mentioned zone is intercepted out from comparison result
Come, after comparing again, carry out design of primers.
The primer designed is screened with the RT-LAMP detection architectures established, obtained satisfactory
Primer, screening obtain 6 primers such as table 2, this 6 primers according to being made into A, B, C 3 shown in table 3
The primer pair combination of the different final concentrations of kind.
The Zika virus RT-LAMP specific primer sequences of table 2
The Zika viruses ABC3 of table 3 combines to primer
Embodiment 2
RNA in-vitro transcriptions
In the embodiment of in-vitro transcription, Zika virus particles template (pGH-new vector) is given birth to by Shanghai JaRa
Thing Engineering Co., Ltd synthesizes, and sequence is that 7621bp-7813bp sections (by taking KU740184.1 as an example) use spy
Specific primer expands to Zika virus particle templates, and amplified production carries out electricity with 1% Ago-Gel
Swimming analysis, determine to be used as in vitro purpose band gel extraction, the DNA product of purifying after size is correct
The template of transcription.
In-vitro transcription uses Promega " RiboMAX Large Scale RNA Production
System-T7 " kits, course of reaction use the method that reagent manufacturer provides.In-vitro transcription RNA is produced
Thing carries out concentration and purity determination using Nanodrop, by unit conversion into copies/ μ L (copy/μ L).
To avoid RNA multigelations, take and be diluted to 1 × 10 in right amount10Copies/ μ L are dispensed, -80 DEG C of juxtaposition
Preserve.
Embodiment 3
Establishing and optimization
Using Zika viral RNAs fragment as template, the system based on the system in table 1 is anti-by optimizing
Answer Tween in system (0%, 10%, 30%), dNTP (1.0,1.4,1.6mM), glycine betaine (0,0.4,
0.8M) set three horizontal memories orthogonal with the ultimate density of magnesium ion (6,8,10mM), four factors
Experiment, devises 9 conditional combinations such as table 4.Real-time fluorogenic quantitative detections system uses SYTO 9
As dyestuff, the path setting for gathering fluorescence is SYBR Green I.
Reaction method is as follows with step：
62 DEG C, 120s, 1 time circulations,
62 DEG C, 60s, 60 times circulations, gather fluorescence
The path setting for gathering fluorescence is SYBR Green I, uses the dyestuffs of SYTO 9.
The Zika viral systems optimal conditions of table 4 combine
Real-time fluorogenic quantitative detections result such as Fig. 1 that differential responses condition expands to LAMP in system
It is shown, the fluorescent amplification curve change procedure in course of reaction to be analyzed, reaction progress is fastest,
The minimum conditional combination of Cycle Time values (CT values) is as optimum combination.
Embodiment 4
Sensitivity and specificity are assessed
Use the H without RNase2It is dilute that the RNA templates that O obtains to Zika virus in-vitro transcriptions respectively carry out gradient
Release, obtain concentration and be followed successively by 1 × 106Copies/ μ L, 1 × 105Copies/ μ L, 1 × 104copies/
μ L, 1 × 103Copies/ μ L, 1 × 102Copies/ μ L, 1 × 101Copies/ μ L and 1 × 100copi
Es/ μ L RNA templates, are detected using the Optimal system in embodiment 3.It is as follows to detect reactions steps：
62 DEG C, 120s, 1 time circulations,
62 DEG C, 60s, 60 times circulations, gather fluorescence
The path setting for gathering fluorescence is SYBR Green I, uses the dyestuffs of SYTO 9.
Sensitivity results are as shown in Fig. 2 " logarithmic curve " occurred represents Zika viruses under the concentration
There is amplification.Wherein：Amplification curve is followed successively by 1 × 10 from left to right6Copies/ μ L,
1×105Copies/ μ L, 1 × 104Copies/ μ L, 1 × 103Copies/ μ L, 1 × 102copies/μL
With 1 × 101copies/μL。
Concentration is 1 × 100Copies/ μ L RNA templates and negative control does not have amplification curve, shows LAMP
Minimum detectable the 1 × 10 of system1Copies/ μ L, because applied sample amount is 5 μ L, so this method is most
Low detection line is 5 × 102copies/μL。
The Zika viral RNAs fragment used in embodiment is inverted in vitro by Institut Pasteur of Shanghai of the Chinese Academy of Sciences
Record synthesis.The Virus Standard strain of other negative controls includes：Dengue fever virus 4 hypotypes (Dengue-1,
Dengue-2, Dengue-3, Dengue-4) and common several Respirovirus RSV A (VR-26),
RSVB (VR-1580), Influenza A (VR-99), Influenza B (VR-789), PIV-3
(VR-93), Adenovirus (VR-930), Rhinovirus (VR-1162), HCoV-229E (VR-740)
(above Strain provides by Institut Pasteur of Shanghai, public purchased from ATCC with HCoV-OC43 (VR-1558)
Department).
Above-mentioned virus is examined using step (b) method in the Optimal system in embodiment 3 and embodiment 1
Survey, as a result as shown in fig. 3 and table 5, test result indicates that, the Zika virus primers of LAMP System Designs
Group has very high specificity.
As a result visible, the specificity of the Zika virus LAMP primer sets of design is good, for non-Zika diseases
Poison detection is negative.
The Zika LAMP of table 5 specific outcome
Embodiment 5
The visual and real-time monitoring of course of reaction
Respectively using 5 μ L concentration as 1 × 106Copies/ μ L, 1 × 105Copies/ μ L,
1×104Copies/ μ L, 1 × 103Copies/ μ L, 1 × 102Copies/ μ L, 1 × 101Copies/ μ L,
1×100Copies/ μ L Zika viral RNAs and water (negative control) are used as template, with following reaction systems
Reaction determines the validity of primer sets in PCR instrument and quantitative real time PCR Instrument respectively.
Using the Optimal system in embodiment 3, the detection method that visually and in real time monitoring is reacted is as follows：
(1) visual detection reactions steps are as follows：
Visual detection is 62 DEG C of isothermal reactions 60 minutes, and then range estimation and scanning is taken pictures.Use HNB dyestuffs
With Calcein dyestuffs；
(2) it is as follows to detect reactions steps in real time for fluorescent quantitation：
62 DEG C, 120s, 1 time circulations,
62 DEG C, 60s, 60 times circulations, gather fluorescence
The path setting for gathering fluorescence is SYBR Green I, uses the dyestuffs of SYTO 9.
Visual detection result and fluorescent quantitation real-time testing result such as Fig. 4, shown in Fig. 5, it is seen that 3 kinds of methods exist
Result is consistent after LAMP reacts 60 minutes：Concentration is 1 × 105Copies/ μ L, 1 × 104Copies/ μ L,
1×103Copies/ μ L, 1 × 102Copies/ μ L, 1 × 101Copies/ μ L Zika viral RNAs are sun
Property；And concentration 1 × 100Copies/ μ L Zika viral RNAs and water (negative control) result are feminine gender.It can be seen that
The testing result of LAMP systems is stable, and method is reproducible, and accuracy rate is very high.
Embodiment 6
Detection to clinical sample
Because the Zika viruses clinical sample of the country is very limited, therefore Zika is synthesized by the way of artificial gene synthesis
The 7621bp-7813bp sections (by taking KU740184.1 as an example) of virus, and reverse transcription is into RNA fragments, as
The positive sample of Zika viruses.The nose swab of the respiratory tract of 16 parts of flu children is collected from Shanghai City Nanxiang District hospital
Sample.It is 1 × 10 by 2 μ L concentration8Zika viral RNA fragments artificial synthesized copies/ μ L are added to 2mL
There was only 7 parts of addition Zika viral RNA fragments in part nose swab sample, in 16 parts of samples, remaining 9 parts additions
It is water, mixes for nucleic acid extraction.Extracted and tried with QIAamp Viral RNA Mini Kit (Qiagen) RNA
Viral RNA in agent box extraction sample leachate, and with without RNase water elution.Eluent can be used as reaction mould
Plate uses.
As a result as shown in Figure 6 and Figure 7, visual detection result (HNB dyestuffs) and fluorescent quantitation detect (SYTO in real time
9 dyestuffs) 2 kinds of methods are consistent：It is Zika virus-positives to have 7 parts in 16 parts of nose swab samples, and 9 parts are Zika
It is viral negative.As a result show, the accuracy rate of LAMP systems of the invention and method is high, and Detection results are good.
The positive sample of virus using the urine of 1 infection Zika virus patient as Zika.It is uninfected by with 2
Negative sample of the urine of the people of Zika viruses as Zika viruses.The above-mentioned μ L of 3 kinds of urines 140 are taken respectively
The disease in sample leachate is extracted with QIAamp Viral RNA Mini Kit (Qiagen) RNA extracts kits
Malicious RNA, and with without RNase water elution.Eluent uses as reaction template, while also (is added using RT-QPCR
Electrophoresis) method verifies Zika LAMP testing result.
LAMP results are as shown in figure 8, there is Zika virus amplifications in the urine of Zika virus patients, and other 2
Individual Zika viruses negative sample does not expand.As shown in figure 9, Zika LAMP results and RT-QPCR (electrophoresis)
As a result it is consistent.Result above shows the Zika LAMP systems of the present invention and the sensitivity height of method, and accuracy rate is high,
Detection results are good.
Comparative example
Before the primer combination of the application is obtained, applicant carried out the substantial amounts of primer for being directed to zika virus
Screening operation.By taking one group of primer therein as an example, illustrate.
Using method similar to Example 1, devise for zika virus NS5 genes 9731bp-9923
The LAMP amplimers combination (a) of bp sections (by taking KU740184.1 as an example), shown in specific primer table 6：
The Zika virus RT-LAMPs primer sequence (comparative example) of table 6
Combined respectively with the primer shown in table 6 and combine with the primer shown in the table 2 of embodiment 1 that (the application's draws
Thing combines), detection 1 × 103The zika virus in copies/ μ L people source, 3 repetitions of every group of carry out.
As a result as shown in Figure 10, with shown in table 2 primer combination detected, sensitivity be significantly better than with
Primer shown in table 6 is detected.
All it is incorporated as referring in this application in all documents that the present invention refers to, just as each document
It is individually recited as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read,
Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen
Please appended claims limited range.
Claims (10)
- A kind of 1. primer collection for detecting zika virus, it is characterised in that the primer that the primer collection includes The NS4B genes of zika virus can be specifically bound to, and for expanding the specificity corresponding to the gene Amplified production.
- 2. primer collection as claimed in claim 1, it is characterised in that the primer collection includes one be selected from the group Kind or a variety of primer pairs：First primer pair：F3 primers：Sequence such as SEQ ID NO:Shown in 1；B3 primers：Sequence such as SEQ ID NO:Shown in 2；Second primer pair：FIP primers：Sequence such as SEQ ID NO:Shown in 3；BIP primers：Sequence such as SEQ ID NO:Shown in 4；Three-primer pair：LF primers：Sequence such as SEQ ID NO:Shown in 5；LB primers：Sequence such as SEQ ID NO:Shown in 6.
- 3. primer collection as claimed in claim 1, it is characterised in that described primer collection is used for zika virus Ring mediation isothermal duplication (LAMP).
- 4. a kind of PCR amplification system, it is characterised in that described system includes：Buffering for amplification Primer collection described in system and claim 1 in the system.
- A kind of 5. kit for detecting zika virus, it is characterised in that the kit includes (i) container, (ii) is located at the primer collection as claimed in claim 1 in the container.
- 6. kit as claimed in claim 5, it is characterised in that described kit includes：First container, and the first primer pair in first container；Second container, and the second primer pair in the second container；And/or3rd container, and the three-primer pair in the 3rd container；Wherein, the first described container, second container, the 3rd container can be each independent or be same Container (including two or three containers be same container).
- 7. a kind of isothermal amplification method based on ring mediation, it is characterised in that including step：(a) using the primer collection described in claim 1, to the isothermal duplication of detection sample progress ring mediation.
- A kind of 8. method for detecting zika virus, it is characterised in that including step：(a) using the primer collection described in claim 1, detection sample is expanded；(b) amplification situation is detected, so as to judge the presence or absence of zika virus in the detection sample.
- 9. method as claimed in claim 8, it is characterised in that in step (b), if specificity Amplified production produces, then zika virus be present in explanation detection sample；If produced without specific amplification products It is raw, then zika virus is not present in explanation detection sample.
- 10. the purposes of the primer collection described in a kind of claim 1, it is characterised in that detect stockaded village for preparing Block the kit of virus.
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| CN114107571A (en) * | 2022-01-18 | 2022-03-01 | 复旦大学附属中山医院 | Kit for LAMP detection of BK virus |
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