CN107400735A - Detect the real-time fluorescent quantitative RT-PCR method of zika virus - Google Patents
Detect the real-time fluorescent quantitative RT-PCR method of zika virus Download PDFInfo
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Abstract
The present invention relates to one kind detection zika virus (also referred to as fork clip virus, Zika virs, ZIKV) real-time quantitative RT PCR methods and its primer, probe and kit.The primer and TaqMan probe are designed according to zika virus conserved sequence, the inventive method specificity is good, high sensitivity, reproducible, simple, convenient quick, can be well applied to the nucleic acid copies for quantitatively detecting zika virus in sample.
Description
Technical field
The present invention relates to field of biological detection, relates more specifically to a kind of real time fluorescent quantitative RT- for detecting zika virus
PCR method.
Background technology
Zika virus (Zika virus, ZIKV) is a kind of arboviruse propagated by mosquito, and host is indefinite,
Main raw primate out of office and mosquito in the tree is inhabited, as circulated in aedes africanus.The virus is most even earlier than nineteen forty-seven
So found by yellow fever monitoring network in the rhesus macaque of stockaded village of Uganda card jungle, with after nineteen fifty-two in Uganda and Tan Sang
Found in the crowd of Buddhist nun Asia.The viral activity compares concealment always, only under the line around Africa, America, Asia and the Pacific Ocean
Area has zika virus to infect Sporadic cases.An earliest outbreak of epidemic is to occur on Western Pacific Mi Keluoni subgroups island for 2007
Ya Pu islands, bigger be once popular in occur in Oceanian French Polynesia, infected about for -2014 years 2013
32000 people.Yellow-fever mosquito also propagates other three kinds of virus in flaviviridae, including dengue fever virus, chikungunya virus and Huang heat
Virus is also mainly popular in subtropical and tropical zones.Decades ago, African researcher notices her mosquito-borne stockaded village's card disease
Malicious epidemic situation somehow or other is followed after yellow-fever mosquito propagation chikungunya virus epidemic situation.Similar rule starts from 2013, works as base
When hole is agreed refined virus and propagated from west to east, zika virus is closelyed follow.
In the prior art, the detection method of stockaded village's card mainly includes Virus culture separation, enzyme linked immunosorbent detection, fluorescent quantitation
The nucleic acid detection methods such as PCR detection techniques.
The sample that Virus culture technology is collected is co-cultured with cell, and disease is detected by observing cytopathy or other method
The amplification of poison determines whether there is viral infection in sample.Although the method is virus infection detection " golden standard " once,
But the virion with infection ability in sample can only be detected, sensitivity deficiency.And operation sequence is complicated, to field experiment
Ground and equipment have bio-safety class requirement, detection process length (at least wanting more than one week), it is impossible to be used in Site Detection, to difference
The testing result of virus is different.
Enzyme mark detection based on antibody, directly with indirect fluorescent detection and collaurum quick detection using antigen with resisting
Specific bond between body, the antibody by being coupled colour developing group realize the Visual retrieval to pathogen.Antibody test is not
Large scale equipment is relied on, and testing result can obtain within 30 minutes.But the process that antibody test is amplified due to no signal,
Sensitivity is lower than Molecular Detection, and false positive and false negative easily occurs in result.Because the manufacturing cycle of new antibodies is longer,
This method is difficult to the new detection for virus occur, and for the detectability of new virus subtype as caused by antigenic shift
Also it is doubtful.The World Health Organization (WHO) research shows that ELISA is difficult to distinguish Zika viruses and dengue fever virus, most
Viral RNA can only be detected using the method for nucleic acid amplification eventually.
At present, zika virus has become global threat, ends on January 26th, 2016, has 24 countries and regions to have epidemic disease
Information road, wherein 22 in America, Europe is multinational at present also has been reported that there is the gesture in the sprawling whole world.End in March, 2016, China
8 Introduced cases zika virus cases of infection are had been acknowledged.Therefore, in order to realize the quantitative inspection of zika virus in clinical sample
Survey, the virus load in quantitative analysis person and other biological specimens, establish the quantitative detecting method of zika virus, and develop phase
The detection reagent answered becomes very urgent.
The content of the invention
It is an object of the invention to provide a kind of real-time fluorescent quantitative RT-PCR method and reagent for detecting zika virus
Box.
In the first aspect of the present invention, there is provided a kind of primer pair, the primer pair specifically bind to zika virus
NS1 genes, and for expanding the specific amplification products corresponding to the gene, also, the primer pair includes：
Sense primer：Sequence such as SEQ ID NO:Shown in 1, and
Anti-sense primer：Sequence such as SEQ ID NO:Shown in 2.
In another preference, the zika virus behaviour zika virus.
In another preference, described primer pair is used for the real-time fluorescence quantitative RT-PCR of zika virus.
In the second aspect of the present invention, there is provided a kind of Nucleic acid combinations, the Nucleic acid combinations include first aspect present invention
Described primer pair and TaqMan probe.
In another preference, described TaqMan probe includes target gene TaqMan probe and reference gene TaqMan
Probe.
In another preference, described target gene is the specific binding region of primer pair described in first aspect present invention
Domain.
In another preference, the sequence such as SEQ ID NO. of described target gene:Shown in 7.
In another preference, described reference gene is artificial synthesized.
In another preference, the sequence such as SEQ ID NO. of described reference gene:Shown in 5.
In another preference, the TaqMan probe is the probe by fluorescence labeling.
In another preference, 5 ' ends of the TaqMan probe are labeled as fluorescent emission group.
In another preference, the fluorescent emission group of the target gene TaqMan probe is FAM.
In another preference, the fluorescent emission group of the reference gene TaqMan probe is HEX.
In another preference, 3 ' ends of the TaqMan probe are labeled as fluorescent quenching group.
In another preference, the fluorescent quenching group of described target gene TaqMan probe is MGB.
In another preference, the fluorescent quenching group of the reference gene TaqMan probe is BHQ1.
In another preference, the sequence such as SEQ ID NO. of the target gene TaqMan probe:Shown in 3.
In another preference, the sequence such as SEQ ID NO. of the reference gene TaqMan probe:Shown in 4.
In the third aspect of the present invention, there is provided a kind of PCR amplification system, described system include：For the slow of amplification
Rush system and the primer pair described in first aspect present invention in the system.
In another preference, the amplification includes real-time fluorescence quantitative RT-PCR.
In another preference, described primer pair includes：
Sense primer：Sequence such as SEQ ID NO:Shown in 1, and
Anti-sense primer：Sequence such as SEQ ID NO:Shown in 2.
In another preference, the concentration of described amplification system middle and upper reaches primer or anti-sense primer is 200-600nM, compared with
It is 300-500nM goodly.
In another preference, TaqMan probe is also included in the amplification system.
In another preference, described TaqMan probe includes target gene TaqMan probe and reference gene TaqMan
Probe.
In another preference, target gene TaqMan probe or reference gene TaqMan probe in described amplification system
Concentration be 100-300nM, preferably 180-220nM.
In another preference, reference gene is also included in described amplification system.
In another preference, the concentration of reference gene is 1-1000 copies/μ L in described amplification system, preferably
1.5-100 copies/μ L, more preferably copy/10 μ L for 2-10.
In another preference, amplification enzyme reverse transcriptase (0.35 μ L/25 μ L) and heat are also included in described amplification system
Start Taq polymerase (1 μ L/25 μ L).
In another preference, dyestuff is also included in described amplification system, such as SYTO 9 (10 μM).
In the fourth aspect of the present invention, there is provided a kind of kit for detecting zika virus, the kit include (i)
First container, and (ii) are located at the primer pair as described in the first aspect of the invention in first container.
In another preference, described kit also includes (i) second container, and (ii) is located in the second container
TaqMan probe.
In another preference, the first container, second container are same containers；And described primer pair and TaqMan are visited
Pin is mixed together, and obtains a mixtures of nucleic acids.
In another preference, described kit also contain for expand reagent, including amplification enzyme, buffer solution,
DNTP, and the reagent for detection, such as the dyestuffs of SYTO 9.
In another preference, described kit also includes the examination criteria product of zika virus.
In another preference, described kit also includes reference gene.
In another preference, the kit is also including the use of specification.
In the fifth aspect of the present invention, there is provided a kind of amplification method based on real-time fluorescence quantitative RT-PCR, including step
Suddenly：
(a) using the primer pair described in first aspect present invention, real-time fluorescence quantitative RT-PCR is carried out to detection sample.
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 method for detecting zika virus, including step：
(a) using the primer pair 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 in explanation detection sample
Zika virus be present；If produced without specific amplification products, illustrate zika virus is not present in detection sample.
In another preference, in step (a), described amplification is real-time fluorescence quantitative RT-PCR.
In another preference, in step (a), using the amplification system described in third aspect present invention to detecting sample
Real-time fluorescence quantitative RT-PCR is carried out, so as to obtain the reactant mixture comprising amplified production.
In another preference, in step (a), the annealing temperature of described real-time fluorescence quantitative RT-PCR is 50-56
DEG C, preferably 53-55 DEG C.
In another preference, the reaction condition of described real-time fluorescence quantitative RT-PCR is：50 DEG C of reverse transcription 30min, 1
Individual circulation；92 DEG C of 3min, 1 circulation；92 DEG C of 10s, 55 DEG C of 20s, 68 DEG C, 20s, 45 circulations, extend phase acquisition at 68 DEG C
Fluorescence signal.
In another preference, described method is nondiagnostic method.
In another preference, described method is in-vitro method
In the seventh aspect of the present invention, there is provided primer pair or second party of the present invention described in a kind of first aspect present invention
The purposes of Nucleic acid combinations described in face, for preparing the kit of detection zika virus.
Present invention also offers the primer and TaqMan probe of a kind of real-time fluorescence quantitative PCR detection zika virus.
The invention provides primer and TaqMan probe can specifically bind to Zika virus NS1 genes, be used for
Quantitatively detect the nucleic acid copies of zika virus in sample.
Described primer, it is characterised in that：Upstream primer sequence such as SEQ ID NO:Shown in 1, downstream primer sequence such as SEQ
ID NO:Shown in 2.
Described target gene TaqMan probe sequence such as SEQ ID NO:Shown in 3, it is characterised in that：5 ' end mark fluorescents
Transmitting group is FAM, and 3 ' end mark fluorescent quenching groups are MGB；Described reference gene TaqMan probe sequence such as SEQ ID
NO:Shown in 4, it is characterised in that：5 ' the end mark fluorescent transmitting group is HEX, and 3 ' end mark fluorescent quenching groups are BHQ1.
Described real-time fluorescence quantitative PCR reaction system includes：25 μ L real-time fluorescence quantitative PCR reaction systems include：On
Swim primer (400nM), anti-sense primer (400nM), target gene TaqMan probe (200nM), reference gene TaqMan probe
(200nM), reference gene (5.6copies/25 μ L), 2 × Quant One Step Probe qRT-PCR Master
Mix12.5 μ L, the μ L/25 μ L of reverse transcriptase (Quant RTase) 0.35, the μ L/25 μ L of thermal starting Taq polymerase 1
(HotMastertaqpolymerase) (TIANGEN Biotech (Beijing) Co., Ltd.).
Described reference gene sequence such as SEQ ID NO:Shown in 5, it is characterised in that the sequence is artificial synthesized design
One section of sequence, reference gene are the RNA of external reverse transcription, and concentration is 5.6copies/25 μ L.
Described RT-QPCR steps and method, the optimal reaction temperature of amplification and time is：50 DEG C of reverse transcription 30min, 1
Individual circulation；92 DEG C of 3min, 1 circulation；92 DEG C of 10s, 55 DEG C of 20s, 68 DEG C, 20s, 45 circulations, extend phase acquisition at 68 DEG C
Fluorescence signal.
The invention provides a kind of kit for being detected to Zika viruses.
Kit provided by the invention, including it is above-mentioned for drawing to zika virus progress real-time fluorescence quantitative PCR detection
Thing and TaqMan probe.
It is nondiagnostic method in method of the present invention.
It is in-vitro method in method of the present invention.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to form new or preferable technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Fig. 1 shows ZikaRT-QPCR primer screening results.
Fig. 2 shows ZikaRT-QPCR probe comparative results.Wherein, red curve is FAM-MGB probe amplification knots
Fruit, the curve of black is FAM-BHQ1 probe amplification results.
Fig. 3 shows ZikaRT-QPCR primer concentration optimum results.
Fig. 4 shows ZikaRT-QPCR annealing temperature optimum results.
Fig. 5 shows ZikaRT-QPCR concentration and probe concentration optimum results.
Fig. 6 shows ZikaRT-QPCR internal reference concentration optimization results.
Fig. 7 shows ZikaRT-QPCR sensitivity technique results.
Fig. 8 shows ZikaRT-QPCR specific detection results.
Fig. 9 A show ZikaRT-QPCR standard curves.
Fig. 9 B show ZikaRT-QPCR pattern detection results.
Figure 10 shows ZikaRT-QPCR standard curves and pattern detection electrophoretogram.
Embodiment
The present inventor by extensively and in-depth study and experiment, disclose first a kind of zika virus (Zika virus,
ZIKV real-time fluorescence quantitative PCR detection method).By comparing screening, have found for the good primer of Zika specificity, institute
State primer and be directed to and specific amplification does not occur containing the nucleic acid beyond Zika.The method of the present invention can be well applied to identify
Zika compositions, and with good repeatability, sensitivity.
The present invention is in the quality and Imported and exported animals inspection and quarantine field of control people and non-human's associated biomolecule product
With larger practical significance, the quality of associated biomolecule product can be ensured, the propagation of zika virus is controlled, available for clinical disease
The calibrating of zika virus, has a extensive future in people and biological products.
Specifically, the present inventor have found in many Zika viruses bases by the analysis to a large amount of Zika genome sequences
Because of NS1 sequence sections relatively conservative in group, based on the screening of this progress primer, obtain can specificity identification Zika primer and
TaqMan probe, specific amplification occurs for Zika RNA in it, and specific amplification does not occur to other viral nucleic acid.
The present invention use Real-Time Fluorescent Quantitative PCR Technique, in the design pair of primers of NS1 conservative regions viral Zika,
When PCR has amplification, probe will be combined with amplified production, and the 5 prime excision enzyme activity of Taq enzyme 5 ' → 3 ' will cut off probe, glimmering
Light group will will produce fluorescence signal, the progress of reaction can be monitored in real time by QPCR instruments away from quenching group,
After PCR reactions terminate, there is serpentine amplification curve, then show that amplification is positive；There is no serpentine amplification curve, then show that amplification is cloudy
Property.
Present invention also offers a kind of identification Zika method, methods described includes：Using the RNA of testing sample as template,
Expanded with specific amplification Zika primer and TaqMan probe；If generation specific amplification, shows in testing sample
Include Zika.It is highly preferred that based on the specific primer and TaqMan probe provided by the present invention for being applied to identification Zika, institute
The method of stating includes：Using the RNA of testing sample as template, expanded with primer and TaqMan probe mix primer；Ruo Fashengte
Specific amplification, then show to include Zika in testing sample.
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 can be used, this kind of kit is those skilled in the art
It is well known.
The invention further relates to a kind of kit for being used to identify Zika, contain in the kit and carry out QPCR for Zika
The primer and TaqMan probe of amplification.
In addition, described kit also contains other identification Zika reagent, such as (but not limited to)：Inspection containing Zika
Survey standard items；Sense primer；Anti-sense primer；Target gene TaqMan probe；Reference gene TaqMan probe；Reference gene；2×
Quant One Step Probe qRT-PCR Master Mix；Reverse transcriptase (Quant RTase)；Thermal starting Taq polymerase
(HotMastertaqpolymerase) (TIANGEN Biotech (Beijing) Co., Ltd.)；RNA extracts reagents；Illustrate to identify stockaded village's card
The operation instructions of viral methods.
In addition, operation instructions and S.O.P. in described kit also containing identification Zika.
Kit of the present invention can realize quick detection, batch detection Zika purpose.
Real-Time Fluorescent Quantitative PCR Technique
Real-Time Fluorescent Quantitative PCR Technique with the advantages that its sensitivity height, high specificity, fast speed gene expression dose,
Mutation and polymorphic Journal of Sex Research, pathogen it is qualitative and quantitative detection etc. be used widely.Sonde method real time fluorescent quantitative
PCR principle：PCR expand when in system contain primer on the basis of add a specific fluorescence probe, the probe
Essence be oligonucleotides, both ends one fluorophor of mark and a quenching group respectively.When PCR is not expanded,
Fluorophor and quenching group are combined together, and the fluorescence signal of fluorophor, which is quenched group and absorbed, to be quenched；When PCR has expansion
When increasing, probe will be combined with amplified production, and the 5 prime excision enzyme activity of Taq enzyme 5 ' → 3 ' will cut off probe, fluorophor
Fluorescence signal will will be produced away from quenching group.
Zika virus
As used herein, term " Zika viruses " and " zika virus " are used interchangeably.
Zika virus (Zika virus, ZIKV) belongs to flaviviridae, Flavivirus, single strand plus RNA virus, diameter
20nm, it is a kind of arboviruse propagated by mosquito, host is indefinite, main raw primate out of office and inhabites
Mosquito on tree, as circulated in aedes africanus.
The incubation period of zika virus disease is unclear (from time that symptom occur is touched), may be a couple of days.Stockaded village's card disease
In malicious the infected, only about 20% can show light symptoms, low-heat of the typical symptom including Acute onset, maculopapule, joint
Pain (mainly involving hand, sufficient Minor articulus), conjunctivitis, other symptoms are bitterly and powerless including myalgia, headache, eye socket.It is rare in addition
Symptom include stomachache, Nausea and vomiting, mucosa ulcer and pruitus.Symptom is generally relatively gentle, continue less than one week, it is necessary to
The serious illness of hospitalization is not common.2013 and 2015 respectively in French Polynesia and Brazilian stockaded village's card epidemic situation phase
Between, it has been reported that zika virus disease is likely to result in nerve and self immune system complication.
Neonate's (neonate's head circumference of birth that many microcephaluses are found that in Brazilian stockaded village's card outbreak of epidemic in 2015
Identical sex and the child in pregnant age ratio, subaverage have exceeded two standard deviations with matching).- 2016 years in Mays, 2015
Between January, 4000 pregnant woman childbirths for infecting zika virus microcephalus youngster, the ratio phase with former years microcephalus are reported altogether
Than rising 20 times.There is diffusivity brain tissue calcification in the neonatal head CT of 35 microcephaluses and head ultrasound prompting, main
Occur by telocoele, by parenchymal tissue and thalamus region, Basal ganglia region.The ventricles of the brain caused by atrophy wither under cortex and cortex
Contracting can also be seen.There is the contracture of joint in fraction baby, prompts surrounding and central nervous system involvement.Stockaded village's card epidemic situation is carried out and adjusted
Discovery is looked into, increasing evidence, which shows to have between zika virus and microcephaly, to be associated.However, explain baby's microcephaly with
Still need to make more investigation before relation between zika virus.
Main advantages of the present invention include：
(a) disclose first it is a kind of can specificity identification Zika primer and TaqMan probe, described primer specificity is good
It is good, it can realize specific amplification for Zika.
(b) described primer or the detection kit containing the primer are utilized, can quickly, detect Zika in large quantity,
Zika is rapidly and accurately distinguished from testing sample, and required sample size is few, it is simple to operate.
(c) primer of the invention has good sensitivity, repeatability, result reliable and stable.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as J. Pehanorm Brookers etc. are write, 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, anticipated known to all specialties used in text and scientific words and one skilled in the art
Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the present invention.Described in text
Preferable implementation only present a demonstration and be used with material.
I. experiment material and method
Viral nucleic acid extracting method
The Zika viral RNAs fragment used in embodiment is synthesized by the external reverse transcription of Institut Pasteur of Shanghai of the Chinese Academy of Sciences.
Nucleic acid in other samples is extracted using QIAamp Viral RNA Mini Kit (Qiagen) RNA extracts kits, and uses nothing
RNase water elution, eluent can use as reaction template.
Reaction buffer
μ L (the Tiangeng biochemical technologies (Beijing) of 2 × Quant One Step Probe qRT-PCR Master Mix 12.5
Co., Ltd).
Reaction enzyme
The μ L/25 μ L of reverse transcriptase (Quant RTase) 0.35, the μ L/25 μ L (HotMaster of thermal starting Taq polymerase 1
Taq polymerase) (TIANGEN Biotech (Beijing) Co., Ltd.).
RT-QPCR reacts
RT-QPCR reaction systems such as table 1.
The ZikaRT-QPCR of table 1 reacts primary structure
It is as follows to react general step：
50 DEG C of reverse transcription 30min, 1 circulation；92 DEG C of 3min, 1 circulation；92 DEG C of 10s, 60 DEG C of 20s, 68 DEG C, 20s, 45
Individual circulation, extend phase acquisition fluorescence signal at 68 DEG C.
II. embodiment
Embodiment 1
The screening of primer and probe
All Zika of GenBank whole genome sequence is obtained, carries out Multiple sequence alignments and sequence analysis, finds and protects
Defending zone domain, it is higher in the conservative of viral (by taking AY632535.2 as an example) sequence 3335bp-3626bp sections of Zika, it is adapted as
Design of primers region.Above-mentioned zone is intercepted from comparison result and come out, after comparing again, carries out design of primers.
The primer designed is screened, met with RT-QPCR (SYTO9 dye methods) detection architecture established
It is required that primer, screening obtains 6 sense primers, and 6 anti-sense primers, and sequence is as shown in table 2, altogether combination obtain 36 (6 ×
6) to primer, according to the system of table 3, optimal primer combination is screened according to amplification curve.
2 ZikaRT-QPCR of table, 6 sense primers (F1-F6) and 6 anti-sense primers (R1-R6)
The ZikaRT-QPCR of table 3 reaction screening primer systems
Reactions steps are as follows：
50 DEG C of reverse transcription 30min, 1 circulation；92 DEG C of 3min, 1 circulation；92 DEG C of 10s, 60 DEG C of 20s, 68 DEG C, 20s, 30
Individual circulation, extend phase acquisition fluorescence signal at 68 DEG C, the path setting for gathering fluorescence is SYBR Green I passages.
The experimental result of 36 pairs of primer combinations (6 × 6) is as shown in Figure 1, the results showed that, sense primer (SEQ ID NO:1)
With anti-sense primer (SEQ ID NO:2) this to primer combination amplification curve it is best, amplification efficiency is best, intend using this to primer
Carry out further Optimal Experimental.
SEQ ID NO. sequence remarks
SEQ ID NO:1CAACTACTGCAAGTGGAAGGGT
SEQ ID NO:2AAGTGGTCCATATGATCGGTTGA
SEQ ID NO:3TGGTATGGAATGGAGATAAGGC 5 end FAM, 3 end MGB
SEQ ID NO:6GGCTGCTGGTATGGAATGGAGATAAGGC 5 end FAM, 3 end BHQ1
With sense primer (SEQ ID NO:And anti-sense primer (SEQ ID NO 1):2) to 2 probes：(SEQ ID NO:3)
(SEQ ID NO:6) screening is compared with the RT-QPCR systems of table 1, concentration is 10 respectively4, 103, 102, 101,
100Copies/ μ L Zika RNA screen optimal probe according to amplification curve as template.
The comparative experiments result of 2 probes is as shown in Figure 2, the results showed that, FAM-MGB probes can amplify 104, 103,
102The template (red curve) of 3 kinds of concentration, and FAM-BHQ1 probes can only amplify 104, 103The template of 2 kinds of concentration is (black
The curve of color), illustrate FAM-MGB probes (SEQ ID NO:3) than FAM-BHQ1 probe (SEQ ID NO:6) amplification efficiency compared with
It is good, so intending using MGB probes (SEQ ID NO:3) further Optimal Experimental is carried out.
Embodiment 2
RNA in-vitro transcriptions
In the embodiment of in-vitro transcription, Zika virus particles template (pGH-new vector) is by Shanghai JaRa bioengineering
Co., Ltd synthesizes, and sequence is 3335bp-3626bp sections (by taking AY632535.2 as an example), sequence such as SEQ ID NO:Shown in 7,
Zika virus particle templates are expanded with specific primer, amplified production carries out electrophoretic analysis with 1% Ago-Gel,
Determine size after correct to purpose band gel extraction, the DNA product of purifying can be as the template of in-vitro transcription.
In-vitro transcription uses Promega " RiboMAX Large Scale RNA Production System-T7 " examinations
Agent box, course of reaction use the method that reagent manufacturer provides.In-vitro transcription RNA products carry out concentration and pure using Nanodrop
Degree measurement, by unit conversion into copies/ μ L (copy/μ L).To avoid RNA multigelations, take it is appropriate be diluted to 1 ×
1010Copies/ μ L are dispensed, -80 DEG C of preservations of juxtaposition.
Reference gene template (pGH-new vector) sequence such as SEQ ID NO:Shown in 5, equally also according to the method described above
In-vitro transcription preserves into RNA, -80 DEG C of juxtaposition.
Embodiment 3, Establishing and condition optimizing
Using Zika RNA fragments as template, the system based on the system in table 1, by being separately optimized 10 in reaction system
μM upstream and downstream primer usage amount (0.25,0.5,0.75,1,1.25,1.5 μ L), annealing temperature (55,51,52.9,55.3,
57.9th, 60.7,63.4,65.8 DEG C), 10 μM of probe usage amount (0.1,0.2,0.3,0.4,0.5,0.6,0.8,1.0 μ L) is interior
Join usage amount (2.8 × 103、1.4×103、7×102、2.5×102、1.25×102、6.2×101、3.1×101copies/μ
L optimal reaction condition) is finally screened according to amplification curve.
In system different primers concentration to real-time fluorescent quantitation QPCR testing results as shown in figure 3, to course of reaction
In fluorescent amplification curve change procedure analyzed, reaction carry out it is fastest, Cycle Time values (Cq values) minimum, fluorescence
It is worth the condition of highest as optimal conditions.As a result show：Cq values are minimum when 10 μM of upstream and downstream primer usage amount is 1 μ L, expand
Increasing Efficiency is best.
Different annealing temperature is as shown in Figure 4 to real-time fluorescent quantitation QPCR testing results in system, the results showed that：
Cq values are minimum when annealing temperature is in the range of 50-55 DEG C, and amplification efficiency is best.
Different probe concentration is as shown in Figure 5 to real-time fluorescent quantitation QPCR testing results in system, the results showed that：
Cq values are minimum when 10 μM of probe usage amount is 0.2-0.5 μ L, and amplification efficiency is best.
Internal reference concentration is as shown in Figure 6 to real-time fluorescent quantitation QPCR testing results in system, the results showed that：Internal reference
Concentration 2.8 × 103—3.1×101Lower in concentration range, CT values are smaller when expanding purpose fragment, and amplification efficiency is better.
By a series of above-mentioned condition optimizings, RT-QPCR Optimal systems are obtained, as shown in table 4：
The ZikaRT-QPCR of table 4 reacts Optimal system
Optimum detection reactions steps are as follows：
50 DEG C of reverse transcription 30min, 1 circulation；92 DEG C of 3min, 1 circulation；92 DEG C of 10s, 55 DEG C of 20s, 68 DEG C, 20s, 45
Individual circulation, extend phase acquisition fluorescence signal at 68 DEG C.The path setting for gathering fluorescence is FAM and HEX.
Embodiment 4
Sensitivity and specificity are assessed
Use the H without RNase2The RNA templates that O obtains to Zika virus in-vitro transcriptions respectively carry out gradient dilution, obtain
Concentration is followed successively by 50copies/ μ L, 25copies/ μ L, 10copies/ μ L, 5copies/ μ L, 1copies/ μ L RNA templates,
Each concentration is repeated 10 times, and is detected using the Optimal system in embodiment 3 and step (in order to reduce internal reference to purpose fragment
Amplification competition performance, the internal reference in table 4 replaces with without RNase water).
Simultaneously using the Optimal system in embodiment 3 and step to Zika viral RNAs fragment and the disease of other negative controls
Malicious type strain：4 hypotypes (Dengue-1, Dengue-2, Dengue-3, Dengue-4) of dengue fever virus and common several
Kind 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) and
HCoV-OC43 (VR-1558) (above Strain provides by Institut Pasteur of Shanghai, purchased from ATCC companies) is detected.
Sensitivity results are as shown in fig. 7, Cq values ＜ 45, fluorescence threshold more than 0.05, " logarithmic curve S " the shapes expansion of appearance
Increase curve, represent that Zika viruses have amplification under the concentration.As a result 50copies/ μ L, 25copies/ μ L, 10copies/ are shown
(the one two three row) has amplification under μ L concentration, and concentration is to have 9 to have amplification in 5copies/ μ L 10 repetitions of RNA templates,
Concentration is to have 6 to have amplification in 1copies/ μ L 10 repetitions of RNA templates, test result indicates that, the Zika RT- of design
Primer and TaqMan probe in QPCR systems have very high sensitivity.
Specific outcome as shown in Fig. 8 and table 6, test result indicates that, the primer in the Zika RT-QPCR systems of design
There is very high specificity with TaqMan probe.
The Zika RT-QPCR of table 6 specific outcome
Embodiment 5
Detection to clinical sample
Due to the country Zika viruses clinical sample it is very limited, we using 1 infection Zika virus patients urine as
The positive sample of Zika viruses.Negative sample of the urine for the people for being uninfected by Zika viruses using 2 as Zika viruses.Take respectively
The above-mentioned μ L of 3 kinds of urines 140 are leached with QIAamp Viral RNA Mini Kit (Qiagen) RNA extracts kits extraction sample
Viral RNA in liquid, and with without RNase water elution.Eluent is used as reaction template, and RT-QPCR result is also used simultaneously
Electrophoresis detection is verified.
RT-QPCR results are as shown in Figure 9, the results showed that Cq values=37,16.5copy/25 of the positive sample of Zika viruses
μ L reaction systems, the viral level that conversion is infected in the urine of Zika virus patients is 1155copy/1000 μ L (urine) sample.
As shown in Figure 10, RT-QPCR results are consistent with electrophoresis result.Result above shows the RT-QPCR systems and method of the present invention
Sensitivity is high, and accuracy rate is high, and Detection results are good.
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document
It is incorporated 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
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (10)
- A kind of 1. primer pair, it is characterised in that the primer pair specifically binds to the genes of NS 1 of zika virus, and for expanding Increase the specific amplification products corresponding to the gene, also, the primer pair includes：Sense primer：Sequence such as SEQ ID NO:Shown in 1, andAnti-sense primer：Sequence such as SEQ ID NO:Shown in 2.
- 2. primer pair as claimed in claim 1, it is characterised in that the zika virus behaviour source zika virus.
- 3. primer pair as claimed in claim 1, it is characterised in that the real-time fluorescence that described primer pair is used for zika virus is determined Measure RT-PCR.
- 4. a kind of Nucleic acid combinations, it is characterised in that the Nucleic acid combinations include the primer pair and TaqMan spies described in claim 1 Pin.
- 5. Nucleic acid combinations as claimed in claim 4, it is characterised in that described TaqMan probe includes target gene TaqMan Probe and reference gene TaqMan probe.
- 6. Nucleic acid combinations as claimed in claim 5, it is characterised in that the sequence such as SEQ of the target gene TaqMan probe ID NO.:Shown in 3.
- 7. Nucleic acid combinations as claimed in claim 5, it is characterised in that the sequence such as SEQ of the reference gene TaqMan probe ID NO.:Shown in 4.
- 8. a kind of PCR amplification system, it is characterised in that described system includes：For the buffer system of amplification and positioned at institute State the primer pair described in the claim 1 in system.
- 9. a kind of kit for detecting zika virus, it is characterised in that the kit includes (i) first container, and (i i) Primer pair as claimed in claim 1 in first container.
- A kind of 10. method for detecting zika virus, it is characterised in that including step：(a) using the primer pair 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.
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