CN107988429A - A kind of reagent for detecting rabies viruses and its application - Google Patents
A kind of reagent for detecting rabies viruses and its application Download PDFInfo
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- CN107988429A CN107988429A CN201711205134.7A CN201711205134A CN107988429A CN 107988429 A CN107988429 A CN 107988429A CN 201711205134 A CN201711205134 A CN 201711205134A CN 107988429 A CN107988429 A CN 107988429A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The present invention relates to animal doctor's the pathogenic microorganism examination technical field, discloses a kind of reagent for detecting rabies viruses and its application.Reagent of the present invention is by SEQ ID NO:Sense primer, the SEQ ID NO of nucleotide sequence shown in 1:Anti-sense primer, the SEQ ID NO of nucleotide sequence shown in 2:The probe composition of nucleotide sequence shown in 3.The present invention provides the primer and probe reagent that one group is suitable for RPA, the reagent can accurately detect rabies viruses, and possess compared with high specific, sensitivity and accuracy, compensate for the defects of existing RPA detection techniques lack excellent effect detection reagent.
Description
Technical field
The present invention relates to animal doctor's the pathogenic microorganism examination technical field, and in particular to it is a kind of detect rabies viruses reagent and
It is applied.
Background technology
Rabies viruses (Rabies virus, RV) belongs to Rhabdoviridae (Rhabdoviridae) Lyssavirus
(Lyssavirus).Shape is in shape is played, and nucleocapsid is helically symmetrical, and surface has coating, inside contains single stranded RNA.Be cause it is mad
The pathogen of dog disease.Rubies epidemiology scope is very wide, and the nearly all area in the whole world has been reported that.Because of the patient of rabies death
99% be distributed in Africa and Asia, wherein 56% in Asia 44% in Africa.China is rabic severe epidemic area, near several
Number of falling ill is in rising trend over year, and therefore, urgent need quickly makes a definite diagnosis the sick detection technique, for rabic field experiment diagnosis and
Epidemiology survey provides new method.
The main method of viral nucleic acid detection at present is PCR method, and conventional PCR detections need special thermal cycler instrument
And cumbersome test procedure, it is difficult to meet the detection needs under scene or field condition.In recent years, some nucleic acid isothermal amplifications
Technology gradually grows up, the PCR instrument that these technologies need not be expensive, and can amplify mesh by rapid, high volume in a short time
Fragment, there is simple, quick, high sensitivity.RPA technologies therein are more known as substituting the nucleic acid of PCR
Detection technique.
RPA technologies depend on three kinds of enzymes：Recombinase, the single stranded DNA of single-chain nucleic acid (Oligonucleolide primers) can be combined
Associated proteins (SSB) and strand displacement archaeal dna polymerase.The mixture of these three enzymes is also active at normal temperatures, optimal reaction temperature
At 37 DEG C or so.RPA technologies are to simulate DNA replication dna in organism, based on the polymerase-mediated amplification principle development of recombinase
.It can be such that target gene increases with exponential within the very short time, if coordinating the probe and fluorescence of fluorescent marker
Signal detecting and measuring apparatus can realize the real-time monitoring to template amplification.RPA technologies greatly reduce detection time, simplify reaction interval
Sequence, with reference to scene or Fields detection is very suitable for after Rapid nucleic acid extractive technique and portable real-time fluorescence detection device, has
Have broad application prospects.There is great clinical value using RPA technology for detection rabies viruses.
The key of RPA analyses is the design of amplimer and probe, the design of its primer and probe unlike normal PCR that
Sample is ripe, and PCR primer is not suitable for RPA mostly yet.The general following points of RPA design of primers principles：
(1) 5 ' 3 to 5 nucleotide in end avoid poly- guanine, preferably cytimidine, can promote restructuring；(2) 3 ' 3 cores in end
Thuja acid is the stability that G or C contributes to polymerase；(3) poly- purine and pyrimidine not occur；(4) G/C content is between 30-70%
Avoid the formation of secondary structure, hairpin structure etc.；(5) repeat element is avoided in targeting regions.
Since currently without the primer-design software for RPA, above-mentioned design of primers principle is also only a kind of recapitulative
Design principle, in terms of obtaining preferable expanding effect, such as specificity, sensitivity if desired, then needs technical staff artificial
The condition of groping is designed optimization.
The content of the invention
In view of this, it is an object of the invention to provide a kind of reagent for detecting rabies viruses so that the reagent can
Suitable for RPA, and there is higher specificity, sensitivity and accuracy；
Another object of the present invention is in the application in providing mentioned reagent and detecting rabies viruses kit in preparation.
To achieve these goals, the present invention provides following technical solution：
A kind of reagent for detecting rabies viruses, by SEQ ID NO:Sense primer, the SEQ ID of nucleotide sequence shown in 1
NO:Anti-sense primer, the SEQ ID NO of nucleotide sequence shown in 2:The probe composition of nucleotide sequence shown in 3.
In the specific embodiment of the invention, primer and probe sequence is as follows in the reagent：
Sense primer CTCTGGTGGAGATAAAACGTACTGATGTAG (SEQ ID NO:1)；
Anti-sense primer CTCAACCTATACAGACTCAAGAGAAGACCG (SEQ ID NO:2)；
Probe AGGCATGGAACTGACAAGAGACCCCACTG (BHQ1-dT) C (THF) C (FAM-dT)
GAGCATGCGTCCTTA(SEQ ID NO:3)；
Wherein, BHQ1-dT represents to carry the thymidylic acid of fluorescent quenching group BHQ1, and THF represents four
Hydrogen furans connexon, FAM-dT represent to carry the thymidylic acid of fluorescein base group FAM.
It is used for quickly detecting with reagent of the present invention, with domestic separated rabies viruses CVS-11 plants, JX-08-45 plants
The amplification curve that may be significantly for template with BD06 plants of nucleic acid RNA, other etiology nucleic acids such as canine distemper virus nucleic acid, dog
Coronavirus nucleic acid, dog rotavirus nucleic acid and canine parvovirus nucleic acid and Healthy Dogs throat swab total nucleic acid carry out RPA for template
Reaction does not have amplification curve.
In addition, in sensitivity test, viral nucleic acid 10 is diluted to multiple gradients again, detection is as a result measured and is limited to 102
A copy, has higher sensitivity.Meanwhile sample in domestic 12 parts of proficiency testing test samples is detected；Use
It is nucleic acid-templated in the QIAGEN RNeasy Mini Kit extraction measuring samples of Qiagen companies, then detected with RPA methods
Whether rabies viruses, and the conventional RT-PCR method pair with reference to World Organization for Animal Health (OIE) recommended are contained in these samples
12 parts of samples carry out Parallel testing.As a result the testing result of two kinds of detection methods is consistent.The above results show the reagent of the present invention
In the result that is obtained of primer sets and probe and its detection method it is consistent with the result of RT-PCR, it was demonstrated that of the invention is reliable
Property.
Based on above-mentioned excellent technique effect, the present invention proposes the reagent in detection rabies viruses kit is prepared
Application.Wherein, in the specific embodiment of the invention, the rabies viruses is CVS-11 plants of rabies viruses and BD06 plants of mad dogs
Virus.
According to above application, the present invention provides a kind of kit for detecting rabies viruses, including reagent of the present invention.
In order to further improve the kit, it can also include rabies viruses RNA extracts reagents and RPA amplified reactions
Reagent.
The rabies viruses RNA extracts reagents may include reagent of this area conventionally used for extracting rabies virus strain RNA
One or more, in the specific embodiment of the invention, rabies viruses RNA extracts reagents of the present invention are using Roche companies
Various reagents in High Pure PCR Template Preparation kit.
The RPA amplification reaction reagents may include the one or more of the reagent in the routine RPA reaction systems of this area, example
As the rehydration buffer solution in TwistAmp exo RT (TwistDx, Cambridge, UK) kit, DEPC water, RPA are freezed
Enzyme ball and magnesium acetate solution etc..
In the specific embodiment of the invention, RPA reaction systems (totally 50 μ l) are in RPA augmentation detections instrument or fluorescent quantitation
In PCR instrument, 37 DEG C are reacted 15 minutes, and system composition is as follows：
Rehydration buffer solution 29.5 μ l, 10 μM of upstream and downstream primer each 2.1 μ l, 10 μM of 0.6 μ l of probe, template ribonucleic acid 2
11.2 μ l of μ l and DEPC water, are vortexed after mixing of short duration centrifugation, foregoing mixed liquor are added in the reaction tube of RPA lyophozyme balls,
Add the 2.5 μ l of magnesium acetate solution of 280mM；
From above technical scheme, the present invention provides the primer and probe reagent that one group is suitable for RPA, the reagent
It can accurately detect rabies viruses, and possess compared with high specific, sensitivity and accuracy, compensate for existing RPA detection techniques
The defects of lacking excellent effect detection reagent.
Brief description of the drawings
Fig. 1 show the real time fluorescent quantitative result that different sample of nucleic acid are expanded using primer and probe of the present invention；
Wherein, curve A represents that rabies viruses CVS-11 vaccine strains nucleic acid RNA, curve B represent BD06 street strain nucleic acid RNA；C-G is it
His etiology nucleic acid：Canine distemper virus nucleic acid, canine coronavirus nucleic acid, dog rotavirus nucleic acid and canine parvovirus nucleic acid and health
Dog throat swab RNA；
Fig. 2 show the real time fluorescent quantitative result of the wild type strains BD06 nucleic acid RNA of different copy numbers；Wherein, it is bent
Representative copy number is followed successively by 10 to line from top to bottom5Copy/μ l, 104Copy/μ l, 103Copy/μ l and 102Copy/μ l.
Embodiment
The invention discloses a kind of reagent for detecting rabies viruses and its application, those skilled in the art can use for reference herein
Content, is suitably modified technological parameter realization.In particular, all similar substitutions and modifications are to people in the art
It is it will be apparent that they are considered as being included in the present invention for member.Reagent of the present invention and application are by preferable
Embodiment is described, related personnel substantially can not depart from present invention, in spirit and scope to examination as described herein
Agent and application are modified or suitably change with combining, to realize and using the technology of the present invention.
Just a kind of reagent for detecting rabies viruses provided by the present invention and its application are described further below.
Embodiment 1：Reagent of the present invention
RV RPA FP (sense primer)：CTCTGGTGGAGATAAAACGTACTGATGTAG(SEQ ID NO:1)；
RV RPA RP (anti-sense primer)：CTCAACCTATACAGACTCAAGAGAAGACCG(SEQ ID NO:2)；
RV RPA P (probe)：AGGCATGGAACTGACAAGAGACCCCACTG(BHQ1-dT)C(THF)C(FAM-dT)
GAGCATGCGTCCTTA(SEQ ID NO:2)；
Embodiment 2：Reagent specificity and sensitivity test of the present invention
Material：CVS-11 plants, JX-08-45 plants and BD06 plants of rabies viruses, canine distemper virus nucleic acid, canine coronavirus core
Acid, dog rotavirus nucleic acid and canine parvovirus nucleic acid, Healthy Dogs throat swab.1 primer and probe of embodiment is by Shanghai life work life
Thing Technology Co., Ltd. synthesizes, other reagents are biological pure reagent.
Nucleic acid extraction：200 μ l cultures or the tissue homogenate after 2000g is centrifuged are taken, uses QIAGEN companies
RNeasy Mini kit extract the RNA in a variety of materials according to product description.
In-vitro transcription prepares BD06 plants of RNA of rabies viruses template extraction rabies viruses virus：With primer
GACCATCRGCCCAATCAATTAAAAAGGG and CTGAGTCRGTGATGCTATGGTACC carries out RT-PCR amplifications, and PCR is produced
Thing is connected on pGEM-T carriers after purification, and behind the identified direction of recombinant plasmid, extraction plasmid uses Restriction Enzyme digestion
It is linear, then in-vitro transcription, product are carried out according to the specification of In vitro Transcription T7Kit (Takara)
Digest, after the sodium acetate ethanol precipitation of 3M, be dissolved in the water of no RNase through DNaseI, correspondence is calculated after measured concentration and is copied
Shellfish number, successively from 105Copy/μ l are with 10 times of doubling dilutions to 100A copy/μ l, -70 DEG C of preservations.
RPA amplifications are using TwistAmp exo RT (TwistDx, Cambridge, UK) kits in 50 μ l reaction systems
RPA experiments are carried out, add rehydration buffer solution 29.5 μ l first in 1.5ml centrifuge tubes, 10 μM of primer each 2.1 μ l, 10 μM
0.6 μ l of RV RPA P probes, 2 μ l and DEPC water of template ribonucleic acid, 11.2 μ l, be vortexed after mixing of short duration centrifugation, by foregoing mixed liquor
It is added in the reaction tube of RPA lyophozyme balls, adds the 2.5 μ l of magnesium acetate solution of 280mM, mix, reaction tube is placed in RPA expands
Increase in detector or fluorescence quantitative PCR instrument, 39 DEG C are reacted 25 minutes.Using different types of etiology nucleic acid and Healthy Dogs total nucleic acid as
Template carries out specific detection, is detected by the sensitivity that template is reacted of the RNA standards of different dilution factors.
It is used for quickly detecting with the primer and probe designed by the present invention, with CVS-11 plants of rabies viruses and BD06 plants of nucleic acid
The amplification curve that RNA may be significantly for template, other etiology nucleic acids such as canine distemper virus nucleic acid, canine coronavirus core
Acid, dog rotavirus nucleic acid and canine parvovirus nucleic acid and Healthy Dogs throat swab total nucleic acid carry out RPA reactions for template not to be had
Amplification curve (Fig. 1).
Viral nucleic acid 10 is diluted to multiple gradients again, detection is as a result measured and is limited to 102A copy, has higher sensitive
Spend (Fig. 2).
Embodiment 3：Reagent accuracy testing of the present invention
Sample in domestic 12 parts of proficiency testing test samples is detected using the RPA methods established in embodiment 2.Make
With nucleic acid-templated in the QIAGEN RNeasy Mini Kit of Qiagen companies extraction measuring samples, then examined with RPA methods
Survey in these samples and whether contain rabies viruses.The conventional RT-PCR side recommended referring concurrently to World Organization for Animal Health (OIE)
Method carries out Parallel testing to 12 parts of samples.The results show that the testing result of two kinds of detection methods is consistent.The above results show this hair
The result that bright primer sets and probe and its detection method are obtained is consistent with the result of RT-PCR, it was demonstrated that of the invention is reliable
Property.
Embodiment 4：With the contrast of other primed probes
The present invention is according to RPA primer general design principles, 9 primers (4 upstreams according to rabies viruses genome design
Primer and 5 anti-sense primers) and 1 probe, different primers is combined using rabies viruses BD06 strain virus nucleic acid as template into
Row test.There are 16 pairs of primers to obtain amplification curve in 20 groups of test results, but universal fluorescence signal is weaker, and this
The pair of primers (F4 and R4) of invention not only expands that the departure time is short and fluorescence signal is strong, and effect is more preferable more than other primers.
Table 1
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Sequence table
<110>Enterprises of Futian District animal epidemic prevention supervision institute;In Shenzhen Entry-Exit Inspection and Quarantine Bureau animals and plants inspection and quarantine technology
The heart
<120>A kind of reagent for detecting rabies viruses and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ctctggtgga gataaaacgt actgatgtag 30
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctcaacctat acagactcaa gagaagaccg 30
<210> 3
<211> 49
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(49)
<223> n(30)=BHQ1-dT;n(32)=THF;n(34)=FAM-dT
<400> 3
aggcatggaa ctgacaagag accccactgn cncngagcat gcgtcctta 49
Claims (6)
1. a kind of reagent for detecting rabies viruses, it is characterised in that by SEQ ID NO:Draw the upstream of nucleotide sequence shown in 1
Thing, SEQ ID NO:Anti-sense primer, the SEQ ID NO of nucleotide sequence shown in 2:The probe composition of nucleotide sequence shown in 3.
2. reagent described in claim 1 is preparing the application in detecting rabies viruses kit.
3. apply according to claim 2, it is characterised in that the rabies viruses is CVS-11 plants of rabies viruses, JX-08-
45 plants of rabies viruses and BD06 plants of rabies viruses.
4. a kind of kit for detecting rabies viruses, it is characterised in that including reagent described in claim 1.
5. kit according to claim 4, it is characterised in that further include：Rabies viruses RNA extracts reagents and RPA amplifications
Reaction reagent.
6. kit according to claim 5, it is characterised in that the RPA amplification reaction reagents include：
29.5 μ l of rehydration buffer solution, DEPC water, RPA lyophozymes ball and magnesium acetate solution.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711205134.7A CN107988429B (en) | 2017-11-27 | 2017-11-27 | Reagent for detecting rabies virus and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711205134.7A CN107988429B (en) | 2017-11-27 | 2017-11-27 | Reagent for detecting rabies virus and application thereof |
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| Publication Number | Publication Date |
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| CN107988429A true CN107988429A (en) | 2018-05-04 |
| CN107988429B CN107988429B (en) | 2020-04-28 |
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| CN201711205134.7A Expired - Fee Related CN107988429B (en) | 2017-11-27 | 2017-11-27 | Reagent for detecting rabies virus and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295260A (en) * | 2018-11-09 | 2019-02-01 | 辽宁佰昊生物科技有限公司 | For detecting and/or assisting detection to cause primer sets, reagent and the kit and detection method of hand-foot-and-mouth disease poison EV71 |
Citations (3)
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|---|---|---|---|---|
| CN101153344A (en) * | 2007-09-30 | 2008-04-02 | 中国人民解放军军事医学科学院军事兽医研究所 | Series RT-PCR detection method and detection reagent kit for rabies viruses |
| CN101413948A (en) * | 2008-11-13 | 2009-04-22 | 浙江大学 | Rabies virus NP-ELISA antibody detection reagent kit |
| CN103361445A (en) * | 2013-07-30 | 2013-10-23 | 广西壮族自治区兽医研究所 | LAMP primers for detecting rabies virus, and kit thereof |
-
2017
- 2017-11-27 CN CN201711205134.7A patent/CN107988429B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153344A (en) * | 2007-09-30 | 2008-04-02 | 中国人民解放军军事医学科学院军事兽医研究所 | Series RT-PCR detection method and detection reagent kit for rabies viruses |
| CN101413948A (en) * | 2008-11-13 | 2009-04-22 | 浙江大学 | Rabies virus NP-ELISA antibody detection reagent kit |
| CN103361445A (en) * | 2013-07-30 | 2013-10-23 | 广西壮族自治区兽医研究所 | LAMP primers for detecting rabies virus, and kit thereof |
Non-Patent Citations (4)
| Title |
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| KORE SCHLOTTAU等: ""Development of molecular confirmation tools for swift and easy rabies diagnostics"", 《VIROLOGY JOURNAL》 * |
| 周萍等: ""狂犬病病毒RT-PCR检测方法的建立"", 《广东畜牧兽医科技》 * |
| 孙丽娜等: ""重组人源抗狂犬病毒单克隆抗体鸡尾酒暴露后预防效果评价"", 《病毒学报》 * |
| 陈继章等: ""新疆羊狂犬病病毒的鉴定及N基因序列分析"", 《中国人兽共患病学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295260A (en) * | 2018-11-09 | 2019-02-01 | 辽宁佰昊生物科技有限公司 | For detecting and/or assisting detection to cause primer sets, reagent and the kit and detection method of hand-foot-and-mouth disease poison EV71 |
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