CN102296064A - Specific primer for detecting enterovirus 71 (EV71) and application thereof - Google Patents
Specific primer for detecting enterovirus 71 (EV71) and application thereof Download PDFInfo
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Abstract
The invention discloses a specific primer for detecting enterovirus 71 (EV71) and application thereof. The specific primer provided in the invention comprises DNAs represented by Sequence 1 to 4 in a sequence table, preferably comprises DNAs represented by Sequence 1 to 6 in the sequence table. The specific primer in the invention is applicable to specific detection and quantitative analysis of EV71. The advantages of the invention are as follows: (1) amplification can be carried out at a constant temperature without need of special equipment; (2) high specificity is obtained; (3) amplification can be finished within 60 minutes (if six primers are used, it only needs 35 minutes), being rapid and efficient; (4) high sensitivity is realized; (5) convenient and simple identification is achieved. The invention is especially suitable for rapid detection of clinical specimens.
Description
Technical field
The present invention relates to detect the primer special and the application thereof of enterovirns type 71.
Background technology
Enterovirns type 71 (EV71) is to suffer from from one in California first in 1969 to separate the infant faeces sample of central nervous system disease and come.Clinical symptom after the different subtype EV71 infected person is incomplete same, usually (hand-foot-mouth disease is HFMD) with the multiple diseases relevant with neural system such as paralysis of aseptic meningitis, BBE and poliomyelitis sample can to cause patient's hand foot mouth disease.This disease disable and case fatality rate higher, especially infant when morbidity complication and mortality ratio are higher.Since the sixties in 20th century clear and definite gradually its pathogenic agent, hand foot mouth disease has repeatedly been broken out in worldwide or is popular, and it is extensive, populous to involve the region.Recent years, China's enterovirus EV 71 infects popular being on the rise of hand foot mouth disease that causes.Traditional common RT-PCR and real-time fluorescent quantitative RT-PCR method etc. often need take a long time or need specific instrument, are difficult to satisfy the needs that break out the epidemic situation quick diagnosis.
(loop-mediated isothermal amplification, LAMP) method has the advantages that to get final product high speed, quick, high special, high-sensitive amplified target sequence under isothermal condition to the ring mediated isothermal amplification method.This method utilizes 4 special primers (F3, FIP, B3 and BIP) under the effect of high reactivity strand displacement archaeal dna polymerase, constantly duplicates amplification of nucleic acid.Can add 2 ring-type primer LF and LB in reaction system, they also combine the startup strand displacement respectively with loop-stem structure synthetic, goes round and begins again.The final product of amplification is the mixture with different number loop-stem structures, different lengths DNA.
Summary of the invention
The purpose of this invention is to provide the primer special and the application thereof that detect enterovirns type 71.
Primer special provided by the invention comprises that the sequence 1 of sequence table is to the DNA shown in the sequence 4.
Described primer special also can comprise DNA shown in the sequence 6 of DNA shown in the sequence 5 of sequence table and/or sequence table.
Described primer special preferably is made up of to DNA shown in the sequence 6 sequence 1 of sequence table.
F3 (sequence 1): 5 '-CTTCCCGGTCTCAGCACA-3 ';
B3 (sequence 2): 5 '-GCGGTGTATAGGCTATGAGC-3 ';
FIP (sequence 3): 5 '-TGACCCAGCAAGGTGGATTGCTTTTAGCAGGGAAAGGTGAGCT-3 ';
BIP (sequence 4): 5 '-TGCGGGTACTACACCCAATGGTTTTTAGCCATGAAGGACCCAGTA-3 ';
LF (sequence 5): 5 '-CGGCTCTGAACACCGCACAC-3 ';
LB (sequence 6): 5 '-GGATCATTAGAAGTCACCTTCATGT-3 '.
In the described primer special, 5 ' end and/or the 3 ' end of F3, B3, FIP, BIP, LF, LB also can prolong according to target sequence; In the described primer special, F3, B3, FIP, BIP, LF, LB also can carry out replacement and/or the replacement and/or the insertion of base, and the homology of maintenance 85% gets final product; Described primer special thing also can be made up of the reverse complementary sequence of F3, B3, FIP, BIP, LF, LB.
The present invention also protects the application of described primer special in the assistant identification enterovirns type 71.
The present invention also protects a kind of method of assistant identification enterovirns type 71, comprises the steps:
(1) RNA of extraction virus to be measured;
(2) with described primer special carry out ring mediated isothermal amplification (loop-mediated isothermalamplification, LAMP);
(3) detect amplified production and determine whether virus to be measured is enterovirns type 71.
Described loop-mediated isothermal amplification condition can be: 60～65 ℃ of reactions 30～60min, 75 ℃ of 2min termination reactions then.Described loop-mediated isothermal amplification condition specifically can be: 63 ℃ of reactions 35-60min, 75 ℃ of 2min termination reactions then.In the described loop-mediated isothermal amplification system, the concentration of F3 and B3 is 5pmol/L, and the concentration of BIP and FIP is 40pmol/L, and the concentration of LB and LF is 20pmol/L.
Described enterovirns type 71 specifically can be BrCr strain or F8-Henan-08 strain.
The present invention also protects the application of described primer special in the test kit of preparation assistant identification enterovirns type 71.
RNA extracts can adopt ordinary method, as using Qiagen RNeasy Mini kit or other similar test kits.
The method of the result being analyzed judgement is as follows:
(1) judges by the turbidity of reaction solution
When nucleic acid was synthetic in a large number, dNTP can separate out a large amount of pyrophosphate ions, the Mg in pyrophosphate ion and the LAMP system
2+(or Mn
2+) combination, produce magnesium pyrophosphate or manganese pyrophosphate precipitation, thereby the turbidity of the liquid that induces reaction changes; Based on this principle, can change having judged whether that nucleic acid is synthetic in a large number according to the turbidity of reaction solution, thereby judge whether template is target sequence; Can adopt the turbidity of real-time turbidimeter and the real-time detection reaction liquid of supporting program software thereof to change,, show that then sample to be tested is from enterovirns type 71 if observe typical amplification curve on the turbidimeter in real time.
(2) judge by the colour-change of fluorescence dye
When application present method is judged, need fluorexon, Mg in the LAMP system
2+And Mn
2+Fluorexon and Mg when reacting initial
2+In conjunction with, it is orange that reaction solution shows transparency, and when nucleic acid is synthetic in a large number, can generate the by product pyrophosphate, and the existence of pyrophosphate ion makes Mg
2+Dissociate out fluorexon and Mn
2+In conjunction with, reaction solution shows little turbid yellow-green colour; Based on this principle, can judge whether that nucleic acid is synthetic in a large number according to the colour-change of reaction solution, thereby judge whether template is target sequence; Under the natural light, if reaction solution shows then that by transparent orange little turbid yellow-green colour that becomes sample to be tested is from enterovirns type 71; Under the UV-light, if reaction solution shows hyperfluorescence then shows that sample to be tested is from enterovirns type 71.
The present invention is applicable to enterovirns type 71 (EV71) is carried out specific detection and quantitative analysis.Advantage of the present invention: (1) only need not need specific installation in steady temperature with regard to the energy amplified reaction; (2) specificity height; (3) rapidly and efficiently, amplified reaction can be finished (as only needing 35 minutes with 6 primers) in 60 minutes; (4) highly sensitive; (5) evaluation is convenient and simple.The present invention is specially adapted to the rapid detection of clinical samples.
Description of drawings
Fig. 1 is at natural light (A) and ultraviolet lamp (B) the color comparative result of each sample down among the embodiment 3; 1 to 6 is followed successively by sample 1 to sample 6.
Fig. 2 is the real-time turbidimeter amplification curve of embodiment 3.
Fig. 3 is the real-time turbidimeter amplification curve of embodiment 4.
Fig. 4 is the real-time turbidimeter amplification curve of embodiment 6.
Fig. 5 is the real-time turbidimeter amplification curve of embodiment 7.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, as the molecular cloning operational manual, or the condition of advising according to manufacturer.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Used inorganic chemical reagent and organic reagent all meet the requirement of molecular biology experiment.Primer is synthetic by Shanghai Ying Jun technology company, RT-LAMP test kit: RNA Amplification Kit (RT-LAMP), Japanese Rong Yan company, CodeNo:LMP244.LAMP luciferase assay reagent: Fluorescent Detection Reagent, Japanese Rong Yan company, CodeNo:LMP221.RNA extracts test kit: Rneasy Mini Kit, QIAgen company product, Cat No.74014.RNA enzyme inhibitors (Ribonuclease inhibitor): precious biological (TAKARA) company in Dalian product, CatNo.D2310.The real-time turbidimeter of LA-320C, Japanese Rong Yan company product.% among the following embodiment if no special instructions, is the quality percentage composition.
Coxsackie virus A 16, Coxsackie B virus 3, poliovirus are all available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
Enterovirns type 71 F8-Henan-08 strain: Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL guarantees and can meet country and army's relevant regulations and provide to the public after relevant departments' approval; Reference: Han Jianfeng, Ankang, etc. Xinxiang, Henan hand foot mouth disease pathogen separation in 2008 is identified and the virogene stack features. institute of Military Medical Science Institute periodical, 2008,32 (6): 523-524..
Enterovirns type 71 BrCr strain: Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL guarantees and can meet country and army's relevant regulations and provide to the public after relevant departments' approval; Reference: Arita M, Shimizu H, Nagata N, et al.Temperature-sensitive mutants of enterovirus 71show attenuation in cynomolgus monkeys.J Gen Virol.2005.86 (PT 5): 1391-1401.
The design of embodiment 1, primer special and the preparation of each primer special
As follows according to enterovirns type 71 (EV71) Chinese epidemic strain VP3 albumen design specific primer sequence:
F3 (sequence 1): 5 '-CTTCCCGGTCTCAGCACA-3 ';
B3 (sequence 2): 5 '-GCGGTGTATAGGCTATGAGC-3 ';
FTP (sequence 3): 5 '-TGACCCAGCAAGGTGGATTGCTTTTAGCAGGGAAAGGTGAGCT-3 ';
BIP (sequence 4): 5 '-TGCGGGTACTACACCCAATGGTTTTTAGCCATGAAGGACCCAGTA-3 ';
LF (sequence 5): 5 '-CGGCTCTGAACACCGCACAC-3 ';
LB (sequence 6): 5 '-GGATCATTAGAAGTCACCTTCATGT-3 '.
Be used for the detection of embodiment 3 to embodiment 6.
The preparation of embodiment 2, Virus Sample
The viral liquid of enterovirns type 71 F8-Henan-08 strain calculates infection titer (PFU/ml) by plaque forming unit.Use the DMEM substratum that viral liquid is carried out gradient dilution, (concentration is respectively 3 * 10 to obtain the viral liquid of 5 kinds of different concns
2PFU/ml, 3 * 10
1PFU/ml, 3PFU/ml, 0.3PFU/ml, 0.03PFU/ml).5 kinds of viral liquid obtaining and DMEM substratum (negative control) as six samples, are carried out the detection of embodiment 3 and embodiment 4 respectively.
Sample 1: concentration is 3 * 10
2The F8-Henan-08 virus liquid of PFU/ml.
Sample 2: concentration is 3 * 10
1The F8-Henan-08 virus liquid of PFU/ml.
Sample 3: concentration is the F8-Henan-08 virus liquid of 3PFU/ml.
Sample 4: concentration is the F8-Henan-08 virus liquid of 0.3PFU/ml.
Sample 5: concentration is the F8-Henan-08 virus liquid of 0.03PFU/ml.
Sample 6:DMEM substratum.
The application of embodiment 3, primer special first (detecting the F8-Henan-08 strain)
Detect with 6 kinds of Virus Samples of primer special first embodiment 2 preparations.
One, the extraction of virus genome RNA (adopting RNA to extract test kit)
210 μ l samples are gone in the new 1.5ml centrifuge tube, add 700 μ l RLT solution and 7 μ l beta-mercaptoethanols, add 490 μ l dehydrated alcohols behind the vibration mixing, add at twice behind the thermal agitation in the silicagel column, carry out silicagel column and filter.
Silicagel column filters: with the silicagel column behind the application of sample 10, and the centrifugal 15s of 000g; Add 700 μ l RW1 damping fluids, 10, the centrifugal 15s of 000g; Wash silicagel column twice with the RPE damping fluid is centrifugal (each 500 μ l); Silicagel column is gone in the new centrifuge tube, 10, the centrifugal 2min of 000g; Silicagel column is gone in the new centrifuge tube, add 50 μ l nuclease free water, room temperature leaves standstill 2min, and then 10, the centrifugal 2min of 000g collects elutriant; Add 1 μ l RNA enzyme inhibitors in elutriant ,-70 ℃ of preservations are RNA to be measured.RNA to be measured is used for the detection of step 2 or step 3.
Two, application specific primer first detects (adopting the RT-LAMP test kit)
Detect the RNA to be measured that step 1 obtains with the primer special first among the embodiment 1, in test tube, react.
LAMP reaction system (25 μ l): the concentration of F3 and B3 is 5pmol/L, and the concentration of BIP and FIP is 40pmol/L, and the concentration of LB and LF is 20pmol/L, and 2 * Enzyme Buffer 12.5ul (contains Mg
2+And Mn
2+), Enzyme 1ul, Fluorecent Detection Reagent 1ul (the yellowish green element of calcic), 1 μ l RNA to be measured, the deionized water constant volume of nuclease free and DNAzyme; Above concentration is the final concentration in the system.
The LAMP reaction system is 63 ℃ of reactions 35min, 75 ℃ of 2min termination reactions then.
After the termination reaction, the photo of test tube is seen Figure 1A under the natural light, after the termination reaction under the ultra violet lamp photo of test tube see Figure 1B.Among Figure 1A, sample 1 is positive to sample 3, and sample 4 is negative to sample 6.Among Figure 1B, sample 1 is positive to sample 3, and sample 4 is negative to sample 6.
Adopt visual inspection, the sensitivity of detection method is 3PFU/ml.Concentration=the 3PFU/ml of viral RNA * 210 * 10 among the RNA to be measured
-3Ml ÷ 50 μ l ≈ 0.01PFU/ μ l.The content of viral RNA in each LAMP reaction system=0.01PFU/ μ l * 1 μ l=0.01PFU.Be that sensitivity is the 0.01PFU/ reaction system.
Adopt ultra violet lamp to observe down, the sensitivity of detection method is the 0.01PFU/ reaction system.
Three, detect EV71 virus (turbidimeter in real time) with the primer special first
Do not contain Fluorecent Detection Reagent in the LAMP reaction system, other is with the LAMP reaction system of step 2.
The LAMP reaction system is placed the real-time turbidimeter of LA320C, 63 ℃ of reactions 35min, 75 ℃ of 2min termination reactions then.
The turbidimeter amplification curve is seen Fig. 2 in real time.Sample 1 is positive to sample 3, and sample 4 is negative to sample 6.
The sensitivity that turbidimeter is observed is the 0.01PFU/ reaction system.
The application of embodiment 4, primer special second, primer special third and primer special fourth (detecting the F8-Henan-08 strain)
6 kinds of Virus Samples with primer special second and third couple of embodiment of primer special, 2 preparations detect respectively, and method is with embodiment 3.Detect with 6 kinds of Virus Samples of primer special fourth to embodiment 2 preparations, in the turbidimeter, the reaction times is 60min in real time, and other is with embodiment 3.
Adopt primer special second: macroscopic sensitivity is the 0.01PFU/ reaction system, and the sensitivity that ultra violet lamp is observed down is the 0.01PFU/ reaction system.The sensitivity that turbidimeter is observed is the 0.01PFU/ reaction system.
Adopt primer special third: macroscopic sensitivity is the 0.01PFU/ reaction system, and the sensitivity that ultra violet lamp is observed down is the 0.01PFU/ reaction system.The sensitivity of observing with turbidimeter is the 0.01PFU/ reaction system.
Adopt the primer special fourth: macroscopic sensitivity is the 0.01PFU/ reaction system, and the sensitivity that ultra violet lamp is observed down is the 0.01PFU/ reaction system.The sensitivity of observing with turbidimeter is the 0.01PFU/ reaction system.The turbidimeter amplification curve is seen Fig. 3 in real time.
The result shows: primer special second, primer special third, primer special fourth and primer special first only when using real-time turbidimeter to detect to judge, the reaction times variant (adopt the reaction of primer special first only to need 35 minutes, adopt the primer special fourth to need 35 minutes approximately); Primer special second, primer special third, primer special fourth and primer special first are when using, and detection sensitivity does not have difference; Each organizes primer special when using different detection determination methods (using real-time turbidimeter, visual inspection or ultraviolet lamp to observe), and detection sensitivity does not have difference.
One, the preparation of Virus Sample
Replace enterovirns type 71 F8-Henan-08 strain with enterovirns type 71 BrCr strain, other is with embodiment 2.
Two, use various primer specials and detect the BrCr strain
Detect with primer special first, primer special second, primer special third and primer special fourth 6 kinds of Virus Samples to the step 1 preparation respectively, method is with embodiment 3.
Adopt primer special third: macroscopic sensitivity is the 0.01PFU/ reaction system, and the sensitivity that ultra violet lamp is observed down is the 0.01PFU/ reaction system.The sensitivity of observing with turbidimeter is the 0.01PFU/ reaction system.
Adopt primer special first, primer special second, primer special third and primer special fourth: macroscopic sensitivity is the 0.01PFU/ reaction system, and the sensitivity that ultra violet lamp is observed down is the 0.01PFU/ reaction system.The sensitivity of observing with turbidimeter is the 0.01PFU/ reaction system.Primer special second, primer special third, primer special fourth and primer special first only when using real-time turbidimeter to detect to judge, the reaction times variant (adopt the reaction of primer special first only to need 35 minutes, adopt the primer special fourth to need 60 minutes approximately).
Adopt the primer special first respectively enterovirns type 71 BrCr strain and similar syndrome strain coxsackie virus A 16, Coxsackie B virus 3, poliovirus to be detected that (concentration of viral liquid to be measured is 3 * 10
2PFU/ml).
Method is with embodiment 3.Coxsackie virus A 16, Coxsackie B virus 5, poliovirus are negative findings, and the positive result of enterovirns type 71 BrCr strain illustrates that the inventive method has good specificity.The turbidimeter amplification curve is seen Fig. 4 in real time.
Detect 25 volunteers' ight soil, ight soil dissolving back is detected with the primer special first as sample, method is with embodiment 3.18 positive results (amplification curve is arranged) wherein, 7 negative results (not having amplification curve).The turbidimeter amplification curve is seen Fig. 5 in real time.Illustrate that the LAMP method can be used for detecting the EV71 virus that the clinical case sample comprises.
Sequence table
＜110〉Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL
＜120〉primer special and the application thereof of detection enterovirns type 71
<130>CGGNARY102428
<160>6
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
cttcccggtc?tcagcaca 18
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
gcggtgtata?ggctatgagc 20
<210>3
<211>43
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
tgacccagca?aggtggattg?cttttagcag?ggaaaggtga?gct 43
<210>4
<211>45
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
tgcgggtact?acacccaatg?gtttttagcc?atgaaggacc?cagta 45
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
cggctctgaa?caccgcacac 20
<210>6
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
ggatcattag?aagtcacctt?catgt 25
Claims (10)
1. primer special, the sequence 1 that comprises sequence table is to the DNA shown in the sequence 4.
2. primer special as claimed in claim 1 is characterized in that: described primer special also comprises DNA shown in the sequence 6 of DNA shown in the sequence 5 of sequence table and/or sequence table.
3. primer special as claimed in claim 2 is characterized in that: described primer special is made up of to DNA shown in the sequence 6 sequence 1 of sequence table.
4. the application of arbitrary described primer special in the assistant identification enterovirns type 71 in the claim 1 to 3.
5. the method for assistant identification enterovirns type 71 comprises the steps:
(1) RNA of extraction virus to be measured;
(2) carry out ring mediated isothermal amplification with arbitrary described primer special in the claim 1 to 3;
(3) detect amplified production and determine whether virus to be measured is enterovirns type 71.
6. method as claimed in claim 5 is characterized in that: described loop-mediated isothermal amplification condition is: 60～65 ℃ of reactions 30～60min, 75 ℃ of 2min termination reactions then.
7. method as claimed in claim 6 is characterized in that: described loop-mediated isothermal amplification condition is: 63 ℃ of reactions 35-60min, 75 ℃ of 2min termination reactions then.
8. as claim 5 or 6 described methods, it is characterized in that: in the reaction system of described ring mediated isothermal amplification, the concentration of F3 and B3 is 5pmol/L, and the concentration of BIP and FIP is 40pmol/L, and the concentration of LB and LF is 20pmol/L.
9. as arbitrary described method in the claim 5 to 8, it is characterized in that: described enterovirns type 71 is BrCr strain or F8-Henan-08 strain.
10. the application of arbitrary described primer special in the test kit of preparation assistant identification enterovirns type 71 in the claim 1 to 3.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103388033A (en) * | 2012-05-07 | 2013-11-13 | 上海仁度生物科技有限公司 | Enterovirus type 71 (EV71) real-time fluorescent nucleic acid isothermal amplification detection kit |
Citations (3)
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|---|---|---|---|---|
| WO2009098789A1 (en) * | 2008-02-07 | 2009-08-13 | University Of Miyazaki | Method of detecting pathogenic virus in crustacean by lamp method and reagent kit for detection |
| CN101671748A (en) * | 2009-10-21 | 2010-03-17 | 中山大学 | Primer for detecting infectious bursal disease viruses and method and kit thereof |
| CN101696453A (en) * | 2009-11-02 | 2010-04-21 | 东北农业大学 | Method for detecting bovine viral diarrhea viruses as well as preparation method and use method of reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction kit |
-
2010
- 2010-06-22 CN CN 201010215834 patent/CN102296064B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009098789A1 (en) * | 2008-02-07 | 2009-08-13 | University Of Miyazaki | Method of detecting pathogenic virus in crustacean by lamp method and reagent kit for detection |
| CN101671748A (en) * | 2009-10-21 | 2010-03-17 | 中山大学 | Primer for detecting infectious bursal disease viruses and method and kit thereof |
| CN101696453A (en) * | 2009-11-02 | 2010-04-21 | 东北农业大学 | Method for detecting bovine viral diarrhea viruses as well as preparation method and use method of reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction kit |
Non-Patent Citations (1)
| Title |
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| 《BMC infect. dis》 20091231 Arita,M.,et al. Development of a reverse transcription-loop-mediated isothermal amplification(RT-PCR) system for a highly sensitive detection of enterovirs in the stool samples of acute flaccid paralysis cases. 208 第9卷, * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103388033A (en) * | 2012-05-07 | 2013-11-13 | 上海仁度生物科技有限公司 | Enterovirus type 71 (EV71) real-time fluorescent nucleic acid isothermal amplification detection kit |
| CN103388033B (en) * | 2012-05-07 | 2016-08-10 | 上海仁度生物科技有限公司 | A kind of enterovirns type 71 (EV71) real-time fluorescence nucleic acid isothermal amplification detection kit |
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