CN110042176A - A kind of primer sets, kit and its detection method and application detecting 4 type of aviadenovirus serum - Google Patents
A kind of primer sets, kit and its detection method and application detecting 4 type of aviadenovirus serum Download PDFInfo
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Abstract
The invention discloses a kind of primer sets, kit and its detection methods and application for detecting 4 type of aviadenovirus serum, the primer sets include outer primer and inner primer, wherein outer primer is F3 and B3, inner primer is FIP and BIP, the nucleotide sequence of the F3 is as shown in SEQ ID NO.1, and the nucleotide sequence of the B3 is as shown in SEQ ID NO.2；The nucleotide sequence of the FIP is as shown in SEQ ID NO.3, and for the nucleotide sequence of the BIP as shown in SEQ ID NO.4, the FIP is the sequence of 5 end biotin labelings, and the BIP is the sequence of 5 end marked by fluorescein isothiocyanate.Present invention combination loop-mediated isothermal amplification technique and lateral flow Lateral Flow Strip, a set of primer is designed for FAdV-4 important factor 52k gene, establish the LAMP-LFD Fast Detection Technique of FAdV-4, LAMP-LFD kit provided by the invention is easy to operate, securely and reliably, sensitive efficient, specific good, it is particularly suitable for quick visualization on site and detects.
Description
Technical field
The invention belongs to 4 type detection technique fields of aviadenovirus serum, and in particular to a kind of 4 type of detection aviadenovirus serum
Primer sets, kit and its detection method and application.
Background technique
Aviadenovirus is the member of Adenoviridae Adenovirus, and according to restriction enzyme enzyme test, aviadenovirus is divided into five
A subspecies (A-E), according to serum cross neutralization, it is divided into 12 serotypes (1-8a, 8b-11).Aviadenovirus is danger
One of the cause of disease of the feeding birds of evil, is mainly caused in farm: fowl gizzard erosion (adenoviral gizzard erosion,
AGE), inclusion body hepatitis (inclusion body hepatitis, IBH), hydropericardium syndrome (hydropericardium
Syndrome, HPS) etc..In these diseases, mainly the hydropericardium as caused by 4 type of aviadenovirus serum (FAdV-4) is comprehensive
The disease death rate reaches 10%-100% etc., easy infection 3-5 week old broiler chicken, and pericardial fluid aggregation, liver occurs in cardia
Inflammation, ephritis.In the monitoring discovery for a long time to Chinese aviadenovirus, year always there is fragmentary aviadenovirus from 2006 to 2014
The case where infection, but do not cause to break out on a large scale.Since the middle ten days in 2015, the multiple regional scale chicken house outbursts of China
Inclusion body hepatitis-hydropericardium syndrome and the trend quickly spread is presented, has seriously endangered the development of poultry farming.Cause
This, anxious accurate, sensitive, quick, the free of contamination clinical pathogenic microorganism method of inspection to be developed.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) is Notomi etc.
People's constant temperature nucleic acid amplification technology new in one kind of invention in 2000.Traditional round pcr be unable to do without this consumption of heating and cooling repeatedly
When the process that consumes energy, response procedures setting is cumbersome, and needs expensive instrument and equipment.In contrast, LAMP technology is directed to target base
Special primer is designed in 6 regions of cause, acts on 30- in 60-65 DEG C of thermostat water bath using strand displacement archaeal dna polymerase
60min can be realized and expand to 109-1010 times of target gene, compared with Standard PCR, be not necessarily to alternating temperature, electrophoresis interpretation etc. repeatedly
Process, can match in excellence or beauty even higher than round pcr in the technical indicators such as sensitivity, specificity and detection range, and not need valuableness
Instrument and equipment, reaction result view mode can directly visually observe precipitating or addition fluorescent dye discoloration etc., cost is far low
In quantitative fluorescent PCR.Therefore, which has and does not need PCR instrument, visually can determine whether that result, reaction time are short, high specificity,
The advantages that high sensitivity, is particularly suitable for the detection of pathogenic microorganism.
Monitoring LAMP reaction method have observation white precipitate, agarose gel electrophoresis (AGE), addition SYBR Green or
Calcein carries out visualization chromogenic reaction.However, the generation for observing by the naked eye white precipitate is sometimes very fuzzy and insufficient
With judgement；Step of uncapping is carried out when being detected with AGE or SYBR Green, be will cause Aerosol Pollution sometimes and generated vacation
Positive reaction；And LAMP amplified reaction can be inhibited when in calcein addition reaction system to a certain extent, and then reduce anti-
Answer sensitivity.Lateral flow test strips (LFD) technology is a kind of method of novel, efficient, accurate detection LAMP product.The technology
Hybridizing for specificity is carried out with the LAMP amplified production of biotin labeling using the probe of fluorescein isothiocynate (FITC) label,
And be combined to form ternary complex with the anti-FITC antibody containing gold nano grain, it is incorporated in matching with biotin for LFD
In the detection line of body；The antibody of the anti-FITC of non-hybridized FITC label probe and colloid gold label, which is formed, is free of the two of biotin
First compound, by detection line, in conjunction on the control line.Judging result is rapidly performed by by the way that whether detection line develops the color.It should
Detection technique avoids AGE or SYBR Green color and visually observes the false positive as caused by non-specific amplification, also avoids calcium
Reaction system, which is added, in yellowish green element reduces sensitivity, and further improves the specificity and sensitivity of reaction, and be swift in response, grasp
Make handy and safe.
Summary of the invention
In order to solve the above problems existing in the present technology, it is an object of that present invention to provide a kind of detection aviadenovirus serum
Primer sets, kit and its detection method of 4 types and application.
The technical scheme adopted by the invention is as follows: a kind of primer sets detecting 4 type of aviadenovirus serum, the primer sets packet
Outer primer and inner primer are included, wherein outer primer is F3 and B3, and inner primer is FIP and BIP, the nucleotide sequence of the F3 such as SEQ
Shown in ID NO.1, the nucleotide sequence of the B3 is as shown in SEQ ID NO.2；The nucleotide sequence of the FIP such as SEQ ID
Shown in NO.3, for the nucleotide sequence of the BIP as shown in SEQ ID NO.4, the FIP is the sequence of 5 end biotin labelings, institute
State the sequence that BIP is 5 end marked by fluorescein isothiocyanate.
Invention additionally discloses a kind of LAMP-LFD kits for detecting 4 type of aviadenovirus serum, including amplifing reagent, the positive
Reference substance, negative controls, lateral flow test strips and primer sets above-mentioned, the positive reference substance are aviadenovirus SC-
Neijiang, the negative controls are distilled water.
Optionally, the amplifing reagent include concentration be 10 × Isothermal Amplification Buffer, dense
Spending the 2.0 WarmStart archaeal dna polymerase of Bst for being 8000U/mL, the dNTPs MIX that concentration is 10mmol/L and concentration is
The MgSO of 100mmol/L4。
Optionally, the concentration be 10 × Isothermal Amplification Buffer in comprising concentration be
The Tris-HCl of the 200mmol/L, (NH that concentration is 100mmol/L4)2SO4, concentration be 500mmol/L KCl, concentration be
The MgSO of 20mmol/L4The Tween 20, the Isothermal Amplification for being 0.1% with percentage by volume
The pH of Buffer is 8.8.
The invention also discloses it is a kind of with aforementioned LAMP-LFD kit detection 4 type of aviadenovirus serum detection method,
The following steps are included:
S1, the DNA for extracting sample to be tested；
S2, prepare LAMP-LFD reaction system；
S3, reaction system in step S2 is formulated in PCR pipe, while positive controls and negative control group is set；
S4, LAMP-LFD amplified reaction: being centrifuged after the PCR pipe containing reaction system is mixed, carry out amplification reaction, and obtains
Reaction solution；
S5, it mixes from drawing 8 μ l hybridization solutions in reaction solution and be added in 100 μ l Buffer, LFD test strips is immersed it
In, testing result is read in reaction after five minutes.
Optionally, the reaction condition of amplified reaction is 65 DEG C of isothermal reaction 40min in the step S4.
Optionally, the volume of LAMP-LFD reaction system is 25 μ l in the step S2；Each component adds in reaction system
Sample amount is respectively as follows: 10 × Isothermal Amplification Buffer be 2.5 μ l, 100mmol/L MgSO4For 1 μ
The 2.0 WarmStart archaeal dna polymerase of Bst that the dNTPs MIX of l, 10mmol/L are 3 μ l, 8000U/mL is 1 μ l, 10 μm of ol/
The F3 and B3 of L is respectively that 0.5 μ l, the FIP and BIP of 10 μm of ol/L is respectively 4 μ l, and sample DNA is 2 μ l, 6.5 μ l of distilled water.
The invention also discloses above-mentioned primer sets in detection 4 type of aviadenovirus serum or to prepare in LAMP-LFD kit
Using.
The invention has the benefit that present invention combination loop-mediated isothermal amplification technique (LAMP) and lateral flow test strips
(LFD) technology designs a set of primer for FAdV-4 important factor 52k gene, and the LAMP-LFD for establishing FAdV-4 is quickly examined
Survey technology, LAMP-LFD kit provided by the invention is easy to operate, safe and reliable, sensitive efficient, specific good, is particularly suitable for
Quick visualization detects on site.
Detailed description of the invention
Fig. 1 is detected through gel electrophoresis result in test 1.
Fig. 2 is detected through gel electrophoresis result in test 2.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiment of the present invention, instead of all the embodiments.The present invention being usually described and illustrated herein in the accompanying drawings is implemented
The component of example can be arranged and be designed with a variety of different configurations.Therefore, below to the reality of the invention provided in the accompanying drawings
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
With reference to the accompanying drawing and specific embodiment the present invention is further elaborated.
Embodiment 1
The purpose of the present embodiment is that providing two pairs of specific primers for detecting 4 type of aviadenovirus serum.
According to the genome sequence of 4 type of aviadenovirus serum, set according to design of primers principle and LAMP amplification technique principle
Two pairs of primers, including outer primer and inner primer are counted, wherein outer primer is F3 and B3, and inner primer is FIP and BIP, the core of the F3
Nucleotide sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the B3 is as shown in SEQ ID NO.2；The nucleotide of the FIP
Sequence is as shown in SEQ ID NO.3, and for the nucleotide sequence of the BIP as shown in SEQ ID NO.4, the FIP is 5 end biotins
The sequence of label, the BIP are the sequence of 5 end marked by fluorescein isothiocyanate.The sequence of F3/B3/FIP/BIP is specifically such as table 1
It is shown:
Table 1
Embodiment 2
The present embodiment provides a kind of LAMP-LFD kits for detecting 4 type of aviadenovirus serum, including amplifing reagent, the positive
Primer sets in reference substance, negative controls, lateral flow test strips and embodiment 1, the positive reference substance are fowl adenopathy
Malicious SC-Neijiang, the negative controls are distilled water.
Wherein, the amplifing reagent include concentration be 10 × Isothermal Amplification Buffer, concentration
For the 2.0 WarmStart archaeal dna polymerase of Bst of 8000U/mL, concentration be 10mmol/L dNTPs MIX and concentration be
The MgSO of 100mmol/L4。
Wherein, the concentration be 10 × Isothermal Amplification Buffer in comprising concentration be
The Tris-HCl of the 200mmol/L, (NH that concentration is 100mmol/L4)2SO4, concentration be 500mmol/L KCl, concentration be
The MgSO of 20mmol/L4The Tween 20, the Isothermal Amplification for being 0.1% with percentage by volume
The pH of Buffer is 8.8.
Embodiment 3
The present embodiment provides a kind of detection sides with the LAMP-LFD kit implemented in 2 detection 4 type of aviadenovirus serum
Method, comprising the following steps:
S1, the DNA for extracting sample to be tested；
S2, prepare LAMP-LFD reaction system；
S3, reaction system in step S2 is formulated in PCR pipe, while positive controls and negative control group is set；
S4, LAMP-LFD amplified reaction: being centrifuged after the PCR pipe containing reaction system is mixed, carry out amplification reaction, and obtains
Reaction solution；
S5, it mixes from drawing 8 μ l hybridization solutions in reaction solution and be added in 100 μ l Buffer, LFD test strips is immersed it
In, testing result is read in reaction after five minutes.
Optionally, the reaction condition of amplified reaction is 65 DEG C of isothermal reaction 40min in the step S4.
Optionally, the volume of LAMP-LFD reaction system is 25 μ l in the step S2；Each component adds in reaction system
Sample amount is respectively as follows: 10 × Isothermal Amplification Buffer be 2.5 μ l, 100mmol/L MgSO4For 1 μ
The 2.0 WarmStart archaeal dna polymerase of Bst that the dNTPs MIX of l, 10mmol/L are 3 μ l, 8000U/mL is 1 μ l, 10 μm of ol/
The F3 and B3 of L is respectively that 0.5 μ l, the FIP and BIP of 10 μm of ol/L is respectively 4 μ l, and sample DNA is 2 μ l, 6.5 μ l of distilled water.
Test 1
The purpose of this test is the specificity using kit in the method validation embodiment 2 in embodiment 3.
Specific step is as follows:
(1) test sample DNA is extracted: extraction or bacterial genomes kit extract the DNA of sample to be tested according to a conventional method；
Sample to be tested includes: 1 for 4 type of aviadenovirus serum, 2 is aviadenovirus serum 7-type, 3 is aviadenovirus serum 8b
Type, 4 be chicken Marek's disease virus, 5 be infectious bursa of Fabricius virus, 6 be bird pox virus.
(2) reaction solution is prepared:
Sample to be tested group: taking 6 PCR reaction tubes, is 1-6 by sample to be tested number, the sample to be tested DNA difference in 1-6
It is followed successively by 4 type of aviadenovirus serum, aviadenovirus serum 7-type, chicken Marek's disease virus, infects aviadenovirus serum 8b type
The DNA of characteristic of disease bursa of Fabricius virus, bird pox virus；Into PCR reaction tube be added embodiment 1 primer sets (including inner primer and
Outer primer), amplifing reagent in embodiment 2 (including concentration be 10 × Isothermal Amplification Buffer,
The dNTPs MIX and concentration that 2.0 WarmStart archaeal dna polymerase of Bst that concentration is 8000U/mL, concentration are 10mmol/L be
The MgSO of 100mmol/L4), total volume is 25 μ L, surplus distilled water polishing；
Negative control group: taking 1 PCR reaction tube, and marked as 7, the reagent being added into PCR reaction tube includes embodiment 1
Primer sets (including inner primer and outer primer) and embodiment 2 amplifing reagent (including concentration be 10 × Isothermal
2.0 WarmStart archaeal dna polymerase of Bst that Amplification Buffer, concentration are 8000U/mL, concentration are
The dNTPs MIX and concentration of 10mmol/L is the MgSO of 100mmol/L4), total volume is 25 μ L, surplus distilled water polishing；
(3) amplified reaction: mixing centrifugation for two groups of reaction solutions prepared in step (2) respectively, expanded, respectively
To two groups of reaction solutions, amplification condition is 65 DEG C of reaction 40min of isoperibol；
(4) it mixes from drawing 8 μ l hybridization solutions in reaction solution and be added in 100 μ l Buffer, LFD test strips is immersed it
In, testing result is read in reaction after five minutes.
(5) result judgement:
As a result as shown in Figure 1, being detected through gel electrophoresis result, the results showed that established LAMP-LFD system is to aviadenovirus
Positive reaction is presented in 4 types, and negative reaction is presented in other viruses, illustrates established method specificity preferably.
Test 2
The purpose of this test is to carry out sensitivity study, test to kit in embodiment 2 using the method in embodiment 3
As a result as shown in Figure 2, wherein 1-10 is 1.5 × 102—1.5×10-710 times of diluted FAdV-4DNA of ng/ul, 1,2 be sky
White control.A is LFD testing result, and nature controlling line and detection line have red line to be shown to be positive, an only nature controlling line, no detection line,
It as a result is feminine gender.Established LAMP-LFD system is higher to FAdV-4 sensitivity, can detect 1.5 × 10-5ng/ul。
The present invention is not limited to above-mentioned optional embodiment, anyone can show that other are various under the inspiration of the present invention
The product of form, however, make any variation in its shape or structure, it is all to fall into the claims in the present invention confining spectrum
Technical solution, be within the scope of the present invention.
Sequence table
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<212> DNA
<213>artificial sequence (Artificial Sequence)
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tgcaccccca agtccag 17
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tcgtgcacac cgccgatacc atgatcgtga ccgacccg 38
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Claims (8)
1. a kind of primer sets for detecting 4 type of aviadenovirus serum, it is characterised in that: the primer sets include outer primer and interior draw
Object, wherein outer primer is F3 and B3, and inner primer is FIP and BIP, and the nucleotide sequence of the F3 is as shown in SEQ ID NO.1, institute
The nucleotide sequence of B3 is stated as shown in SEQ ID NO.2；The nucleotide sequence of the FIP is described as shown in SEQ ID NO.3
For the nucleotide sequence of BIP as shown in SEQ ID NO.4, the FIP is the sequence of 5 end biotin labelings, and the BIP is that 5 ends are different
The fluorescein-labeled sequence of thiocyanic acid.
2. a kind of LAMP-LFD kit for detecting 4 type of aviadenovirus serum, it is characterised in that: right including amplifing reagent, the positive
According to product, negative controls, lateral flow test strips and primer sets as described in claim 1, the positive reference substance is fowl
Adenovirus SC-Neijiang, the negative controls are distilled water.
3. the LAMP-LFD kit of detection 4 type of aviadenovirus serum according to claim 2, it is characterised in that: described
Amplifing reagent include concentration be 10 × Isothermal Amplification Buffer, the Bst that concentration is 8000U/mL
The MgSO that the dNTPs MIX and concentration that 2.0 WarmStart archaeal dna polymerases, concentration are 10mmol/L are 100mmol/L4。
4. the LAMP-LFD kit of detection 4 type of aviadenovirus serum according to claim 3, it is characterised in that: described
Concentration is 10 × Isothermal Amplification Buffer in be 200mmol/L comprising concentration Tris-HCl, dense
Degree is the (NH of 100mmol/L4)2SO4, concentration be 500mmol/L KCl, concentration be 20mmol/L MgSO4And volume basis
The pH of the Tween 20 that number is 0.1%, the Isothermal Amplification Buffer are 8.8.
5. a kind of detection of the detection of the LAMP-LFD kit described in claim 2-3 any one 4 type of aviadenovirus serum
Method, which comprises the following steps:
S1, the DNA for extracting sample to be tested；
S2, prepare LAMP-LFD reaction system；
S3, reaction system in step S2 is formulated in PCR pipe, while positive controls and negative control group is set；
S4, LAMP-LFD amplified reaction: it is centrifuged after the PCR pipe containing reaction system is mixed, carries out amplification reaction, reacted
Liquid；
S5, it mixes from drawing 8 μ l hybridization solutions in reaction solution and be added in 100 μ l Buffer, LFD test strips is immersed, instead
Testing result should be read after five minutes.
6. detection method according to claim 5, it is characterised in that: the reaction condition of amplified reaction in the step S4
For 65 DEG C of isothermal reaction 40min.
7. detection method according to claim 5, it is characterised in that: the body of LAMP-LFD reaction system in the step S2
Product is 25 μ l；In reaction system the sample-adding amount of each component be respectively as follows: 10 × Isothermal Amplification Buffer
For the MgSO of 2.5 μ l, 100mmol/L4DNTPs MIX for 1 μ l, 10mmol/L is the Bst 2.0 of 3 μ l, 8000U/mL
It is respectively 4 μ l that WarmStart archaeal dna polymerase, which is respectively 0.5 μ l, the FIP and BIP of 10 μm of ol/L for 1 μ l, the F3 and B3 of 10 μm of ol/L,
Sample DNA is 2 μ l, 6.5 μ l of distilled water.
8. a kind of primer sets described in claim 1 are in detection 4 type of aviadenovirus serum or prepare in LAMP-LFD kit
Using.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373499A (en) * | 2019-07-24 | 2019-10-25 | 安徽科技学院 | A kind of universal two warm formula PCR primers and method of I group I fowl adenovirus of quick detection |
| CN110616278A (en) * | 2019-08-09 | 2019-12-27 | 山东省农业科学院家禽研究所 | Specific primer and kit for detecting FAV-8 and FAV-11 |
| CN114990262A (en) * | 2022-06-20 | 2022-09-02 | 云南省畜牧兽医科学院 | Primer group for LAMP (loop-mediated isothermal amplification) detection of avian adenovirus serotype 4 and application of primer group |
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2019
- 2019-05-22 CN CN201910430677.1A patent/CN110042176A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373499A (en) * | 2019-07-24 | 2019-10-25 | 安徽科技学院 | A kind of universal two warm formula PCR primers and method of I group I fowl adenovirus of quick detection |
| CN110616278A (en) * | 2019-08-09 | 2019-12-27 | 山东省农业科学院家禽研究所 | Specific primer and kit for detecting FAV-8 and FAV-11 |
| CN110616278B (en) * | 2019-08-09 | 2022-08-12 | 山东省农业科学院家禽研究所 | Specific primer and kit for detecting FAV-8 and FAV-11 |
| CN114990262A (en) * | 2022-06-20 | 2022-09-02 | 云南省畜牧兽医科学院 | Primer group for LAMP (loop-mediated isothermal amplification) detection of avian adenovirus serotype 4 and application of primer group |
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Application publication date: 20190723 |