CN109457039A - A kind of freeze-drying PCR reagent detecting shrimp seedling liver sausage born of the same parents worm - Google Patents
A kind of freeze-drying PCR reagent detecting shrimp seedling liver sausage born of the same parents worm Download PDFInfo
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Abstract
The present invention discloses a kind of freeze-drying PCR reagent for detecting shrimp seedling liver sausage born of the same parents worm; including Taq DNA polymerase, freeze drying protectant, oligonucleotide upstream primer and oligonucleotide downstream primer and oligonucleotide fluorescence probe; oligonucleotide upstream primer sequence is 5 '-CACTGTAAACCTTAAAGCA-3 '; oligonucleotide downstream primer sequence is 5 '-TCTCCAACTGTATTTGAAAG-3 ', and oligonucleotide fluorescence probe sequence is 5 '-AGAGACGATATTTACACAGACACAGCA-3 '.The freeze-drying PCR reagent of detection shrimp seedling liver sausage born of the same parents worm of the invention is resistant to higher temperature, and transport, storage are convenient, can realize sensitive, special, quick and easy detection for shrimp seedling liver sausage born of the same parents worm, have a good application prospect.
Description
Technical field
The present invention relates to marine organisms Pathogen test fields, and in particular, to a kind of freeze-drying for detecting shrimp seedling liver sausage born of the same parents worm
PCR reagent and its redissolution liquid and application.
Background technique
Liver sausage born of the same parents worm (Enterocytozoon hepatopenaei, EHP) is micro- spore of the discovered in recent years in prawn
Sub- worm, main infection litopenaeus vannamei (Litopenaeus vannamei), Penaeus monodon (Penaeus monodon) etc. are important
Shrimps in culture.Liver sausage born of the same parents worm is separated and is named for the first time in the Penaeus monodon of the slow growth in Thailand's cultivation in 2009.Infection
Cause prawn slow growth after liver sausage born of the same parents worm because illness prawn is yet eating always feed, cause raiser can when
Between and feed waste supported at these with little prawn, so that more difficult obtain effective economic well-being of workers and staff.Prevent having for anti-liver sausage born of the same parents worm
Efficacious prescriptions method is the stringent selective mechanisms since seed, and the shrimp seedling for carrying pathogen is avoided to be mixed into cultivating pool.Therefore, spirit is established
Quick, special, quick and easy liver sausage born of the same parents' worm the Methods of Detection of Pathogens is the important channel for reducing shrimp seedling liver sausage born of the same parents worm and occurring and endangering.
The detection of liver sausage born of the same parents worm can be carried out in terms of specific antigen and specific gene two, with liver sausage born of the same parents' worm specific antigen
Relevant detection method has ELISA method, colloidal gold strip method etc., but these detection methods are since detection sensitivity is low, very
It is applied in the detection of liver sausage born of the same parents worm less.Detection method relevant to liver sausage born of the same parents' worm specific gene has PCR (polymerase
Chain reaction) method, LAMP (loop-mediated isothermo amplification) method etc., PCR and LAMP
Sensitivity all with higher, can detect the minimal amount of liver sausage born of the same parents worm that preclinical shrimp seedling carries, therefore from sensitivity angle
For degree to analyze, PCR and LAMP technology are all the alternative approach of preferable shrimp seedling liver sausage born of the same parents worm detection.In addition, though LAMP technology
Specificity is low compared with PCR, still, the characteristics of due to LAMP technology constant-temperature amplification, cheap instrument can be used and carry out gene magnification,
Also there is certain application value under special circumstances.And PCR technology has a wide range of application since development time is long, cures the desease original in people
The detection fields such as microorganism have very extensive application, are a kind of highly developed technical methods.But round pcr is to experimental situation
Harshness is required with operation, product is easy pollution, leads to false positive.Compared with general round pcr, fluorescent PCR detection technique simplifies
Operating procedure, and cross contamination caused by amplified production can be eliminated, reduce the generation of false positive.In the prior art, CN
106048022 A, CN 104846098 A and 106636471 A of CN disclose be related to using real-time fluorescent PCR technology examine
The technical solution of shrimp liver sausage born of the same parents worm is surveyed, the PCR amplification used time needs 2~3.5 hours.Therefore, time-consuming, higher cost limitation
Extensive use of the real-time fluorescence PCR in the conventional pathogen detection of aquiculture animal.
In addition, prawn culturing field is generally in remote rural area, traffic is very not convenient, and sample transport is past to down town
It is past to need to expend more time, and sample, during long-distance transport, the degradation of DNA will have the possibility of detection failure.Often
In the liquid PCR reagent of rule, due to needing cold chain transportation containing ingredients such as temperature sensitive archaeal dna polymerase, primer, probes, increase
While reagent adding cost, it is most likely that cold chain fails in transit, to cause reagent deterioration failure.
Summary of the invention
The object of the present invention is to provide a kind of freeze-drying PCR reagent for detecting shrimp seedling liver sausage born of the same parents worm and its liquid and application are redissolved,
At least one of to solve the above technical problems.
According to an aspect of the present invention, a kind of freeze-drying PCR reagent detecting shrimp seedling liver sausage born of the same parents worm, including Taq are provided
Archaeal dna polymerase, freeze drying protectant, oligonucleotide upstream primer and oligonucleotide downstream primer and oligonucleotide fluorescence are visited
Needle, oligonucleotide upstream primer sequence are 5 '-CACTGTAAACCTTAAAGCA-3 ' (SEQ ID NO:1), oligonucleotide
Downstream primer sequence is 5 '-TCTCCAACTGTATTTGAAAG-3 ' (SEQ ID NO:2), oligonucleotide fluorescence probe sequence
For 5 '-AGAGACGATATTTACACA GACACAGCA-3 ' (SEQ ID No:3).
Preferably, oligonucleotide fluorescence probe both ends difference mark fluorescent group and quenching group, fluorophor are
One of FAM, HEX, JOE, ROX or other have the characteristics of luminescence fluorophor, quenching group BHQ1, BHQ2, BHQ3,
One of MGB, TAMRA or other have the chemical group of fluorescent quenching function.
Preferably, every pipe freeze-drying PCR reagent includes: 1-20pmol oligonucleotide upstream primer, 1-20pmol oligomerization core
Thuja acid downstream primer, 0.5-10pmol oligonucleotide fluorescence probe, 1-20 unit Taq archaeal dna polymerase and m/v are 2-15%
Freeze drying protectant, 0~2.5mM magnesium ion, 0~200mM potassium chloride, 0~1% (m/v) non-ionic detergent, 0~
200mM Tris, 0~200mM ammonium sulfate.
Preferably, every pipe freeze-drying PCR reagent includes: under 5pmol oligonucleotide upstream primer, 5pmol oligonucleotide
Swim the freeze drying protectant that primer, 2.5pmol oligonucleotide fluorescence probe, 7.5 unit Taq archaeal dna polymerases and m/v are 7.2%.
Preferably, freeze drying protectant be trehalose, sucrose, glucose, fucose, sorbierite, mannitol, dextran,
One of polyethylene glycol or a variety of mixing.
Preferably, the freeze-drying PCR reagent for detecting shrimp seedling liver sausage born of the same parents worm is prepared by freeze-drying response procedures as follows: -45 DEG C,
2h；- 45 DEG C, 20Pa, 2h；- 45 DEG C, 10Pa, 16h；25 DEG C, 10Pa, 2h.
According to another aspect of the present invention, a kind of kit detecting shrimp seedling liver sausage born of the same parents worm, including above-mentioned freeze-drying are provided
PCR reagent and for redissolve freeze-drying PCR reagent redissolution liquid, redissolve liquid include following components: 0~0.2 μm of ol magnesium ion, 0
The non-ionic detergent that~2.5 μm of ol Tris, 0~5 μm of ol potassium ion and mass volume ratio are 0~2%.
Preferably, redissolving liquid includes following components: 0~0.1 μm of ol magnesium ion, 0~2.5 μm of ol Tris, 0~2.5 μ
The non-ionic detergent that mol potassium ion and mass volume ratio are 0~0.1%.
Preferably, non-ionic detergent is one of triton x-100, polysorbas20, Tween 80, NP40 etc. or a variety of
Combination.
The present invention is using liver sausage born of the same parents worm SWP1 (spore wall protein 1) gene conserved sequence as drone design oligomerization
Nucleotide primer and oligonucleotide fluorescence probe, obtained genetic fragment have suitable length and specific base sequence,
A possibility that reducing sequence while capable of guaranteeing sequence-specific there are non-aim sequence sites.Utilize thus obtained oligomerization
Nucleotide primer and oligonucleotide fluorescence probe production freeze-drying PCR reagent can specifically to liver sausage born of the same parents worm SWP1 base
Because being expanded, expanded without the shrimp seedling DNA to no infection liver sausage born of the same parents worm.
The present invention selects suitable freeze drying protectant to prepare freeze-drying PCR reagent, so that the archaeal dna polymerase wherein contained, widow
Poly-nucleotide primer, oligonucleotide fluorescence probe can coexist in room temperature even high temperatures, so that prepared freeze-drying
Its function and effect can still be maintained in PCR reagent after experience is transported at room temperature, so that production, transport, the storage of freeze-drying PCR reagent
While cost substantially reduces, the accuracy of the freeze-drying PCR reagent on-site test is also improved, is conducive to real-time fluorescence PCR and exists
It is universal in the conventional pathogen detection of aquiculture animal.
Oligonucleotide primer concentration, oligonucleotide fluorescence probe concentration and Mg2+Concentration is all to influence PCR reaction effect
An important factor for rate, then, the present invention, pass through to oligonucleotide primer content, oligonucleotide fluorescence in freeze-drying PCR reagent
Mg in probe content and redissolution liquid2+The limitation of content, thus utility freeze-drying PCR reagent provided by the invention and redissolution
Liquid can quickly and efficiently complete PCR amplification.
To sum up, a kind of freeze-drying PCR reagent detecting shrimp seedling liver sausage born of the same parents worm provided by the invention and its redissolution liquid and application, energy
Sensitive, special, quick and easy detection enough is realized for shrimp seedling liver sausage born of the same parents worm, is had a good application prospect.
Detailed description of the invention
Fig. 1 is the corresponding PCR amplification curve graph of 2 test result of embodiment；
Fig. 2 is the amplification curve diagram of the freeze-drying PCR reagent after the corresponding high-temperature process of 3 test result of embodiment.
Specific embodiment
It in order to enable those skilled in the art to better understand the solution of the present invention, below will be to the skill in the embodiment of the present invention
Art scheme is clearly and completely described, it is clear that and the described embodiment is only a part of the embodiment of the present invention, without
It is whole embodiments.
Embodiment 1
The freeze-drying PCR reagent of preparation detection shrimp seedling liver sausage born of the same parents worm
1. design of primers
Applicant using the conserved sequence of liver sausage born of the same parents worm SWP1 (spore wall protein 1) gene order as target,
With primer-design software " Beacon designer 8 " design primer and oligonucleotide fluorescence probe: oligonucleotide upstream
Primer sequence is " 5 '-CACTGTAAACCTTAAAGCA-3 ' ", and oligonucleotide downstream primer sequence is " 5 '-
TCTCCAACTGTATTTGAAAG-3 ' ", oligonucleotide fluorescence probe sequence are " 5 '-
5 ' the ends of AGAGACGATATTTACACAGACACAGCA-3 ' ", oligonucleotide fluorescence probe are marked using FAM, and 3 ' ends use
BHQ1 label.Wherein, FAM indicates Fluoresceincarboxylic acid (6-carboxy-fluorescein), the oligonucleotides as the present embodiment
The fluorophor of sour fluorescence probe, BHQ1 indicate Black Hole Quencher 1, and the oligonucleotide as the present embodiment is glimmering
The quenching group of light probe.
Be compared with specificity of the Primer-BLAST to designed primer (https:// Www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi? LINK_LOC=BlastHome): due to this
Invention will avoid primer amplification from going out healthy shrimp to be capable of detecting when whether shrimp seedling infects liver sausage born of the same parents worm as purpose design primer
Segment in seedling DNA compares this section of primer sequence amplification with the litopenaeus vannamei of health and Penaeus monodon DNA respectively
Right, amplification comparing result is shown cannot base sequence similar with the amplified production of above-mentioned primer；Again in the same way
This section of primer sequence amplification is compared with the DNA of liver sausage born of the same parents worm, amplification display can be right using above-mentioned primer
The SWP1 gene order of liver sausage born of the same parents worm is effectively expanded, and applicant proves also by many experiments, utilizes above-mentioned primer amplification
Product and the SWP1 gene order of liver sausage born of the same parents worm be identical.Therefore, it can be deduced that the primer tool that the present embodiment designs
There is excellent specificity.
2. preparing PCR reagent to be lyophilized
Every pipe PCR reagent to be lyophilized is prepared according to formula as below: 7.5 unit Taq DNA polymerases, 5pmol oligomerization core
Thuja acid upstream primer, 5pmol oligonucleotide downstream primer, 2.5pmol oligonucleotide fluorescence probe, m/v are 7.2% sea
Algae sugar.Wherein, m/v refers to mass concentration, indicates quality is how many in per unit volume, unit is " kg/L " or " kg/m3”。
3. freeze-drying reaction
PCR reagent to be lyophilized is added in PCR pipe, into preparation freeze-drying PCR reagent in freeze dryer, reaction interval is lyophilized
Sequence are as follows: -45 DEG C, 2h；- 45 DEG C, 20Pa, 2h；45 DEG C, 10Pa, 16h；25 DEG C, 10Pa, 2h.
Embodiment 2
The detection of shrimp seedling liver sausage born of the same parents worm
1.DNA is extracted
Prepare three parts of shrimp seedling samples and a health young shrimp sample for having infected liver sausage born of the same parents worm, takes 30mg shrimp seedling to nothing respectively
In bacterium centrifuge tube, with Aquatic animals virus DNA/RNA extracts kit (centrifugal column method) (the limited public affairs of Guangzhou Hua Feng biotechnology
Department) DNA is extracted, the DNA extracted is successively labeled as EHP sample 1, EHP sample 2 and EHP sample 3.
2. PCR reagent is lyophilized to redissolve
It is prepared according to formula as below and redissolves liquid: 0.05 μm of ol magnesium ion, 1.25 μm of ol Tris, 2.25 μm of ol potassium ions,
0.1% (w/v) polysorbas20.
The freeze-drying PCR reagent that the present embodiment uses is tried for the freeze-drying PCR of detection shrimp seedling liver sausage born of the same parents worm obtained by embodiment 1
Agent.It is added into freeze-drying PCR reagent and redissolves liquid, freeze-dried reagent is made to reform into solution state, the volume after redissolution is 20-
24.5 μ l are added 22.5 μ l into freeze-drying PCR reagent and redissolve liquid in the present embodiment.
3. the setting of experimental group
The freeze-drying PCR reagent after the redissolution in the manner described above of 5 parts of same volumes is taken, DNA sample or nothing are separately added into
Comparative experiments is arranged in bacterium water, and after sample-adding, overall solution volume is 25 μ l, and specific set-up mode is as shown in table 1.
The specific setting of 1 experimental group of table
| Group | Sample loading alternative |
| Blank control group | 2.5 μ l sterile waters |
| Negative control group | The 2.5 extracted DNA of μ l health young shrimp |
| Test 1 group | 2.5 μ l EHP samples 1 |
| Test 2 groups | 2.5 μ l EHP samples 2 |
| Test 3 groups | 2.5 μ l EHP samples 3 |
4.PCR rapid amplifying
So that the experimental group for completing to be arranged is carried out PCR amplification by following procedure: 95 DEG C are reacted 1 minute；95 DEG C are reacted 5 seconds
Clock, 60 DEG C are reacted 20 seconds, and totally 40 recycle.
5. test result
As shown in Figure 1, horizontal amplification curve is presented in the test result of blank control group and negative control group, and test 1
Group, 2 groups of experiment and test 3 groups of test result typically " S " amplification type amplification curve be presented, in conjunction with design of primers as a result,
Further explaining the freeze-drying PCR reagent that embodiment 1 is prepared can be realized the inspection effective, special to shrimp seedling liver sausage born of the same parents worm
It surveys.In addition, can see from " S " type curve in Fig. 1, expanded using freeze-drying PCR reagent made from embodiment 1 according to above-mentioned PCR
Increase program and PCR amplification is carried out to liver sausage born of the same parents' worm gene order, expands 40 circulations, amplification curve has reached substantially to platform
Phase, it was demonstrated that amplification procedure has been basically completed at this time, and the overall process used time is no more than 30 minutes, compared with prior art, PCR amplification
The process used time is obviously shortened, and detection speed is remarkably enhanced.
Embodiment 3
PCR reagent long term stability tests are lyophilized
The freeze-drying PCR reagent that the present embodiment uses is the freeze-drying PCR reagent being prepared according to embodiment 1.By 45 DEG C
Store one, two, surrounding and 25 DEG C of freeze-drying PCR reagents for storing three, six, 12 months and freshly prepared freeze-drying PCR reagent point
The redissolution liquid that 22.5 μ l are prepared according to embodiment 2 is not added, then is separately added into 2.5 μ l liver sausage born of the same parents worm DNA, carries out PCR expansion
Increase, PCR amplification program are as follows: 95 DEG C are reacted 1 minute；95 DEG C are reacted 5 seconds, and 60 DEG C are reacted 20 seconds, and totally 40 recycle.
Test results are shown in figure 2 for PCR amplification, and the amplification curve for using freshly prepared freeze-drying PCR reagent to obtain is made
For control, the amplification curve for the freeze-drying PCR reagent for storing storage one, two week in three, six, 12 months and 45 DEG C using 25 DEG C is in
The existing very high goodness of fit, and although there is deviation in the amplification curve for the freeze-drying PCR reagent for storing surrounding using 45 DEG C, but it is bent
Typical " S " type PCR amplification curve is still presented in wire shaped, still can be realized effective detection of prawn liver sausage born of the same parents worm.As a result,
Conclusion out, the freeze-drying PCR reagent that embodiment 1 provides are resistant to 25 DEG C of long term storages, 45 DEG C of short term storeds.
SEQUENCE LISTING
<110>Guangzhou Huafeng Biotech Co., Ltd.
<120>a kind of freeze-drying PCR reagent for detecting shrimp seedling liver sausage born of the same parents worm
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<400> 1
cactgtaaaccttaaagca 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
tctccaactgtatttgaaag 20
<210> 3
<211> 27
<212> DNA
<213>artificial sequence
<400> 3
agagacgatatttacacagacacagca 27
Claims (9)
1. a kind of freeze-drying PCR reagent for detecting shrimp seedling liver sausage born of the same parents worm, including Taq archaeal dna polymerase, freeze drying protectant, oligonucleotides
Sour upstream primer and oligonucleotide downstream primer and oligonucleotide fluorescence probe, the oligonucleotide upstream primer sequence
For 5 '-CACTGTAAACCTTAAAGCA-3 ', the oligonucleotide downstream primer sequence is 5 '-
TCTCCAACTGTATTTGAAAG-3 ', the oligonucleotide fluorescence probe sequence are 5 '-
AGAGACGATATTTACACAGACACAGCA-3’。
2. the freeze-drying PCR reagent of detection shrimp seedling liver sausage born of the same parents worm as described in claim 1, which is characterized in that the oligonucleotide
Mark fluorescent group and quenching group are distinguished in fluorescence probe both ends, and the fluorophor is one of FAM, HEX, JOE, ROX
Or other fluorophors with the characteristics of luminescence, the quenching group be one of BHQ1, BHQ2, BHQ3, MGB, TAMRA or
Other chemical groups with fluorescent quenching function.
3. the freeze-drying PCR reagent of detection shrimp seedling liver sausage born of the same parents worm as described in claim 1, which is characterized in that PCR is lyophilized described in every pipe
Reagent includes: oligonucleotide downstream primer, 0.5- described in oligonucleotide upstream primer, 1-20pmol described in 1-20pmol
Oligonucleotide fluorescence probe described in 10pmol, 1-20 unit Taq archaeal dna polymerase and m/v are the freeze drying protectant of 2-15%, 0
~2.5mM magnesium ion, 0~200mM potassium chloride, m/v be 0~1% non-ionic detergent, 0~200mM Tris, 0~
200mM ammonium sulfate.
4. the freeze-drying PCR reagent of detection shrimp seedling liver sausage born of the same parents worm as claimed in claim 3, which is characterized in that PCR is lyophilized described in every pipe
Reagent includes: oligonucleotide downstream primer described in oligonucleotide upstream primer, 5pmol described in 5pmol, described in 2.5pmol
The freeze drying protectant that Taq archaeal dna polymerase described in oligonucleotide fluorescence probe, 7.5 units and m/v are 7.2%.
5. the freeze-drying PCR reagent of detection shrimp seedling liver sausage born of the same parents worm, feature exist as claimed in claim 3: the freeze drying protectant is
One of trehalose, sucrose, glucose, fucose, sorbierite, mannitol, dextran, polyethylene glycol are a variety of mixed
It closes.
6. the freeze-drying PCR reagent of detection shrimp seedling liver sausage born of the same parents worm as claimed in claim 3, which is characterized in that anti-by being lyophilized as follows
Program is answered to prepare: -45 DEG C, 2h；- 45 DEG C, 20Pa, 2h；- 45 DEG C, 10Pa, 16h；25 DEG C, 10Pa, 2h.
7. it is a kind of detect shrimp seedling liver sausage born of the same parents worm kit, including freeze-drying PCR reagent described in any one of claims 1-6 and
For redissolving the redissolution liquid of the freeze-drying PCR reagent, the redissolution liquid includes following components: 0~0.2 μm of ol magnesium ion, 0~
2.5 μm of ol Tris, 0~5 μm of ol potassium ion and mass volume ratio are the non-ionic detergent of 0-2%.
8. the kit of detection shrimp seedling liver sausage born of the same parents worm as claimed in claim 7, which is characterized in that the redissolution liquid includes with the following group
Point: 0~0.1 μm of ol magnesium ion, 0~2.5 μm of ol Tris, 0~2.5 μm of ol potassium ion and mass volume ratio are 0~0.1%
Non-ionic detergent.
9. the kit of detection shrimp seedling liver sausage born of the same parents worm as claimed in claim 7, it is characterised in that: the non-ionic detergent
For one of triton x-100, polysorbas20, Tween 80, NP40 or a variety of combinations.
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| CN110468239A (en) * | 2019-09-22 | 2019-11-19 | 山东森芃生物科技有限公司 | A kind of quick Q-PCR detection method of freeze-dried type African swine fever virus and kit |
| CN111647688A (en) * | 2020-06-18 | 2020-09-11 | 安徽国泰国瑞医疗科技有限公司 | Freeze-dried PCR reagent for detecting COVID-19, FluA and FluB viruses and preparation method thereof |
| CN114231605A (en) * | 2021-12-27 | 2022-03-25 | 南京诺唯赞生物科技股份有限公司 | Freeze-dried composition for nucleic acid amplification |
| CN114231605B (en) * | 2021-12-27 | 2022-08-26 | 南京诺唯赞生物科技股份有限公司 | Freeze-dried composition for nucleic acid amplification |
| CN114214434A (en) * | 2022-02-22 | 2022-03-22 | 中国肉类食品综合研究中心 | Multiple RT-PCR premixed reagent freeze-dried ball for synchronously identifying various animal-derived components in food and preparation method and application thereof |
| CN114752662A (en) * | 2022-02-25 | 2022-07-15 | 福州迈新生物技术开发有限公司 | Fluorescent PCR (polymerase chain reaction) freeze-drying protective agent and application thereof |
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