CN109609701A - Detect CPA primer sets, reagent, kit and the detection method and application of aleutian disease virus - Google Patents
Detect CPA primer sets, reagent, kit and the detection method and application of aleutian disease virus Download PDFInfo
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Abstract
The invention discloses a kind of CPA primer sets, reagent, kit and detection methods and application for detecting aleutian disease virus, are related to viral molecular biology technical field.The CPA primer sets include that removing primer pair and Characteristics for Single Staggered primer pair, the nucleotides sequence for removing primer pair are classified as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotides sequence of Characteristics for Single Staggered primer pair is classified as SEQ ID NO.3 and SEQ ID NO.4.The specificity of the CPA primer sets is good, and high sensitivity can effectively detect aleutian disease virus.The invention also discloses CPA reagent, CPA kit and the detection method of detection aleutian disease virus, CPA kit can quickly, sensitively detect aleutian disease virus, Monitoring lower-cut is down to 1.82 × 102For copies/ μ L, it can be achieved that quick visualization qualitative detection, the detection method is easy, quick, efficient, does not need complex device, is suitble to field quick detection.
Description
Technical field
The present invention relates to viral molecular biology technical fields, in particular to a kind of CPA for detecting aleutian disease virus
Primer sets, reagent, kit and detection method and application.
Background technique
Aleutian disease virus (Amdoparvovirus) belongs to Parvoviridae (Parvoviridae), parvovirus subfamily
(Parvovirinae), aleutian disease virus category.Aleutian disease virus main infection mustelid, skunk section and Little Bear cat family etc. are extensive
Host, aleutian disease virus be the aleutian disease of mink, ferret, otter and skunk and lesser panda etc. cause of disease and Canidae it is dynamic
The potential cause of disease of object Vulpes and racoon dog etc..There may be new virus types for aleutian disease virus category, infect wider host.Wherein Ah
Shen mink disease viral (Aleutian mink disease virus, AMDV) is stayed to will lead to Aleutian disease, to mink farming
Industry harm is serious.
The method of currently used detection aleutian disease virus mainly has conventional PCR detection, CIEP detection and ELISA inspection
It surveys.Conventional virus PCR detection, needs to extract nucleic acid, provides accurate reaction condition, electrophoresis detection nucleic acid amplification specific band,
It is mainly completed in special laboratory, time-consuming, experiment condition requires height, and application is limited at the scene.CIEP passes through between detection antibody
Determining virus infection is connect, virus cannot be directly detected.The antibody of the quantitative virus of ELISA detection needs professional precision instrument, reagent
Higher cost, application above has considerable restraint at the scene.
In view of this, the present invention is specifically proposed at least one of to solve the above technical problems.
Summary of the invention
The purpose of the present invention is to provide CPA primer sets, reagent, kit and the detection sides for detecting aleutian disease virus
Method, with solve to lack in the prior art it is a kind of it is fast and convenient, result is accurate, on-site test aleutian disease virus at low cost and suitable
Detection method.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
In a first aspect, the present invention provides a kind of CPA primer sets for detecting aleutian disease virus, including removing primer pair and list
Cross primer pair；
It is described removing primer pair nucleotide sequence include:
BL-F:5 '-ctgtMacagaaaccaaccaaggta-3 ' (SEQ ID NO.1)；
BL-R:5 '-ttgaatgttaggRtatctgttgta-3 ' (SEQ ID NO.2)；
The nucleotide sequence of the Characteristics for Single Staggered primer pair includes:
JC-F:5 '-gttggtttggttgctctccaaggactccagctgcgccgttgg-3 ' (SEQ ID NO.3)；
JC-R:5 '-actccagctgcgccgttggttggtttggttgctctccaagga-3 ' (SEQ ID NO.4).
Second aspect, the present invention provides a kind of reagents for detecting aleutian disease virus, including the detection aleutian disease
The CPA primer sets of poison.
The third aspect, the present invention provides the CPA kits of detection aleutian disease virus, including the detection aleutian disease
The reagent of the CPA primer sets of poison or the detection aleutian disease virus.
Further, on the basis of technical solution provided by the invention, the CPA kit further includes releasing agent, DNA poly-
Synthase, buffer, dNTPs and color developing agent；
Preferably, the CPA kit further includes positive control template；
Preferably, the positive control template is the plasmid comprising the removing primer amplification section.
Further, on the basis of technical solution provided by the invention, the releasing agent includes two kinds of reagents of A and B；Institute
Stating A reagent includes KOH 0.4~0.6mol/L and TritonX-100 0.1% (v/v)；The B reagent include HCl 0.4~
0.6mol/L；
Preferably, the archaeal dna polymerase is Bst 2.0WarmStart archaeal dna polymerase；
Preferably, the color developing agent is SYBR Green I.
Fourth aspect, the present invention provides a kind of detection method of aleutian disease virus, using the CPA primer sets or
The reagent or the CPA kit carry out constant-temperature amplification using sample to be tested treated DNA as template, detect Ah staying
Shen virus.
Further, on the basis of technical solution provided by the invention, the sample to be tested include fluid test sample or
Solid sample to be tested；The fluid test sample includes blood, serum or urine；The solid sample to be tested includes tissue or excrement
Just.
Further, on the basis of technical solution provided by the invention, the processing of fluid test sample the following steps are included:
Every 1~2 μ L fluid test sample and 2.5~3.5 μ L A reagents mix, and act on 8~10min, add 2.0~3.0 μ L B examination
Agent is mixed to get template DNA；
Preferably, the processing of solid sample to be tested the following steps are included: freeze thawing 2 after solid sample to be tested is mixed with water~
It is stood after 3 homogenate and obtains supernatant, taken 1~2 μ L supernatant and 2.5~3.5 μ L releasing agent A to mix, act on 8~10min,
It adds 2.0~3.0 μ L releasing agent B and is mixed to get template DNA；
Preferably, the mass volume ratio (g/mL) of the solid sample to be tested and water is 1:(3~5).
Further, on the basis of technical solution provided by the invention, the condition of the constant-temperature amplification include: temperature 59~
64 DEG C, 50~70min of reaction time；
Preferably, the condition of constant-temperature amplification includes: 62~64 DEG C of temperature, 50~60min of reaction time.
5th aspect, the present invention provides the detection aleutian disease virus CPA primer sets or the detection aleutian disease virus
Reagent or it is described detection aleutian disease virus CPA kit or the aleutian disease virus detection method detection aleutian disease virus
In application.
Compared with prior art, the invention has the benefit that
(1) the CPA primer of detection aleutian disease virus provided by the invention, specific good, high sensitivity, stable amplification result
Reliably, aleutian disease virus can effectively be detected.
(2) the CPA kit of detection aleutian disease virus provided by the invention, detection is quick, sensitive, and Monitoring lower-cut can be low
To 1.82 × 102Copies/ μ L is, it can be achieved that fast qualitative detection to aleutian disease virus, and production cost is low, operation letter
Just, it is suitble to carry out rapid screening and detection in laboratory or other places, expands the scope of application.
(3) detection method of aleutian disease virus provided by the invention, the detection method is easy, quick, efficient, amplification and inspection
Complex instrument equipment is not needed when survey, can judge disease by the way that visualization color developing agent observation color change is added after amplified reaction
The presence or absence of poison, is very suitable to base and field quick detection.Meanwhile the detection method simplifies detection process, reduces
The profession of operator is required, so that layman can also smoothly complete detection work, there is biggish application prospect.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the agarose gel electrophoresis figure of the CPA amplified production of embodiment 1 and embodiment 3；
Fig. 2 is the agarose gel electrophoresis figure of the CPA amplified production of embodiment 2；
Fig. 3 is the agarose gel electrophoresis figure of the regular-PCR amplified production of embodiment 3.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
In the present invention, if without particularly illustrating, all embodiments mentioned in this article and preferred implementation method
It can be combined with each other to form new technical solution.
In the present invention, if without particularly illustrating, all technical characteristics and preferred feature mentioned in this article can be with
Intercombination forms new technical solution.
In the present invention, if percentage (%) or part refer to the volume relative to composition without particularly illustrating
Percentage.
In the present invention, if related each component or its preferred ingredient can be combined with each other shape without particularly illustrating
The technical solution of Cheng Xin.
In the present invention, unless otherwise indicated, numberical range " a~b " indicates the contracting of any real combinings between a to b
Sketch form shows that wherein a and b is real number.Such as numberical range " 6~22 " indicate herein all listed " 6~22 " it
Between whole real numbers, " 6~22 " be these combinations of values breviary indicate.
" range " disclosed in this invention can be respectively one or more lower limits and one in the form of lower and upper limit
A or multiple upper limits.
In the present invention, unless otherwise indicated, each reaction or operating procedure can be carried out sequentially, can not also be in sequence
It carries out.Preferably, reaction method herein is that sequence carries out.
Unless otherwise indicated, profession used herein and meaning phase known to scientific term and one skilled in the art
Together.In addition, any method similar to or equal to what is recorded or material can also be applied in the present invention.
According to the first aspect of the invention, a kind of CPA primer sets for detecting aleutian disease virus are provided, including removing is drawn
Object to and Characteristics for Single Staggered primer pair；
Removing primer pair nucleotide sequence include:
BL-F:5 '-ctgtMacagaaaccaaccaaggta-3 ' (SEQ ID NO.1)；
BL-R:5 '-ttgaatgttaggRtatctgttgta-3 ' (SEQ ID NO.2).
The BL-F in primer pair is wherein removed, using base M is annexed, indicates that the site is that adenine A or cytimidine C are equal
It can；The BL-R in primer pair is removed, using base R is annexed, indicates that the site is adenine A or cytimidine G.
The nucleotide sequence of Characteristics for Single Staggered primer pair includes:
JC-F:5 '-gttggtttggttgctctccaaggactccagctgcgccgttgg-3 ' (SEQ ID NO.3)；
JC-R:5 '-actccagctgcgccgttggttggtttggttgctctccaagga-3 ' (SEQ ID NO.4).
Cross primer isothermal amplification technology (Cross priming amplification, CPA) is a kind of novel constant temperature
Nucleic acid amplification method is designed specific primer for target gene, is reacted under constant temperature conditions using strand displacement archaeal dna polymerase certain
Nucleic acid amplification reaction can be completed in time.By marking specific probe to enable its amplified production that disposable nucleic acid detection apparatus is used
It is detected, amplified production bring is effectively prevent to pollute, reduce false positive results.
CPA technology is as a kind of basic methods, when being applied to specific test object, needs for test object gene sequence
The characteristics of column, pointedly selects target gene and amplification site, rationally designs cross primer and removing primer with improvement method
Specificity and sensitivity.
Removing primer refers to the short strand primer positioned at Characteristics for Single Staggered primer rear, and effect is in strand displacement archaeal dna polymerase
Under effect, the extended chain of amplification is peeled from a template.
Characteristics for Single Staggered primer refer to for intersect amplification main primer, wherein 5 ' end sequences of forward primer with reversely draw
The complementary series of object is identical, and 5 ' end sequences of reverse primer are identical as the complementary series of forward primer, therefore is expanding
Two primers of this in journey introduce mutually the complementary series of other side, increase the hybridization site of primer, promote amplified reaction.
The CPA primer of above-mentioned detection aleutian disease virus includes removing primer pair and Characteristics for Single Staggered primer pair, CPA primer sets
Specificity it is good, high sensitivity, stable amplification result is reliable, can effectively detect aleutian disease virus.
According to the second aspect of the invention, a kind of reagent for detecting aleutian disease virus is provided, including above-mentioned detection
The CPA primer sets of aleutian disease virus.
To detection aleutian disease virus reagent specific type without limit, including being removed in above-mentioned CPA primer sets
Primer and Characteristics for Single Staggered primer, can be used for detecting aleutian disease virus.The reagent of above-mentioned aleutian disease virus is due to containing above-mentioned
CPA primer sets, therefore there is specific good, high sensitivity, the reliable advantage of stable amplification result.
According to the third aspect of the present invention, a kind of CPA kit for detecting aleutian disease virus is provided, including above-mentioned
The reagent of CPA primer sets or above-mentioned detection aleutian disease virus.
The CPA kit of the detection aleutian disease virus is able to detect 6 Ah staying having found outside ash disposal fox aleutian disease virus
Shen virus kind, is not only applicable in the detection of Aleutian Mink Disease Parvovirus (AMDV), racoon dog, fox Aleutian mink disease (RFAV), it can also be used to
Detect the other kinds of aleutian disease virus infection of new host.
The CPA kit of above-mentioned detection aleutian disease virus, detect it is quick, sensitive, Monitoring lower-cut can down to 1.82 ×
102Copies/ μ L is, it can be achieved that fast qualitative detection to aleutian disease virus, and production cost is low, easy to operate, is suitble to
Laboratory or other places carry out rapid screening and detection, expand the scope of application.
It is preferably carried out in mode in one kind, the CPA kit for detecting aleutian disease virus further includes releasing agent, DNA polymerization
Enzyme, buffer, dNTPs and color developing agent.
A reagent provides strong basicity environment in releasing agent, can be with the structural proteins of lytic cell and virus, to sufficiently discharge
Nucleic acid；In releasing agent B reagent be acid, A reagent effect after be added B reagent can in and A reagent alkalinity, it is simple through releasing agent
Processing sample can make nucleic acid release be suitable for making CPA reaction template.
Archaeal dna polymerase is important toolenzyme during DNA constant-temperature amplification, catalytic dna synthesis.Archaeal dna polymerase needs herein
Meet the following conditions: with 5 ' → 3 ' polymerase activity and strong strand-displacement activity, but without 5 ' → 3 ' Exonucleolytic enzyme activity
Property.To the type of archaeal dna polymerase without limiting, above-mentioned condition, preferably Bst 2.0WarmStart DNA can satisfy
Polymerase.
The effect of buffer is to keep the pH of the reaction system solution of constant-temperature amplification more stable, reduces pH variation to constant-temperature amplification
The influence of reaction.
Color developing agent can be in conjunction with the DNA after constant-temperature amplification, and visualization color developing agent, which is added, to develop the color under visible light, leads to
Direct visual color variation is crossed to judge whether target gene sufficiently expands；Or it is observed using the laboratory facilities of this field routine
With the presence or absence of target gene.To the type of color developing agent without limiting, preferably can directly develop the color under visible light in conjunction with DNA
Color developing agent, further preferred SYBR Green I.
It is preferred that detecting the type of archaeal dna polymerase and color developing agent in the CPA kit of aleutian disease virus, CPA reagent can be made
Box detect aleutian disease virus more rapidly with sensitive, and more convenient operation.
Using CPA amplification technique, 4 primer combinations are specifically designed for aleutian disease virus, will discharge reagent, constant-temperature amplification
Reagent and reaction result visualization determine that indicator is combined into kit, realize simplification, the reaction temperature of the nucleic acid extraction of sample
Condition is easy to get to be visualized with result judgement, can satisfy live easy-to-use, detectable major part aleutian disease virus.
It is preferably carried out in mode in one kind, above-mentioned CPA kit further includes positive control template.
To the specific type of positive control template without limiting, if meet can after CPA reacts can it is obvious and
Accurate detection is to aleutian disease virus.It is preferred that positive control template is the plasmid comprising removing primer amplification section, such as examine
It surveys Aleutian Mink Disease Parvovirus (AMDV), positive control template preferably comprises the AMDV-G 836bp segment of removing primer amplification section
Plasmid.
Positive control template is added in CPA kit can make the testing result of sample and the testing result of positive control
It is significantly compared, convenient for users to preferably identifying in sample to be tested with the presence or absence of aleutian disease virus.It is preferred that positive control
Template can make positive findings more stable and reliable, improve the accuracy of CPA kit.
It is preferably carried out in mode in one kind, above-mentioned releasing agent includes two kinds of reagents of A and B；The A reagent includes KOH
0.4~0.6mol/L and TritonX-100 0.1% (v/v)；The B reagent includes 0.4~0.6mol/L of HCl.
In A reagent, the typical but non-limiting concentration of KOH is, for example, 0.4mol/L, 0.5mol/L or 0.6mol/L；B
In reagent, the typical but non-limiting concentration of HCL is, for example, 0.4mol/L, 0.5mol/L or 0.6mol/L.
It should be noted that KOH is identical as the concentration of HCL in B reagent in A reagent when using release-agent-treated sample.
It is preferred that releasing agent can make in sample to be tested treatment process, the nucleic acid of sample is easier to discharge, and reduces to nucleic acid
Damage, improve the detection accuracy of sample.
It is preferably carried out in mode in one kind, 1 × buffer includes following component: 18~22mM Tris-HCl, 8~
12mM(NH)2SO4, 2~3.5mmol/L MgSO4, Tween-20 0.1% (v/v) and 0.35~0.45mol/L Betaine,
The pH8.8 of buffer.
The component and concentration of preferred buffer, can make the buffering effect of buffer more preferable, keep the pH of reaction system steady
It is fixed, make the detection of CPA kit more rapidly, it is sensitive and accurate.
According to the fourth aspect of the present invention, a kind of detection method of aleutian disease virus is provided, is drawn using above-mentioned CPA
Object group or above-mentioned reagent or above-mentioned CPA kit carry out constant-temperature amplification, inspection using sample to be tested treated DNA as template
Survey aleutian disease virus.
Preferably, sample to be tested includes fluid test sample or solid sample to be tested, wherein fluid test sample includes blood
Liquid, serum or urine；Solid sample to be tested includes tissue or excrement.
The aleutian disease virus detection method is easy, quick, efficient, does not need complex instrument equipment when amplification is with detection,
It is very suitable to base and field quick detection.
It is preferably carried out in mode in one kind, A Liushen detection method including the following steps: processing sample to be tested is matched
CPA reaction system, CPA reaction and result identification processed.
Handle sample to be tested
The processing of preferred liquid sample to be tested, comprising the following steps: every 1~2 μ L fluid test sample and 2.5~3.5 μ L
A reagent mixes, and acts on 8~10min, adds 2.0~3.0 μ L B reagents and be mixed to get template DNA；
It is preferred that the processing of solid sample to be tested, comprising the following steps: freeze thawing 2~3 after mixing solid sample to be tested with water
It is stood after secondary homogenate and obtains supernatant, 1~2 μ L supernatant and 2.5~3.5 μ L A reagents is taken to mix, act on 8~10min, then
2.0~3.0 μ L B reagents are added and are mixed to get template DNA；It is preferred that the mass volume ratio (g/mL) of solid sample to be tested and water is
1:(3~5).
When handling sample to be tested, preferably the additional amount ratio A reagent of B reagent lacks 0.5 μ L, releasing agent can be made to play better
Effect.
It should be noted that in fluid test sample or solid sample to be tested treatment process, can according to actual needs and
It is reasonably adjusted referring to the ratio and action time of above-mentioned sample and releasing agent, when being not limited to aforementioned proportion and effect
Between.
Prepare CPA reaction system
It is preferred that CPA reaction system include: each 0.12~0.28 μm of ol/L of removing primer pair, Characteristics for Single Staggered primer pair each 0.32~
0.64 μm of ol/L, 7.5~8.5U of Bst 2.0DNA polymerase, 10 × buffer 2.5~3.5 μ L and dNTPs 0.3~
0.5mmol/L, template DNA add water to volume and are 30 μ L and mix.
10 × buffer refers to that above-mentioned 1 × buffer each component concentration expands 10 times.
In CPA reaction system, typical but non-limiting each concentration for removing primer is, for example, 0.14 μm of ol/L, 0.16 μ
Mol/L, 0.18 μm of ol/L, 0.2 μm of ol/L, 0.23 μm of ol/L, 0.25 μm of ol/L or 0.28 μm of ol/L；Each Characteristics for Single Staggered primer
Typical but non-limiting concentration is, for example, 0.32 μm of ol/L, 0.38 μm of ol/L, 0.4 μm of ol/L, 0.45 μm of ol/L, 0.5 μ
Mol/L, 0.55 μm of ol/L, 0.6 μm of ol/L or 0.64 μm of ol/L；The concentration of archaeal dna polymerase is typical but non-limiting to be, for example,
7.5U, 7.8U, 8U, 8.2U or 8.5U；The typical but non-limiting volume of 10 × buffer is, for example, 2.5 μ L, 2.8 μ L, 3
μ L, 3.2 μ L or 3.5 μ L；The concentration of dNTPs it is typical but non-limiting be, for example, 0.3mmol/L, 0.4mmol/L or
0.5mmol/L。
It is preferred that the concentration ratio for removing primer pair and Characteristics for Single Staggered primer pair is (2~2.4): 1 in CPA reaction system；Into one
The concentration of preferably each removing primer of step is 0.2 μm of ol/L, and the concentration of each Characteristics for Single Staggered primer is 0.4 μm of ol/L.
It should be noted that the accuracy in order to prove experimental result, CPA reaction system can also increase water as template work
DNA for negative control and aleutian disease virus is template as positive control.
CPA reaction
It is preferred that the temperature of constant-temperature amplification (CPA reaction) is 59~64 DEG C, the time is 50~70min；Further preferred constant temperature
The temperature of amplification is 62~64 DEG C, and the time is 50~60min.
The typical but non-limiting temperature of constant-temperature amplification is, for example, 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C or 64 DEG C；
The typical but non-limiting time of constant-temperature amplification is, for example, 50min, 55min, 60min, 65min or 70min.
CPA reaction can be carried out in vacuum cup or thermostat water bath or other equipment for being capable of providing above-mentioned temperature in
It carries out, it is simple and efficient, it is not necessarily to complex device, is very suitable to base and field quick detection.
As a result it identifies
After the reaction was completed, color developing agent is added in CPA, after mixing observation colour developing result.
After CPA reaction amplification generates DNA, it is preferably added to visualization color developing agent, such as SYBR Green I, which is added
After DNA, visual results can be carried out after brief centrifugation mixes, feminine gender be it is orange, the positive is green.Amplified reaction
Afterwards, the presence or absence of virus can be judged by the way that visualization color developing agent observation color change is added, be very suitable to base and scene
Quickly detection.
It should be noted that be added the macroscopic metachromasia of color developing agent SYBR Green I lower limit be 1.82 ×
102copies/μL。
In addition, other color developing agents can also be added and carry out agarose gel electrophoresis experiment after CPA reaction amplification generates DNA,
Testing result or other technological means commonly used in the art that can show result are observed by ultraviolet irradiation.
The detection method of the aleutian disease virus is easy, quick, efficient, does not need complex instrument when amplification is with detection and sets
It is standby, the presence or absence of virus can be judged by the way that visualization color developing agent observation color change is added after amplified reaction, be very suitable to
Base and field quick detection.Meanwhile the detection method simplifies detection process, reduces and requires the profession of operator,
So that layman can also smoothly complete detection work, there is biggish application prospect.
A method of aleutian disease virus preferably being detected using above-mentioned CPA kit, comprising the following steps:
(a) sample treatment: every 1~2 μ L fluid test sample and 2.5~3.5 μ L A reagents mix, and act on 8~10min,
It adds 2.0~3.0 μ L B reagents and is mixed to get template DNA；
Or, the processing of solid sample to be tested is the following steps are included: freeze thawing 2~3 times after solid sample to be tested is mixed with water
It is stood after homogenate and obtains supernatant, taken 1~2 μ L supernatant and 2.5~3.5 μ L A reagents to mix, act on 8~10min, add
2.0~3.0 μ L B reagents are mixed to get template DNA；The mass volume ratio (g/mL) of solid sample to be tested and water is 1:(3~5)；
(b) PCA reaction system is prepared: each 0.12~0.28 μm of ol/L of removing primer pair, Characteristics for Single Staggered primer pair each 0.32~
0.64 μm of ol/L, 7.5~8.5U of Bst 2.0DNA polymerase, 10 × buffer 2.5~3.0 μ L and dNTPs 0.3~
0.5mmol/L, template DNA add water to volume and are 30 μ L and mix；
(c) constant-temperature amplification: reaction system obtains reaction product in 59~64 DEG C of 50~70min of reaction；
(d) result is identified: color developing agent being added in the reaction product that step (c) obtains, after mixing observation colour developing knot
Fruit.
The detection method of the aleutian disease virus uses above-mentioned CPA kit, section explicitly defines specific in detection process
Operating method, step and reaction condition etc. optimize the concentration of other substances in each primer concentration ratio and reaction system, excellent
Reaction condition is changed, has made testing result more rapidly and accurately.
According to the fifth aspect of the present invention, above-mentioned CPA primer sets or mentioned reagent or above-mentioned CPA kit are provided
Or application of the above-mentioned detection method in detection aleutian disease virus.
In order to be more clear goal of the invention of the invention, technical solution and its advantageous effects, following embodiment and
Comparative example, the present invention will be described in further detail.It should be understood that embodiment described in this specification merely to
It explains the present invention, is not intended to limit the present invention.Each raw material of the present invention can pass through commercially available acquisition.
1 aleutian disease virus CPA design of primers of embodiment and detection
1, design of primers
With reference to AMDV gene order (NC_001662) is compared, the VP2 gene for designing and filtering out AMDV is the CPA of target
Primer sets, including removing primer pair and Characteristics for Single Staggered primer pair, sequence difference are as follows:
Removing primer pair nucleotide sequence include:
BL-F:5 '-ctgtMacagaaaccaaccaaggta-3 ' (SEQ ID NO.1)；
BL-R:5 '-ttgaatgttaggRtatctgttgta-3 ' (SEQ ID NO.2)；
The nucleotide sequence of Characteristics for Single Staggered primer pair includes:
JC-F:5 '-gttggtttggttgctctccaaggactccagctgcgccgttgg-3 ' (SEQ ID NO.3)；
JC-R:5 '-actccagctgcgccgttggttggtttggttgctctccaagga-3 ' (SEQ ID NO.4).
2, CPA amplified reaction
Using AMDV genomic DNA as positive template, AMDV template quantity is respectively 5.38 × 101 copies/μL、5.38×
102copies/μL、5.38×103copies/μL、5.38×104copies/μL、 5.38×105copies/μL、5.38×
106Copies/ μ L and 5.38 × 107Copies/ μ L, ddH2O is that negative control carries out CPA amplified reaction.
CPA reaction system (30 μ L): each 0.2 μm of ol/L of removing primer pair, Characteristics for Single Staggered primer pair each 0.48 μm of ol/L, Bst
2.0DNA polymerase 8U, 10 × buffer, 3 μ L and dNTPs 0.4mmol/L, template DNA add water to volume and are 30 μ L and mix.
CPA reaction condition are as follows: 63 DEG C, 60min amplification terminates reaction.
3, result is identified
(1) 10 μ L amplified productions are taken to carry out agarose gel electrophoresis respectively, in the electrophoretogram of Fig. 1, M is represented
DL2000Marker, 101、102、103、104、105、106With 107Respectively representing AMDV template quantity is 5.38 × 101copies/μ
L、5.38×102copies/μL、5.38×103copies/μL、 5.38×104copies/μL、5.38×105copies/μ
L、5.38×106Copies/ μ L and 5.38 × 107The positive control of copies/ μ L ,-represent negative control.
The electrophoresis result of Fig. 1 shows that the distinctive ladder of CPA is presented without obvious band, the product of each positive template in negative control
Shape band.
(2) reaction result is judged using easy visual detection: SYBR Green I is added in amplified production, as a result
Negative tube is shown in orange, each positive pipe is in green.
2 CPA primer sets specific test of embodiment
1, DNA and the mink, blue fox of hepatitis infectiosa canis virus, canine parvovirus, canine distemper virus and mink enteritis virus are selected
DNA with Wusuli Racoon Doy tissue is as DNA profiling, using AMDV as positive template, ddH2O is negative control, utilizes 1 institute of embodiment
The amplification system and method stated carry out CPA amplified reaction, then carry out agarose gel electrophoresis, as a result see Fig. 2.
2, result is identified
In the agarose electrophoresis figure of Fig. 2, M represents DL2000Marker, and 1~4 to respectively represent template be that hepatitis infectiosa canis virus, dog are thin
The DNA of small virus, canine distemper virus and mink enteritis virus, 5~7 respectively represent template as mink, blue fox and Wusuli Racoon Doy group
The DNA knitted ,+positive control is represented ,-represent negative control；
The agarose electrophoresis of Fig. 2 is the results show that be only added AMDV positive control template has the distinctive trapezoidal item of CPA
Band illustrates that the specificity of above-mentioned CPA primer and detection method is good.
3 CPA primer sets sensitivity test of embodiment
1, the DNA concentration for testing AMDV genome extracting solution is 5.4 × 107Copies/ μ L dilutes 10 times, until dilute step by step
Releasing concentration is 5.4 × 102Copies/ μ L, the template as regular-PCR.
2, CPA reaction system is identical with embodiment 1, and common PCR reaction condition is as follows:
30 μ L reaction systems: 1 μ L template, 1 × PCR buffer, removing respectively 0.3~0.6 μM of primer, Hot Star
TaqTM2~3U of polymerase to 30 μ L is mixed.
Response procedures: 95 DEG C of initial denaturation 3min, 95 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 30sec, into
Row 35 circulations, last 72 DEG C of extensions 5min.
3, result is identified
10 μ L regular-PCR products are taken to carry out agarose gel electrophoresis, in the electrophoretogram of Fig. 3, M represents DL2000Marker,
102、103、104、105With 106Respectively representing AMDV template quantity is 5.4 × 102copies/μL、5.4×103copies/μL、
5.4×104copies/μL、5.4×105Copies/ μ L and 5.4 × 106The positive control of copies/ μ L ,-represent it is negative right
According to ,+to represent AMDV template quantity be 5.4 × 106copies/μL。
As shown in Figure 3: regular-PCR product product band is about 223bp, regular-PCR template concentrations be about 5.4 ×
102When copies/ μ L, electrophoretic band is very faint.
CPA agarose gel electrophoresis results in Fig. 1, CPA are 5.38 × 10 in template concentrations2It is still visible when copies/ μ L
Amplification condition, it is seen that the sensibility of the CPA detection method of aleutian disease virus of the invention is significantly higher than regular-PCR.
The foundation of 4 CPA detection kit of embodiment
1, a kind of CPA detection kit for detecting AMDV, following reagent is contained in the kit: CPA primer sets are released
Put agent, Bst 2.0WarmStart archaeal dna polymerase, 10 × buffer, 10mM dNTPs, SYBR Green I (× 60) and 5 ×
103Copies/ μ L AMDV-G 836bp plasmid.
Releasing agent: KOH 0.5mol/L, TritonX-100 0.1% (v/v) and HCL 0.5mol/L；
10 × buffer: 200mM Tris-HCl, 100mM (NH)2SO4、20mmol/L MgSO4, 1%Tween-20 and
4mol/L Betaine, pH of buffer 8.8.
The method that embodiment 5 detects aleutian disease virus using CPA kit
Aleutian disease virus is detected using the CPA kit of embodiment 4, specific detection method includes the following steps:
(1) sample treatment: 1~2 μ L blood and 2.5~3.5 μ L A reagents are mixed, and are acted on 8~10min, are added
The mixing of 2.0~3.0 μ L B reagents, obtains template DNA；
(2) 30 μ L PCA reaction systems are prepared: each 0.2 μm of ol/L of removing primer pair, each 0.48 μm of ol/ of Characteristics for Single Staggered primer pair
L, Bst 2.0DNA polymerase 8U, 10 × buffer, 3 μ L and dNTPs 0.4mmol/L, template DNA, adding water to volume is 30 μ L
And it mixes；
(3) constant-temperature amplification: reaction system obtains reaction product in 63 DEG C of reaction 60min；
(4) result is identified: 1 μ L SYBR Green I being added in the reaction product that step (3) obtains, after mixing
Observation colour developing as a result, it is negative be it is orange, the positive is green.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Gao Yun
<120>CPA primer sets, reagent, kit and the detection method and application of aleutian disease virus are detected
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213>artificial sequence
<400> 1
ctgtmacaga aaccaaccaa ggta 24
<210> 2
<211> 24
<212> DNA
<213>artificial sequence
<400> 2
ttgaatgtta ggrtatctgt tgta 24
<210> 3
<211> 42
<212> DNA
<213>artificial sequence
<400> 3
gttggtttgg ttgctctcca aggactccag ctgcgccgtt gg 42
<210> 4
<211> 42
<212> DNA
<213>artificial sequence
<400> 4
actccagctg cgccgttggt tggtttggtt gctctccaag ga 42
Claims (10)
1. a kind of CPA primer sets for detecting aleutian disease virus, which is characterized in that including removing primer pair and Characteristics for Single Staggered primer pair；
It is described removing primer pair nucleotide sequence include:
BL-F:5 '-ctgtMacagaaaccaaccaaggta-3 ' (SEQ ID NO.1)；
BL-R:5 '-ttgaatgttaggRtatctgttgta-3 ' (SEQ ID NO.2)；
The nucleotide sequence of the Characteristics for Single Staggered primer pair includes:
JC-F:5 '-gttggtttggttgctctccaaggactccagctgcgccgttgg-3 ' (SEQ ID NO.3)；
JC-R:5 '-actccagctgcgccgttggttggtttggttgctctccaagga-3 ' (SEQ ID NO.4).
2. a kind of reagent for detecting aleutian disease virus, which is characterized in that including detection aleutian disease virus described in claim 1
CPA primer sets.
3. a kind of CPA kit for detecting aleutian disease virus, which is characterized in that including detection A Liushen described in claim 1
The reagent of the CPA primer sets of virus or detection aleutian disease virus as claimed in claim 2.
4. the CPA kit of detection aleutian disease virus described in accordance with the claim 3, which is characterized in that the CPA kit is also
Including releasing agent, archaeal dna polymerase, buffer, dNTPs and color developing agent；
Preferably, the CPA kit further includes positive control template；
Preferably, the positive control template is the plasmid comprising the removing primer amplification section.
5. detecting the CPA kit of aleutian disease virus according to claim 4, which is characterized in that the releasing agent includes A
With two kinds of reagents of B；The A reagent includes KOH 0.4~0.6mol/L and TritonX-100 0.1% (v/v)；The B reagent
Including 0.4~0.6mol/L of HCl；
Preferably, the archaeal dna polymerase is Bst 2.0WarmStart archaeal dna polymerase；
Preferably, the color developing agent is SYBR Green I.
6. a kind of detection method of aleutian disease virus, which is characterized in that apply CPA primer sets described in claim 1 or right
It is required that reagent described in 2 or the described in any item CPA kits of claim 3-5, using sample to be tested treated DNA as mould
Plate carries out constant-temperature amplification, detects aleutian disease virus.
7. the detection method of aleutian disease virus according to claim 6, which is characterized in that the sample to be tested includes liquid
Sample to be tested or solid sample to be tested；
The fluid test sample includes blood, serum or urine；
The solid sample to be tested includes tissue or excrement.
8. the detection method of aleutian disease virus according to claim 7, which is characterized in that the processing packet of fluid test sample
Include following steps: every 1~2 μ L fluid test sample and 2.5~3.5 μ L A reagents mix, and act on 8~10min, add 2.0
~3.0 μ L B reagents are mixed to get template DNA；
Preferably, the processing of solid sample to be tested is the following steps are included: freeze thawing 2~3 times after solid sample to be tested is mixed with water
It is stood after homogenate and obtains supernatant, taken 1~2 μ L supernatant and 2.5~3.5 μ L A reagents to mix, act on 8~10min, add
2.0~3.0 μ L B reagents are mixed to get template DNA；
Preferably, the mass volume ratio (g/mL) of the solid sample to be tested and water is 1:(3~5).
9. according to the detection method of the described in any item aleutian disease virus of claim 6-8, which is characterized in that the constant-temperature amplification
Condition include: 59~64 DEG C of temperature, 50~70min of reaction time；
Preferably, the condition of constant-temperature amplification includes: 62~64 DEG C of temperature, 50~60min of reaction time.
10. detecting detection aleutian disease virus described in the CPA primer sets or claim 2 of aleutian disease virus described in claim 1
Described in the CPA kit or claim any one of 6-9 of reagent or the described in any item detection aleutian disease virus of claim 3-5
Aleutian disease virus detection method detection aleutian disease virus in application.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638685A (en) * | 2008-07-29 | 2010-02-03 | 杭州优思达生物技术有限公司 | Method for amplifying target nucleic acid sequence by using cross primer and kit for amplifying target nucleic acid sequence and application thereof |
-
2019
- 2019-02-21 CN CN201910130378.6A patent/CN109609701A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638685A (en) * | 2008-07-29 | 2010-02-03 | 杭州优思达生物技术有限公司 | Method for amplifying target nucleic acid sequence by using cross primer and kit for amplifying target nucleic acid sequence and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 韦韬 等: "毛皮兽阿留申病毒SYBR Green I-qPCR检测方法的建立及病毒种鉴别", 《中国兽医科学》 * |
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