CN109880936A - A kind of detection primer and application of source of mouse TTV virus - Google Patents
A kind of detection primer and application of source of mouse TTV virus Download PDFInfo
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- CN109880936A CN109880936A CN201910304336.XA CN201910304336A CN109880936A CN 109880936 A CN109880936 A CN 109880936A CN 201910304336 A CN201910304336 A CN 201910304336A CN 109880936 A CN109880936 A CN 109880936A
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Abstract
The present invention provides a kind of detection primers of source of mouse TTV virus, belong to technical field of medical examination.Source of mouse TTV viral diagnosis primer provided by the invention can accurately, specifically detect that source of mouse TTV virus, detection sensitivity reach 1 × 101Copy, sensitivity with higher；The accuracy of detection can be improved using detection primer detection source of mouse TTV virus, and the kit of application detection primer preparation detection source of mouse TTV virus can be convenient application, detect fast and flexible.
Description
Technical field
The present invention relates to the detection primers and application of a kind of source of mouse TTV virus, belong to technical field of medical detection.
Background technique
TTV virus (Torque teno virus, TTV), belongs to finger ring Viraceae, finger ring Tobamovirus is a kind of no capsule
Film, single stranded circle DNA virus.It is separated in the serum of Japanese patients for the first time earliest, is related to hidden originality hepatitis infection
Most current virus, this virus can infection plant and vertebrate, by the way that the human serum of the TTV positive is injected into chimpanzee body
On, it was demonstrated that TTV can have found TTV virus in Britain for propagation to primate, 2014 in rodent for the first time, be one
The virus of kind mouse, is named as RoTTV, currently, not yet establishing the detection method for being directed to source of mouse TTV virus.
Summary of the invention
In view of the deficiencies in the prior art, the present invention provides a kind of detection primer of source of mouse TTV virus.
The technical solution adopted by the present invention is as follows:
The present invention designs upstream primer at the 259-278bp of source of mouse TTV virus genom DNA, sets at 537-557bp
Downstream primer is counted, the sequence of upstream primer is as shown in SEQ ID No:1, and the sequence of downstream primer is as shown in SEQ ID No:2.It should
Primer is used for the specific detection of source of mouse TTV virus.
The application method of the primer includes carrying out PCR reaction by template of sample to be examined；PCR response procedures are as follows: 94 DEG C, in advance
It is denaturalized 5min；94 DEG C, it is denaturalized 15s；56~60 DEG C, anneal 30s；72 DEG C, extend 30s；35 circulations；72 DEG C, 10min.
The present invention also provides a kind of detection kit of source of mouse TTV virus, the detection kit includes SEQ ID
Upstream and downstream primer shown in No:1 and SEQ ID No:2.
It further include PCR reaction buffer, Taq archaeal dna polymerase, SYBR Premix Ex Taq in kit of the present invention
II, Taq PCR MasterMix, target fragment standard items and Mg2+At least one of.
The application method of kit of the present invention is to carry out PCR reaction by template of sample to be examined；PCR response procedures are as follows: 95
DEG C, initial denaturation 30s；95 DEG C, it is denaturalized 5s；56~60 DEG C, anneal 30s；72 DEG C of collection fluorescence signal 15s, 39 circulations.
The present invention also specifically provides the extracting method of source of mouse TTV virus genom DNA:
1) the 200 μ L of blood plasma or tissue of the host animal of infection source of mouse TTV virus are taken；
2) the Proteinase K solution of 20 μ L is added, mixes；
3) the Buffer RLV of 200 μ L is added, whirlpool shakes 15s；
4) it is incubated for 15min under the conditions of 56 DEG C, is centrifuged, the solution of tube wall is collected into tube bottom；
5) dehydrated alcohol of 250 μ L is added, whirlpool shakes 15s, is incubated for 5min, centrifugation；
6) solution of step 5) is all added in adsorption column, 12000rpm is centrifuged 1min, waste liquid is outwelled, by adsorption column
Place back in collecting pipe；
7) the Buffer RW1,12000rpm that 500 μ L are added into adsorption column are centrifuged 1min, outwell useless in collecting pipe
Adsorption column is placed back in collecting pipe by liquid；
8) the Buffer RW2,12000rpm that 500 μ L are added into adsorption column are centrifuged 1min, outwell useless in collecting pipe
Adsorption column is placed back in collecting pipe by liquid；
9) dehydrated alcohol of 500 μ L is added into adsorption column, 12000rpm is centrifuged 1min, outwells the waste liquid in collecting pipe,
Adsorption column is placed back in into collecting pipe；
10) 1min is centrifuged with 12000rpm, outwells the waste liquid in collecting pipe, adsorption column is stood into 5-10min, is thoroughly dried in the air
It is dry；
11) adsorption column is placed in the centrifuge tube of nuclease free, 50 μ L nuclease-free waters, room is vacantly added dropwise into adsorption column
Temperature places 5min, and 12000rpm is centrifuged 1min, collects DNA solution, and -80 DEG C of preservations are spare.
Compared with prior art, the beneficial effects of the present invention are:
Source of mouse TTV viral diagnosis primer provided by the invention can accurately, specifically detect source of mouse TTV virus, concentration
Down to 1 × 101Copy/μ L can still detect, sensitivity with higher；Using detection primer detection source of mouse TTV virus
The accuracy of detection can be improved, and the kit of application detection primer preparation detection source of mouse TTV virus can be convenient application, examine
Survey fast and flexible.The present invention screens the extracting method of TTV virus genom DNA, shortens viral DNA extraction time,
Operating process is simplified, detection sensitivity and accuracy is improved.
Detailed description of the invention
Fig. 1 is the electrophoresis result figure of the embodiment of the present invention 1；In figure, M:D2000DNA molecular mass standard, 1: specificity expands
Increase production object, 2: specific amplification products compare, and 3: negative control；
Fig. 2 is the fluorescent quantitative PCR solubility curve figure that the embodiment of the present invention 4 provides；In figure, 2~8: concentration 107、106、
105、104、103、102、101copies/μL；
Fig. 3 is the canonical plotting that the embodiment of the present invention 4 provides；
Fig. 4 is the experimental group fluorescence signal figure that the embodiment of the present invention 4 provides.
Specific embodiment
Below by specific embodiment, invention is further described in detail.
Experimental method used in the embodiment of the present invention is conventional method unless otherwise specified.
Material used in the embodiment of the present invention, reagent etc., are commercially available unless otherwise specified.
SYBR Premix Ex Taq II (Tli RNaseH Plus) (2 ×), is purchased from Japanese Takara company, 2 × Taq
It is raw that PCR MasterMix, zero background quick clone kit of D2000Marker, pLB and plasmid extraction kit are purchased from Tiangeng
Change scientific and technological (Beijing) Co., Ltd, bacillus coli DH 5 alpha competent cell, pillar DNA plastic recovery kit are purchased from Tiangeng biochemistry section
Skill (Beijing) Co., Ltd.Remaining goes out conventional reagent and is purchased from Chinese medicines group.
Embodiment l
Upstream primer (5 '-is designed at the 259-278bp of source of mouse TTV virus genom DNA
GATACTACCGACCAAGGAGA-3 ', SEQ ID No:1), downstream primer (5 '-is designed at 537-557bp
ATCCAGCAAGGTAAGATGTCC-3 ', SEQ ID No:2), which is used for the specific detection of source of mouse TTV virus.
The application method of the detection primer of source of mouse TTV virus:
1) using 1 μ L sample to be examined as template, be added 2 × Taq PCR MasterMix, 25 μ L, it is provided by the invention up and down
Each 1 μ L of primer is swum, ddH is added2O complements to 50 μ L；
2) PCR reaction, program are carried out are as follows: 95 DEG C, initial denaturation 5min；94 DEG C, it is denaturalized 15s；56 DEG C, anneal 30s；72 DEG C,
Extend 30s；35 circulations；72 DEG C, 10min.
3) after reaction, reaction product carries out agarose gel electrophoresis, passes through the band judging result of electrophoresis.
Experimental result is as shown in Figure 1, it can be seen from the figure that the size 299bp of purpose band and experimental design size one
It causes, and band is single bright；Illustrate that the primer amplification effect of design is preferable, meet expection, and there is preferable specificity.
Embodiment 2
A kind of detection kit of source of mouse TTV virus, kit contain the detection primer of the offer of embodiment 1.Certainly, this examination
Agent box further includes at least one reagent that can directly carry out PCR reaction, such as PCR reaction buffer, Taq archaeal dna polymerase, Taq
PCR MasterMix, SYBR Premix Ex Taq II, target fragment standard items or Mg2+。
It is specific as follows the present embodiment provides the application method of kit:
Regular-PCR
1) using 1 μ L sample to be examined as template, be added 2 × Taq PCR MasterMix, 25 μ L, it is provided by the invention up and down
Each 1 μ L of primer is swum, ddH is added2O is supplied to 50 μ L；
2) PCR response procedures are carried out are as follows: 95 DEG C, initial denaturation 5min；94 DEG C, it is denaturalized 15s；59 DEG C, anneal 30s；72 DEG C, prolong
Stretch 30s；35 circulations；72 DEG C, 10min.
Quantitative fluorescent PCR
1) reaction system of quantitative fluorescent PCR: using 2 μ L sample to be examined as template, SYBR Premix Ex Taq II (Tli
RNaseH Plus) (2 ×) 12.5 μ L, upstream and downstream primer each 1 μ L, ddH provided by the invention2O is supplied to 25 μ L；
2) response procedures of quantitative fluorescent PCR: 95 DEG C, initial denaturation 30s；95 DEG C, it is denaturalized 5s；60 DEG C, anneal 30s；72℃
Collect fluorescence signal 15s, 39 circulations.
According to the data and melt curve analysis of fluorescent quantitative PCR experiment, obtain in sample whether containing source of mouse TTV virus with
And the amount containing source of mouse TTV virus.
Embodiment 3
The present embodiment provides the extracting methods of viral DNA:
1) the 200 μ L of blood plasma or tissue for taking the host animal of infection source of mouse TTV virus at room temperature, if supplying 200 μ L,
It can be supplied with 0.9% physiological saline；
2) the Proteinase K solution of 20 μ L is added, mixes；
3) the Buffer RLV of 200 μ L is added, whirlpool shakes 15s；
4) 15min is incubated under the conditions of 56 DEG C, the solution of tube wall is collected into tube bottom by of short duration centrifugation；
5) dehydrated alcohol of 250 μ L is added, whirlpool shakes 15s, is incubated at room temperature 5min, of short duration centrifugation；
6) solution of step 1.5 is all added in adsorption column, 12000rpm is centrifuged 1min, outwells waste liquid, will adsorb
Column places back in collecting pipe；
7) the Buffer RW1,12000rpm that 500 μ L are added into adsorption column are centrifuged 1min, outwell useless in collecting pipe
Adsorption column is placed back in collecting pipe by liquid；
8) the Buffer RW2,12000rpm that 500 μ L are added into adsorption column are centrifuged 1min, outwell useless in collecting pipe
Adsorption column is placed back in collecting pipe by liquid；
9) dehydrated alcohol of 500 μ L is added into adsorption column, 12000rpm is centrifuged 1min, outwells the waste liquid in collecting pipe,
Adsorption column is placed back in into collecting pipe；
10) 1min is centrifuged with 12000rpm, outwells the waste liquid in collecting pipe, adsorption column is placed in room temperature 5-10min, thoroughly
It dries；
11) adsorption column is placed in the centrifuge tube of nuclease free, is vacantly added dropwise into adsorption column
50 μ LRNase-free Water (water of nuclease free) are placed at room temperature for 5min, and 12000rpm is centrifuged 1min, collect
DNA solution, -80 DEG C of preservations are spare.
Embodiment 4
The present embodiment provides the preparation methods of target fragment standard items:
1) source of mouse TTV virus genom DNA is obtained in Example 3, carries out PCR amplification；
2) by glue recovery purifying pcr amplification product, the target fragment purified；
3) 2.5 μ L of target fragment is connect with 1 μ L of carrier through T4DNA ligase；
4) the bacillus coli DH 5 alpha competent cell that the connection product in step 2.3 is transferred to 50 μ L is mixed, ice bath
30min, 42 DEG C of heat shock 90s shift ice bath and place 2-3min on ice；
5) the fresh not antibiotic LB liquid medium of 700 μ L is added, in 150rpm shake culture on 37 DEG C of shaking tables
45min recovery thallus；
6) hickie is selected on the LB plate containing ampicillin, and carries out amplification cultivation and extract to contain target fragment
The recombinant plasmid of standard items.
Bacterium colony PCR identification, sequence verification are carried out to the successful bacterium colony of conversion and carried out with restriction enzyme Hind III
Digestion identification.The stripe size 299bp and purpose band of electrophoresis amplification are in the same size, and band is single clear, tie with expected
Fruit is consistent.
Embodiment 5
The present embodiment carries out the detection of sensitivity and the foundation of standard curve, specific method to kit provided by the invention
It is as follows:
1) by the recombinant plasmid extracted in embodiment 4 according to 1 × 101Copy/μ L, 1 × 102Copy/μ L, 1 × 103Copy/
μL、1×104Copy/μ L, 1 × 105Copy/μ L, 1 × 106Copy/μ L and 1 × 107Copy/μ L concentration is diluted；
2) using the recombinant plasmid of 7 concentration gradients in step 1) as template, and blank group and negative control group are set, carried out
Fluorescent quantitative PCR, the operating method reference implementation example 2 of quantitative fluorescent PCR；
Experimental result is as shown in fig. 2, it can be seen that when recombinant plasmid concentration is 1 × 101Copy/μ L, still there is amplification
Solubility curve illustrates that the primer of design has good sensitivity.
Using cycle-index (Ct) value of the intersection point with baseline as ordinate, the logarithm of plasmid copy number is abscissa, system
Make standard curve；Standard curve is as shown in figure 3, R2=1.002.The source of mouse of arbitrary sample can be calculated by standard curve
TTV viral level.
Adjustment baseline is subject to blank group and negative control group curve does not occur, and 57 DEG C -95 DEG C of experimental group of fluorescence is taken to believe
Number；As a result it as shown in figure 4, the template reaction curve of 7 concentration of experimental group is almost the same, is identical, further illustrates this hair
The primer specificity of bright design is good；Each concentration can detected fluorescence signal, and the primer for illustrating that the present invention designs also has
There is preferable sensitivity.
Embodiment 6
2 parts of samples that the present embodiment obtains clinic are tested.
The doubtful source of mouse TTV virus infection sample for picking up from Haikou Area home mouse is detected, is provided using embodiment 2
Kit carries out sample process, and carries out Standard PCR and quantitative fluorescent PCR identification, Standard PCR and fluorescent quantitative PCR experiment side
Method is referring to embodiment 2.
By identification, detect that a sample is positive sample.
In conclusion the detection primer of source of mouse TTV virus provided in an embodiment of the present invention have preferable specificity and compared with
High sensitivity is applied to detection source of mouse TTV virus using the detection primer, and is applied to preparation detection source of mouse TTV virus
Kit, all there is preferable specificity and higher sensitivity；The kit of detection source of mouse TTV virus facilitates extraction virus
Nucleic acid and identified, practicability with higher and higher application value.
The above content is specific embodiment is combined, further detailed description of the invention, and it cannot be said that this hair
Bright specific implementation is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, it is not taking off
Under the premise of from present inventive concept, a number of simple deductions or replacements can also be made.
Sequence table
<110>Hainan Medical College
<120>a kind of detection primer and application of source of mouse TTV virus
<141> 2019-04-16
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gatactaccg accaaggaga 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atccagcaag gtaagatgtcc 21
Claims (6)
1. a kind of detection primer of source of mouse TTV virus, which is characterized in that in the 259-278bp of source of mouse TTV virus genom DNA
Place's design upstream primer, designs downstream primer, the sequence of upstream primer is as shown in SEQ ID No:1, downstream at 537-557bp
The sequence of primer is as shown in SEQ ID No:2.
2. the application method of the detection primer of source of mouse TTV virus described in claim 1, which is characterized in that including to sample
This is that template carries out PCR reaction；
PCR response procedures are as follows: 94 DEG C, initial denaturation 5min；94 DEG C, it is denaturalized 15s；56~60 DEG C, anneal 30s；72 DEG C, extend 30s；
35 circulations；72 DEG C, 10min.
3. a kind of detection kit of source of mouse TTV virus, which is characterized in that the detection kit includes described in claim 1
Detection primer.
4. the detection kit of source of mouse TTV virus according to claim 3, which is characterized in that further include PCR reaction buffering
Liquid, Taq archaeal dna polymerase, SYBR Premix Ex Taq II, Taq PCR MasterMix, target fragment standard items and Mg2+
At least one of.
5. the application method of the detection kit of source of mouse TTV virus as claimed in claim 4, which is characterized in that including with to be checked
Sample is that template carries out PCR reaction；
PCR response procedures are as follows: 95 DEG C, initial denaturation 30s；95 DEG C, it is denaturalized 5s；56~60 DEG C, anneal 30s；72 DEG C of collection fluorescence letters
Number 15s, 39 circulations.
6. the application method of the detection kit of source of mouse TTV virus according to claim 5, which is characterized in that source of mouse TTV
The extracting method of virus genom DNA are as follows:
1) the 200 μ L of blood plasma or tissue of the host animal of infection source of mouse TTV virus are taken；
2) the Proteinase K solution of 20 μ L is added, mixes；
3) the Buffer RLV of 200 μ L is added, whirlpool shakes 15s；
4) it is incubated for 15min under the conditions of 56 DEG C, is centrifuged, the solution of tube wall is collected into tube bottom；
5) dehydrated alcohol of 250 μ L is added, whirlpool shakes 15s, is incubated for 5min, centrifugation；
6) solution of step 5) is all added in adsorption column, 12000rpm is centrifuged 1min, outwells waste liquid, again by adsorption column
Put back to collecting pipe；
7) Buffer RW1,12000rpm the centrifugation 1min of 500 μ L is added into adsorption column, outwells the waste liquid in collecting pipe, it will
Adsorption column places back in collecting pipe；
8) Buffer RW2,12000rpm the centrifugation 1min of 500 μ L is added into adsorption column, outwells the waste liquid in collecting pipe, it will
Adsorption column places back in collecting pipe；
9) dehydrated alcohol of 500 μ L is added into adsorption column, 12000rpm is centrifuged 1min, outwells the waste liquid in collecting pipe, will inhale
Attached column places back in collecting pipe；
10) 1min is centrifuged with 12000rpm, outwells the waste liquid in collecting pipe, adsorption column is stood into 5-10min, is thoroughly dried；
11) adsorption column is placed in the centrifuge tube of nuclease free, 50 μ L nuclease-free waters is vacantly added dropwise into adsorption column, room temperature is put
5min is set, 12000rpm is centrifuged 1min, collects DNA solution, and -80 DEG C of preservations are spare.
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| CN103205511A (en) * | 2013-04-27 | 2013-07-17 | 金陵科技学院 | Primer pair for detecting pigeon torque teno viruses and application of primer pair |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205511A (en) * | 2013-04-27 | 2013-07-17 | 金陵科技学院 | Primer pair for detecting pigeon torque teno viruses and application of primer pair |
| CN107586888A (en) * | 2017-11-03 | 2018-01-16 | 福建省农业科学院畜牧兽医研究所 | One breeding pigeon TTV real-time fluorescence quantitative PCR detection kits |
| CN107604098A (en) * | 2017-11-03 | 2018-01-19 | 福建省农业科学院畜牧兽医研究所 | For detecting dove TTV EvaGreen real-time fluorescence quantitative PCRs primer and kit |
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| QIAAMP: "《QiAamp MinElute virus Spin Kit Handbook》", 30 April 2010 * |
| WU,Y. 等: "MF688246.1 Rodent Torque teno virus 3 isolate RoTTV3HMU1,complete genome", 《GENBANK》 * |
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| 吴悦 等: "鼠源TTV病毒海口株的基因组分析及real-time PCR检测方法的建立", 《海南医学院学报》 * |
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