CN108866156A - The preparation method and applications of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system - Google Patents
The preparation method and applications of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
" preparation method and applications of the single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system " of the invention belong to kit detection technique field.The preparation method includes：Reaction solution is prepared according to the following formulation：20~50mM of Tris-HCl reaction buffer, the MgCl of pH value 8.3~8.721~3mM, 40~50mM of KCl, 50~200 μM of dNTPs, 100~400 μM of dUTP, 100~200U/ of reverse transcriptase μ l, 0.8~2.8U/ of Taq enzyme μ l, RNase inhibitor 10.5U/T~12.5U/T, 0.1~1 μM of primer, 0.01~0.2 μM of probe, final concentration of 1~20Mm or volume ratio 0.05%~10% increased response agent, remaining be sterile nuclease-free water.
Description
Technical field
The invention belongs to kit detection technique field, it is related to the single mass system enzymatic amplification for POCT fluorescence RT-PCR system
The preparation method and applications of composition.
Background technique
Detection kit mainly has enzyme linked immunosorbent assay (ELISA) (ELISA) detection kit, General polymeric enzyme chain anti-at present
Answer (PCR) kit, real-time fluorescent reverse transcription polymerase chain reaction (Real-time FQ RT-PCR) kit.It is viral, thin
Bacterium, the routine clinical of helminth and laboratory testing identification method have microscopy, pathogen separation, immunohistochemistry, serodiagnosis with
And molecular biology method.Wherein real-time fluorescent reverse transcription polymerase chain reaction (Real-time FQ RT-PCR) kit
Have the advantages that time-consuming short, high sensitivity, high specificity.
Common real-time fluorescent reverse transcription polymerase chain reaction (Real-time FQ RT-PCR) expands currently on the market
Kit includes reaction solution (containing primed probe), enzyme mixation, sterile nuclease-free water, carries out real-time fluorescent reverse transcription polymerase
When chain reaction (RT-PCR) expands, first ratio to specifications prepares the mixed liquor of various reagents, is then dispensed into again each
In reaction tube.The real-time fluorescent reverse transcription polymerase chain reaction (Real-time FQ RT-PCR) applied currently on the market is expanded
Increase kit to have the following disadvantages：One, amplification system prepares mistake, leads to result mistake；Two, reagent constituents include reaction solution
(contain primed probe), enzyme mixation, sterile nuclease-free water etc. ratio must prepare premixed liquid to specifications before detection, then into
Row packing.Be easy to cause with liquid mistake, pollution, and premixed liquid need it is ready-to-use, it is cumbersome and take a long time；Three, common
The real-time fluorescent reverse transcription polymerase chain reaction amplification kit reaction time usually require 1~2 hour, time-consuming, it is difficult to
Meets the needs of mass detection.
POCT (point-of-care testing), refers to real-time test, on-site rapid inspection, refers to and carries out beside patient
Clinical detection and bedside detection (bedside testing), be not necessarily clinical examination teacher usually to carry out.It is existing in sampling
Field is analyzed at once, saves complex process program of the sample in laboratory inspection, and the one kind for quickly obtaining inspection result is new
Method.
POCT is unified on the basis of being repeatedly expounded through peer review by Chinese medicine equipment association POCT equipment technology Professional Committee
Name, and be defined as：It is being carried out in sampling location, detection knot is quickly obtained using portable analysers and matched reagent
A kind of detection mode of fruit.POCT meaning can be understood in terms of two：Spatially, the inspection carried out at one's side in patient, i.e. " bed
It examines on side "；On time, it can carry out " real-time test ".
Currently, limitation POCT detection pattern has in the main restricting factor of the application of PCR detection field：Standard PCR is glimmering
Light PCR need scene with liquid, it is cumbersome, take a long time, be unstable, is easy to pollute etc..
Therefore, need a kind of good stability, long shelf-life, it is easy to operate, be not required to (extract template and be directly added into expansion with liquid
Increasing system), the preparation method of the single mass system enzymatic amplification composition of the short POCT fluorescence RT-PCR system of detection time.
Summary of the invention
It is a kind of for POCT fluorescence RT- the technical problem to be solved by the present invention is in view of the deficiencies of the prior art, provide
Single mass system enzymatic amplification composition of PCR system and preparation method thereof, single mass system enzymatic amplification composition stability made from this method
Good, long shelf-life, it is easy to operate, be not required to liquid (extracted template and be directly added into amplification system), and RT-PCR proliferation time is only
30 minutes (the RT-PCR proliferation time of conventional kit was at 70 minutes or more) are needed, reduce detection time compared to conventional detection
75%~80%, and a possibility that test malfunctions and pollutes can be greatly reduced, it is highly suitable for POCT detection pattern.
Technical scheme is as follows：
The preparation method of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system, which is characterized in that including：
(1) primer and probe of specificity is designed and synthesized for target sequence；
(2) reaction solution is prepared according to the following formulation：
20~50mM of Tris-HCl reaction buffer, the MgCl of pH value 8.3~8.721~3mM, 40~50mM of KCl,
50~200 μM of dNTPs, 100~400 μM of dUTP, 100~200U/ of reverse transcriptase μ l, 0.8~2.8U/ of Taq enzyme μ l, RNA enzyme
Inhibitor 10.5U/T~12.5U/T, 0.1~1 μM of primer, 0.01~0.2 μM of probe, final concentration of 1~20Mm or volume ratio
0.05%~10% increased response agent, remaining be sterile nuclease-free water；
(3) above-mentioned reaction solution is dispensed by part, every a i.e. single mass system enzymatic amplification composition.
The reaction solution further includes 0.1~1 μM of general internal control primer, 0.01~0.2 μM of internal reference probe；General internal reference is preferred
Xeno RNA Control。
The preferred PrimeScript of reverse transcriptaseTMRT Enzyme；The preferred Takara E of Taq enzymeX TaqTM HS
Enzyme.
The increased response agent, which is selected from, has following one of following substances of final concentration in the reaction solution, 10-
Glycerol that formamide or glycine betaine that the ammonium sulfate of 20mM, volumetric concentration are 1%~5%, volumetric concentration are 1%~10%, body
Product concentration be 0.05%~0.1% Tween-20, volumetric concentration be 0.05%~0.1% BSA, or, 1mM~2mM DTT
Or gelatin.
The total volume of 1 part of single mass system enzymatic amplification composition be selected from 12~14 μ l, 14~16 μ l, 16~18 μ l, 18~20 μ l,
20~22 μ l, 22~24 μ l or 24~26 μ l.
The single mass system enzymatic amplification composition that the preparation method is prepared.
Kit for POCT fluorescence RT-PCR system, which is characterized in that expand including single mass system enzyme described at least 1 part
Increase composition.
The single mass system enzymatic amplification composition is several pieces.
The single mass system enzymatic amplification composition is stored in PCR amplification pipe；It is stored described in 1 part in each PCR amplification pipe
Single mass system enzymatic amplification composition.
The present invention provides a kind of preparation method of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system,
It is characterized in that, the single mass system enzymatic amplification composition of 1 part of quick real-time fluorescent reverse transcription polymerase chain reaction kit includes
Reaction buffer, dNTPs, dUTP, increased response agent, reverse transcriptase, Taq enzyme, RNA inhibitor, primer, probe, amplification PCR
Pipe.
One aspect of the present invention, its spy of the preparation method of the single mass system enzymatic amplification composition of POCT fluorescence RT-PCR system
Sign is that the single mass system enzymatic amplification composition chosen is 1 part of amplified reaction.
Still another aspect of the invention, the preparation method of the single mass system enzymatic amplification composition of POCT fluorescence RT-PCR system its
It is characterized in that the primed probe chosen is the primed probe that can arbitrarily carry out inverse transcription polymerase chain reaction.
In a specific embodiment of the invention, selected primed probe is that can expand canine distemper virus and internal reference.
Still another aspect of the invention, the preparation method of the single mass system enzymatic amplification composition of POCT fluorescence RT-PCR system its
It is characterized in that the amplification chosen with PCR pipe is the amplification pipe that can be applied to different fluorescent PCR instrument.
In a specific embodiment of the invention, selected amplification is 25 μ l of Cepheid tubes, fluorescence with PCR pipe
PCR instrument is Anheal T8.
The present invention also provides the purposes of single mass system enzymatic amplification composition described above, are used as POCT fluorescence RT-PCR body
The part of system.Single mass system enzymatic amplification composition of the invention can be placed in inverse transcription polymerase chain reaction kit.
In a specific embodiment of the invention, POCT fluorescence RT-PCR system used includes but is not limited to hundstaupe pyreticosis
Malicious quickly real-time fluorescent reverse transcription polymerase chain reaction kit.
Compared with prior art, preparation method of the invention can prepare the fluorescence for being very suitable for POCT detection pattern
PCR single mass system enzymatic compositions, have filled up the blank of POCT fluorescence RT-PCR detection field.The single mass system enzyme of the method for the present invention preparation
Amplification object stability is good, long shelf-life, it is easy to operate, be not required to keep away with liquid (extracted template and be directly added into amplification system)
Exempt from matching while using liquid in the prior art, it is cumbersome, the shortcomings that malfunctioning and polluting is easy, it can be achieved that examining in time.The present invention
Preparation method and its POCT fluorescence RT-PCR single mass system enzymatic compositions and kit prepared through repetition test, sensitivity
Up to 101.5TCID50, accuracy rate be up to 100%, long shelf-life and foreshorten to 30 minutes up to 18 months, RT-PCR proliferation time, phase
A possibility that shortening 75%~80% than routine operation, substantially reducing test error and pollution.
Detailed description of the invention
Fig. 1 is to use existing conventional means：The Real-time FQ RT-PCR that normal fluorescence RT-PCR amplification system carries out
Amplification；
Fig. 2 is that the single mass system enzymatic amplification composition provided using one embodiment of the present of invention carries out Real-time FQ
The amplification of RT-PCR；
Fig. 3 be after the single mass system enzymatic amplification composition that provides of one embodiment of the present of invention is placed 0 month be drawn off into
The amplification of row Real-time FQ RT-PCR；
Fig. 4 be after the single mass system enzymatic amplification composition that provides of one embodiment of the present of invention is placed 6 months be drawn off into
The amplification of row Real-time FQ RT-PCR；
Fig. 5 is after the single mass system enzymatic amplification composition that one embodiment of the present of invention provides is placed 12 months, to be drawn off
Carry out the amplification of Real-time FQ RT-PCR；
Fig. 6 is after the single mass system enzymatic amplification composition that one embodiment of the present of invention provides is placed 18 months, to be drawn off
Carry out the amplification of Real-time FQ RT-PCR；
Fig. 7 is the specific test result for the single mass system enzymatic amplification composition that one embodiment of the present of invention provides：Wherein,
1：CPIV；2：AIV；3：CPV；4：CCV；5：CAV-1；6：CAV-2；7：CDV.
Fig. 8 is the PCR sensitivity tests result for the single mass system enzymatic amplification composition that one embodiment of the present of invention provides：Its
In, 1：105.5TCID50；2：104.5TCID50；3：103.5TCID50；4：102.5TCID50；5：101.5TCID50；6：100.5TCID50；
Fig. 9 is the application test result for the single mass system enzymatic amplification composition that one embodiment of the present of invention provides：1~6 is
Clinical sample detection；7~12 detect internal reference for clinical sample.
Figure 10 is the quick Real-time FQ RT-PCR kit of canine distemper virus that an experimental example of the invention provides
Sensitivity verifying；Wherein 1 is 105.5TCID50；2 be 104.5TCID50；3 be 103.5TCID50；4 be 102.5TCID50；5 are
101.5TCID50；6 be 100.5TCID50。
Figure 11 is that the porcine reproductive and respiratory syndrome virus that an experimental example of the invention provides and swine fever virus are multi-joint fast
The expanding effect of fast Real-time FQ RT-PCR kit porcine reproductive and respiratory syndrome virus.
Figure 12 is that the porcine reproductive and respiratory syndrome virus that an experimental example of the invention provides and swine fever virus are multi-joint fast
The expanding effect of fast Real-time FQ RT-PCR kit swine fever virus.
Figure 13 is an experimental example porcine reproductive and respiratory syndrome virus of the invention and the multi-joint quick Real- of swine fever virus
Internal reference expanding effect in time FQ RT-PCR kit.
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 should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Technology or conditions person, described technology or conditions are (yellow such as with reference to the work such as J. Pehanorm Brooker according to the literature in the art
What training hall etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Examination used
Production firm person is not specified in agent or instrument, and being can be with conventional products that are commercially available.
1st group of embodiment, single mass system enzymatic amplification preparation method of composition of the invention
This group of embodiment provides a kind of preparation side of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system
Method, which is characterized in that including：
(1) primer and probe of specificity is designed and synthesized for target sequence；
(2) reaction solution is prepared by following formulas：
20~50mM of Tris-HCl reaction buffer, the MgCl of pH value 8.3~8.721~3mM, 40~50mM of KCl,
DNTPs50~200 μM, 100~400 μM of dUTP, 100~200U/ of reverse transcriptase μ l, 0.8~2.8U/ of Taq enzyme μ l, RNA enzyme suppression
Preparation 10.5U/T~12.5U/T, 0.1~1 μM of primer, 0.01~0.2 μM of probe, final concentration of 1~20mM or volume ratio
0.05%~10% increased response agent, remaining be sterile nuclease-free water；
Or, any group of formula of according to the form below 1 prepares reaction solution：
Table 1
(3) above-mentioned reaction solution is dispensed by part, the every 1 part i.e. single mass system enzymatic amplification composition.
In a further embodiment, the reaction solution further includes 0.1~1 μM of general internal control primer, internal reference probe 0.01
~0.2 μM μM；The general preferred Xeno RNA Control of internal reference, it is commercially available.
In a further embodiment, the preferred PrimeScriptTM RT Enzyme of the reverse transcriptase；The Taq enzyme
It is preferred that Takara EX TaqTM HS enzyme.Both enzymes are the enzyme for having carried out the commercialization of enzyme activity optimization；Wherein
PrimeScriptTM RT Enzyme reverse transcriptase is lacked by gene modification and the RNase H activity of recombinant technique clonal expression
It loses, improves thermal stability, while enhancing the tolerance to RNA complexity secondary structure, shorten the reaction time；Taq enzyme (Takara
EX TaqTM HS enzyme) it is the thermal starting enzyme based on antibody act modification, after genetic engineering techniques, when can shorten reaction
Between.
In some embodiments, the increased response agent component is pressed shown in following formulas,
Ingredient is ammonium sulfate 10-20mM, volumetric concentration is 1%-5% formamide or glycine betaine, volumetric concentration 1%-
BSA, DTT that Tween-20 that 10% glycerol, volumetric concentration are 0.05%-0.1%, volumetric concentration are 0.05%-0.1% or
Gelatin 1mM-2mM.
Alternatively, the increased response agent has following final concentrations each in the reaction solution selected from as shown in table 2 below
One of substance：
Table 2
In the particular embodiment, the total volume of 1 part of single mass system enzymatic amplification composition be selected from 12~14 μ l, 14~16 μ l,
16~18 μ l, 18~20 μ l, 20~22 μ l, 22~24 μ l or 24~26 μ l.
It is the concrete operation step during preparation method test of the present invention below：
One, primer and probe designs
Two, nucleic acid extraction
(member henry extracts kit)
Three, prepared by standard items
Carrier T conversion bacillus coli DH 5 alpha containing amplified fragments extracts plasmid after converting, being enriched with, and uses spectrophotometer
It dilutes and quantifies to 1 × 1010Copy/μ l is placed in -80 DEG C of preservations after packing.
Four, reaction medium optimizes
Reaction system each component is arranged：
Template DNA/RNA 15,30,45,60,75,90,120, nine 150,180ng gradients.
0.3,0.4,0.5,0.6,0.7,0.9,1.2,1.4,1.8 μM of six concentration gradient of ol/L of random primer.
0.01,0.05,0.10,0.15,0.2 μM of five gradient of probe.
Taq enzyme 0.5,1,1.5,2, five 2.5,3U concentration gradients.
50,100,150,200 μM of four concentration gradients of dNTPs.
100,150,200,250,300,350,400 μM of six concentration gradients of dUTP.
Reverse transcriptase 100,120,140,160, six 180,200U/ μ l concentration gradients.
RNase inhibitor 10.5,11.5, tetra- 12.5,13.5U/T concentration gradients.
Mg2+3.5,3.0,2.5,2,1.5,1, eight 0.5,0.025mmol/L concentration gradients.
Five, temperature optimization
Reverse transcriptase
Enzyme solution is mixed with the 10 μ l of RNA extracted, respectively in PCR instrument be arranged 41 DEG C, 42 DEG C, 43 DEG C, 44 DEG C,
45 DEG C, 46 DEG C, 47 DEG C, 48 DEG C, 49 DEG C, 50 DEG C, 51 DEG C, 52 DEG C, while carrying out warm bath.Reverse transcription product takes 2 μ l to carry out PCR,
All PCR products are subjected to 1.5%~2% agarose electrophoresis, the observation of ethidium bromide stained gel imaging system after reaction.
2.Taq enzyme
In the case where other conditions are constant, 32 DEG C, 34 DEG C, 36 DEG C, 38 DEG C, 40 DEG C are set by annealing temperature, are compared
Fluorescent quantitative PCR result.
Six, pH value optimizes
PH of buffer is adjusted, is set as 8.0,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8 pH value gradient, at random
It chooses primer and carries out quantitative fluorescent PCR, comparing result.
Seven, freezing-thawing test
Using multigelation method, 4 parts of single mass system Amplification objects is taken to freeze 2h in -20 DEG C of refrigerators, melting 1h is one
Circulation, freeze thawing 0,5,10,20 time.After completing freeze thawing one by one, the single mass system Amplification object of freeze thawing does not occur in -20 DEG C of refrigerators
As control, randomly selects primer and carry out quantitative fluorescent PCR contrasting detection.
2nd group of embodiment, single mass system enzymatic amplification composition of the invention
This group of embodiment provides a kind of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system, feature
It is that the single mass system enzymatic amplification composition is prepared by the 1st group of described in any item preparation methods of embodiment.
3rd group of embodiment, kit of the invention
This group of embodiment provides a kind of kit for POCT fluorescence RT-PCR system, which is characterized in that including at least 1
The 2nd group of described in any item single mass system enzymatic amplification compositions of embodiment of part.
In the particular embodiment, the single mass system enzymatic amplification composition is several pieces.
In a further embodiment, the single mass system enzymatic amplification composition is stored in amplification pipe；Each amplification pipe
1 part of single mass system enzymatic amplification composition of middle storage.
Experimental example 1：The comparison of single mass system enzymatic amplification composition
The present inventor buys Commercial optical RT-PCR amplification system, and designs the primed probe of amplification canine distemper virus, leads to
Comparability test is crossed, the preparation method and amplification condition of single mass system enzymatic amplification composition are screened.
Normal fluorescence RT-PCR amplification system and amplification condition, matching while using
| Reaction reagent | Volume (μ L) | Final concentration |
| Reaction buffer (contains MgCl2And dNTP) | 12.5 | 1× |
| Hot start Taq polymerase | 0.5 | 0.1U/μL |
| Efficient reverse transcriptase | 0.5 | —— |
| 10pmoL/ μ L primer pair | 1.5 | 400nM |
| 10pmoL/ μ L probe | 0.5 | 200nM |
| ROX dyestuff | 0.5 | 1× |
| Total serum IgE | 2 | —— |
| ddH2O | 7 | —— |
| Total volume | 25 | —— |
Amplification condition
Single mass system enzymatic amplification composition preparation method and amplification condition
According to being prepared above, after prepared reaction system is mixed well, each Cepheid is dispensed
Each 15 μ L, as single mass system enzymatic amplification composition, freeze in -20 DEG C in tubes reaction tube.
Amplification condition
Sensibility and specificity result is consistent, but single mass system enzymatic amplification composition stability is good, long shelf-life, operation letter
Just, it is not required to only need 30 minutes with liquid (extracted template and be directly added into amplification system), proliferation time；Compared to conventional commercial fluorescence
70 minutes or more the time required to RT-PCR amplification system and method, reaction duration of the invention effectively shortens 75% or more.
As a result as depicted in figs. 1 and 2 respectively.
Experimental example 2：Single mass system enzymatic amplification composition storage life
The storage life experimental result for the single mass system enzymatic amplification composition that preparation method of the present invention is prepared such as Fig. 3-Fig. 6 institute
Show, when long shelf-life was up to 18 months, taking-up can still provide for amplified reaction, and obtain excellent result.
Experimental example 3：The quick POCT fluorescence RT-PCR system reagent box composition (10 parts/box) of canine distemper virus
1. reaction tube：1 part of canine distemper virus single mass system enzymatic amplification composition, 15 μ L/ branch, 10, -20 DEG C of preservations；
2.RNA internal reference：300 μ L/ branch, 1, -20 DEG C of preservations；
3. lysate：6mL/ bottles, 1 bottle, room temperature preservation.
4. washing lotion：12mL/ bottles, 1 bottle, room temperature preservation.
5. eluent：1mL/ branch, 1, room temperature preservation.
6. adsorption column and collecting pipe：10 sets/bag, 1 bag, room temperature preservation.
The application method of kit prepared by experimental example 4, experimental example 3
1 sample preparation
1.1 bedew sterile cotton swab with sterile saline, and overturning is embrocated in conjunctival sac or at schneiderian membrance, acquire sample
This, or lavage of trachea sample is dipped with sterile swab.Swab is inserted into sterile saline again, the concussion that is vortexed mixes, and then will
Swab extracts liquid in tube wall, is put into thimerosal and discards, and takes 200 μ L of supernatant in 1.5mL sterile centrifugation tube.
1.2EDTA anticoagulation, cell culture fluid take 200 μ L of supernatant in 1.5mL sterile centrifugation tube.
The extraction of 2 viral RNAs
The sample handled well is separately added into 20 μ L RNA internal references by 2.1, is added 500 μ L of lysate, is sufficiently mixed by inversion,
It is stored at room temperature 3min.Draw liquid into adsorption column that (adsorption column will put on collecting pipe, and sucking of trying not when sucking liquid suspends
Impurity, in order to avoid adsorption column is blocked when centrifugation), 13000rpm is centrifuged 30s.
2.2 discard liquid in collecting pipe, draw 500 μ L of washing lotion and adsorption column is added, 13000rpm is centrifuged 30s.
2.3 discard liquid in collecting pipe, draw 500 μ L of washing lotion and adsorption column is added, 13000rpm centrifugation 2min (is asked when taking out
Pay attention to that adsorption column is avoided to encounter following liquid) to remove remaining washing lotion.
2.4 move into adsorption column in 1.5mL centrifuge tube, and 50 μ L eluents, 13000rpm centrifugation is added to adsorption column center
30s, liquid is template ribonucleic acid in centrifuge tube.
The operation of 3 real-time fluorescence RT-PCRs
It takes reaction tube to be placed in room temperature thawing, draws 5 μ L of template ribonucleic acid with pipettor and be added in reaction tube, be centrifuged with centrifuge
30s is put into machine, carries out augmentation detection using program CDV.
【Result judgement】
According to automatically analyzing, simultaneously there is specific amplification curve for the CDV positive in value≤36 Ct；When Ct value > 36, and occur
Specific amplification curve need to resample and extract RNA, and result judgement is carried out after amplification, such as specific amplification curve still occur, can
It is determined as the positive；For certain not shown S type curves, but the higher sample of background, it should be negative.
The application of kit prepared by experimental example 5, experimental example 3
1 specific test
Specific test result is shown in Fig. 7.As seen from Figure 7, the primed probe of design can only amplify mesh from CDV culture
Segment, other virus amplification results that can infect dog be feminine gender.
2 sensitivity tests results
Sensitivity tests result is shown in Fig. 8.It as seen from Figure 8, can be from doing 10 using the PCR method of this test5.5TCID50、
104.5TCID50、103.5TCID50、102.5TCID50、101.5TCID50Positive amplification result is obtained in diluted 5 μ L sample, it is minimum
Detected level is 101.5TCID50.7500 Instrumental results are as shown in Figure 10, and 1 in figure is 105.5TCID50；2 are
104.5TCID50；3 be 103.5TCID50；4 be 102.5TCID50；5 be 101.5TCID50；6 be 100.5TCID50。
3 application test results
Preliminary experiment results are shown in Fig. 9.As seen from Figure 9,4 parts of positive expansions can be obtained from 6 parts of doubtful samples using PCR method
Increase result.Accuracy rate is 100%
The detection kit performance verification of the present invention of experimental example 6, other a few class difference detection targets
Above-mentioned preparation method according to the invention, can be prepared：Porcine reproductive and respiratory syndrome virus and swine fever virus
Multi-joint quick POCT fluorescence RT-PCR system reagent box POCT fluorescence RT-PCR system, preparation method are identical as experimental example 3.
Classes of agents box expanding effect is good, the quick POCT fluorescence RT-PCR system examination of porcine reproductive and respiratory syndrome virus
The expanding effect of agent box is as shown in figure 11；Expanding effect such as Figure 12 of the quick POCT fluorescence RT-PCR system reagent box of swine fever virus
It is shown；Internal reference expanding effect is as shown in figure 13.
The storage life of mentioned reagent box can reach 18 months or more, and carry out POCT fluorescence using above-mentioned classes of agents box
The reaction time of RT-PCR system controls within 40 minutes, and sensitivity can reach 101.5TCID50。
Claims (9)
1. the preparation method of the single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system, which is characterized in that including：
(1) primer and probe of specificity is designed and synthesized for target sequence；
(2) reaction solution is prepared according to the following formulation：
20~50mM of Tris-HCl reaction buffer, the MgCl of pH value 8.3~8.721~3mM, 40~50mM of KCl, dNTPs
50~200 μM, 100~400 μM of dUTP, 100~200U/ of reverse transcriptase μ l, 0.8~2.8U/ of Taq enzyme μ l, RNase inhibitor
10.5U/T~12.5U/T, 0.1~1 μM of primer, 0.01~0.2 μM of probe, final concentration of 1~20Mm or volume ratio 0.05%~
10% increased response agent, remaining be sterile nuclease-free water；
(3) above-mentioned reaction solution is dispensed by part, every a i.e. single mass system enzymatic amplification composition.
2. preparation method according to claim 1, which is characterized in that the reaction solution further includes general internal control primer 0.1
~1 μM, 0.01~0.2 μM of internal reference probe；The general preferred Xeno RNA Control of internal reference.
3. preparation method according to claim 1, which is characterized in that the preferred PrimeScript of reverse transcriptaseTMRT
Enzyme；The preferred Takara E of Taq enzymeX TaqTMHS enzyme.
4. preparation method according to claim 1, which is characterized in that the increased response agent is selected from following described
One of following substances of final concentration in reaction solution, the ammonium sulfate of 10-20mM, the formamide or sweet tea that volumetric concentration is 1%~5%
Tween-20 that glycerol that dish alkali, volumetric concentration are 1%~10%, volumetric concentration are 0.05%~0.1%, volumetric concentration are
0.05%~0.1% BSA, or, 1mM~2mM DTT or gelatin.
5. preparation method according to claim 1 to 3, which is characterized in that 1 part of single mass system enzymatic amplification composition it is total
Volume is selected from 12~14 μ l, 14~16 μ l, 16~18 μ l, 18~20 μ l, 20~22 μ l, 22~24 μ l or 24~26 μ l.
6. the single mass system enzymatic amplification composition that any preparation method of claim 1-5 is prepared.
7. being used for the kit of POCT fluorescence RT-PCR system, which is characterized in that including at least 1 part of list as claimed in claim 6
System enzymatic amplification composition.
8. kit according to claim 7, which is characterized in that the single mass system enzymatic amplification composition is several pieces.
9. according to the kit of claim 7 or 8, which is characterized in that the single mass system enzymatic amplification composition is stored in PCR expansion
Increase in pipe；1 part of single mass system enzymatic amplification composition is stored in each PCR amplification pipe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810794915.2A CN108866156A (en) | 2018-07-19 | 2018-07-19 | The preparation method and applications of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system |
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