CN109337899A - A kind of nucleic acid extraction and PCR detection kit - Google Patents
A kind of nucleic acid extraction and PCR detection kit Download PDFInfo
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- CN109337899A CN109337899A CN201811247216.2A CN201811247216A CN109337899A CN 109337899 A CN109337899 A CN 109337899A CN 201811247216 A CN201811247216 A CN 201811247216A CN 109337899 A CN109337899 A CN 109337899A
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Abstract
The present invention proposes that a kind of nucleic acid extraction and PCR detection kit, the nucleic acid extraction and PCR detection kit are made of cracking liquid kit and PCR pipe.Nucleic acid cleavage liquid is held in cracking liquid kit, sample to be tested is added after nucleic acid cleavage liquid, viral nucleic acid is released by the effect of the nucleic acid cleavage liquid, and realizes purifying, gains can be added directly into PCR pipe realization PCR detection after nucleic acid cleavage liquid is added in sample.The freezing dry powder PCR amplification system reagent for being directly used in quantitative fluorescent PCR measurement is placed in the PCR pipe, the freezing dry powder PCR amplification system reagent is the gains prepared as raw material by freeze drying process using PCR reaction buffer, archaeal dna polymerase, atopic primer, probe and freeze drying protectant.The pipe shaft of the PCR pipe includes reacting part, buffer part and seal pipe lid, and the buffer locations are in the top of the pipe shaft, and in the lower part of the pipe shaft, both buffer part and reacting part are connected to the reactive site by conduit.
Description
Technical field
The present invention relates to technical field of biological more particularly to a kind of nucleic acid extractions and PCR detection kit.
Background technique
Real-Time Fluorescent Quantitative PCR Technique is widely used to hereditary disease molecular diagnosis, clinical examination, animals and plants inlet and outlet inspection
The various fields such as epidemic disease, food safety monitoring, edaphon detection and paternity test.Due in blood, food, soil equal samples
Containing a large amount of inhibiting factors, such as hemoglobin, protoferriheme, lactoferrin, humic acid, to conventional Taq archaeal dna polymerase
Apparent inhibiting effect will be generated.Therefore, it is necessary to be subsequently used for PCR amplification first from these sample to be tested separation and Extraction nucleic acid.Core
It is one of key method in the detection of nucleic acids first step and molecular biology that acid, which extracts,.Basis is provided for downstream nucleic acid detection, is mentioned
Quality and integrality is taken to directly affect clinical research or diagnosis.
General nucleic acid extraction process includes two steps: the cracking and purifying of sample.Traditional extraction reagent is due to acquirement rate phase
To lower, and step is more many and diverse, to extracting work belt centainly to influence, it is difficult to obtain qualified nucleic acid samples.Paramagnetic particle method is close
The nucleic acid extraction mode to grow up for several years, the nucleic acid purity of extraction is high, yield is big.Obviously, operated in accordance with conventional methods step it is more, at
It is this height, larger to the requirement of sample and easily cause cross contamination, it is unfavorable for client's rapidly extracting purification of nucleic acid sample.
In addition, reagent is not mostly instant in the most widely used real-time fluorescence PCR technology of current detection of nucleic acids
, it causes PCR to detect the problems such as generally existing complicated for operation, bothersome, required cost is high, complicated configuration work is needed before use
Make, be easy to cause the error even failure of test.Different personnel's operations also cause experimental error；Certain reagents save at normal temperature
It is unstable；It cannot accomplish one-step method rapidly extracting nucleic acid；PCR detection reagent needs on-site manual to configure, and multiple steps need people
The detecting instrument of work label, each stage is independent, is unfavorable for full automatic treatment a large number of experiments sample.
Summary of the invention
In view of above-mentioned disadvantages described above existing in the prior art, the invention proposes a kind of nucleic acid extractions and PCR detection to try
Agent box.The nucleic acid extraction and PCR detection kit, which use, directly extracts viral nucleic acid in simultaneously amplified reaction detection clinical sample
Real-time fluorescence PCR reaction system, by sample to be tested be added lysate realize nucleic acid cleavage and purifying, crack product after purification
PCR detection can be directly carried out, and realizes that amplified reaction and PCR detect with the freeze-dried powder reagent of instant, without being prepared before surveying,
Detection thoroughly solves the problems, such as that the nucleic acid DNA dependent on purifying is the bottleneck that template carries out PCR amplification, and applicable sample includes blood
Liquid, serum, blood plasma, saliva, urine, tissue fluid, sperm, juice, pus and the breathing polymorphic types sample such as liquid and mucus.
The present invention provides a kind of nucleic acid extraction and PCR detection kit characterized by comprising cracking liquid kit with
And PCR pipe, it cracks in liquid kit and holds nucleic acid cleavage liquid, release viral nucleic acid by the effect of the nucleic acid cleavage liquid
Come；The freezing dry powder PCR amplification system reagent for being directly used in quantitative fluorescent PCR measurement is placed in the PCR pipe, the freezing is dry
Powder PCR amplification system reagent is with PCR reaction buffer, archaeal dna polymerase, atopic primer, probe and frozen-dried protective
Agent is the gains that raw material is prepared by freeze drying process；
The pipe shaft of the PCR pipe includes reacting part, buffer part and seal pipe lid, and the buffer locations are upper in the pipe shaft
Portion, in the lower part of the pipe shaft, both buffer part and reacting part are connected to the reactive site by conduit.
Wherein, the nucleic acid cleavage liquid include CuHCl, NaOH, ethylenediamine tetra-acetic acid, trishydroxymethylaminomethane, NaCl,
Triton X-100, surfactant, phenol, isoamyl alcohol.Wherein, the concentration of CuHCl is 5.6-5.85g/10ML；Second
Ethylenediamine tetraacetic acid (EDTA) 0.03-0.04g/10ML；Trishydroxymethylaminomethane 0.06-0.07g/10ML；NaCl 0.01-0.015g/
10ML；Triton X-100 2%-10%(volume ratio)；Surfactant 0.075-0.1%(volume ratio)；Nucleic acid cleavage
1%-2.5%(volume ratio is added in liquid) phenol, isoamyl alcohol mixed liquor, wherein phenol, isoamyl alcohol volume ratio 20:1-25:1.
The surfactant using it is following any one: lauryl sodium sulfate, hexadecyl first triamine, poly- second
Alkene pyrrolidone, lauroyl propylhomoserin sodium.
Wherein, the freezing dry powder PCR amplification system reagent is prepared in the following way: by PCR reaction buffer, DNA
After polymerase, atopic primer, probe are mixed with frozen-dried protective agent solution according to predetermined ratio, freeze-drying.
Wherein, the freeze-drying is divided into precooling, main freeze-drying, reinforces dry three phases, and the first stage is
Precooling, slow cooling is to -35 to -40 degrees Celsius, and cooling duration is greater than 1 hour, and holding 2.5 is small under the conditions of the temperature
When；Second stage is the main freeze-drying stage, and temperature is kept for -15 to -20 degrees Celsius, is kept for 5 hours, wherein air pressure is maintained at
0.1mbar；Phase III is to reinforce drying, and temperature is kept for 15-25 degrees Celsius, and drying time 2 hours, air pressure kept 0.1mbar.
Wherein, preparing the freeze drying protectant that the freezing dry powder PCR amplification system reagent uses includes: mannitol, poly- second
Glycol, maltose, ascorbic acid and polyvinylpyrrolidone.Wherein, mannitol additional amount 3-5g/10ML；Polyethylene glycol adds
Enter amount 2.5-3g/10ML；Maltose additional amount 15-18g/10ML；Ascorbic acid additional amount 0.4-0.6g/10ML；Polyethylene pyrrole
Pyrrolidone 0.8-1.5g/ML.
Wherein, the frozen-dried protective agent solution is prepared with the deionized water that pyrophosphatase is added.Wherein, disulfurase plus
Entering amount is 1.1-1.6g/10ML.
Wherein, preparing the PCR reaction buffer that the freezing dry powder PCR amplification system reagent uses includes: deoxyribose
Ribonucleoside triphosphote, magnesium chloride.
It wherein, also include transient metal sulfide in the freezing dry powder PCR amplification system reagent.
Invention enhances nucleic acid cleavage effectiveness and degree of purification, precipitate without nucleic acid, and lysate can be detected directly,
Realize that amplified reaction and PCR detect with the freeze-dried powder reagent of instant, without preparing before surveying, detection is thoroughly solved dependent on pure
The nucleic acid DNA of change is the bottleneck that template carries out PCR amplification problem, and applicable sample includes blood, serum, blood plasma, saliva, urine
Liquid, tissue fluid, sperm, juice, pus and the breathing polymorphic types sample such as liquid and mucus.
Figure of description
Fig. 1 is the PCR pipe structural schematic diagram of the embodiment of the present invention.
Specific embodiment
Below by embodiment, technical solution of the present invention is described in further detail.
The present invention provides a kind of nucleic acid extraction and PCR detection kit, and the nucleic acid extraction and PCR detection kit are by cracking
Liquid kit and PCR pipe composition.Hold nucleic acid cleavage liquid in cracking liquid kit, sample to be tested be added nucleic acid cleavage liquid it
Afterwards, release viral nucleic acid by the effect of the nucleic acid cleavage liquid, and realize purifying, sample be added nucleic acid cleavage liquid it
Gains can be added directly into PCR pipe and realize PCR detection afterwards.It is placed in the PCR pipe and is directly used in quantitative fluorescent PCR measurement
Freezing dry powder PCR amplification system reagent, the freezing dry powder PCR amplification system reagent is poly- with PCR reaction buffer, DNA
Synthase, atopic primer, probe and freeze drying protectant are the gains that raw material is prepared by freeze drying process.
(1) nucleic acid cleavage liquid
Specifically, the nucleic acid cleavage liquid includes CuHCl, ethylenediamine tetra-acetic acid, trishydroxymethylaminomethane, NaCl, poly- second
Glycol octyl phenyl ether, surfactant, phenol, isoamyl alcohol.Wherein, the concentration of CuHCl is 5.6-5.85g/10ML, preferably
5.75g/10ML.Ethylenediamine tetra-acetic acid 0.03-0.04g/10ML, preferably 0.038g/10ML.Trishydroxymethylaminomethane 0.06-
0.07g/10ML, preferably 0.062g/10ML.NaCl 0.01-0.015g/10ML, preferably 0.011g/10ML.Polyethylene glycol is pungent
Base phenyl ether 2%-10%(volume ratio), preferably 2.7%.Surfactant can be using any one or more following mixing:
Lauryl sodium sulfate, hexadecyl first triamine, polyvinylpyrrolidone, lauroyl propylhomoserin sodium；Surfactant
0.075-0.1%(volume ratio), preferably 0.85%.1%-2.5%(volume ratio is added in nucleic acid cleavage liquid) phenol, isoamyl alcohol it is mixed
Liquid is closed, wherein phenol, isoamyl alcohol volume ratio 20:1-25:1.Lysate pH value is 4-4.5.It is produced obtained by nucleic acid cleavage to realize
The subsequent Direct PCR detection of object, lysate of the invention can produce the nucleic acid product of high-purity and high integrality, will be in cell
The ingredients such as protein, polysaccharide remove completely, and reagent noresidue, unstressed configuration depression effect, retain nucleic acid molecules base itself
The integrality of sequence.The present invention realizes in cracking process that CuHCl and NaCl mixed solution provide the ring for promoting nucleic acid cleavage release
Border, the effect for protecting nucleic acid not degraded by nuclease are prominent；Surfactant and protein, polysaccharide form slightly solubility sediment,
Realize the separation of impurity and nucleic acid；Phenol, isoamyl alcohol further promote the separation of protein and nucleic acid compositions, and wherein phenol makes egg
The denaturation of white matter ingredient, the protein of denaturation is retained in organic phase, and nucleic acid is left water phase, and isoamyl alcohol promotes organic phase and water
Mutually separate；Lysate is without nucleic acid precipitate components such as ethyl alcohol, isopropanols, therefore nucleic acid compositions are maintained in lysate, Ke Yizhi
Tap into row PCR detection.Wherein, CuHCl, NaCl, surfactant, phenol, the concentration of isoamyl alcohol and lysate pH value all bases
It is optimized in experiment, to realize the maximization of nucleic acid extraction yield.
The lysate embodiment 1-3 and its experiment effect that lower mask body introduction is realized according to the present invention.
Embodiment 1:
Scheme according to the invention prepares nucleic acid cleavage liquid 40ML, including CuHCl 5.6g/10ML, ethylenediamine tetra-acetic acid
0.03g/10ML, trishydroxymethylaminomethane 0.06g/10ML, NaCl 0.01g/10ML, Triton X-100 2%(body
Product ratio), surfactant can use lauryl sodium sulfate, surfactant 0.075%(volume ratio), 1%(volume ratio
Example) phenol, isoamyl alcohol mixed liquor, wherein phenol, isoamyl alcohol volume ratio 20:1, adjusting lysate pH value is 4.
Embodiment 2
Scheme according to the invention prepares nucleic acid cleavage liquid 40ML, including CuHCl 5.85g/10ML, ethylenediamine tetra-acetic acid
0.04g/10ML, trishydroxymethylaminomethane 0.07g/10ML, NaCl 0.015g/10ML, Triton X-100 10%
(volume ratio), surfactant can be mixed using polyvinylpyrrolidone, lauroyl propylhomoserin sodium, surfactant 0.1%
(volume ratio), 2.5%(volume ratio) phenol, isoamyl alcohol mixed liquor, wherein phenol, isoamyl alcohol volume ratio 25:1 adjust cracking
Liquid pH value is 4.5.
Embodiment 3
Scheme according to the invention prepares nucleic acid cleavage liquid 40ML, including CuHCl 5.75g/10ML, ethylenediamine tetra-acetic acid
0.038g/10ML, trishydroxymethylaminomethane 0.062g/10ML, NaCl 0.011g/10ML, Triton X-100
For 2.7%(volume ratio), surfactant uses hexadecyl first triamine, surfactant 0.85%(volume ratio), nucleic acid
2.5%(volume ratio is added in lysate) phenol, isoamyl alcohol mixed liquor, wherein phenol, isoamyl alcohol volume ratio 22:1 are adjusted
Lysate pH value is 4.
Take each 0.5ML of the nucleic acid cleavage liquid of above three embodiments, and take viral plasma sample 0.1ML, by sample with split
Solution liquid is mixed to centrifuge tube, and centrifuge tube is reversed 3-5 times, and nucleic acid is made to realize sufficiently cracking release.It is micro- using NanoDrop 1000
The nucleic acid extractive yield measured after nucleic acid analyzer measurement is sufficiently reacted in lysate is as follows: embodiment 1 is 12.3 μ g, embodiment
2 be 11.7 μ g, and embodiment 3 is 14.8 μ g, it is seen that nucleic acid cleavage output effect is good, and lysate nucleic acid concentration can be used for PCR
Directly measure.
(2) freezing dry powder PCR amplification system
The freezing dry powder PCR amplification system reagent for being directly used in quantitative fluorescent PCR measurement is placed in the PCR pipe, it will be to test sample
This addition lysate and sufficiently crack after resulting liquid product and go RNA water be directly added into the PCR pipe realize amplification and PCR
Fluorometric investigation.
The pipe shaft of the PCR pipe is described as shown in Figure 1, include reacting part 101, buffer part 102 and seal pipe lid 103
Buffer part 102 is located at the top of the pipe shaft, and the reacting part 101 is located at the lower part of the pipe shaft, buffer part and reacting part two
Person is connected to by conduit 104.During the test, need for lysate to be added the PCR pipe, lysate is first stored in the buffering
Then portion progresses into reacting part through the conduit, reacted with freezing dry powder PCR amplification system, and reaction effect can be improved
Rate.
The freezing dry powder PCR amplification system reagent is prepared in the following way: PCR reaction buffer, DNA are polymerize
After enzyme, atopic primer, probe are mixed with frozen-dried protective agent solution according to predetermined ratio, three phases are divided to carry out freezing dry
It is dry.Wherein, the first stage is precooling, and slow cooling is to -35 to -40 degrees Celsius, and cooling duration is greater than 1 hour, and in the temperature
It is kept for 2.5 hours under the conditions of degree；Second stage is the freeze-drying stage, and temperature is kept for -15 to -20 degrees Celsius, is kept for 5 hours,
Wherein air pressure is maintained at 0.1mbar；Phase III is to reinforce drying, 15-25 degrees Celsius of temperature holding, drying time 2 hours, gas
Pressure keeps 0.1mbar.Wherein, the effect of freeze drying protectant is the activity of enzyme needed for keeping PCR to react in freeze-drying process, preparation
The freeze drying protectant that the freezing dry powder PCR amplification system reagent uses includes: mannitol, polyethylene glycol, maltose, Vitamin C
Acid and polyvinylpyrrolidone；It is molten as the freeze drying protectant that deionized water pyrophosphatase is added prepares mentioned component
Liquid.Wherein, mannitol additional amount 3-5g/10ML；Polyethylene glycol additional amount 2.5-3g/10ML；Maltose additional amount 15-18g/
10ML, preferably 16.4g/10ML；Ascorbic acid additional amount 0.4-0.6g/10ML；Polyvinylpyrrolidone 0.8-1.5g/ML.It is burnt
The additional amount of sulfatase is 1.1-1.6g/10ML.Mannitol and polyethylene glycol mixing are used as excipient to have good effect, protect
The viscosity for holding freezing dry powder, avoids surface porosity.Maltose, ascorbic acid and polyvinylpyrrolidone are protected as mixed type
Agent can substitute the water and protein composition hydrogen bond of loss in freeze-drying process, play a protective role, keep protein active.
Wherein, preferably, it prepares in the reagent of the freezing dry powder PCR amplification system also comprising transient metal sulfide
MoS2, additional amount are 55 μ g/10ML, can promote the sensitivity and specificity of detection.Also, prepare the freezing dry powder PCR
The PCR reaction buffer that amplification system reagent uses includes: deoxyribonucleoside triphosphate, magnesium chloride, is conducive to amplification effect
The realization of fruit.
The freezing dry powder PCR amplification system reagent 1-3 and its experiment effect that lower mask body introduction is realized according to the present invention.
Embodiment 1
Frozen-dried protective agent solution 10ML is prepared, specific ingredient is as follows: mannitol additional amount 3g/10ML, polyethylene glycol additional amount
2.5g/10ML；Maltose additional amount 15g/10ML, ascorbic acid additional amount 0.4g/10ML, polyvinylpyrrolidone 0.8g/ML,
Disulfurase 1.1g/10ML.By PCR reaction buffer, archaeal dna polymerase, atopic primer, probe and above-mentioned frozen-dried protective
After agent solution is mixed according to predetermined ratio, three phases is divided to be freeze-dried.Wherein, the first stage is precooling, is slowly dropped
To -35 to -40 degrees Celsius, cooling duration is greater than 1 hour temperature, and is kept for 2.5 hours under the conditions of the temperature；Based on second stage
It is freeze-dried the stage, temperature is kept for -15 to -20 degrees Celsius, is kept for 5 hours, wherein air pressure is maintained at 0.1mbar；Third rank
Section is reinforces drying, and temperature is kept for 15-25 degrees Celsius, and drying time 2 hours, air pressure kept 0.1mbar.
Embodiment 2
Frozen-dried protective agent solution 10ML is prepared, specific ingredient is as follows: mannitol additional amount 5g/10ML, polyethylene glycol additional amount 3g/
10ML；Maltose additional amount 18g/10ML, ascorbic acid additional amount 0.6g/10ML, polyvinylpyrrolidone 1.5g/ML, burnt sulphur
Sour enzyme 1.6g/10ML.PCR reaction buffer, the archaeal dna polymerase, reaction spy that deoxyribonucleoside triphosphate, magnesium chloride are mixed
After specific primer, probe are mixed with above-mentioned frozen-dried protective agent solution according to predetermined ratio, three phases is divided to be freeze-dried.Its
In, the first stage is precooling, and slow cooling is to -35 to -40 degrees Celsius, and cooling duration is greater than 1 hour, and in the temperature condition
It is lower to be kept for 2.5 hours；Second stage is the freeze-drying stage, and temperature is kept for -15 to -20 degrees Celsius, is kept for 5 hours, wherein gas
Pressure is maintained at 0.1mbar；Phase III is to reinforce drying, and temperature is kept for 15-25 degrees Celsius, and drying time 2 hours, air pressure was kept
0.1mbar。
Embodiment 3
Frozen-dried protective agent solution 10ML is prepared, specific ingredient is as follows: mannitol additional amount 4.5g/10ML, polyethylene glycol additional amount
2.8g/10ML；Maltose additional amount 16.5g/10ML, ascorbic acid additional amount 0.55g/10ML, polyvinylpyrrolidone 1.1g/
ML, disulfurase 1.35g/10ML.By PCR reaction buffer, archaeal dna polymerase, atopic primer, probe and above-mentioned freeze-drying
After protection agent solution is mixed according to predetermined ratio, three phases is divided to be freeze-dried.Wherein, the first stage is precooling, is delayed
Slowly -35 to -40 degrees Celsius are cooled to, cooling duration is greater than 1 hour, and is kept for 2.5 hours under the conditions of the temperature；Second stage
To be freeze-dried the stage, temperature is kept for -15 to -20 degrees Celsius, is kept for 5 hours, wherein air pressure is maintained at 0.1mbar；Third rank
Section is reinforces drying, and temperature is kept for 15-25 degrees Celsius, and drying time 2 hours, air pressure kept 0.1mbar.
Freezing dry powder PCR amplification system reagent prepared by three above embodiment is respectively divided into 5 parts, in 37 degrees Celsius of rings
It is placed under border 0,3,7,10,20 day, is then loaded test agent made from lysate embodiment 1-3, concussion mixes, upper machine after centrifugation
PCR test is carried out, embodiment 1- embodiment 3 is 10 in 0,3,7,10 day measurement sensitivity4, sensitivity in 20 days is 105.It can
Measurement sensitivity can be kept the long period by seeing.
As it can be seen that being precipitated without nucleic acid, lysate can be direct invention enhances nucleic acid cleavage effectiveness and degree of purification
Detection realizes that amplified reaction and PCR detect with the freeze-dried powder reagent of instant, without being prepared before surveying, detection thoroughly solve according to
The nucleic acid DNA of Lai Yu purifying is the bottleneck that template carries out PCR amplification problem, and applicable sample includes blood, serum, blood plasma, saliva
Liquid, urine, tissue fluid, sperm, juice, pus and the breathing polymorphic types sample such as liquid and mucus.
Above embodiments are merely to illustrate the present invention, and not limitation of the present invention, the common skill in relation to technical field
Art personnel can also make a variety of changes and modification without departing from the spirit and scope of the present invention, therefore all etc.
Same technical solution also belongs to scope of the invention, and scope of patent protection of the invention should be defined by the claims.
Claims (10)
1. a kind of nucleic acid extraction and PCR detection kit characterized by comprising cracking liquid kit and PCR pipe, cracking
Nucleic acid cleavage liquid is held in liquid kit, releases viral nucleic acid by the effect of the nucleic acid cleavage liquid；The PCR pipe
Interior placement is directly used in the freezing dry powder PCR amplification system reagent of quantitative fluorescent PCR measurement, the freezing dry powder PCR amplification body
It is reagent is passed through using PCR reaction buffer, archaeal dna polymerase, atopic primer, probe and freeze drying protectant as raw material
The gains of freeze drying process preparation；
The pipe shaft of the PCR pipe includes reacting part, buffer part and seal pipe lid, and the buffer locations are upper in the pipe shaft
Portion, in the lower part of the pipe shaft, both buffer part and reacting part are connected to the reactive site by conduit.
2. nucleic acid extraction according to claim 1 and PCR detection kit, which is characterized in that wherein, the nucleic acid cleavage
Liquid includes CuHCl, NaOH, ethylenediamine tetra-acetic acid, trishydroxymethylaminomethane, NaCl, Triton X-100, surface work
Property agent, phenol, isoamyl alcohol.
3. nucleic acid extraction according to claim 2 and PCR detection kit, which is characterized in that in the nucleic acid cleavage liquid,
The concentration of CuHCl is 5.6-5.85g/10ML；Ethylenediamine tetra-acetic acid 0.03-0.04g/10ML；Trishydroxymethylaminomethane 0.06-
0.07g/10ML；NaCl 0.01-0.015g/10ML；Triton X-100 2%-10%(volume ratio)；Surface-active
Agent 0.075-0.1%(volume ratio)；In nucleic acid cleavage liquid be added 1%-2.5%(volume ratio) phenol, isoamyl alcohol mixed liquor,
Middle phenol, isoamyl alcohol volume ratio 20:1-25:1.
4. nucleic acid extraction according to claim 3 and PCR detection kit, which is characterized in that the surfactant is adopted
With it is following any one: lauryl sodium sulfate, hexadecyl first triamine, polyvinylpyrrolidone, lauroyl propylhomoserin
Sodium.
5. nucleic acid extraction according to claim 1 and PCR detection kit, which is characterized in that the freezing dry powder PCR expands
Increase system reagent to prepare in the following way: by PCR reaction buffer, archaeal dna polymerase, atopic primer, probe and jelly
After dry protection agent solution is mixed according to predetermined ratio, freeze-drying；The freeze-drying be divided into precooling, main freeze-drying,
Reinforce dry three phases, the first stage is precooling, and to -35 to -40 degrees Celsius, cooling duration is greater than 1 hour slow cooling,
And it is kept for 2.5 hours under the conditions of the temperature；Second stage is the main freeze-drying stage, and temperature holding -15 to -20 is Celsius
Degree is kept for 5 hours, and wherein air pressure is maintained at 0.1mbar；Phase III is to reinforce drying, and temperature is kept for 15-25 degrees Celsius, is done
Dry time 2 h, air pressure keep 0.1mbar.
6. nucleic acid extraction according to claim 5 and PCR detection kit, which is characterized in that prepare the freezing dry powder
The freeze drying protectant that PCR amplification system reagent uses includes: mannitol, polyethylene glycol, maltose, ascorbic acid and polyethylene
Pyrrolidones.
7. nucleic acid extraction according to claim 6 and PCR detection kit, which is characterized in that mannitol additional amount 3-5g/
10ML；Polyethylene glycol additional amount 2.5-3g/10ML；Maltose additional amount 15-18g/10ML；Ascorbic acid additional amount 0.4-
0.6g/10ML；Polyvinylpyrrolidone 0.8-1.5g/ML.
8. nucleic acid extraction according to claim 7 and PCR detection kit, which is characterized in that pyrophosphatase is added
Deionized water prepares the frozen-dried protective agent solution.
9. nucleic acid extraction according to claim 8 and PCR detection kit, which is characterized in that prepare the freezing dry powder
The PCR reaction buffer that PCR amplification system reagent uses includes: deoxyribonucleoside triphosphate, magnesium chloride.
10. nucleic acid extraction according to claim 9 and PCR detection kit, which is characterized in that the freezing dry powder PCR
It also include transient metal sulfide in amplification system reagent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111118222A (en) * | 2020-02-26 | 2020-05-08 | 珠海丽珠试剂股份有限公司 | HBV detection kit and using method and application thereof |
| CN116287106A (en) * | 2022-12-30 | 2023-06-23 | 上海碧云天生物技术有限公司 | Method and reagent for enhancing firefly luciferase ATP bioluminescence detection performance |
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