CN105420416A - Direct real-time quantitative fluorescent PCR method of layered packaging tape indicator - Google Patents
Direct real-time quantitative fluorescent PCR method of layered packaging tape indicator Download PDFInfo
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Abstract
The invention provides a direct real-time quantitative fluorescent PCR method of a layered packaging tape indicator. Hot-start DNA polymerase and PCR amplification reaction liquid are both packaged in a PCR amplification tube in layers. Nucleic acid lysis liquid and a sample to be detected are added when the method is used so that real-time quantitative fluorescent PCR can be carried out. By means of the method, the virus quantity lower to 20IU/ml can be detected, and the preferential detection range is 30-2*10<9>IU/ml. Detection results are not limited by the professional level of operators and experiment conditions in a laboratory, and the method has the advantages of being easy, convenient and quick to operate.
Description
Technical field
The present invention relates to biology field, particularly a kind of direct real-time quantitative fluorescence PCR method of layering strap indicator.
Background technology
Polymerase chain reaction (PolymeraseChainReaction, PCR) is a kind of technology of the external rapid amplifying specific dna sequence designed according to DNA replication dna character in organism.PCR reaction system is primarily of nucleic acid primer, 4 kinds of dNTP, archaeal dna polymerase, template DNA and PCR reaction buffer system compositions.From U.S. Cetus company human inheritance room KaryMullis and worked together since 1985 invention polymerase chain reaction (PCR), round pcr and deriving technology thereof just by fast Development out, and obtain and use widely in multiple nucleic acids detects.Especially the detection of virus or other pathogenic agent, when knowing a certain specific gene fragment of cause of disease to be checked, the target dna of trace in specific primer pair sample can be utilized to carry out pcr amplification, it is made to reach detection limit, detected by suitable detection means, the existence of pathogenic agent can be determined whether.
Real-Time Fluorescent Quantitative PCR Technique, refers to and add fluorophor in PCR reaction system, utilize the whole PCR process of the accumulation Real-Time Monitoring of fluorescent signal in DNA cloning process, finally by typical curve, unknown template is carried out to the method for quantitative analysis.Compared with common PCR, Real-Time Fluorescent Quantitative PCR Technique have high specificity, highly sensitive, reproducible, quantitatively accurately, the advantage such as fast, the totally-enclosed reaction of speed, achieve the quantitative analysis of PCR.
At present, no matter clinical labororatory is detected or epidemic control, and the nucleic acid fluorescent PCR detection technique of infectious disease pathogens, all needs through nucleic acid extraction or purge process, the laboratory environment that this process need is special and relevant device, and experiment operator need through professional training; In addition, such operation adds laboratory operating procedures, and extends detection time, adds the probability of pollution.Meanwhile, archaeal dna polymerase contacts with the mixing of PCR reaction reagent, also can cause the reduction of its activity, thus reduces the susceptibility of real-time fluorescence quantitative PCR.
Therefore, need to find a kind of good, easy and simple to handle, the quantitative real-time quantitative fluorescence PCR method accurately of reagent stability.
Summary of the invention
The technical problem to be solved in the present invention extracts complicated operation for real-time quantitative fluorescence PCR process amplifying nucleic acid, easy generation is polluted, and the shortcoming that PCR sensitivity is low, provide a kind of good, easy and simple to handle, the quantitative direct real-time quantitative fluorescence PCR method accurately of reagent stability.
In order to solve the problems of the technologies described above, technical scheme provided by the invention is:
A direct real-time quantitative fluorescence PCR method for layering strap indicator, is packaged in pcr amplification pipe by warm start archaeal dna polymerase and each self demixing of pcr amplification reaction liquid, adds nucleic acid cleavage liquid and testing sample, carry out real-time quantitative fluorescence PCR during use.
The concrete implementation step of technical solution of the present invention is:
1) with fusing point be the paraffin oil mixture bag of 50 DEG C ~ 55 DEG C by warm start Taq DNA polymerase, and be placed at the bottom of pcr amplification pipe pipe;
2) mix primer, probe, Tris alkali, magnesium chloride, Repone K, malachite green and dNTP, obtained fluorescent PCR amplifing reagent is in paraffin oil mixture upper strata;
3) close described fluorescent PCR amplifing reagent in step 1 with the paraffin oil that fusing point is 40 DEG C ~ 45 DEG C) pcr amplification pipe in;
4), after paraffin oil condensation, nucleic acid cleaving reagent and testing sample is added;
5) sample is detected.
In the inventive solutions, warm start archaeal dna polymerase and pcr amplification reaction liquid are used paraffin oil mixture bag quilt by method that contriver adopts layering to pack respectively, the two is made to be separated from each other and not mix, ensure the activity of polysaccharase with this, the activity preventing polysaccharase from causing because directly mixing with pcr amplification reaction liquid reduces.
As preferably, described warm start archaeal dna polymerase is Taq DNA polymerase.
As preferably, described pcr amplification reaction liquid comprises primer, probe, Tris alkali, magnesium chloride, Repone K and dNTP.
As preferably, in an embodiment of the invention, contriver utilizes paraffin oil mixture to carry out layering packaging to described warm start archaeal dna polymerase and pcr amplification reaction liquid.As preferably, described paraffin oil mixture comprises paraffin oil and resin.More preferably, described paraffin oil is high-quality paraffin oil, and described resin is st-yrax.
As preferably, in an embodiment of the invention, the fusing point of described paraffin oil mixture is not less than 40 DEG C.Meanwhile, the blending ratio of paraffin oil and resin is regulated paraffin oil mixture to be mixed with the paraffin oil mixture that fusing point is 50 DEG C ~ 55 DEG C and 40 DEG C ~ 45 DEG C.The paraffin oil mixture that described warm start archaeal dna polymerase is 50 DEG C ~ 55 DEG C by fusing point is packaged in bottom pcr amplification pipe, and described pcr amplification reaction liquid is packaged in the top of warm start archaeal dna polymerase by the paraffin oil mixture that fusing point is 40 DEG C ~ 45 DEG C.Warm start archaeal dna polymerase and pcr amplification reaction liquid are used respectively the paraffin oil mixture bag quilt of different melting points, PCR reaction reagent can be made first to discharge when heating and fully mix with cracked sample, and now can not affect the activity of polysaccharase, the activity preventing polysaccharase from causing because directly mixing with pcr amplification reaction liquid reduces.To be heated to 72 DEG C time, polysaccharase fully discharges, and fully mixes with liquid, to ensure carrying out smoothly of subsequent experimental.
For the ease of observing the effect of the layering packaging of paraffin oil, as preferably, in an embodiment of the invention, containing indicator in described pcr amplification reaction liquid.More preferably, described indicator be selected from tetrabromophenol sulfonphthalein, malachite green, methyl red, tropeolin-D, Luo Danming, tetrabromo-mcresolsulfonphthalein, magenta, xylenol orange, pentanoic or Congo red in one.
Simultaneously as preferably, in an embodiment of the invention, also containing indicator in described nucleic acid cleavage liquid, and indicator in pcr amplification reaction liquid is different from the indicator in nucleic acid cleavage liquid.The layering packaging effect of paraffin oil mixture can not only be ensured like this, simultaneously also can ensure the accuracy that sample adds, prevent from adding, mistake adds, leakage adds.
More preferably, in yet another embodiment of the present invention, the add-on of described indicator is lower than 0.01wt%.
In the present invention, in the consumption of described warm start archaeal dna polymerase and pcr amplification reaction liquid, the ratio of each component can be arranged as required.As preferably, in an embodiment of the invention, described warm start archaeal dna polymerase concentration is 2.5 ~ 3.5U/ μ L; The probe of upstream and downstream primer that concentration is 30 ~ 50nmol/ μ L and 10 ~ 30nmol/ μ L, the Tris alkali of 10 ~ 30nmol/ μ L, 10 ~ 30mmol/ μ L magnesium chloride, 100 ~ 200mmol/ μ L Repone K, the dNTP of 150 ~ 250umol/ μ L and the malachite green indicator of no more than 0.001wt% is comprised in described pcr amplification reaction liquid.
In the present invention, the Packaging Method of described pcr amplification reaction liquid is top pcr amplification reaction liquid described in 20 ~ 30 μ L being joined packaged warm start archaeal dna polymerase, add the paraffin oil mixture that 15 ~ 25 μ L fusing points are 40 ~ 45 DEG C, carry out closed pcr amplification reaction liquid.
After described pcr amplification reaction liquid has been packed, add nucleic acid cleavage liquid above it.The pcr amplification pipe containing warm start archaeal dna polymerase, pcr amplification reaction liquid and nucleic acid cleavage liquid obtained can use immediately and also can encapsulate rear stored refrigerated.
As preferably, in an embodiment of the invention, described nucleic acid cleavage liquid is made up of 0.2 ~ 0.4N sodium hydroxide, 0.3 ~ 0.6M Repone K, 0.01 ~ 0.05%N-sodium lauroyl sareosine, 5mMEDTA, 0.3 ~ 0.6MTris-HCL and 1 ~ 2% triton x-100.
More preferably, in yet another embodiment of the present invention, described nucleic acid cleavage liquid is made up of 0.3N sodium hydroxide, 0.45M Repone K, 0.03%N-sodium lauroyl sareosine, 0.45MTris-HCL, 5mMEDTA and 1% triton x-100.
The liquid of nucleic acid cleavage described in the present invention can be arranged arbitrarily as required.As preferably, in an embodiment of the invention, the add-on of described nucleic acid cleavage liquid is 15 ~ 25 μ L, is more preferably 20 μ L.
As preferably, in an embodiment of the invention, to described pcr amplification pipe 37 DEG C heating 5min after adding nucleic acid cleavage liquid and testing sample, then under 72 DEG C of conditions, heat 30s, after 500rpm is centrifugal, carry out real-time quantitative fluorescence PCR.
More specifically, utilize the method for direct real-time fluorescence quantitative PCR of the present invention to carry out PCR detection by quantitative, comprise the steps:
1) 15 ~ 25 μ L samples to be tested are joined in the pcr amplification pipe containing warm start archaeal dna polymerase, pcr amplification reaction liquid and nucleic acid cleavage liquid, shake up gently;
2) above-mentioned pcr amplification pipe is placed on the xeothermic device with refrigerating function and runs 37 DEG C, 5min; 72 DEG C, 30s, then puts upside down mixing by layering reagent in pipe, 500rpm horizontal centrifugal 30 second.
3) gained pcr amplification pipe is positioned in PCR instrument, and Real-Time Monitoring fluorescent signal.
In order to better lysate sample, can before carrying out pcr amplification low-speed centrifugal pcr amplification pipe, to reduce tube wall and pipe covers the nucleic acid cleavage liquid sticked, thus abundant lysate sample, the rotating speed of described low-speed centrifugal can be 100 ~ 500rpm/min.
In the present invention, contriver utilize fusing point be the paraffin oil mixture of 40 DEG C ~ 45 DEG C and 50 DEG C ~ 55 DEG C by respectively warm start archaeal dna polymerase, pcr amplification reaction liquid and nucleic acid cleavage liquid being isolated, ensure that the activity that warm start archaeal dna polymerase can not cause because mixing with PCR reaction solution reduces with this.Meanwhile, nucleic acid cleavage liquid level, in the upper strata of mixture, after adding measuring samples, the xeothermic device with refrigerating function runs 37 DEG C, 5min.This step not only can ensure that nucleic acid cleavage liquid fully can mix with measuring samples under 37 DEG C of environment, and can accelerate lysis efficiency, realizes thorough cracking and the release of sample nucleic.Preservation state and the activity of PCR reaction reagent and warm start archaeal dna polymerase can not be affected simultaneously under 37 DEG C of environment, realize the differential responses in a pipe.The xeothermic device with refrigerating function runs 72 DEG C, in 30s step, warm start archaeal dna polymerase, pcr amplification reaction liquid can be made, the nucleic acid that discharges fully mixes, and is giving security smoothly of subsequent experimental.Carry out the thawing program of paraffin oil at xeothermic device after, layering reagent in pipe is put upside down mixing, 500rpm centrifugal 30 seconds, directly carry out fluorescent PCR amplification.
As preferably, described testing sample includes but not limited to cultured cells, bacterium, serum, blood plasma, saliva, urine or ascites pleural fluid etc., is preferably serum or blood plasma.Described testing sample volume is 15 ~ 25 μ L, is preferably 20 μ L, after described sample adds nucleic acid cleavage liquid, rocks gently and makes it fully mix with detection sample.
The test item that the present invention is suitable for comprises the DNA virus such as hepatitis B virus, CMV, EBV and human serum, blood plasma genomic nucleic acids detects.
Real-time quantitative fluorescence PCR reaction conditions in the present invention can be arranged as required.As preferably, in an embodiment of the invention, described pcr amplification program is: 37 DEG C, 2min; 95 DEG C, 10min; Then with 95 DEG C, 10s and 61 DEG C, 45s carry out 45 ~ 50 circulations.Fluorescent signal is detected at 61 DEG C.
Beneficial effect of the present invention is:
The method of direct real-time fluorescence quantitative PCR of the present invention, adopts the paraffin oil of different melting points gradient to split warm start archaeal dna polymerase, pcr amplification reaction liquid and nucleic acid cleavage liquid respectively.This method not only can make environment residing for warm start DNA enzymatic and pcr amplification reaction liquid separate, ensure the activity of warm start archaeal dna polymerase, simultaneously for the abundant reaction of nucleic acid cleavage liquid and measuring samples provide one independently environment to ensure that the nucleic acid in measuring samples fully discharges.Meanwhile, adopt the indicator of different colours to distinguish pcr amplification reaction liquid and nucleic acid cleavage liquid, ensure that the validity of paraffin oil segmentation and follow-up serum/plasma equal samples add the reliability fully mixed.After the previously prepared packaging of reagent, the trace samples such as the serum of collection, blood plasma, saliva, urine or ascites pleural fluid only need directly be added in PCR reaction tubes by user, can directly detect on fluorescent PCR instrument through an of short duration warm handling procedure.Temperature handling procedure not only can realize the abundant reaction of lysate and measuring samples, and acceleration cracking also makes nucleic acid thoroughly discharge, also can achieve differential responses state in a pipe namely in 37 DEG C of environment warm start archaeal dna polymerase and pcr amplification reaction liquid unaffected.In addition, sample application amount can reach more than 20 microlitres in the present invention.Some all ensure that high detection susceptibility of the present invention above, and utilize the present invention can detect the virus quantity being low to moderate 20IU/ml, preferred sensing range is 30 ~ 2 × 10
9iU/ml.Detected result, by the restriction of operator's professional standards and laboratory experiment condition, has easy and simple to handle, advantage fast.
Accompanying drawing explanation
Fig. 1 is the real-time quantitative fluorescence PCR detected result figure to HBVDNA in embodiment 1;
Fig. 2 is the real-time quantitative fluorescence PCR detected result figure to HBVDNA in embodiment 2;
Fig. 3 is the repeated detected result figure of the inventive method.
Embodiment
The invention discloses a kind of direct real-time quantitative fluorescence PCR method of layering strap indicator, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.It needs to be noted, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention, and related personnel obviously can change content described herein on the basis not departing from content of the present invention, spirit and scope or suitably change and combination, realizes and applies the technology of the present invention.
In the present invention, unless otherwise stated, Science and Technology noun used herein has the implication that those skilled in the art understand usually.
In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1: the real-time quantitative fluorescence PCR of HBVDNA is detected
1. the bag quilt of warm start archaeal dna polymerase:
Getting the warm start archaeal dna polymerase of 1 μ L (2.5 ~ 3.5U/ μ L), is that the paraffin oil mixture of 50 DEG C ~ 55 DEG C is coated at the bottom of pcr amplification pipe through melting and condensation process with 10 ~ 15 μ L fusing points.
2. the preparation of fluorescent PCR amplification reaction solution:
Concentration is 40nmol/ μ L upstream and downstream primer and 30nmol/ μ L probe; The Tris alkali of 20nmol/ μ L; 20mmol/ μ L magnesium chloride; 150mmol/ μ L Repone K, the dNTP of 200umol/ μ L and the malachite green indicator of no more than 0.001wt% obtain fluorescent PCR amplification reaction solution.
3. the bag quilt of fluorescent PCR amplification reaction solution:
Be dispensed into by fluorescent PCR amplifing reagent described in 27 μ L at the bottom of the pcr amplification pipe pipe of step 1, adding 25 μ l fusing points is that the solid paraffin oil of 40 ~ 45 DEG C carrys out closed fluorescent PCR amplification reaction solution.
4. the adding of nucleic acid cleaving reagent:
20 μ L nucleic acid cleavage liquid (lilac) are joined the paraffin oil upper strata of wrapping by pcr amplification pipe, and capping refrigeration is for subsequent use.Described nucleic acid cleavage liquid comprises 0.3N sodium hydroxide, 0.45M Repone K, 0.03%N-sodium lauroyl sareosine, 5mMEDTA, 0.45MTris-HCL and 1% triton x-100 composition.
The detection of 5.HBVDNA
Getting clinical HBVDNA detection by quantitative value is 2.0 × 10
9the Serum of Patients with Hepatitis B of IU/ml, adopts HBVDNA to be detected as negative sample as diluent, then carries out 10 times of dilutions successively, namely obtain HBVDNA concentration from 2.0 × 10
9iU/mL ~ 2.0 × 10
1the sample of IU/mL.
1) 20 μ L samples are joined in the pcr amplification pipe of above preparation, shake pcr amplification pipe gently, the nucleic acid cleavage liquid in pipe is mixed with the serum added.Room temperature leaves standstill 10 minutes.
2) PCR pipe is positioned in PCR instrument increases;
Pcr amplification program is: 56 DEG C, 5min; 95 DEG C, 10 minutes; 95 DEG C that carry out 50 circulations, 10 seconds and 61 DEG C, 30 second pcr amplification.Fluorescent signal is detected at 61 DEG C.
As shown in Figure 1, result shows that the sensitivity of the inventive method is 20IU/mL to test-results.
Embodiment 2: the real-time quantitative fluorescence PCR of HBVDNA is detected
1. the bag quilt of warm start archaeal dna polymerase:
Getting the warm start Taq DNA polymerase of 1 μ L1.5U/ μ L, is that the paraffin oil mixture of 50 DEG C ~ 55 DEG C is coated at the bottom of pcr amplification pipe through melting and condensation process with 10 ~ 15 μ L fusing points.
2. the preparation of fluorescent PCR amplification reaction solution:
Each 1 μ L concentration is 20 μm of ol/L upstream and downstream primers and 1 μ L10 μm ol/L probe; The Tris alkali of 20nmol/ μ L; 4 μ l25mmol/L magnesium chlorides; 2 μ L500mmol/L Repone K, the dNTP of each 10mmol/L of 1 μ L and the malachite green indicator of no more than 0.001wt% obtain fluorescent PCR amplification reaction solution.
3. the bag quilt of fluorescent PCR amplification reaction solution:
Be dispensed into by fluorescent PCR amplifing reagent described in 20 μ L at the bottom of the pcr amplification pipe pipe of step 1, adding 30 μ L fusing points is that the solid paraffin oil of 40 ~ 45 DEG C carrys out closed fluorescent PCR amplification reaction solution.
4. the adding of nucleic acid cleaving reagent:
25 μ L nucleic acid cleavage liquid (lilac) are joined the paraffin oil upper strata of wrapping by pcr amplification pipe, and capping refrigeration is for subsequent use.Described nucleic acid cleavage liquid comprises sodium hydroxide and 1% TritonX-100 of 0.25N.
The detection of 5.HBVDNA
Getting clinical HBVDNA detection by quantitative value is 1.0 × 10
9the Serum of Patients with Hepatitis B of IU/mL, adopts HBVDNA to be detected as negative sample as diluent, then carries out 10 times of dilutions successively, namely obtain HBVDNA concentration from 1.0 × 10
9iU/mL ~ 1.0 × 10
2the sample of IU/mL.
1) 5 μ L samples are joined in the above-mentioned PCR pipe of embodiment, shake pcr amplification pipe gently, the nucleic acid cleavage liquid in pipe is mixed with the test serum added.Then so that 200g/min is centrifugal, liquid is thrown to bottom.
2) PCR pipe is positioned in PCR instrument increases;
Pcr amplification program is: 56 DEG C, 5min; 95 DEG C, 10 minutes; 95 DEG C that carry out 50 circulations, 10 seconds and 61 DEG C, 30 second pcr amplification.Fluorescent signal is detected at 61 DEG C.
Experimental result as shown in Figure 2.Experimental result shows, detects by the inventive method, and its whole conventional efficient is in higher level, detects 1.0 × 10
2iU/mL-1.0 × 10
9the virus quantity of IU/mL, Ct value scope, 10 ~ 34 circulation left and right, while raising experimental implementation efficiency, effectively ensure that the true and reliable of experimental result.
Embodiment 3: reperformance test is detected to the real-time quantitative fluorescence PCR of HBVDNA
Except using clinical HBVDNA detection by quantitative value to be 2.0 × 10 respectively
3iU/ml, 2.0 × 10
5iU/ml and 2.0 × 10
8beyond the Serum of Patients with Hepatitis B of IU/ml, the method identical with embodiment 1 is utilized to carry out real-time quantitative fluorescence PCR detection.Carry out simultaneous test with commercially available reagent box, described commercially available reagent is Roche COBAS Automatic nucleic acid Testing system simultaneously.As shown in Figure 3 and Table 1, result shows experimental result, the detection of the inventive method repeatability CV% value and commercialization Roche COBAS reagent basically identical.
The repeatability of table 1 the inventive method and Roche COBAS reagent contrasts
Embodiment 4: the convenience of the inventive method and pollution-proof performance
Experimental technique is identical with embodiment 1, and difference is 1 × 10 for only preparing HBVDNA concentration
8patients serum's sample of IU/mL.Adopt calf serum as negative sample, two samples are spaced respectively does 48 multiple hole, totally 96 holes.Then calculate and pollute probability, time used.Experimental result is as shown in table 2, and result shows, operation steps of the present invention is simple, whole experiment handing time is short, and without the need to training professionals, and sample not easily pollutes, experimentation is not subject to the impact of laboratory condition, and HBV detection method conventional on market has qualitative leap.
The convenience of table 2 the inventive method
| Project category | Performance characteristics is analyzed |
| Need reagent | 8-15 minute |
| Operation steps | 2 steps |
| Special Equipment | Only need xeothermic device |
| Professional | Do not need special special training |
| Be applicable to environment | The unit being applicable to various laboratory condition uses |
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the direct real-time quantitative fluorescence PCR method of a layering strap indicator, it is characterized in that, warm start archaeal dna polymerase and each self demixing of pcr amplification reaction liquid are packaged in pcr amplification pipe, add nucleic acid cleavage liquid and testing sample during use, carry out real-time quantitative fluorescence PCR.
2. method according to claim 1, is characterized in that, described warm start archaeal dna polymerase is Taq DNA polymerase.
3. method according to claim 1, is characterized in that, described pcr amplification reaction liquid comprises primer, probe, Tris alkali, magnesium chloride, Repone K and dNTP.
4. method according to claim 1, is characterized in that, described layering packaging utilizes the paraffin oil mixture of different melting points to pack respectively described warm start archaeal dna polymerase and pcr amplification reaction liquid.
5. method according to claim 4, it is characterized in that, described paraffin oil mixture comprises paraffin oil and resin, the paraffin oil mixture that described warm start archaeal dna polymerase is 50 DEG C ~ 55 DEG C by fusing point is packaged in bottom pcr amplification pipe, and described pcr amplification reaction liquid is packaged in the top of warm start archaeal dna polymerase by the paraffin oil mixture that fusing point is 40 DEG C ~ 45 DEG C.
6. method according to claim 1, is characterized in that, to described pcr amplification pipe 37 DEG C heating 5min after adding nucleic acid cleavage liquid and testing sample, then under 72 DEG C of conditions, heats 30s, carries out real-time quantitative fluorescence PCR after 500rpm is centrifugal.
7. method according to claim 1, is characterized in that, containing indicator in described pcr amplification reaction liquid and described nucleic acid cleavage liquid, and indicator in pcr amplification reaction liquid is different from the indicator in nucleic acid cleavage liquid.
8. the method according to claim 1,6 or 7, it is characterized in that, described nucleic acid cleavage liquid is made up of 0.2 ~ 0.4N sodium hydroxide, 0.3 ~ 0.6M Repone K, 0.01 ~ 0.05%N-sodium lauroyl sareosine, 5mMEDTA, 0.3 ~ 0.6MTris-HCL and 1 ~ 2% triton x-100.
9. method according to claim 8, is characterized in that, described nucleic acid cleavage liquid is made up of 0.3N sodium hydroxide, 0.45M Repone K, 0.03%N-sodium lauroyl sareosine, 0.45MTris-HCL, 5mMEDTA and 1% triton x-100.
10. method according to claim 1, is characterized in that, described testing sample is cell, bacterium, serum, blood plasma, saliva, urine or ascites pleural fluid, is preferably serum or blood plasma.
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| NARENDRA P.SINGH等: "X-ray-induced DNA double-strand breaks in human sperm", 《MUTAGENESIS》 * |
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| CN113736868B (en) * | 2021-09-16 | 2023-01-24 | 广州生凌医疗科技有限公司 | RNA nucleic acid detection reagent storage method, kit and use method |
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