CN110819626A - Lysis solution for extracting HCMV nucleic acid by paramagnetic particle method and application thereof - Google Patents
Lysis solution for extracting HCMV nucleic acid by paramagnetic particle method and application thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
The invention provides a lysis solution for extracting HCMV nucleic acid by a paramagnetic particle method, which at least comprises the following components, wherein the content of each component is 1-3mol/L of guanidinium isothiocyanate, 0.05-0.1% (w/v) of sodium dodecyl sulfate, 401-5% (w/v) of NP, 10-80mmol/L of Tris-HCl, 5-15mmol/L of EDTA, 0.1-0.5% (v/v) of β -mercaptoethanol, and the solvent is water, based on the total amount of the lysis solution.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to lysis solution for extracting HCMV nucleic acid by a paramagnetic particle method and application thereof.
Background
HCMV is a ubiquitous double-stranded DNA virus that is transmitted primarily by sexual contact or direct contact with the blood, urine, or saliva of an infected person with a mean latency of approximately 28-60 days. The immunity of pregnant women in gestation period is reduced, so that the immunity of pregnant women in gestation period can be reduced, on one hand, the inflammatory reaction of organisms can be inhibited, and the tolerance of the pregnant women to fetal antigens is increased, and on the other hand, the susceptibility of the pregnant women and fetuses to certain infectious diseases can be increased. After the pregnant woman has primary HCMV infection, the intrauterine fetal infection rate reaches 30-40%.
The types of HCMV detection kits on the market are various at present, and the detection can be divided into antibody detection and nucleic acid detection according to the target substance to be detected.
HCMV infection first induces the body to produce specific IgM antibodies followed by the appearance of IgG antibodies. The HCMV primary infection of the pregnant woman can be realized by antibody detection, and the method comprises the following steps:
(1) detecting the serum antibody level of the pregnant woman, and repeating the measurement after 3-4 weeks. Diagnostic criteria included seroconversion phenomena (primary sero-antibody negative pregnant women show specific IgG antibodies), or a 4-fold increase in IgG antibody titers;
(2) IgG antibody affinity assay: the affinity index is less than 30%, which indicates that the HCMV infection of the pregnant women is primary infection within 2-4 months.
However, the detection of antibodies did not respond to the load of HCMV in the current sample and there was no direct evidence of congenital infection of the fetus. HCMV can be excreted into the amniotic fluid through the fetal urine, and detection of HCMV-DNA is currently considered to be the most direct evidence of prenatal fetal HCMV intrauterine infection.
At present, 15 kinds of HCVM nucleic acid detection for obtaining the registration certificate of the medical device are found from the inquiry site of the medical device of the official website of the national drug administration, and the covered sample types comprise serum, plasma, urine, milk and lymphocyte, wherein most of the samples are quantitative products.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a lysis solution for nucleic acid extraction by a magnetic bead method and use thereof.
In order to achieve the above objects and other related objects, a first aspect of the present invention provides a lysis solution for nucleic acid extraction by a magnetic bead method, the lysis solution at least comprising the following components, wherein the content of each component is, based on the total amount of the lysis solution:
the second aspect of the present invention provides a method for preparing a lysate for extraction of HCMV nucleic acid by a paramagnetic particle method, comprising at least the following steps:
mixing guanidinium isothiocyanate, sodium dodecyl sulfate, NP40, Tris-HCl, EDTA and water, adjusting the pH value of the lysate, and adding β -mercaptoethanol to obtain the lysate.
In a third aspect, the invention provides a use of the lysis solution for magnetic bead method HCMV nucleic acid extraction for preparing an HCMV nucleic acid extraction product in a sample or extracting HCMV nucleic acid in amniotic fluid.
The invention provides a kit for extracting HCMV nucleic acid by a paramagnetic particle method, which comprises the lysis solution, and the kit also comprises one or more of magnetic beads, a washing solution, an eluent, an RNA (ribonucleic acid) precipitation aid and proteinase K.
In a fifth aspect, the present invention provides a method for extracting HCMV nucleic acid, comprising at least the following steps:
(1) adding a sample into the lysate and magnetic bead mixed solution, uniformly mixing, adding an RNA (ribonucleic acid) precipitation aid and proteinase K, and forming a magnetic bead-nucleic acid compound under the action of an external magnetic field;
(2) adding a first washing solution into the magnetic bead-nucleic acid compound, uniformly mixing, carrying out magnetic separation under the action of an external magnetic field, and discarding waste liquid to obtain a first mixture;
(3) adding a second washing solution into the first mixture, uniformly mixing, and carrying out magnetic separation under the action of an external magnetic field to obtain a second mixture;
(4) and adding the eluent into the second precipitate, uniformly mixing, and carrying out magnetic separation under the action of an external magnetic field, wherein the supernatant is the nucleic acid solution.
As described above, the lysis solution for HCMV nucleic acid extraction by a magnetic bead method and the application thereof have the following beneficial effects:
the invention firstly solves the current situation that no HCMV quantitative detection reagent for amniotic fluid samples exists in the market, and the amniotic fluid samples have complex components and contain a plurality of unknown inhibitors. The invention successfully realizes the high-efficiency extraction of the amniotic fluid sample by using a paramagnetic particle method, and makes the quantification of HCMV in the amniotic fluid sample possible. This also makes it possible to screen for congenital cytomegalovirus infection clinically. The "detection of HCMV-DNA is currently considered to be the most direct evidence of prenatal fetal HCMV intrauterine infection" is clearly indicated in the congenital cytomegalovirus infection screening and clinical intervention guidelines.
Drawings
FIG. 1: schematic diagram of extraction reagent prepackage plate of kit.
FIG. 2: PCR amplification profiles for comparison of extraction results (saline dilution, blue solid line representing the extraction reagent of example 3 of the present invention, and red dotted line representing the comparative extraction reagent).
FIG. 3: PCR amplification plots for comparison of extraction results (negative amniotic fluid dilution, blue solid line representing the extraction reagent of example 3 of the present invention, and red dotted line representing the comparative extraction reagent).
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples of the invention can be used in the practice of the invention in accordance with the knowledge of one skilled in the art and the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
Various starting materials and reagents were purchased from commercial suppliers without further purification unless otherwise indicated. The raw materials and the reagents which are easy to be affected with damp are stored in a fully sealed bottle and are directly used without special treatment.
The unit w/v referred to in the present invention refers to the ratio of mass to volume, wherein the unit of mass is g and the unit of volume is ml.
The lysis solution for HCMV nucleic acid extraction by the paramagnetic particle method at least comprises the following components, wherein the content of each component is calculated by taking the total amount of the lysis solution as a reference:
specifically, based on the total amount of the lysis solution, in the lysis solution,
the content of guanidinium isothiocyanate may be 1-3mol/L, 1-1.5mol/L, 1.5-2mol/L, 2-2.5mol/L, 2.5-3 mol/L.
The sodium lauryl sulfate may be present in an amount of 0.05-0.1% (w/v), 0.05-0.06% (w/v), 0.06-0.07% (w/v), 0.07-0.08% (w/v), 0.08-0.09% (w/v), 0.09-0.1% (w/v).
The NP40 content may be 1-5% (w/v), 1-2% (w/v), 2-3% (w/v), 3-4% (w/v), 4-5% (w/v).
The content of Tris-HCl can be 10-80mmol/L, 10-20mmol/L, 20-30mmol/L, 30-40mmol/L, 40-50mmol/L, 50-60mmol/L, 60-70mmol/L, 70-80 mmol/L.
The content of EDTA can be 5-15mmol/L, 5-8mmol/L, 8-10mmol/L, 10-12mmol/L, 12-15 mmol/L.
β -mercaptoethanol can be present in a volume percentage of 0.1-0.5%, 0.1-0.2%, 0.2-0.3%, 0.3-0.4%, 0.4-0.5%.
Wherein NP40 is ethyl phenyl polyethylene glycol.
The solvent may be process water.
In one embodiment, the lysis solution comprises at least the following components, wherein the content of each component is based on the total amount of the lysis solution:
the lysate can be used for extracting HCMV nucleic acid from amniotic fluid samples.
The pH value of the lysate is 8.5 +/-0.1.
The preparation method of the lysis solution for HCMV nucleic acid extraction by the paramagnetic particle method at least comprises the following steps:
mixing guanidinium isothiocyanate, sodium dodecyl sulfate, NP40, Tris-HCL, EDTA and water, adjusting the pH value of the lysate, and adding β -mercaptoethanol to obtain the lysate.
Further, the pH value of the lysate is adjusted to 8.5 +/-0.1.
The invention provides application of a lysate for extracting HCMV nucleic acid by a paramagnetic particle method to preparation of an HCMV nucleic acid extraction product in a sample or extraction of HCMV nucleic acid in amniotic fluid.
The HCMV nucleic acid sample may be selected from amniotic fluid samples.
When extracting HCMV nucleic acid from a sample, the sample is required to be used in an amount of at least 200. mu.L. Further, it can be 200-.
The kit for extracting HCMV nucleic acid by a paramagnetic particle method comprises the lysis solution, and the kit comprises one or more of magnetic beads, washing solution, eluent, RNA (ribonucleic acid) precipitation-assisting agent and proteinase K.
The RNA adjuvant may be a commercially available product and proteinase K may be a commercially available product, and may be, for example: RNA precipitation aid (Glycogen,20mgml), proteinase K, Tiangen Biochemical technology (Beijing) Ltd., cat # RT 403.
In one embodiment, the magnetic beads are used at a concentration of 50-100 mg/mL.
The magnetic bead can be in the model of Orrunn SM 1-050.
In one embodiment, the washing solution comprises a first washing solution, the first washing solution comprises at least the following components, wherein the content of each component is as follows based on the total amount of the first washing solution: guanidine isothiocyanate: 0.5-2mol/L, sodium chloride: 0.1-0.5mol/L, triton X-100: 0.5-1% (w/v), the volume percentage of absolute ethyl alcohol is 30-50%, Tris-HCl: 2-8mmol/L, and the solvent is water. The first washing liquid is used for washing away impurities of proteins in the sample, and the washing effect is good.
In one embodiment, the washing solution further comprises a second washing solution, wherein the second washing solution comprises at least the following components in the following content based on the total amount of the second washing solution: anhydrous ethanol: 75 percent by volume and the solvent is water. The second washing liquid is used for washing salt impurities, and the washing effect is good.
In one embodiment, the eluent comprises at least the following components, wherein the content of each component is based on the total amount of the eluent: 10-80mmol/L, 0.5-25mmol/L EDTA, and water as solvent. The eluent is used for eluting nucleic acid. The eluent may also be a commercially available product.
The pH of the first wash solution was 8.0. + -. 0.1.
The pH of the eluent is 8.0 plus or minus 0.1.
When in use, the volume ratio of the lysis solution to the magnetic beads is (300-: 20 mu L of the solution; .
The lysis solution and the magnetic beads can be mixed and then are pre-loaded in a 96-well plate. The 96-well plate is selected from a 96-deep-well plate.
In one embodiment, as shown in FIG. 1, the lysate is pre-loaded with magnetic beads in row A of a 96-well plate at a volume of 500. mu.L. + -. 50. mu.L.
The first washing solution, the second washing solution and the eluent can be respectively pre-filled in a 96-well plate.
In one embodiment, the first wash solution is pre-loaded in a 96-well plate in row B, C and the volume is controlled to 600. mu.L. + -. 50. mu.L.
In one embodiment, the second wash solution is pre-loaded in a 96-well plate in rows D, E, at volumes controlled to 600 μ L. + -. 50 μ L and 500 μ L. + -. 50 μ L, respectively.
In one embodiment, the eluate is prepackaged in a 96-well plate in H rows and the volume is controlled at 50 μ L. + -. 5 μ L.
The kit can be used for extracting HCMV nucleic acid in an amniotic fluid sample.
The method for extracting HCMV nucleic acid at least comprises the following steps:
(1) adding a sample into the lysate and magnetic bead mixed solution, uniformly mixing, adding an RNA (ribonucleic acid) precipitation aid and proteinase K, and forming a magnetic bead-nucleic acid compound under the action of an external magnetic field;
(2) adding a first washing solution into the magnetic bead-nucleic acid compound, uniformly mixing, carrying out magnetic separation under the action of an external magnetic field, and discarding waste liquid to obtain a first mixture;
(3) adding a second washing solution into the first mixture, uniformly mixing, and carrying out magnetic separation under the action of an external magnetic field to obtain a second mixture;
(4) and adding the eluent into the second precipitate, uniformly mixing, and carrying out magnetic separation under the action of an external magnetic field, wherein the supernatant is the nucleic acid solution.
The nucleic acid can be extracted by using a full-automatic nucleic acid extractor (model: EX3600/2400) of Shanghai river biological medicine science and technology Limited, before extraction, 200uL of sample/quality control product is added into each hole of the line A, 1 uL of internal standard, 6 uLRNA settling agent and 20 uL of protease K are added, the full-automatic nucleic acid extractor is placed into a deep hole plate, the program ZJ Blood-gDNA is operated, and after the program is operated, the DNA template solution of the line H is taken out by a liquid transfer gun for later use.
In one embodiment, the sample is an amniotic fluid sample.
Example 1
The kit for extracting HCMV nucleic acid by a paramagnetic particle method is prepared by the following formula and comprises lysis solution, magnetic beads, first washing solution, second washing solution, eluent, RNA precipitation aid and proteinase K, wherein the lysis solution, the magnetic beads, the first washing solution, the second washing solution, the eluent, the RNA precipitation aid and the proteinase K are mixed to prepare the kit for extracting HCMV nucleic acid by a paramagnetic particle method
Based on the total amount of the cracking solution, the content of each component of the cracking solution is as follows:
based on the total amount of the first washing liquid, the content of each component of the first washing liquid is as follows: guanidine isothiocyanate: 2mol/L, sodium chloride: 0.5mol/L, triton X-100: 1% (w/v), absolute ethanol: 50% (v/v), Tris-HCl: 2mmol/L, and the solvent is water.
And the content of each component of the second washing liquid is as follows by taking the total amount of the second washing liquid as a reference: anhydrous ethanol: 75% (v/v) and the solvent is water.
Based on the total amount of the eluent, the contents of all the components of the eluent are as follows: 40mmol/L of EDTA, 10mmol/L of EDTA and water as solvent.
As shown in FIG. 1, the lysate and magnetic beads are pre-loaded in line A of a 96-deep well plate, and the volume is controlled to 500. mu.L. + -. 50. mu.L.
The first wash was pre-loaded in 96-well plates on lines B, C and the volume was controlled at 600. mu.L. + -. 50. mu.L.
The second wash was pre-loaded in 96-well plates at row D, E, at volumes controlled at 600. mu.L. + -. 50. mu.L and 500. mu.L. + -. 50. mu.L, respectively.
The eluent is pre-loaded in H line of 96 deep-well plate, and the volume is controlled at 50 mu L +/-5 mu L.
Example 2
1. Experimental methods
1.1 detection of objects
Taking a high-concentration HCMV positive amniotic fluid sample, and diluting three gradients by using the HCMV negative amniotic fluid sample and normal saline respectively. Three concentration samples diluted by HCMV negative amniotic fluid samples are respectively defined as AF-1, AF-2 and AF-3, and three concentration samples diluted by physiological saline are respectively defined as NC-1, NC-2 and NC-3.
1.2 sample nucleic acid extraction
The Kit for extracting HCMV nucleic acid by the paramagnetic particle method and a comparative extraction reagent (QIAamp circle R DNA mini Kit (QIAGEN company) extraction reagent in the market) described in the embodiment 1 of the invention are used for sample extraction, and each extraction is repeated for two times. The comparative extraction reagents were used according to the instructions, and the kit for magnetic bead method HCMV nucleic acid extraction was used according to the method of example 1, with two wells per sample. Add 1. mu.l internal standard, 6. mu. LRNA deposition aid and 20. mu.L proteinase K into the line A of the prepackage plate, and then add AF-1, AF-2, AF-3, NC-1, NC-2, NC-3 and negative quality control article into each well in turn. And putting the deep-hole plate into a full-automatic nucleic acid extractor, operating a program ZJ Blood-gDNA, and taking out the DNA template solution in the row H by using a pipette gun for later use after the program is operated.
1.3 sample detection
When preparing the PCR reaction solution, 30 mul of HCMV PCR detection mixed solution is taken and mixed with 0.5 mul of PCR enzyme to obtain the PCR reaction mixed solution, the mixture is evenly mixed for a plurality of seconds by oscillation, and the mixture is centrifuged for a plurality of seconds at 3000rpm to obtain the mixed solution. And (3) placing 30 mu l of the mixed solution into a PCR tube, then adding 20 mu l of the sample, the DNA template solution of the negative quality control product and the calibrator into the PCR reaction tube, covering the PCR reaction tube, and immediately carrying out PCR amplification reaction.
The cycle parameters are set as: 2min at 37 ℃; 94 ℃ for 2 min; 45 cycles of 93 ℃ X15 sec and 60 ℃ X60 sec were alternately repeated. Single-point fluorescence detection was performed at 60 ℃ in a 50. mu.l reaction system.
The fluorescence channel was selected as in table 1 below.
TABLE 1 selection of fluorescent channels
| |
1 | 2 |
| ABI7500 | FAM | |
| SLAN | Channel | |
| 1 | |
Note: if an ABI Prism7500 real-time fluorescent PCR instrument is used, both the passive reference and the query will need to be selected as "none".
2. Results of the experiment
The amplification curve of the amniotic fluid sample diluted by the physiological saline is shown in figure 2, the amplification curve of the amniotic fluid sample diluted by the HCMV negative amniotic fluid sample is shown in figure 3, and the Ct value of the detection result is statistically shown in the following table 2:
TABLE 2 comparative test results of extracted reagents
According to fig. 2, the Ct value data of table 2 show that there is no significant difference between the two extraction reagents for the sample extraction efficiency of saline dilution.
According to fig. 3, the Ct value data of table 2 show that the efficiency of the extraction reagent of the present invention for extracting a sample diluted by an amniotic fluid sample is significantly higher than that of the comparative extraction reagent, and the Ct value is advanced by about 2 Ct.
The above results show that the extraction efficiency of the two extraction reagents is not obviously different for the sample diluted by normal saline, but the extraction effect of the extraction reagent of the invention is obviously better than that of other commercial extraction reagents for the sample diluted by amniotic fluid. The fact that PCR inhibiting components exist in the amniotic fluid sample is shown, but the extraction reagent can well remove the inhibitor in the extraction step, and the obtained DNA template sample has a better PCR amplification effect.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and alterations without departing from the spirit and scope of the present invention, and all equivalent changes, modifications and alterations to the present invention are equivalent embodiments of the present invention; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Claims (10)
1. The lysis solution for extracting HCMV nucleic acid by a paramagnetic particle method is characterized by at least comprising the following components in percentage by weight based on the total amount of the lysis solution:
2. a lysate used for extracting HCMV nucleic acid by a paramagnetic particle method according to claim 1, wherein the lysate at least comprises the following components in percentage by weight based on the total amount of the lysate:
3. a lysate used for extracting HCMV nucleic acid by a paramagnetic particle method according to claim 3, wherein the HCMV nucleic acid extracted by the lysate is derived from an amniotic fluid sample.
4. A method for preparing a lysate for HCMV nucleic acid extraction by using the magnetic bead method as described in any one of claims 1 to 3, comprising at least the steps of:
mixing guanidinium isothiocyanate, sodium dodecyl sulfate, NP40, Tris-HCl, EDTA and water, adjusting the pH value of the lysate, and adding β -mercaptoethanol to obtain the lysate.
5. Use of the lysate for nucleic acid extraction by the paramagnetic particle method according to any one of claims 1 to 3, for preparing an amniotic fluid HCMV nucleic acid extraction product or for extracting an amniotic fluid HCMV nucleic acid.
6. The use according to claim 5, wherein the sample is extracted for HCMV nucleic acid in an amount of at least 200 μ L.
7. A kit for extracting HCMV nucleic acid by a paramagnetic particle method, wherein the kit comprises the lysis solution as defined in any one of claims 1 to 3, and the kit further comprises one or more of magnetic beads, a washing solution, an eluent, an RNA (ribonucleic acid) precipitation promoter and proteinase K.
8. The kit for magnetic bead method HCMV nucleic acid extraction according to claim 7, further comprising one or more of the following characteristics:
(1) the using concentration of the magnetic beads is 50-100 mg/mL;
(2) the washing liquid comprises a first washing liquid, the first washing liquid at least comprises the following components in percentage by weight based on the total amount of the first washing liquid: guanidine isothiocyanate: 0.5-2mol/L, sodium chloride: 0.1-0.5mol/L, triton X-100: 0.5-1% (w/v), absolute ethanol: 30-50% by volume, Tris-HCl: 2-8mmol/L, and the solvent is water;
(3) the washing liquid also comprises a second washing liquid, wherein the second washing liquid at least comprises the following components in percentage by weight based on the total amount of the second washing liquid: anhydrous ethanol: 75 percent by volume and water as solvent;
(4) the eluent at least comprises the following components, wherein the content of each component is calculated by taking the total amount of the eluent as a reference: Tris-HCl: 10-80mmol/L EDTA 0.5-25mmol/L, solvent is water;
(5) when in use, the volume ratio of the lysis solution to the magnetic beads is (300-: 20 mu L of the solution;
(6) mixing the lysis solution with magnetic beads, and pre-loading the lysis solution in a 96-well plate;
(7) the first washing solution, the second washing solution and the eluent are respectively pre-filled in a 96-well plate;
(8) the kit is used for extracting HCMV nucleic acid in an amniotic fluid sample.
9. A method for extracting HCMV nucleic acid at least comprises the following steps:
(1) adding a sample into a lysis solution and magnetic bead mixed solution, uniformly mixing, adding an RNA (ribonucleic acid) precipitation aid and proteinase K, and forming a magnetic bead-nucleic acid compound under the action of an external magnetic field;
(2) adding a first washing solution into the magnetic bead-nucleic acid compound, uniformly mixing, carrying out magnetic separation under the action of an external magnetic field, and discarding waste liquid to obtain a first mixture;
(3) adding a second washing solution into the first mixture, uniformly mixing, and carrying out magnetic separation under the action of an external magnetic field to obtain a second mixture;
(4) and adding the eluent into the second precipitate, uniformly mixing, and carrying out magnetic separation under the action of an external magnetic field, wherein the supernatant is the nucleic acid solution.
10. The method for nucleic acid extraction of HCMV of claim 9, wherein the sample is an amniotic fluid sample.
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| CN112195177A (en) * | 2020-10-28 | 2021-01-08 | 上海慕柏生物医学科技有限公司 | Nucleic acid extraction method and kit |
| CN113699145A (en) * | 2021-09-06 | 2021-11-26 | 上海伯杰医疗科技有限公司 | Lysis binding solution based on paramagnetic particle method pathogen nucleic acid extraction, product and application thereof |
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