CN109810974A - Extract the reagent combination product and its method of human immunodeficiency virus HIV-1 nucleic acid - Google Patents
Extract the reagent combination product and its method of human immunodeficiency virus HIV-1 nucleic acid Download PDFInfo
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- CN109810974A CN109810974A CN201910274778.4A CN201910274778A CN109810974A CN 109810974 A CN109810974 A CN 109810974A CN 201910274778 A CN201910274778 A CN 201910274778A CN 109810974 A CN109810974 A CN 109810974A
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 51
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 51
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 51
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 39
- 241000713772 Human immunodeficiency virus 1 Species 0.000 title claims abstract description 23
- 239000013066 combination product Substances 0.000 title claims abstract description 19
- 229940127555 combination product Drugs 0.000 title claims abstract description 19
- 239000000284 extract Substances 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
- 239000006166 lysate Substances 0.000 claims abstract description 29
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000004140 cleaning Methods 0.000 claims abstract description 24
- 239000003480 eluent Substances 0.000 claims abstract description 22
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000001509 sodium citrate Substances 0.000 claims abstract description 16
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 16
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229960004543 anhydrous citric acid Drugs 0.000 claims abstract description 12
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims abstract description 10
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims abstract description 9
- 239000002736 nonionic surfactant Substances 0.000 claims abstract description 9
- 239000002738 chelating agent Substances 0.000 claims abstract description 8
- 239000003945 anionic surfactant Substances 0.000 claims abstract description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000011324 bead Substances 0.000 claims description 28
- 239000002904 solvent Substances 0.000 claims description 16
- 108010067770 Endopeptidase K Proteins 0.000 claims description 15
- 239000002480 mineral oil Substances 0.000 claims description 15
- 235000010446 mineral oil Nutrition 0.000 claims description 15
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 238000012360 testing method Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 239000005662 Paraffin oil Substances 0.000 claims description 5
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 5
- 229910052710 silicon Inorganic materials 0.000 claims description 5
- 239000010703 silicon Substances 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical group [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 3
- ADXOBFJXQZRQGT-UHFFFAOYSA-N 1,1-diaminopropane-1-sulfonic acid Chemical compound CCC(N)(N)S(O)(=O)=O ADXOBFJXQZRQGT-UHFFFAOYSA-N 0.000 claims description 2
- 150000001450 anions Chemical class 0.000 claims description 2
- DEQXHPXOGUSHDX-UHFFFAOYSA-N methylaminomethanetriol;hydrochloride Chemical compound Cl.CNC(O)(O)O DEQXHPXOGUSHDX-UHFFFAOYSA-N 0.000 claims description 2
- 238000000197 pyrolysis Methods 0.000 claims description 2
- 239000003921 oil Substances 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 22
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
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Abstract
The present invention relates to nucleic acid extraction fields, in particular to extract the reagent combination product and its method of human immunodeficiency virus HIV-1 nucleic acid.The reagent combination product, including lysate: guanidinium isothiocyanate 2-6mol/L, chelating agent 3-20mmol/L, anionic surfactant 1%-10%, nonionic surfactant 100-300mmol/L, isopropanol 1-4mol/L and CTAB 0.1%-5%；Cleaning solution: anhydrous citric acid 15-45mmol/L, two water 10-40mmol/L, Proclin950 0.5-3ml/L, pH 4.7-5.3 of sodium citrate；Eluent: trishydroxymethylaminomethane 10-40mmol/, Tri(Hydroxymethyl) Amino Methane Hydrochloride 1-15mmol/L, Proclin950 0.5-3ml/L, Carrier RNA 0.3-2 μ g/ μ l.Coordinated between each component, sensitivity, Nucleic acid quality and the extraction efficiency of extraction are promoted, and extract good stability.
Description
Technical field
The present invention relates to nucleic acid extraction fields, in particular to extraction human immunodeficiency virus HIV-1 nucleic acid
Reagent combination product and its method.
Background technique
AIDS case was reported for the first time so far from the U.S. in 1981, AIDS has had resulted in the death more than 28,000,000 people,
It is had been cited as global main high-risk anti-since the virus has high lethal and infectiousness, and so far still without effective therapy
One of disease-control poison.
AIDS also known as acquired immunodeficiency disease (Human Immunodeficiency Virus, HIV) are a kind of anti-
RNA virus is transcribed, the main pathogenesis of AIDS is the immune mechanism by destroying human body, and the paralysis of immune system causes respectively
Kind virus cannot be destroyed in time, and normally clinical syndromes, the lethal such as breeding generation tumour are high in human body.AIDS old complaint
It is divided into HIV-1 type and two kinds of HIV-2 type again according to its genotype, relative to HIV-2, HIV-1 has stronger infectiousness and causes a disease
Property.The gene structure of HIV and other retrovirus are essentially identical, are RNA bimolecular, single strand RNA molecule is by 9749 cores
Thuja acid composition, there is 3 groups of totally 9 genes.First group of gene common for retrovirus, i.e. gag, pol and env gene, this three
A gene is the structural gene of virus, encodes virus protein.The HIV-2 virus being found in world wide mainly flows in non-state
Row is largely focused on West Africa area, and the AIDS of China's prevalence is HIV-1 type strain, since AIDS is retrovirus,
Gene necessarily has diversity, therefore the HIV-1 strain of China's different geographical prevalence is not identical, has many hypotypes,
AE, BC recombinant type and B/B ', HIV-1 in the country's mainly M group of HIV-1 is due to the region its gene env, gag, pol
High mutability has powerful mutation ability.Therefore in order to accurately detect AIDS, we are one when sequence selection
Surely the conserved region of height is found, and multiple sections is selected to be designed as far as possible.
The detection method of AIDS is broadly divided into Virus culture detection, immune-gold labeled method, antibody test and detection of nucleic acids
Deng Virus culture detection cycle is too long, and sensitivity is low；Immune-gold labeled method sensitivity is low；Antibody test is also due to it need to be in place
It after the antibody to take on a certain scale in master reaches detection limit, can just be detected, window phase is not also short, about 3-5 weeks, urgent need is known
For the patient of road state of an illness result, this period is very long；And with the progress of Biological Detection technology, HIV detection of nucleic acids
Due to its relatively much shorter window phase (about 1 week), the advantages that quick detection efficiency (about 2h), higher sensitivity, at present
Widely available at home and utilization.
In detection of nucleic acids, it need to be examined simultaneously when test object is to mix nucleic acid using the method for the mixed inspection of nucleic acid, such as blood sieve
Survey the virus of plural number, it is desirable that compatibility；And when test object is single nucleic acid, then it is examined using nucleic acid list, it is desirable that
Specific aim.The step of there are also very big optimization spaces for HIV-1 method for extracting nucleic acid on the market at this stage, such as still show redundancy, it is more
Remaining reaction time, the higher formula of bio-hazard and ratio, coarse reaction details and higher detection limit etc..
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide the reagent combination product for extracting human immunodeficiency virus HIV-1 nucleic acid,
Coordinated between each component, sensitivity, Nucleic acid quality and the extraction efficiency of extraction are promoted, and extract good stability.
The second object of the present invention is to provide a kind of method for extracting human immunodeficiency virus HIV-1 nucleic acid, will split
It solves and combines and plan as a whole to be a step, and washing eluent is optimized, while the operating time stringent control of each step, totally only
It needs 3 steps that purpose nucleic acid can be obtained, substantially increases the extraction efficiency of nucleic acid.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
Extract the reagent combination product of human immunodeficiency virus HIV-1 nucleic acid, comprising:
Lysate: taking water as a solvent, and component includes: guanidinium isothiocyanate 2-6mol/L, chelating agent 3-20mmol/L, yin
Ionic surface active agent mass concentration is 1%-10%, nonionic surfactant 100-300mmol/L, isopropanol 1-4mol/L
It is 0.1%-5% with CTAB mass concentration；
Cleaning solution: taking water as a solvent, and component includes: anhydrous citric acid 15-45mmol/L, two water 10- of sodium citrate
40mmol/L, Proclin950 0.5-3.0ml/L, pH 4.7-5.3；
Eluent: taking water as a solvent, and component includes: trishydroxymethylaminomethane 10-40mmol/, trihydroxy methyl amino
Methane hydrochloride salt 1-15mmol/L, Proclin950 0.5-3.0ml/L, Carrier RNA 0.3-2.0 μ g/ μ l.
The reagent combination product provided by the invention for extracting human immunodeficiency virus HIV-1 nucleic acid, has in lysate
Anion surfactant and CTAB, this two kinds of ingredients make the impurity generated when cell cracking be easier to be digested, improve
Nucleic acid quality and extraction efficiency, also, will crack and combine and plan as a whole to be a step；Coordinated between each reagent, is extracted
Nucleic acid quality is more preferable, and according to clinical verification, true and reliable detection limit is not less than 30IU/ml, relative to mentioning on existing market
Its detection of product is taken to be limited to 60IU/ml, sensitivity is higher.In addition, the good stability that reagent extracts.
Further, the chelating agent is two water of sodium citrate.
Further, the anionic surfactant is ten diamino propane sulfonic acid.
Further, the nonionic surfactant is triton x-100.
Further, the reagent further includes Proteinase K, magnetic bead, mineral oil.
Further, the concentration of the Proteinase K is 20 ± 5mg/ml.
Further, the magnetic bead is silicon substrate magnetic bead.
Further, the partial size of the magnetic bead is 1.2 ± 0.2 μm.
Further, the mineral oil is paraffin oil.
Preferably, the lysate: taking water as a solvent, component includes: 4.2 ± 0.2mol/L of guanidinium isothiocyanate, chela
10 ± 1mmol/L of mixture, anionic surfactant mass concentration be 5% ± 0.5%, nonionic surfactant 167.6 ±
5mmol/L, isopropanol 2.6 ± 0.2mol/L and CTAB mass concentration are 0.5% ± 0.05%.
Preferably, the cleaning solution: taking water as a solvent, component includes: 30.7 ± 2mmol/L of anhydrous citric acid, lemon
Lemon acid sodium two water 19.2 ± 2mmol/L, Proclin950 1 ± 0.2ml/L, pH are 5 ± 0.2.
Preferably, eluent: take water as a solvent, component include: trishydroxymethylaminomethane 22.5 ±
0.5mmol/, Tri(Hydroxymethyl) Amino Methane Hydrochloride 7.4 ± 0.2mmol/L, Proclin950 1 ± 0.2ml/L, Carrier
RNA 1±0.2μg/μl。
The present invention also provides a kind of extracting methods of human immunodeficiency virus HIV-1 nucleic acid, using above-mentioned reagent
Combination product extracts, comprising the following steps:
Proteinase K, internal standard, sample to be tested, lysate, mineral oil and magnetic bead sequentially add, mixed pyrolysis, magnetic suck；
The cleaning solution is added to be rinsed, liquid is removed after magnetic suck；
Then the eluent is added, to the Nucleic Acid Elution being adsorbed on magnetic bead, obtained liquid is to contain to have purpose core
The extracting solution of acid.
It will crack and combine in extraction reagent operation step of the invention and plan as a whole to be a step, and is excellent to washing eluent progress
Change, while the operating time stringent control of each step, it is overall only to need 3 steps that purpose nucleic acid can be obtained, substantially increase nucleic acid
Extraction efficiency.
Preferably, Proteinase K, internal standard (concentration range 500-10000IU/ml, preferably 8000IU/ml), to be measured
Sample, lysate, mineral oil, magnetic bead (concentration is 50 ± 10mg/ml) volume ratio be 8 ± 0.5:1:100 ± 1:96 ± 1:8
±0.5:2±0.2。
In the present invention, sample to be tested is generally blood sample.
The lysate cracking strength that the present invention is matched for HIV nucleic acid is high, and the cracking liquid proportional lower than sample size can be abundant
Cracking.
Further, the mixing is mixed using piping and druming, and the piping and druming time is 120 ± 10s.
Further, magnetic suck is carried out after the completion of piping and druming mixes, the time of the magnetic suck is 360 ± 30s.
Further, the piping and druming volume is the 80% of overall solution volume, and piping and druming mode is that liquid level is followed to blow and beat.
To greatly it reduce using special piping and druming range, piping and druming method and to reaction solution progress mineral oil seal cover fine and closely woven small
The generation of bubble, improves efficiency and stability.
The volume ratio of the cleaning solution and the sample to be tested is 80-150:1, and the piping and druming time is 60 ± 10s, magnetic suck
Time is 60 ± 10s.
Further, the cleaning solution only washed once.
Further, the volume ratio of the eluent and the sample to be tested is 10-15:1, and elution time is 60 ± 10s.
Compared with prior art, the invention has the benefit that
(1) the reagent combination product provided by the invention for extracting human immunodeficiency virus HIV-1 nucleic acid, is examined through clinic
It surveys, true and reliable detection limit is not less than 30IU/ml, and relative to the extraction product on existing market, its detection is limited to 60IU/ml,
Sensitivity is higher.
(2) it is provided by the invention extract human immunodeficiency virus HIV-1 nucleic acid reagent combination product, Nucleic acid quality and
Extraction efficiency is high, the good stability of extraction.
(3) in extraction reagent operation step of the invention will cracking and combine plan as a whole be a step, and to washing eluent into
Row optimization, while the operating time stringent control of each step, it is overall only to need 3 steps that purpose nucleic acid can be obtained, substantially increase core
The extraction efficiency of acid.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is three kinds of extraction agent prescriptions in embodiment 2 to 5 × 104The HIV sample of IU/ml concentration extracts laggard
The curve graph of row real-time fluorescence quantitative PCR；
Fig. 2 is that reagent of the present invention is bent to the PCR after 20 repetition tests of HIV sample progress of 100IU/ml in embodiment 3
Line chart；
Fig. 3 carries out the PCR curve after 20 repetitions are tested for HIV sample of the reagent of the present invention in embodiment 3 to 50IU/ml
Figure；
Fig. 4 carries out the PCR curve after 20 repetitions are tested for HIV sample of the reagent of the present invention in embodiment 3 to 30IU/ml
Figure；
Fig. 5 is the curve that the nucleic acid extracted after 160 μ l lysates is added in embodiment 4 and carries out real-time fluorescence quantitative PCR
Figure；
Fig. 6 is the curve that the nucleic acid extracted after 200 μ l lysates is added in embodiment 4 and carries out real-time fluorescence quantitative PCR
Figure；
Fig. 7 is the curve that the nucleic acid extracted after 300 μ l lysates is added in embodiment 4 and carries out real-time fluorescence quantitative PCR
Figure.
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 is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
The present invention provide a kind of optimization, can be quick, stablize the extraction reagent for extracting HIV-1 nucleic acid in blood.Below
The technology of the present invention is described in the specific embodiment of offer, it is important to note that embodiment is one to summary of the invention
Kind explanation, can not limit, affiliated industry those skilled in the art carry out non-according to summary of the invention as protection of the invention
The adjustment and improvement of matter, still fall within protection scope of the present invention.The step of in following embodiment without being described in detail,
Belong to my industry universal technology.
Embodiment 1
The configuration of nucleic acid extracting reagent
Agent prescription is made of lysate A, cleaning solution B, eluent C, Proteinase K, magnetic bead, mineral oil A:
Lysate component A is as follows: 4.2mol/L guanidinium isothiocyanate, two water of 10mmol/L sodium citrate, 5% ten diaminos
Base propane sulfonic acid, 167.6mmol/L triton x-100,2.6mol/L isopropanol and 0.5%CTAB.
Cleaning solution B component is as follows: 30.7mmol/L anhydrous citric acid, two water of 19.2mmol/L sodium citrate, 1ml's
Proclin950, pH 5.
Eluent component C is as follows: 22.5mmol/L trishydroxymethylaminomethane, 7.4mmol/L trishydroxymethylaminomethane
Hydrochloride, the Proclin950 of 1ml, the Carrier RNA of 1 μ g/ μ l.
Proteinase K: 20mg/ml.
Magnetic bead: silicon substrate magnetic bead, partial size are 1.2 μm, concentration 50mg/ml.
Mineral oil A: paraffin oil.
Internal standard: armor RNA.
Embodiment 2
Configure nucleic acid extraction kit X, Y and Z of three kinds of formulas.
Kit X, Y and Z are by lysate A, cleaning solution B, eluent C, Proteinase K, magnetic bead, mineral oil A and internal standard group
At.
Lysate A, cleaning solution B, eluent C, Proteinase K, magnetic bead, mineral oil A are shown in embodiment 1 in X kit.
Y kit:
Lysate A:3.5mol/L guanidinium isothiocyanate, two water of sodium citrate of 2.5mmol/L, 166.1mmol/L Qula are logical
The isopropanol of X-100,3.3mol/L.
Cleaning solution B:31.1mmol/L anhydrous citric acid, two water of 19.0mmol/L sodium citrate, 1ml Proclin950,
PH value is 4.0.
Cleaning solution C:5.7mmol/L anhydrous citric acid, two water of 3.7mmol/L sodium citrate, 1ml Proclin950, pH
Value is 4.0.
Eluent C:14.8mmol/L trishydroxymethylaminomethane, 5.0mmol/L Pehanorm hydrochloride, 1 μ g/ μ l
Carrier RNA.
Proteinase K: 20mg/ml.
Magnetic bead: silicon substrate magnetic bead, partial size are 1.2 μm, concentration 50mg/ml.
Mineral oil A: paraffin oil.
Internal standard: armor RNA.
Z kit:
Lysate A:2.0mol/L guanidinium isothiocyanate, two water of sodium citrate of 5mmol/L, 150.0mmol/L Qula lead to X-
100, the isopropanol of 3.0mol/L.
Cleaning solution B:32.1mmol/L anhydrous citric acid, two water of 20.0mmol/L sodium citrate, 1ml Proclin950,
PH value is 4.5.
Cleaning solution C:5.7mmol/L anhydrous citric acid, two water of 3.7mmol/L sodium citrate, 1ml Proclin950, pH
Value is 4.5.
Eluent C:14.8mmol/L trishydroxymethylaminomethane, 5.0mmol/L Pehanorm hydrochloride, 1 μ g/ μ l
Carrier RNA.
Proteinase K: 20mg/ml.
Magnetic bead: silicon substrate magnetic bead, partial size are 1.2 μm, concentration 50mg/ml.
Mineral oil A: paraffin oil.
Internal standard: armor RNA.
Using configured kit X, Y, Z is respectively to 5 × 104The HIV-1 sample of IU/ml concentration carries out nucleic acid extraction,
Steps are as follows (it may be noted that X kit, that is, kit washing step of the invention only once):
(1) into 2ml round bottom centrifuge tube by permanent order sequentially add Proteinase K, internal standard, sample to be tested, lysate A,
Mineral oil A, magnetic bead, the addition sequence is immutable, then carries out piping and druming mixing, then carries out in the equipment with magnetic suck
Magnetic suck；
(2) sufficiently to solution cracking in pipe, addition cleaning solution B enters 2ml round bottom centrifuge tube and is rinsed, and sufficiently blows and beats
Afterwards, it is placed on the device with magnetic suck and is adsorbed, rear waste liquid of abandoning retains magnetic bead；
(3) the cleaning solution C for diluting 10 times is added 2ml round bottom centrifuge tube and carries out two by (Y, Z kit are additionally required this step)
Secondary rinsing carries out magnetic suck sufficiently after piping and druming, abandons liquid and retains magnetic bead；
(4) addition eluent C enters 2ml round bottom centrifuge tube and elutes to the nucleic acid being adsorbed on magnetic bead, sufficiently blows and beats
Solution is saved after mixing, obtained solution is A, the corresponding nucleic acid of tri- samples of B, C.
The nucleic acid for the various concentration sample that tri- kits of X, Y and Z are extracted respectively and pre-configured reaction solution
Real-time fluorescence quantitative PCR reaction is carried out after mixing, obtained result is as shown in Figure 1.In Fig. 1, forward CT value is of the invention real
Apply the sample that the reagent of example 1 extracts.
From the PCR curve figure of Fig. 1, it can be seen that the kit PCR curve CT value that the X kit i.e. present invention is assert is most
It is forward, illustrate that the Nucleic acid quality extracted is more preferable.
Embodiment 3
It is compared with main product sensitivity and stability on the market
HIV sample of nucleic acid is chosen, two kinds of concentration, respectively 100IU/ml, 50IU/ml are diluted to.To this two kinds of concentration
Sample using a kind of actual clinical mainstream company (the triumphant outstanding person in Shenzhen) on the market human immunodeficiency virus (hiv-1) nucleic acid quantification
Detection kit extracts detection, carries out 20 repetitions, obtained concentration as shown in table 1 below and CT value.
1 available reagent of table extracts testing result
The detection sensitivity that the clinical reagent box specification indicates the reagent is 100IU/ml, and illustrates limiting case
Can carry out the super quick detection of 50IU/ml sensitivity down, it is actually detected it can be seen from table 1 in, concentration is the sample of 100IU/ml
Should kit can stablize detection really, only an example missing inspection, recall rate reaches 95%, and hypersensitivity recall rate only has
70%, there are 6 missing inspection situations to occur, illustrates that the true and reliable detection limit of the clinical reagent is > 50IU/ml.
Meanwhile the agent prescription sensitivity and stability researched and developed for the verifying present invention, use HIV sample stoste of the same race
Be diluted to 3 kinds of concentration: 100IU/ml, 50IU/ml and 30IU/ml, testing result is as shown in table 2 and Fig. 2-4.
The result that table 2 detects the HIV sample of three kinds of concentration
It is of the invention it can be seen from table 2 and Fig. 2-4 in 20 repetitions of the concentration of 100IU/ml and 50IU/ml
Reagent has 100% recall rate, and curve stability, repeatability are also preferable, and under the concentration of 30IU/ML, there is an example sample not
Detection, recall rate 95%, loosely, therefore, the detection that HIV of the invention extracts agent prescription is limited to > to curve co-insides
30IU/ML.The data very can intuitively find out that reagent of the invention is superior in sensitivity and stability as comparison
Clinical mainstream HIV extracts kit.
Embodiment 4
Lysate Efficiency testing
Using agent prescription provided by the present invention, make the addition volume of lysate as variable, remaining factor is constant, right
Three kinds of concentration 1 × 104, 1 × 103, 1 × 102Sample extract experiment, the HIV-1 sample using addition is 200 μ l as template,
The addition volume of lysate is respectively 160 μ l (8:10), 200 μ l (1:1), 300 μ l (3:2).
For remaining concrete operation step with embodiment 2, every kind of concentration does three repetitions.
The nucleic acid obtained after extraction is mixed with the reaction solution prepared in advance, carries out real-time fluorescence quantitative PCR detection,
Obtained result is successively as illustrated in figs. 5-7.In Fig. 5-7, the curve in addition to internal standard is the result that volume is added in different lysates
Figure.
It can be seen from the figure that fluorescence curve without significant difference, shows that the quality of nucleic acid is consistent, illustrates lysate
Even if additional amount is also abundant enough lower than its lysis efficiency of sample size.
In addition, changing the proportion of the lysate A in kit X, cleaning solution B, eluent C, following reagent is such as selected:
Extract reagent combination 1:
Lysate: taking water as a solvent, component are as follows: guanidinium isothiocyanate 2mol/L, chelating agent 20mmol/L, anion table
Face activating agent mass concentration is 10%, nonionic surfactant 100mmol/L, isopropanol 1mol/L and CTAB mass concentration
It is 0.1%.
Cleaning solution: taking water as a solvent, component are as follows: anhydrous citric acid 15mmol/L, two water 40mmol/L of sodium citrate,
Proclin950 0.5ml/L, pH 4.7.
Eluent: taking water as a solvent, component are as follows: trishydroxymethylaminomethane 10mmol/, trishydroxymethylaminomethane salt
0.3 μ g/ μ l of hydrochlorate 15mmol/L, Proclin950 0.5ml/L, Carrier RNA.
Extract reagent combination 2:
Lysate: taking water as a solvent, component are as follows: guanidinium isothiocyanate 6mol/L, chelating agent 3mmol/L, anionic surface
Activating agent mass concentration is 1%, nonionic surfactant 300mmol/L, isopropanol 4mol/L and CTAB mass concentration are
5%.
Cleaning solution: taking water as a solvent, component are as follows: anhydrous citric acid 45mmol/L, two water 10mmol/L of sodium citrate,
Proclin950 3.0ml/L, pH 5.3.
Eluent: taking water as a solvent, component are as follows: trishydroxymethylaminomethane 40mmol/, trishydroxymethylaminomethane salt
2.0 μ g/ μ l of hydrochlorate 1mmol/L, Proclin950 3.0ml/L, Carrier RNA.
Above-mentioned these reagents combination can also reach the extraction effect almost the same with above-described embodiment.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. extracting the reagent combination product of human immunodeficiency virus HIV-1 nucleic acid characterized by comprising
Lysate: taking water as a solvent, and component includes: guanidinium isothiocyanate 2-6mol/L, chelating agent 3-20mmol/L, anion table
Face activating agent mass concentration is 1%-10%, nonionic surfactant 100-300mmol/L, isopropanol 1-4mol/L and CTAB
Mass concentration is 0.1%-5%；
Cleaning solution: taking water as a solvent, and component includes: anhydrous citric acid 15-45mmol/L, two water 10-40mmol/ of sodium citrate
L, Proclin950 0.5-3.0ml/L, pH 4.7-5.3；
Eluent: taking water as a solvent, and component includes: trishydroxymethylaminomethane 10-40mmol/, trishydroxymethylaminomethane
Hydrochloride 1-15mmol/L, Proclin950 0.5-3.0ml/L, Carrier RNA 0.3-2.0 μ g/ μ l.
2. reagent combination product according to claim 1, which is characterized in that the chelating agent is two water of sodium citrate；
Further, the anionic surfactant is ten diamino propane sulfonic acid；
Further, the nonionic surfactant is triton x-100.
3. reagent combination product according to claim 1, which is characterized in that the reagent combination product further includes protease
K, magnetic bead, mineral oil are any one or more of；
Further, the concentration of the Proteinase K is 20 ± 5mg/ml；
Further, the magnetic bead is silicon substrate magnetic bead；
Further, the partial size of the magnetic bead is 1.2 ± 0.2 μm；
Further, the mineral oil is paraffin oil.
4. reagent combination product according to claim 1-3, which is characterized in that the lysate: being molten with water
Agent, component include: 4.2 ± 0.2mol/L of guanidinium isothiocyanate, 10 ± 1mmol/L of chelating agent, anionic surfactant quality
Concentration is 5% ± 0.5%, 167.6 ± 5mmol/L of nonionic surfactant, isopropanol 2.6 ± 0.2mol/L and CTAB mass
Concentration is 0.5% ± 0.05%.
5. reagent combination product according to claim 1-3, which is characterized in that the cleaning solution: being molten with water
Agent, component include: 30.7 ± 2mmol/L of anhydrous citric acid, two water 19.2 ± 2mmol/L, Proclin950 1 of sodium citrate
± 0.2ml/L, pH are 5 ± 0.2.
6. reagent combination product according to claim 1-3, which is characterized in that eluent: taking water as a solvent,
Component includes: 22.5 ± 0.5mmol/ of trishydroxymethylaminomethane, 7.4 ± 0.2mmol/L of Tri(Hydroxymethyl) Amino Methane Hydrochloride,
1 ± 0.2 μ g/ μ l of Proclin950 1 ± 0.2ml/L, Carrier RNA.
7. a kind of extracting method of human immunodeficiency virus HIV-1 nucleic acid, which is characterized in that any using claim 1-6
Reagent combination product described in extracts, comprising the following steps:
Proteinase K, internal standard, sample to be tested, lysate, mineral oil and magnetic bead sequentially add, mixed pyrolysis, magnetic suck；
The cleaning solution is added to be rinsed, liquid is removed after magnetic suck；
Then the eluent is added, to the Nucleic Acid Elution being adsorbed on magnetic bead, obtains the extracting solution containing purpose nucleic acid.
8. extracting method according to claim 7, which is characterized in that Proteinase K, internal standard, sample to be tested, lysate, mine
Object oil, magnetic bead volume ratio be ± 0.5:2 ± 0.2 8 ± 0.5:1:100 ± 1:96 ± 1:8；
Wherein, interior target concentration range is 500-10000IU/ml, preferably 7000-8000IU/ml；The concentration of magnetic bead be 50 ±
10mg/ml。
9. extracting method according to claim 7, which is characterized in that the mixing is mixed using piping and druming, and the piping and druming time is
120±10s；
Further, magnetic suck is carried out after the completion of piping and druming mixes, the time of the magnetic suck is 360 ± 30s；
Further, the piping and druming volume is the 80% of overall solution volume, and piping and druming mode is that liquid level is followed to blow and beat.
10. according to the described in any item extracting methods of claim 7-9, which is characterized in that the cleaning solution and described to test sample
This volume ratio is 80-150:1, and the piping and druming time is 60 ± 10s, and the magnetic suck time is 60 ± 10s；
Further, the cleaning solution only washed once；
Further, the volume ratio of the eluent and the sample to be tested is 10-15:1, and elution time is 60 ± 10s.
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