CN102505012A - Lysis solution for extracting animal virus ribonucleic acid (RNA) - Google Patents

Abstract

The invention relates to a lysis solution TRIzolLS for extracting animal virus ribonucleic acid (RNA). The lysis solution is prepared from guanidine thiocyanate, ammonium isothiocyanate, sodium acetate, sodium citrate, dithiothreitol (DTT), phenol, nonidet P-40 (NP-40), TritonX-100, glycerol, methyl red and sodium lauryl sulfate (SLS) in a certain proportion. The extracted RNA is analyzed by a spectrophotometric method and a fluorescent reverse transcription-polymerase chain reaction (RT-PCR) method; and the result proves that the quality of the obtained RNA fully meets the requirements of analysis and detection. The lysis solution is sold at affordable price, has stable and reliable performance, and can be used for extracting the animal virus RNA from different samples.

Description

A kind of lysate that is used to extract animal virus RNA

Technical field

The present invention relates to this molecule field of biology, relate in particular to a kind of lysate that is used to extract animal virus RNA.

Background technology

The appearance of round pcr and development have realized the gene in vitro amplification, and the extractive technique of nucleic acid is also fast-developing.Wherein the extraction of RNA is a basic fundamental of modern molecular biology, also is simultaneously the important means of research nucleic acid, and the quarantine for a large amount of virus diseases has far reaching significance especially especially.

The method that is directed against the extraction of RNA at present has guanidinium isothiocyanate-phynol method (TRIzol method), phenol-SDS method, guanidine hydrochloride method etc.TRIzol method wherein, because of it contains the polycomponent centrifugation, and advantage such as easy and simple to handle, weak point consuming time is adopted by most laboratory.Wherein the TRIzol of American I nvitrogen company is that share of market is the highest, but its genus import brand costs an arm and a leg.Therefore, seek the substitute of reliable material benefit,, be significant in biology field with the more favourable promotion experiment detection and the carrying out of scientific effort.

Summary of the invention

In view of this, be necessary to above-mentioned problem, the TRIzol reagent of the stable performance of a kind of animal virus RNA that is used for extracting different samples is provided.

For solving the problems of the technologies described above, the present invention adopts following technical scheme:

A kind of lysate TRIzol LS that is used to extract animal virus RNA is formulated according to following ratio by guanidinium isothiocyanate, isothiocyanic acid amine, sodium acetate, Trisodium Citrate, DTT, phenol, NP-40, TritonX-100, glycerine, methyl red and SLS:

Composition	Working concentration	Content (1L)
			Guanidinium isothiocyanate	9.2%	92.00 g
Isothiocyanic acid amine	3%	30.00 g
			Sodium acetate solution (3M)	0.1 M	33.33 mL
Trisodium Citrate (0. 5 M)	25 mM	50.00 mL
			DTT（5 M）	5 mM	1.00 mL
The phenol that contains 0.1% oxine	38%	380.00 mL
			NP-40	0.5%	5.00 mL
TritonX-100	0.5%	5.00 mL
			Glycerine	5%	50.00 mL
The acetic acid saturated solution of methyl red	0.02%	0.20 mL
			SLS	0.5%	5.00 g

Compared with prior art, the present invention has following beneficial effect: the present invention is identical with existing TRIzol reagent on method of use and consumption, and price material benefit, stable and reliable for performance, can be used to extract the animal virus RNA in the different samples.

Embodiment

The present invention provides a kind of lysate that is used to extract animal virus RNA, and is clearer, clear and definite for making the object of the invention, technical scheme and effect, below to further explain of the present invention.

A kind of lysate TRIzol LS that is used to extract animal virus RNA is formulated according to table one ratio by guanidinium isothiocyanate, isothiocyanic acid amine, sodium acetate, Trisodium Citrate, DTT, phenol, NP-40, TritonX-100, glycerine, methyl red and SLS.

Table one

Composition	Working concentration	Content (1L)
			Guanidinium isothiocyanate	9.2%	92.00 g
Isothiocyanic acid amine	3%	30.00 g
			Sodium acetate solution (3M)	0.1 M	33.33 mL
Trisodium Citrate (0. 5 M)	25 mM	50.00 mL
			DTT（5 M）	5 mM	1.00 mL
The phenol that contains 0.1% oxine	38%	380.00 mL
			NP-40	0.5%	5.00 mL
TritonX-100	0.5%	5.00 mL
			Glycerine	5%	50.00 mL
The acetic acid saturated solution of methyl red	0.02%	0.20 mL
			SLS	0.5%	5.00 g

The present invention is a kind of, and to be used to extract the preparation method of lysate TRIzol LS of animal virus RNA following:

1, prepares the sodium acetate solution of 3M, the DTT solution of 5M and the sodium citrate soln of 0.5M respectively.

2,33.33mL sodium acetate solution, 1mL DTT solution and 50 mL sodium citrate solns are blended in the container; Add 380 mL phenol (0.1% oxine); 5.00 the NP-40 of mL; 5.00 the TritonX-100 of mL, the acetic acid saturated solution of the methyl red of the glycerine of 50.00 mL and 0.20 mL.

3, weighing 92.00g guanidinium isothiocyanate, 30.00 isothiocyanic acid amine g and 5.00g SLS join in the solution of 2 preparations.

4, stirring and dissolving, constant volume is to 1L.

The present invention can extract any RNA viruses of any animal.Because of the process for extracting comparative maturity of RNA, only briefly introduce below:

1, in 1.5 mL Eppendorf pipe, add 600 μ L TRIzol reagent, each 200 μ L of seized sample are through the trichloromethane 200 μ L of-20 ℃ of precoolings, mixing.In 4 ℃, centrifugal 15 min of 12000 r/m.

2, draw supernatant in the step 1 and be transferred to and be added with in the Eppendorf pipe of the Virahol of-20 ℃ of precoolings, put upside down mixing.It is suitable with the supernatant volume that institute adds Virahol.In 4 ℃, centrifugal 10 min of 12000 r/m.

3, abandon supernatant, add 600 μ L, 75% ethanol, put upside down washing, in 4 ℃, centrifugal 10 min of 12000 r/m.

4, abandon supernatant, centrifugal 10 s of 4000 r/min abandon supernatant, drying at room temperature 3 ~ 5 min.

5, on the estimation rna content adds DEPC water, mixing gently, and the RNA on the dissolving tube wall, centrifugal 5 s of 2000 r/m preserve subsequent use on ice.

If seized sample is a liquid sample,, directly gets 200 μ L and be used for the RNA extraction like serum or transudate etc.; If sample is a solid sample, like tissue etc., should in sample, add damping fluid such as saline water to grind or pulverize, carry out the RNA extraction with 200 μ L supernatants after the multigelation three times.

For verifying concrete effect of the present invention; The TRIzol that is produced with Shanghai invitrogen company does controlled trial; To the animal virus RNA that extracts through the present invention, adopt ultraviolet spectrophotometer method to survey its concentration and purity, adopt fluorescence quantitative PCR method to survey its average Ct value; The technology that said method is well known to those skilled in the art is not done introduction at this.Write for ease, represent the present invention to be used to extract the lysate TRIzol LS of animal virus RNA with LS in the hereinafter form; The TRIzol that on behalf of Shanghai invitrogen company, IN produced.

Adopt above-mentioned RNA process for extracting, respectively to the sample extraction animal virus RNA of 3 kinds of forms, the sample of every kind of form has all been selected 1/2/ and 1,/20 two concentration with LS and 2 kinds of TRIzol reagent of IN, and detailed results is referring to table two and table three.

Use ultraviolet spectrophotometer method to measure RNA mean concns and Reinheitszahl that two kinds of TRIzol reagent extract three kinds of samples, the result sees table two.

Table two

Annotate: concentration unit is (ng/ μ L).

RNA concentration and purity result that ultraviolet spectrophotometer method records show that the effect that swab and serum sample use the present invention to obtain is better, and tissue sample adopts the TRIzol effect of Shanghai invitrogen company better.

Use fluorescence quantitative PCR method to measure the average Ct value of RNA that two kinds of TRIzol reagent extract three kinds of samples, the result is referring to table three.

Table three

Fluorescent quantitative PCR result shows that the TRIzol effect of the present invention and Shanghai invitrogen company is close, and in the detection of swab sample and serum sample, TRIzol of the present invention (LS) effect slightly is superior to the TRIzol (IN) of Shanghai invitrogen company.

Can be known that by above result the RNA quality of using the present invention to obtain meets the requirement of analyzing and testing fully, the potential alternative import TRIzol reagent of the present invention is used for the extraction of swab, serum and tissue RNA.

The above embodiment has only expressed several kinds of embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art under the prerequisite that does not break away from the present invention's design, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with accompanying claims.

Claims (1)

1. lysate TRIzol LS who is used to extract animal virus RNA, formulated according to following ratio by guanidinium isothiocyanate, isothiocyanic acid amine, sodium acetate, Trisodium Citrate, DTT, phenol, NP-40, TritonX-100, glycerine, methyl red and SLS:

Composition Working concentration Content (1L) Guanidinium isothiocyanate 9.2% 92.00 g Isothiocyanic acid amine 3% 30.00 g Sodium acetate solution (3M) 0.1 M 33.33 mL Trisodium Citrate (0. 5 M) 25 mM 50.00 mL DTT（5 M） 5 mM 1.00 mL The phenol that contains 0.1% oxine 38% 380.00 mL NP-40 0.5% 5.00 mL TritonX-100 0.5% 5.00 mL Glycerine 5% 50.00 mL The acetic acid saturated solution of methyl red 0.02% 0.20 mL SLS 0.5% 5.00 g

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