WO2021164396A1 - Viral nucleic acid extraction method based on glass fiber filter paper - Google Patents

Abstract

A viral nucleic acid extraction method based on glass fiber filter paper, comprising the following steps: step 1, adding protease K in a biological sample and performing rapid mixing to obtain a mixed solution; step 2, adding a binding buffer and Carrier RNA, and turning upside down to sufficiently mixing same; step 3, dropwise adding the mixed liquid on glass fiber filter paper, fixing the glass fiber filter paper by taking double-loop filter paper as a support, and absorbing the filtered waste liquid by using absorbent paper; step 4, performing cleaning by using a washing buffer, and then absorbing the waste liquid by using absorbent paper; and step 5, dropwise adding an eluting buffer on the glass fiber filter paper, and collecting eluent to obtain a required nucleic acid. According to the method, the glass fiber filter paper is introduced into the nucleic acid extraction method and can be used for enrichment and extraction of nucleic acids in biological samples. The method simplifies the steps of nucleic acid extraction and required instrument equipment, thereby saving operation time, reducing extraction costs, and ensuring a high-efficiency nucleic acid recovery rate at the same time.

Description

Method for extracting virus nucleic acid based on glass fiber filter paper Technical field

The invention belongs to the technical field of sample nucleic acid extraction for medical testing, and specifically relates to a method for rapidly extracting nucleic acid from biological samples, and more specifically to a method for extracting virus nucleic acid based on glass fiber filter paper.

Background technique

Body fluids such as plasma, serum, and tissue fluid can be used for the main detection and analysis samples for a variety of blood-borne infectious diseases. Common blood-borne infectious diseases include malaria, chronic hepatitis B, acquired immunodeficiency syndrome, etc. These diseases are caused by pathogens that are parasitic in human blood and lymph, mainly through contact and exchange of blood and wounds. Pass the disease to another body. Therefore, by extracting and detecting the nucleic acid content of the pathogen in the blood, it can help determine whether the disease is infected, the progress of the infection, and the prognosis status.

Glass fiber filter paper is chemically inert, does not contain binders, and is made of 100% borosilicate glass fiber. With a capillary fiber structure, it can absorb more water than the same cellulose filter paper. It has the characteristics of fast flow rate, high temperature resistance, strong dirt holding capacity, and can filter fine particles. As an important part of glass fiber reinforced plastic products, glass fiber is widely used, and the application of filter paper is very close to daily life. Under a certain ionic environment, nucleic acids can be selectively adsorbed to the surface of silica, silica gel or glass to be separated from other biological molecules.

The traditional nucleic acid extraction methods mainly include: phenol extraction, alkaline lysis, CTAB extraction and EtBr-CsCl gradient centrifugation. These traditional extraction methods can separate nucleic acids from different tissue samples, but these techniques include precipitation and centrifugation and other operation steps, which require a large amount of biological samples, and the extraction steps are more complicated, time-consuming and labor-intensive, and the yield is low. high. New nucleic acid extraction methods based on solid-phase adsorbent carriers, such as spinning spin column extraction method, anion exchange method, nano-magnetic bead extraction method, and glass powder adsorption method. Among them, the spin column extraction method and the nano magnetic bead extraction method have been widely used, but the disadvantage is that the spin column method requires repeated centrifugation to extract nucleic acid and requires equipment such as a centrifuge to meet the needs, while the nano magnetic bead extraction method requires an external magnetic field. , And the required sample is usually 200uL and above, the volume is relatively large, and the enrichment and elution process is relatively cumbersome.

Summary of the invention

In view of the shortcomings of the existing problems, the purpose of the present invention is to provide a method for extracting virus nucleic acid based on glass fiber filter paper, which can realize rapid extraction and purification of nucleic acid on site and is convenient to operate.

The technical solutions adopted by the present invention to solve its technical problems are:

To achieve the above objective, the present invention proposes a nucleic acid extraction method based on glass fiber filter paper, which includes the following steps:

Step 1: Add proteinase K to the biological sample to be extracted nucleic acid, mix quickly to obtain a mixed solution, and proteinase K helps to lyse the virus to release the nucleic acid;

Step 2: Add binding buffer and carrier RNA to the mixed solution obtained in step 1. The binding buffer helps the nucleic acid to be adsorbed on the glass fiber membrane, and the ribonucleic acid carrier helps to enrich the nucleic acid;

Step three, drop the mixed liquid obtained in step two on the glass fiber filter paper, the glass fiber filter paper is fixed with the double-circle filter paper as the support, and the filtered waste liquid is absorbed by absorbent paper. At this time, the required nucleic acid is adsorbed on the glass fiber filter paper ；

Step 4: Wash with washing buffer for 1 to 2 times, and absorb the waste liquid with absorbent paper. The purpose is to remove any residual contaminants that may seriously affect downstream applications;

Step 5: Add elution buffer dropwise on the glass fiber filter paper, and collect the filtered eluate to obtain the desired nucleic acid solution.

Preferably, in the first step, the concentration of proteinase K is 20-200ug/mL.

Preferably, in the second step, the carrier RNA concentration is 1-8 ug/mL, and more preferably, the carrier RNA concentration is 3-6 ug/mL.

Preferably, in the second step, the binding buffer mainly includes 1-8M guanidine hydrochloride, 10-200mM Tris-HCl, 10-100mM EDTA, 100-800mM NaCl, 0.5%-2% SDS;

More preferably, the binding buffer mainly includes 2-8M guanidine hydrochloride, 50-100mM Tris-HCl, 10-80mM EDTA, 100-800mM NaCl, 0.5%-1% SDS.

More preferably, the pH of Tris-HCl in the binding buffer is 6.0-8.0.

Preferably, in the fourth step, the washing buffer mainly includes 1-10 mM EDTA, 10-100 mM Tris-HCl, 10-100 mM NaCl, and 55%-75% ethanol.

Preferably, in the fourth step, the elution buffer mainly includes 10-100 mM Tris-HCl and 1-10 mM EDTA.

More preferably, the elution buffer is composed of 10 mM Tris-HCl and 1 mM EDTA.

Preferably, the glass fiber filter paper is 100% borosilicate glass fiber.

Preferably, in the third step, the glass fiber filter paper is circular, with a diameter of 6-10 mm, and is fixed in the hydrophilic area of the double-circle filter paper with a PET single-sided transparent tape.

More preferably, the diameter of the glass fiber filter paper is 6-8 mm.

Preferably, in the third step, the double-circle filter paper includes a wax-sprayed hydrophobic area and a hydrophilic area, and the hydrophilic area is circular with a diameter of 8-12 mm.

Preferably, the biological sample includes serum, plasma and tissue fluid.

Beneficial effect

Compared with the prior art, the method for extracting virus nucleic acid based on glass fiber filter paper provided by the present invention has the following beneficial effects:

This method introduces glass fiber filter paper to directly adsorb and purify nucleic acid. The extraction method is fast and convenient, does not require bulky equipment, and requires a small sample volume. It can be used to extract nucleic acid from serum, plasma and tissue fluid, making the nucleic acid extraction method suitable for on-site Detection.

Description of the drawings

Figure 1 is a schematic diagram of the comparison of the efficiency of nucleic acid extraction of hepatitis B virus in serum between a nucleic acid extraction method based on glass fiber filter paper and Quan's gold magnetic bead method virus group DNA\RNA kit in Example 1 of the present invention;

2 is a schematic diagram of the comparison of the efficiency of nucleic acid extraction of hepatitis B virus in serum between a nucleic acid extraction method based on glass fiber filter paper and Quan's gold magnetic bead method virus group DNA\RNA kit in Example 2 of the present invention;

FIG. 3 is a schematic diagram of the comparison of the nucleic acid extraction efficiency of the hepatitis B virus nucleic acid in the serum between the nucleic acid extraction method based on glass fiber filter paper and the nucleic acid extraction method of replacing the glass fiber filter paper with double circle filter paper and fusion respectively in Example 3 of the present invention.

Detailed ways

Hereinafter, the present invention will be further described in detail with reference to the embodiments. Reagents or equipment that do not indicate the manufacturer are regarded as conventional products that can be purchased on the market.

Example 1

A nucleic acid extraction method based on glass fiber filter paper, including the following steps:

Step 1. Take two 50uL of serum containing hepatitis B virus (HBV virus) and place them in a 1.5mL centrifuge tube as experimental group 1 (and labeled HBV11, HBV16) as the biological samples to be extracted nucleic acid, and add 5uL proteinase K Then mix quickly to get a mixed solution;

Step 2: Add 80uL binding buffer and 0.7uL Carrier RNA to the mixed solution obtained in step 1, and incubate at room temperature for 10 minutes after mixing;

Step three, drop the mixed liquid obtained in step two onto the glass fiber filter paper, put absorbent paper under the filter paper, and absorb the filtered waste liquid with absorbent paper;

Step 4: Slowly add 125uL washing buffer on the glass fiber filter paper, put absorbent paper under the filter paper, and absorb the filtered waste liquid with absorbent paper, and dry it in a ventilated place for no more than 10 minutes;

Step 5: Add 30uL elution buffer dropwise on the above-mentioned glass fiber filter paper, collect the filtered solution in a 1.5mL centrifuge tube to obtain the required nucleic acid. The required nucleic acid can be carried out in the next experiment or at -20℃ Save; where the proteinase K concentration is 80ug/mL, the carrier RNA concentration is 6ug/mL; the binding buffer is composed of 6M guanidine hydrochloride, 50mM Tris-HCl, 20mM EDTA, 500mM NaCl and 0.5% SDS, and the washing buffer is composed of 10mM EDTA , 50mM Tris-HCl (pH 6.5), 100mM NaCl and 75% ethanol, the elution buffer is composed of 10mM Tris-HCl (pH 8.0) and 1mM EDTA, the glass fiber filter paper is 100% borosilicate glass fiber and the diameter is 6mm.

Set up the control group 1 with the same number as the above experimental group, use the Quan's gold magnetic bead method virus group DNA\RNA kit, the sampling volume is 200uL, the elution volume is 120uL, and the nucleic acid extraction of the control group is completed.

The control group 1 and the amount of nucleic acid extracted by the extraction method of the present invention were determined using Nanodrop 2000 to determine the total amount of nucleic acid. The test results are as follows:

Table 1 Results of total nucleic acid determination

To	Control group 1	To	Invention Experiment Group 1	To
Sample serial number	Nucleic acid concentration (ng/uL)	A260/A280	Nucleic acid concentration (ng/uL)	A260/A280
HBV11	3.5	1.32	53.9	1.62
HBV16	4.6	1.65	50.2	1.21

The 1uL nucleic acid in the above-mentioned experimental group and the control group was selected as a template and detected by polymerase chain reaction (PCR). The detection result is shown in Figure 1, and the length of the amplified target fragment is 1203bp.

Table 2 HBV fluorescence quantitative polymerase chain reaction test results

Sample serial number	Experimental group 1 Ct value	Control group 1 Ct value
HBV11	23.98	26.40
HBV16	21.40	25.03

The 1uL nucleic acid in the above-mentioned experimental group and the control group was selected as a template for detection by HBV fluorescent quantitative polymerase chain reaction (PCR), and the detection results are shown in Table 2 below.

Example 2

A nucleic acid extraction method based on glass fiber filter paper, including the following steps:

Step 1. Take two 50uL serums containing hepatitis B virus (HBV virus) and place them in a 1.5mL centrifuge tube as experimental group 2 (and labeled HBV1 and HBV5) as biological samples to be extracted nucleic acid, and add 5uL proteinase K Then mix quickly to get a mixed solution;

Step 2: Add 80uL binding buffer and 0.7uL Carrier RNA to the mixed solution obtained in step 1, and incubate at room temperature for 10 minutes after mixing;

Step three, drop the mixed liquid obtained in step two on the glass fiber filter paper, the glass fiber filter paper is fixed with the double-circle filter paper as the support, the absorbent paper is placed under the filter paper, and the filtered waste liquid is absorbed with the absorbent paper;

Step 4: Slowly drop 125uL washing buffer on the glass fiber filter paper of step 3, put absorbent paper under the filter paper, and absorb the filtered waste liquid with absorbent paper, and dry it in a ventilated place for no more than 10 minutes;

Step 5: Add 30uL elution buffer dropwise to the glass fiber filter paper of step 4, and collect the filtered solution in a 1.5mL centrifuge tube to obtain the required nucleic acid. The required nucleic acid can be carried out in the next experiment or at -20 Store at ℃.

Among them, the proteinase K concentration is 100ug/mL, the carrier RNA concentration is 6ug/mL, the binding buffer consists of 8M guanidine hydrochloride, 100mM Tris-HCl, 80mM EDTA, 800mM NaCl and 1% SDS, and the washing buffer consists of 10mM EDTA, 100mM It is composed of Tris-HCl (pH 6.5), 100 mM NaCl and 75% ethanol. The elution buffer is composed of 10 mM Tris-HCl (pH 8.5) and 1 mM EDTA. The glass fiber filter paper is 100% borosilicate glass fiber with a diameter of 8 mm.

Set up the control group 2 with the same number as the above experimental group, use the Quan's gold magnetic bead method virus group DNA\RNA kit, the sampling volume is 200uL, the elution volume is 120uL, and the nucleic acid extraction of the control group is completed.

The control group 2 and the amount of nucleic acid extracted by the extraction method of the present invention were determined using Nanodrop 2000 to determine the total amount of nucleic acid, and the test results are as follows:

Table 3 Results of total nucleic acid determination

To	Control group 2	To	Invention Experimental Group 2	To
Sample serial number	Nucleic acid concentration (ng/uL)	A260/A280	Nucleic acid concentration (ng/uL)	A260/A280
HBV1	3.1	1.81	60.7	1.60
HBV5	8.9	1.79	157.5	1.32

The 1uL nucleic acid in the above-mentioned experimental group and the control group was selected as a template and detected by polymerase chain reaction (PCR). The detection result is shown in Figure 2, and the length of the amplified target fragment is 1203bp.

The 1uL nucleic acid in the above-mentioned experimental group and the control group was selected as a template for detection using HBV fluorescent quantitative polymerase chain reaction (PCR). The detection results are shown in Table 4 below.

Table 4 HBV fluorescence quantitative polymerase chain reaction test results

Sample serial number	Experimental group 2 Ct value	Control group 2 Ct value
HBV1	20.92	24.93
HBV5	20.71	23.94

Implementation 1 and implementation 2 combined with Table 1, Table 2, Table 3, Table 4, and Figure 1 and Figure 2 show that the nucleic acid extraction method of the present invention has a higher total amount of nucleic acid than the control reagent, and the actual target nucleic acid amount is higher. The advantages of the present invention are No additional equipment is required, the effect of nucleic acid extraction application is better, and the extraction time is shortened.

Example 3

A nucleic acid extraction method based on glass fiber filter paper, including the following steps:

Step 1. Take a 150uL serum containing hepatitis B virus (HBV virus) and divide it into three evenly and place it in a 1.5mL centrifuge tube (labeled as LZ, BX, F5) as the biological sample for nucleic acid extraction. Mix 5uL proteinase K quickly to obtain a mixed solution;

Step 2: Add 80uL binding buffer and 0.7uL Carrier RNA to the mixed solution obtained in step 1, and incubate at room temperature for 10 minutes after mixing;

Step 3: Drop the mixed solution from step 2 marked LZ on the double-circle filter paper, drop the one marked BX on the glass fiber filter paper, drop the one marked F5 on the Fusion 5 filter membrane, on the three filter papers Put absorbent paper on the bottom pad, and absorb the filtered waste liquid with absorbent paper;

Step 4: Add 125uL washing buffer slowly on the double-circle filter paper, glass fiber filter paper and Fusion 5 filter membrane of step 3, put absorbent paper under the filter paper, and absorb the filtered waste liquid with absorbent paper, and dry it in a ventilated place. More than 10 minutes;

Step 5: Add 30uL elution buffer dropwise on the double-circle filter paper, glass fiber filter paper and Fusion 5 filter membrane of step 4, and collect the filtered solution in a 1.5mL centrifuge tube to obtain the required nucleic acid. You can proceed to the next experiment or store it at -20°C.

Among them, the proteinase K concentration is 20ug/mL, the carrier RNA concentration is 3ug/mL, the binding buffer consists of 2M guanidine hydrochloride, 50mM Tris-HCl, 10mM EDTA, 100mM NaCl and 0.5% SDS, and the washing buffer consists of 1mM EDTA, 10mM It is composed of Tris-HCl (pH 7.0), 100 mM NaCl and 70% ethanol. The elution buffer is composed of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The glass fiber filter paper is 100% borosilicate glass fiber with a diameter of 6 mm.

The nucleic acid extraction method in which the glass fiber filter paper is replaced with double-circle filter paper and fusion 5 and the amount of nucleic acid extracted by the extraction method of the present invention are determined by Nanodrop 2000. The test results are as follows in Table 5:

Table 5 Results of total nucleic acid determination

Sample serial number	Nucleic acid concentration (ng/uL)	A260/A280
F5	4.4	1.93
LZ	6.3	2.88
BX	25.2	2.66

The 1uL nucleic acids labeled as F5, LZ, and BX were selected as templates and detected by polymerase chain reaction (PCR). The detection results are shown in Figure 3, and the length of the amplified target fragment is 1203bp.

Example 3 combined with Table 5 and Figure 3, it can be seen that the total amount of nucleic acid extracted by using glass fiber filter paper is higher than that using double-circle filter paper and Fusion 5 filter membrane, the actual target nucleic acid amount is higher, and the effect of nucleic acid extraction application is better.

The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that those skilled in the art can think of are all included in the present invention, and the appended claims are the protection scope.

Claims (10)

1. A nucleic acid extraction method based on glass fiber filter paper, which is characterized in that it includes the following steps:

   Step 1: Add proteinase K to the biological sample to be extracted nucleic acid and mix quickly to obtain a mixed solution;

   Step two, add binding buffer and carrier RNA to the mixed solution obtained in step one;

   Step three, drop the mixed liquid obtained in step two onto the glass fiber filter paper, the glass fiber filter paper is fixed with the double-circle filter paper as a support, and the filtered waste liquid is absorbed with absorbent paper;

   Step 4: Wash with washing buffer, and then absorb the waste liquid with absorbent paper;

   Step 5: Add elution buffer dropwise on the glass fiber filter paper, and collect the filtered eluate to obtain the desired nucleic acid solution.
2. The nucleic acid extraction method based on glass fiber filter paper according to claim 1, characterized in that: in the third step, the glass fiber filter paper is round with a diameter of 6-10 mm, and is fixed on the double-circle filter paper with a PET single-sided transparent tape. In the hydrophilic area.
3. The method for extracting nucleic acid based on glass fiber filter paper according to claim 1 or 2, characterized in that: in the third step, the double-circle filter paper comprises a wax-sprayed hydrophobic area and a hydrophilic area, and the hydrophilic area is circular with a diameter of 8-12mm.
4. The method for extracting nucleic acid based on glass fiber filter paper according to claim 1 or 2, characterized in that: in the first step, the concentration of proteinase K is 20-100 ug/mL.
5. The nucleic acid extraction method based on glass fiber filter paper according to claim 1 or 2, characterized in that: in the second step, the carrier RNA concentration is 1-8ug/mL.
6. The nucleic acid extraction method based on glass fiber filter paper according to claim 1 or 2, characterized in that: in the second step, the binding buffer mainly includes 1-8M guanidine hydrochloride, 10-200mM Tris-HCl, 10-100mM EDTA , 100-800mM NaCl, 0.5%-2% SDS.
7. The method for extracting nucleic acid based on glass fiber filter paper according to claim 6, wherein the pH of Tris-HCl in the binding buffer is 6.0-7.0.
8. The nucleic acid extraction method based on glass fiber filter paper according to claim 1 or 2, characterized in that: in the fourth step, the washing buffer mainly includes 1-10 mM EDTA, 10-100 mM Tris-HCl, 10-100 mM NaCl and 55%-75% ethanol.
9. The nucleic acid extraction method based on glass fiber filter paper according to claim 1 or 2, characterized in that: in the fourth step, the elution buffer mainly includes 10-100 mM Tris-HCl, 1-10 mM EDTA; preferably, the The pH of the Tris-HCl is 7.0-8.5.
10. The nucleic acid extraction method based on glass fiber filter paper according to claim 1 or 2, characterized in that: in the first step, the biological sample includes serum, plasma and tissue fluid.

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