CN115074356A - New coronavirus nucleic acid extraction promoter and application thereof - Google Patents

Abstract

The invention discloses a novel coronavirus nucleic acid extraction accelerator and an application thereof, belonging to the technical field of biological detection. In order to solve the problem that the detection Ct value of the internal reference will decrease, and some samples cannot even detect the Ct value of the internal reference, in order to solve the problem that the sample is placed for more than 24 hours before extraction. The present invention provides a novel coronavirus nucleic acid extraction accelerator. The components of the accelerator are as follows: a reducing agent, a detergent and a phosphate buffer solution. The experimental results of adding this reagent to the samples collected by the new crown showed that it can increase the content of the internal reference and the new coronavirus nucleic acid in the samples extracted by the magnetic bead method by more than 10 times. The application of the nucleic acid extraction accelerator in the detection of new crowns can significantly improve the detection sensitivity of the virus and improve the detection rate of weakly positive samples of the new crowns.

Description

A novel coronavirus nucleic acid extraction accelerator and its application

technical field

The invention belongs to the technical field of biological detection, and in particular relates to a novel coronavirus nucleic acid extraction accelerator and application thereof.

Background technique

In modern molecular biology experiments, the extraction of DNA and RNA from various sources is an essential process for many experiments. DNase and RNase are enzymes that specifically degrade nucleic acids. These enzymes are widely found in nature. Therefore, nucleic acid molecules exposed to the outside world are easily degraded, especially in tissue cells. The content of these two enzymes is very high. Improperly, it is difficult to extract complete nucleic acid molecules from these raw materials. In a complete viral particle, the viral nucleic acid is bound to various proteins, and the enzymes that degrade DNA and RNA cannot access the nucleic acid. Therefore, the viral nucleic acid can be preserved intact. If viral nucleic acids are released from virus particles, they will be degraded quickly without protective measures. Therefore, the viral nucleic acids obtained in this case are difficult to guarantee the success of various molecular experiments, especially virus detection experiments.

In the new crown test, the samples were all derived from the pharynx or nasal cavity. These samples not only contained a large amount of mucus, but also scraped off a large number of epithelial cells during the sampling process. When these cells rupture, a large amount of RNases are released. If these enzymes are not completely inactivated, the nucleic acid of the new coronavirus will be hydrolyzed quickly after being released. - Positive result detected in PCR. Therefore, at present, a certain amount of guanidine salt is added to the lysate in the new crown detection tube as a denaturant, and these denaturants can effectively inhibit the activity of RNase within a certain period of time. However, we often find in the process of detecting new crown nucleic acid that if the sample is placed for more than 24 hours, the detection Ct value of the internal reference will decrease, and some samples cannot even detect the Ct value of the internal reference. These phenomena indicate that only using guanidine salts as RNase inhibitors is not ideal, and there is still a small amount of biologically active RNases after the samples are lysed in the lysis buffer.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the problem that the detected Ct value of the internal reference will decrease and the internal reference of some samples cannot even detect the Ct value before extraction is performed after the sample is placed for more than 24 hours.

The present invention provides an accelerator for nucleic acid extraction of novel coronavirus. The composition of the accelerator is as follows: a reducing agent, a detergent and a phosphate buffer solution.

Further defined, the reducing agent is DTT.

Further defined, the detergent is SDS.

Further limited, the phosphate buffer is disodium hydrogen phosphate.

To be further defined, the components of the accelerator are as follows: DTT, SDS and disodium hydrogen phosphate.

To be further defined, the composition of the accelerator is as follows: 0.1-10 mM DTT, 0.5-5% SDS and 0.1-1 M disodium hydrogen phosphate, pH 7.2.

Further defined, the composition of the accelerator is as follows: 10 mM DTT, 5% SDS and 1 M disodium hydrogen phosphate, pH 7.2.

To be further defined, the composition of the accelerator is as follows: 0.1 mM DTT, 0.5% SDS and 0.1 M disodium hydrogen phosphate, pH 7.2.

The present invention provides the application of the above-mentioned accelerator in the preparation of a kit for extracting novel coronavirus RNA.

The present invention provides the application of the above-mentioned accelerator in improving the extraction efficiency of novel coronavirus nucleic acid.

Beneficial effect: The formula of the novel coronavirus nucleic acid extraction accelerator mainly contains a reducing agent, a detergent and a phosphate buffer solution, wherein the reducing agent includes DTT, and the detergent includes SDS. The final concentration of reducing agent is between 0.1-10mM, and the best concentration is 1mM. Its function in protective agent is to destroy the disulfide bond in RNase and permanently inactivate RNase; The final concentration is between 0.5-5%, and the recommended final concentration is 0.5%. Its function is to remove the protein component in RNA, especially in the process of magnetic bead extraction, it can effectively separate the connection between nucleic acid and protein, and promote nucleic acid. Binding with magnetic beads; the final concentration of phosphate is 0.1-1M, and the recommended final concentration is 0.1M. Its function in the extraction accelerator is to provide a stable buffer environment for nucleic acids, which is conducive to the stable preservation of nucleic acids. In addition, a salt ion environment is provided in nucleic acid extraction, so that the magnetic beads extract RNA or DNA to carry strong positive ions, and promote the electrostatic binding of nucleic acid and magnetic beads.

Description of drawings

Fig. 1 is the maximum dilution factor that the detection result is positive;

Figure 2 shows the maximum dilution for a positive test result.

Detailed ways

Example 1. Novel coronavirus nucleic acid extraction accelerator

The formula (10x) of the new coronavirus nucleic acid extraction accelerator is experimentally operated as follows:

Extraction accelerator formula: DTT 10mM

SDS 5%

Disodium hydrogen phosphate: 1M

pH 7.2.

Comparative example 1. Novel coronavirus nucleic acid extraction accelerator

Extraction accelerator control formula 1: SDS 5%

Disodium hydrogen phosphate: 1M

pH 7.2.

Comparative example 2. Novel coronavirus nucleic acid extraction accelerator

Extraction Enhancer Control 2 Recipe: DTT 10mM

Disodium hydrogen phosphate: 1M

pH 7.2.

Comparative example 3. Novel coronavirus nucleic acid extraction accelerator

Extraction Enhancer Control Formulation 3: DTT 10mM

SDS 5%

pH 7.2.

Use the following experiments to verify the experimental effect:

New coronavirus RNA extraction method:

1. Take 5 empty sample sampling tubes A1, A2, A3, A4 and B1, add 1.8ml of commercial sample preservation solution to each tube, and add 200µl volume of extraction accelerator (Example 1) to tubes A1 to A4 respectively. ), extraction accelerator (comparative example 1), extraction accelerator (comparative example 2) and extraction accelerator (comparative example 3), add 200 µl of pure water to tube B1 as a control.

2. Take a sampling tube C containing 6 ml of sample preservation solution, and use a sampling swab to collect pharyngeal samples of two healthy people. The collected throat swab is fully stirred in the preservation solution, so that the collected sample is fully released from the swab into the preservation solution.

3. Take 1ml of the sample in tube C and add it to tubes A1-A4 and tube B1 respectively. Since it usually takes 1-4 hours from sample collection to sample delivery to the testing laboratory for testing, in order to simulate this process time, the sampling tubes A1-A4 and B1 into which the samples were added were first placed at room temperature for 24 hours, and then separated from A1. - Take 200ul from A4 and B1 to extract nucleic acid in the sample by magnetic bead method (the kit was purchased from Shuo Shi Company).

4. After the sample is extracted in the automatic extractor, take 5µl from the eluate for fluorescence RT-PCR detection. PCR method). The detection effect was evaluated by the Ct value of the internal reference RNase-P gene detected in the sample.

The results showed that the Ct value of the A1 sample group added with the extraction accelerator was reduced by about 4.80%, 4.86%, 2.67% and 10.36%. These results are shown in Table 1, and various components in the extraction accelerator formulation have the function of significantly reducing the nucleic acid Ct value for nucleic acid detection, which proves that the extracted nucleic acid content is significantly increased. The extracted samples were further tested for changes in nucleic acid content by 10-fold serial dilution or 2-fold dilution. The results showed that the nucleic acid extracted from sample A1 could still be detected as positive when diluted 10,000 times, but the control samples A2, A2, The nucleic acids extracted from A3, A4 and B1 can only be detected positive when they are 4000 times, 4000 times, 2000 times and 1000 times (as shown in Figure 1), and they are negative when they are further diluted. These results show that the nucleic acid extracted from the samples with the addition of the extraction accelerator is more than 10 times higher than that of the samples without any extraction accelerator, and 2.5 times and 2.5 times higher than the samples A2, A3 and A4 of the control group added with various extraction accelerators, respectively. times and more than 5 times (as shown in Table 2). These results clearly show that the extraction enhancer can effectively improve the efficiency of RNA detection in the sample, and various components have the function of improving the nucleic acid extraction efficiency to different degrees, and they together have a positive synergistic effect on the nucleic acid extraction effect.

Table 1

Note: Sensitivity is the ratio of the maximum dilution ratio of the samples tested positive, with Ct value being positive and no Ct value being negative.

Table 2: The multiple improvement of the detection sensitivity of the A1 group compared with other control groups

The following experimental operation is to apply the prepared extraction promoter and various extraction promoters to the extraction of new coronavirus nucleic acid, and the new coronavirus particles are replaced by the pseudovirus prepared by us. The specific operation and experimental results are as follows:

1. Take 5 empty sample sampling tubes C1, C2, C3, C4 and D1, add 1.8ml of commercial sample preservation solution to each tube, and then add 200µl volume of extraction accelerator, Extraction accelerator control 1, extraction accelerator control 2 and extraction accelerator control 3, add 200ul of pure water to the D1 tube as a control.

2. Take a sampling tube E containing 6 ml of sample preservation solution, and use a sampling swab to collect pharyngeal samples from two healthy people. The collected throat swabs were fully stirred in the preservation solution, and 200 ul of pseudovirus solution was added at the same time. After thorough mixing, 1 ml of the liquid in tube E was added to tubes C1, C2, C3, C4 and D1 respectively.

3. Since it usually takes 1-4 hours from sample collection to sample delivery to the testing laboratory, in order to simulate this process time, put all the sampling tubes into which the samples are added at room temperature for 24 hours, and then take 200ul from each tube. The nucleic acid in the sample was extracted by magnetic bead method (the kit was purchased from Shuo Shi Company).

4. After the sample was extracted in the automatic extractor, 5µl was taken from the eluate for fluorescence RT-PCR detection. The detection kit was purchased from Wuhan Mingde Company. The effect of detection was evaluated by the Ct values of the new coronavirus N gene and ORF1ab gene in the sample.

The results show (Tables 3 and 4 below), in the sample C1 added with the extraction promoter, the Ct values of the N gene and ORF1ab gene were higher than those of the extraction promoter control group C2, C3, C4 and the control without any extraction promoter. Group D1 average reduced by 14.93%, 6.34%, 5.59%, and 2.1%. The CT values of the ORF1AB gene were reduced by 13.34%, respectively, and the control of the D1 group without any extraction promotion agent, respectively. 7.41%, 6% and 2.35%.

The extracted nucleic acid of the new coronavirus pseudovirus was diluted 10-fold or 2-fold, and the content of the extracted viral nucleic acid was further analyzed. The results showed that the nucleic acids N and ORF1ab genes extracted from samples C1, C2, C3, C4 and D1 were positive. The maximum dilution ratios of the samples were 12,000, 4,000, 4,000, 2,000 and 1,000 times respectively (as shown in Figure 2). The C1 group with the addition of the extraction promoter was better than the extraction promoter control group C2, C3, C4 and without any extraction promoter. The sample nucleic acids extracted from the D1 group were increased by 3 times, 3 times, 6 times and 12 times respectively (as shown in Table 5). These results show that the nucleic acid extraction accelerator can effectively improve the detection efficiency of 2019-nCoV nucleic acid in samples. Moreover, various components have the function of improving the extraction efficiency of new coronavirus nucleic acid to varying degrees, and together they have a positive synergistic effect on the extraction effect of viral nucleic acid.

table 3

Note: Sensitivity is the ratio of the maximum dilution ratio of the samples tested positive, with Ct value being positive and no Ct value being negative.

Claims (7)

1. An accelerant for extracting nucleic acid of a new coronavirus, which is characterized by comprising the following components: reducing agents, detergents and phosphate buffer solutions; the reducing agent is DTT; the detergent is SDS.

2. The enhancer of claim 1, wherein the phosphate buffer is disodium hydrogen phosphate.

3. Accelerator according to claim 1, characterized in that the composition of the accelerator is as follows: 0.1-10mM DTT, 0.5-5% SDS and 0.1-1M disodium hydrogen phosphate, pH 7.2.

4. Accelerator according to claim 1, characterized in that the composition of the accelerator is as follows: 10mM DTT, 5% SDS by mass and 1M disodium hydrogen phosphate, pH 7.2.

5. Accelerator according to claim 1, characterized in that the composition of the accelerator is as follows: 0.1mM DTT, 0.5% SDS by mass and 0.1M disodium hydrogen phosphate, pH 7.2.

6. Use of the promoter of any one of claims 1 to 5 for the preparation of a kit for the extraction of new coronavirus RNA.

7. Use of the promoter according to any one of claims 1 to 5 for increasing the efficiency of extraction of nucleic acid from a novel coronavirus.

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