CN110684869B - Nucleic acid preservation solution and application thereof, and quality control product containing nucleic acid preservation solution - Google Patents
Nucleic acid preservation solution and application thereof, and quality control product containing nucleic acid preservation solution Download PDFInfo
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Abstract
The invention relates to the technical field of fine nucleic acid, in particular to a nucleic acid preservation solution and application thereof, and a quality control product containing the nucleic acid preservation solution. The nucleic acid preservation solution provided by the invention can effectively prevent degradation of nucleic acid, provide a stable environment for nucleic acid, prolong the preservation time of nucleic acid, have the advantages of low preparation cost, stable performance, easiness in preservation and the like, can effectively monitor factors such as personnel operation, instrument state, effectiveness of a kit and the like, and greatly improve the accuracy and reliability of a nucleic acid detection kit.
Description
Technical Field
The invention relates to the technical field of nucleic acid, in particular to a nucleic acid preservation solution and application thereof, and a quality control product containing the nucleic acid preservation solution.
Background
Respiratory tract infections (Respiratory tract infection, RTI) are the most common type of disease in humans, can occur in any gender, age and territory, and are one of the leading causes of morbidity and mortality in the population worldwide. Clinical symptoms and signs caused by respiratory tract infection are similar, clinical manifestations of the respiratory tract infection mainly include symptoms such as rhinitis, pharyngitis, laryngitis, tonsillitis and the like, and the respiratory tract infection can seriously cause tracheitis, bronchitis, pneumonia and the like, but infections caused by different pathogens are different in treatment method, curative effect and disease course. It has been demonstrated that most respiratory diseases are caused by bacteria, viruses and other pathogens, with respiratory viruses being the most common. Clinically common respiratory pathogens include influenza a virus, influenza b virus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, adenovirus, respiratory infectious enterovirus (enterovirus/rhinovirus), mycoplasma pneumoniae, chlamydia pneumoniae, coronavirus, bocavirus, legionella pneumophila, and the like.
Currently, nucleic acid detection methods for detecting respiratory pathogens mainly include gene chip amplification, isothermal nucleic acid amplification, and real-time reverse transcription polymerase chain reaction (RT-PCR). Based on the method, a plurality of respiratory pathogen gene detection kits have been developed, but the quality control products of the respiratory pathogen gene detection kits are not sold economically, effectively, stably and stably in the market at present, and the existing nucleic acid quality control products have the defects of instability, easy degradation, difficult preservation and the like, and the quality control products are important methods and quality indexes for measuring and evaluating the detection accuracy, stability and other performances of a kit. Therefore, there is an urgent need to provide a nucleic acid quality control product that can be stably preserved.
Disclosure of Invention
In view of the above, the present invention aims to provide a nucleic acid preserving fluid and an application thereof, and a quality control product containing the same, which can effectively prevent degradation of nucleic acid, provide a stable environment for nucleic acid, prolong preservation time of nucleic acid, and make the quality control product of nucleic acid prepared by using the nucleic acid preserving fluid not easy to degrade and can be stably preserved.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
the invention provides a nucleic acid preservation solution, which is prepared from BSA, PEG8000 and NaN 3 And Tricine-buffer.
In some embodiments, the concentration of each component in the nucleic acid preservation solution is:
0.03～0.05mg/ml BSA、0.3-1wt％PEG8000、2～8wt‰NaN 3 and 20-120 mM Tricine salt buffer.
In some embodiments, the concentration of each component in the nucleic acid preservation solution is: 0.04mg/ml BSA, 0.5wt% PEG8000, 3 wt% NaN 3 And 50mM salt buffer.
In some embodiments, the concentration of each component in the nucleic acid preservation solution is: 0.03mg/ml BSA, 0.3 wt% PEG8000, 2 wt% NaN 3 And 20mM Tricine-salt buffer.
In some embodiments, the concentration of each component in the nucleic acid preservation solution is: 0.05mg/ml BSA, 1 wt% PEG8000, 8 wt% NaN 3 And 120mM Tricine-salt buffer.
In some embodiments, the nucleic acid preservation solution provided by the invention is a nucleic acid of a human respiratory pathogen.
Wherein the human respiratory pathogen comprises at least one of influenza A virus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, chlamydia pneumoniae, adenovirus, legionella pneumophila and human metapneumovirus.
In a specific embodiment, the nucleic acid preservation solution is used for preserving nucleic acid of human respiratory pathogens, the degradation rate of the nucleic acid preserved at-80 ℃ and 37 ℃ for two weeks is less than 1%, and the preservation effect is obviously better than that of a control group. The nucleic acid preservation solution can obviously improve the stability of nucleic acid and effectively prevent degradation of the nucleic acid in the preservation process. The nucleic acid quality control product prepared by the nucleic acid preservation solution is not easy to degrade and has higher stability. Therefore, the invention also provides application of the nucleic acid preservation solution in preparing quality control products for detecting human respiratory pathogens.
The invention also provides a nucleic acid quality control product for detecting human respiratory pathogens, comprising a positive quality control product and a negative quality control product;
the positive quality control product consists of the nucleic acid preservation solution and a nucleic acid sequence of human respiratory pathogens;
the negative quality control product consists of the nucleic acid preservation solution and nucleic acid sequences of non-human respiratory pathogens.
Wherein the human respiratory pathogen nucleic acid sequence is a target gene fragment of a human respiratory pathogen comprising at least one of influenza a virus (FluA), influenza b virus (FluB), respiratory Syncytial Virus (RSV), mycoplasma Pneumoniae (MP), chlamydia Pneumoniae (CP), adenovirus (ADV), legionella Pneumophila (LP) and human metapneumovirus (hMPV).
The nucleic acid sequence of the non-human respiratory pathogen is at least one of beta globin gene and human 18S gene.
The invention also provides a preparation method of the nucleic acid quality control product, which comprises the following steps:
step 1: preparation of a cationic quality control product: (1) obtaining target gene fragments of human respiratory pathogens by PCR amplification; (2) respectively carrying out enzyme digestion on the vector and the target gene fragment, then connecting, converting competent cells, screening to obtain positive colonies of target genes of human respiratory pathogens, and sequencing and verifying; (3) performing ultrasonic induction on a colony with a correct sequencing result to prepare a pseudo-virus solution; (4) extracting nucleic acid of the pseudovirus solution, and adding BSA protein, PEG8000, naN3 and Tricine to obtain a cationic quality control product;
step 2: preparation of negative quality control: (1) PCR amplification to obtain a nucleic acid sequence of the non-human respiratory pathogen; (2) respectively carrying out enzyme digestion, connection and transformation of competent cells on the vector and the nucleic acid sequence, screening to obtain positive colonies containing nucleic acid sequences of the eight human respiratory pathogens, and sequencing and verifying; performing ultrasonic induction on a colony with a correct sequencing result to prepare a pseudo-virus solution; (4) extracting nucleic acid to obtain pseudovirus solution, adding BSA, PEG8000 and NaN 3 And Tricine to obtain the negative quality control product.
Wherein the human respiratory pathogen nucleic acid sequence is a target gene fragment of a human respiratory pathogen comprising at least one of influenza a virus (FluA), influenza b virus (FluB), respiratory Syncytial Virus (RSV), mycoplasma Pneumoniae (MP), chlamydia Pneumoniae (CP), adenovirus (ADV), legionella Pneumophila (LP), and human metapneumovirus (hMPV); the nucleic acid sequence of the non-human respiratory pathogen is at least one of beta globin gene and human 18S gene.
The invention also provides application of the quality control product in preparing a kit for detecting human respiratory pathogens.
The nucleic acid preservation solution provided by the invention consists of BSA, PEG8000 and NaN 3 And Tricine buffer groupAnd (3) forming the finished product. The test result shows that the nucleic acid preservation solution is used for preserving the nucleic acid of human respiratory pathogens, the nucleic acid degradation rate is below 1% after being preserved for two weeks at-80 ℃ and 37 ℃, and the nucleic acid degradation rate is obviously lower than that of each control group. The nucleic acid preservation solution can effectively prevent degradation of nucleic acid in the preservation process, remarkably improve the stability of the nucleic acid and prolong the preservation time. The nucleic acid quality control product prepared by the nucleic acid preservation solution is not easy to degrade, and has higher stability and longer shelf life.
Detailed Description
The invention discloses a nucleic acid preservation solution and application thereof, and a quality control product containing the nucleic acid preservation solution, and a person skilled in the art can refer to the content of the nucleic acid preservation solution and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
The composition of the nucleic acid preservation solution of this example is shown in Table 1:
TABLE 1
Example 2
The nucleic acid quality control product comprises a positive quality control product and a negative quality control product.
The positive quality control comprises influenza a virus (FluA) target gene quality control, influenza b virus (FluB) target gene quality control, respiratory Syncytial Virus (RSV) target gene quality control, mycoplasma Pneumoniae (MP) target gene quality control, chlamydia Pneumoniae (CP) target gene quality control, adenovirus (ADV) target gene quality control, legionella Pneumophila (LP) target gene quality control, and human metapneumovirus (hMPV) gene target quality control. Wherein, the quality control product contains target genes of eight corresponding respiratory pathogens and the nucleic acid preservation solution in the nucleic acid preservation solution formula 1 of the embodiment 1.
The negative quality control product comprises a human beta globin gene quality control product and a humanized 18S gene quality control product. The negative reference contains the corresponding DNA molecule and the nucleic acid preservation solution of the nucleic acid preservation solution formula 1 of example 1, respectively.
The preparation method comprises the following steps:
(1) Preparation of a cationic quality control product: (1) preparation of the respiratory pathogen genes of interest: and designing a conserved primer sequence according to the target respiratory pathogen gene nucleic acid sequence in the NCBI database to synthesize the target gene. PCR amplification was performed using a nucleic acid containing the target gene sequence as a template, and the primer sequences are shown in Table 1. And (3) carrying out agarose gel electrophoresis on the PCR product, cutting the band containing the target gene fragment, and recovering the target gene by using an agarose gel recovery kit. (2) Constructing and verifying a plasmid vector containing a target gene: and (3) respectively carrying out double enzyme digestion on the target gene fragment and the vector by using restriction enzymes, carrying out agarose gel electrophoresis detection on enzyme digestion products, and carrying out gel digestion recovery. And (3) connecting the enzyme digestion products to obtain connection products, then converting the connection products into competent cells, coating the competent cells on an LB culture medium plate, culturing the competent cells in an inverted manner at 37 ℃, and selecting single white positive colonies. Extracting DNA of positive colony, PCR verifying, and sequencing colony positive to target geneSyndrome/pattern. (3) Preparation of a target gene pseudovirus solution: single positive colonies confirmed by sequencing are inoculated into LB liquid medium, after being cultured, the culture medium is centrifuged, and the supernatant is discarded, and sediment is left. Adding an ultrasonic buffer solution (50 mmol/L Tris-HCl, pH 8.0;2mmol/L EDTA; glycerol with a volume fraction of 20%; 100mmol/L NaCl; triton X-100 with a volume fraction of 0.1%) to carry out ice bath ultrasonic treatment under the ultrasonic conditions: 350W, intermittent 5s, ultrasonic 5s,30 cycles. And centrifuging the ultrasonic product at 6000rpm for 10 minutes, and collecting the supernatant to obtain the pseudovirus solution. (4) And (3) preparing a quality control product: DNaseI is added to the pseudovirus solution and the DNA in the solution is digested. Extracting nucleic acid from pseudovirus solution by Trizol extraction method, adding BSA, PEG8000 and NaN 3 And Tricine salt to obtain;
(2) Preparation of negative quality control: (1) preparation of non-target respiratory pathogen genes: and designing a conserved primer sequence according to the nucleic acid sequences of the target respiratory pathogen gene human beta globin gene and the humanized 18S gene in the NCBI database to synthesize the target gene. PCR amplification was performed using a nucleic acid containing the target gene sequence as a template, and the primer sequences are shown in Table 1. And (3) carrying out agarose gel electrophoresis on the PCR product, cutting the band containing the target gene fragment, and recovering the target gene by using an agarose gel recovery kit. (2) Constructing and verifying a plasmid vector containing a target gene: and (3) respectively carrying out double enzyme digestion on the target gene fragment and the vector by using restriction enzymes, carrying out agarose gel electrophoresis detection on enzyme digestion products, and carrying out gel digestion recovery. And (3) connecting the enzyme digestion products to obtain connection products, then converting the connection products into competent cells, coating the competent cells on an LB culture medium plate, culturing the competent cells in an inverted manner at 37 ℃, and selecting single white positive colonies. DNA of positive colonies is extracted, PCR verification is performed, and the colonies positive for the target genes are verified by PCR and sequencing verification is performed. (3) Preparation of a target gene pseudovirus solution: single positive colonies confirmed by sequencing are inoculated into LB liquid medium, after being cultured, the culture medium is centrifuged, and the supernatant is discarded, and sediment is left. Adding ultrasonic buffer solution (50 mmol/L Tris-HCl, pH 8.0;2mmol/L EDTA; glycerol with volume fraction of 20%, 100mmol/L NaCl; triton X-100 with volume fraction of 0.1%) into the solution, performing ice bath ultrasonic treatment under ultrasonic conditionsThe method comprises the following steps: 350W, intermittent 5s, ultrasonic 5s,30 cycles. And centrifuging the ultrasonic product at 6000rpm for 10 minutes, and collecting the supernatant to obtain the pseudovirus solution. (4) And (3) preparing a quality control product: DNaseI is added to the pseudovirus solution and the DNA in the solution is digested. Extracting nucleic acid of pseudovirus solution by Trizol extraction method, adding BSA, PEG8000 and NaN 3 And Tricine salt.
TABLE 2
Example 3
This example investigated the effect of different nucleic acid preservation solutions on preservation of nucleic acids.
1. Design of experiment
Taking the amplification of the first-class target gene sequence in example 2 as an example, the preservation effect of different nucleic acid preservation solutions on nucleic acid is studied, and the stability of quality control products prepared from different preservation solutions and nucleic acid under different conditions (-80 ℃ and 37 ℃) is examined.
Test groups 1 to 30 and control groups 1 to 2 are set, wherein test groups 1 to 4 and 16 to 19 contain a first-class target gene sequence and one additive, test groups 5 to 10 and 20 to 24 contain a first-class target gene sequence and two additives, test groups 11 to 13 and 25 to 27 contain a first-class target gene sequence and three additives, test groups 14 to 15 and 28 to 29 contain a first-class target gene sequence and four additives, and control groups 1 to 2 contain a first-class target gene sequence and DEPC water, and the specific is shown in Table 3.
TABLE 3 experimental design
2. Experimental method
A pseudovirus solution containing a first-class target sequence was prepared as in example 2, nucleic acid of the pseudovirus solution was extracted, and an average of 30 groups was divided, and a preservation solution was added to the nucleic acid solution according to the design of Table 2, to prepare a quality control of each group of first-class target genes. Nucleic acid concentration was determined and recorded for each group of a-flow gene references prior to the start of the experiment. And then, storing each group of the nail-flow gene reference products under corresponding conditions, and respectively measuring the size and the concentration of the nucleic acid fragments of each group of nail-flow target gene quality control products by an Agilent 2100 biological analyzer after 2 weeks, so as to detect the stability and degradation condition of each group of nail-flow target gene quality control products. Wherein, the detection of the 800-810bp nucleic acid fragment indicates that the nail-flow target gene quality control product is not completely degraded, and the degradation rate of the nail-flow target gene quality control product is calculated by using the following formula: percent degradation = (pre-experiment nucleic acid concentration-post-experiment nucleic acid concentration)/pre-experiment nucleic acid concentration x 100%.
2. Experimental results
After 2 weeks, the control of the nail flow target gene was tested, and specific experimental results of experimental groups 1 and 2 and control groups 1 and 2 are shown in table 4.
Table 4 Experimental results
As can be seen from the above experimental results, the quality control products (experimental groups 14 and 29) of the present invention have nucleic acid degradation rates of 0.12% and 0.38% at-80℃and 37℃respectively, which are lower than 1%. Under the same preservation conditions, the degradation rate of the nucleic acid in the quality control products of the control groups 1-2 is far higher than that of the quality control products (test group 14 and test group 29) of the invention, and especially under the preservation condition of 37 ℃, the nucleic acid solution in the control group 2 is completely degraded (degradation rate 100%) after 2 weeks and far higher than that of the invention (0.38%). The degradation rate of the nucleic acid of other test group quality control products is between 2.57 and 24.79 percent under the preservation condition of 37 ℃, and is obviously higher than that of the quality control product of the invention.
The preservation effect of the nucleic acid preservation solutions of the formulas 2 and 3 of the example 1 on the amplification of the first-class target gene sequence of the example 2 was examined according to the method of the example 3, and the results show that the nucleic acid degradation rates of the nucleic acid preservation solution of the formula 2 under the conditions of-80 ℃ and 37 ℃ are 0.15% and 0.42%, respectively, and the nucleic acid degradation rates of the nucleic acid preservation solution of the formula 3 under the conditions of-80 ℃ and 37 ℃ are 0.17% and 0.67%, respectively, and are lower than 1%.
The results show that the nucleic acid preservation solution can effectively prevent degradation of nucleic acid in the preservation process, remarkably improve the stability of the nucleic acid and prolong the preservation time, and the nucleic acid quality control product prepared by using the nucleic acid preservation solution is not easy to degrade, and has higher stability and longer shelf life.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. A nucleic acid preserving fluid comprising BSA, PEG8000 and NaN 3 And Tricine buffer; the concentration of each component in the nucleic acid preservation solution is as follows:
0.03～0.05mg/ml BSA、0.3-1wt％PEG8000、2～8wt‰NaN 3 and 20-120 mM Tricine salt buffer.
2. The nucleic acid storage solution according to claim 1, wherein the nucleic acid is a nucleic acid of a human respiratory pathogen.
3. Use of the nucleic acid preservation solution according to any one of claims 1-2 for the preparation of a nucleic acid quality control for detecting human respiratory pathogens.
4. A nucleic acid quality control for detecting a human respiratory pathogen, comprising a positive quality control and a negative quality control;
the positive quality control product consists of the nucleic acid preservation solution according to any one of claims 1-2 and a nucleic acid sequence of a human respiratory pathogen;
the negative quality control product consists of the nucleic acid preservation solution according to any one of claims 1 to 2 and a nucleic acid sequence of a non-human respiratory pathogen.
5. The nucleic acid quality control product of claim 4, wherein the human respiratory pathogen nucleic acid sequence is a target gene fragment of a human respiratory pathogen comprising at least one of influenza a virus (FluA), influenza b virus (FluB), respiratory Syncytial Virus (RSV), mycoplasma Pneumoniae (MP), chlamydia Pneumoniae (CP), adenovirus (ADV), legionella Pneumophila (LP), and human metapneumovirus (hMPV).
6. The nucleic acid quality control product according to claim 4, wherein the nucleic acid sequence of the non-human respiratory pathogen is at least one of a beta globin gene and a human 18S gene.
7. The method for producing a nucleic acid quality control product according to any one of claims 4 to 6, comprising the steps of:
step 1: preparation of a cationic quality control product: (1) obtaining target gene fragments of human respiratory pathogens by PCR amplification; (2) respectively carrying out enzyme digestion on the vector and the target gene fragment, then connecting, converting competent cells, screening to obtain positive colonies of target genes of human respiratory pathogens, and sequencing and verifying; (3) performing ultrasonic induction on a colony with a correct sequencing result to prepare a pseudo-virus solution; (4) extracting nucleic acid of the pseudovirus solution, and adding BSA protein, PEG8000, naN3 and Tricine to obtain a cationic quality control product;
step 2: preparation of negative quality control: (1) PCR amplification to obtain a nucleic acid sequence of the non-human respiratory pathogen; (2) respectively carrying out enzyme digestion, connection and transformation of competent cells on the vector and the nucleic acid sequence, screening to obtain positive colonies containing nucleic acid sequences of the eight human respiratory pathogens, and sequencing and verifying; performing ultrasonic induction on a colony with a correct sequencing result to prepare a pseudo-virus solution; (4) extracting nucleic acid to obtain pseudovirus solution, adding BSA, PEG8000 and NaN 3 And Tricine to obtain the negative quality control product.
8. The method of claim 7, wherein the human respiratory pathogen nucleic acid sequence is a target gene fragment of a human respiratory pathogen comprising at least one of influenza a virus (FluA), influenza b virus (FluB), respiratory Syncytial Virus (RSV), mycoplasma Pneumoniae (MP), chlamydia Pneumoniae (CP), adenovirus (ADV), legionella Pneumophila (LP), and human metapneumovirus (hMPV); the nucleic acid sequence of the non-human respiratory pathogen is at least one of beta globin gene and human 18S gene.
9. Use of a quality control product according to any one of claims 4 to 6 for the preparation of a kit for detecting human respiratory pathogens.
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| CN108998569A (en) * | 2018-08-13 | 2018-12-14 | 郑州安图生物工程股份有限公司 | A kind of hepatitis type B virus serum sample saves dilution and preparation method thereof |
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