阅读说明：本技术 一种常温保护液及其配制方法与应用 (Normal-temperature protection solution and preparation method and application thereof ) 是由 安娜 宋飞 于 2020-04-30 设计创作，主要内容包括：本发明公开了一种常温保护液,包括1.0～1.5g氯化钠、0.03～0.06g氯化钾、0.01～0.03g氯化钙、0.1～0.6ml吐温-20、2～8Mol盐酸胍、10～50mMol Tris-HCl及50～100ml无菌水。常温保护液的配制方法,包括(1)将1.0～1.5g氯化钠、0.03～0.06g氯化钾、0.01～0.03g氯化钙加入50～100ml无菌水中,混合均匀配制成溶解液；(2)再依次向溶解液中加入0.1～0.6ml吐温-20、2～8Mol盐酸胍及10～50mMol Tris-HCl,混合均匀后得到常温保护液。上述的常温保护液作为常温条件下储运DNA病原样品的保护液的应用。(The invention discloses a normal-temperature protection solution which comprises 1.0-1.5 g of sodium chloride, 0.03-0.06 g of potassium chloride, 0.01-0.03 g of calcium chloride, 0.1-0.6 ml of Tween-20, 2-8 Mol of guanidine hydrochloride, 10-50 mMol of Tris-HCl and 50-100 ml of sterile water. A preparation method of a normal-temperature protection solution comprises (1) adding 1.0-1.5 g of sodium chloride, 0.03-0.06 g of potassium chloride and 0.01-0.03 g of calcium chloride into 50-100 ml of sterile water, and uniformly mixing to prepare a solution; (2) and sequentially adding 0.1-0.6 ml of Tween-20, 2-8 Mol of guanidine hydrochloride and 10-50 mMol of Tris-HCl into the dissolved solution, and uniformly mixing to obtain the normal-temperature protection solution. The normal-temperature protective solution is applied as a protective solution for storing and transporting DNA pathogen samples at normal temperature.)

1. A normal temperature protective solution is characterized by comprising:

1.0 to 1.5g of sodium chloride, 0.03 to 0.06g of potassium chloride, 0.01 to 0.03g of calcium chloride, 0.1 to 0.6ml of Tween-20, 2 to 8Mol of guanidine hydrochloride, 10 to 50ml of Tris-HCl and 50 to 100ml of sterile water.

2. A room temperature protective solution according to claim 1, wherein the room temperature is 4 to 37 ℃.

3. A preparation method of a normal-temperature protection solution is characterized by comprising the following specific implementation steps:

(1) respectively adding 1.0-1.5 g of sodium chloride, 0.03-0.06 g of potassium chloride and 0.01-0.03 g of calcium chloride into 50-100 ml of sterile water, fully dissolving and uniformly mixing to prepare a dissolving solution;

(2) and (2) sequentially adding 0.1-0.6 ml of Tween-20, 2-8 Mol of guanidine hydrochloride and 10-50 ml of Tris-HCl into the dissolved solution obtained in the step (1), and uniformly mixing to obtain the normal-temperature protection solution.

4. The preparation method of a room temperature protective solution according to claim 3, wherein the room temperature is 4 to 37 ℃.

5. Use of the ambient protective fluid according to any one of claims 1 to 4 as a protective fluid for storage and transportation of DNA pathogen samples at ambient conditions.

6. The use according to claim 5, wherein the DNA pathogen sample is swine poultry oral fluid.

7. The application of claim 6, wherein when the normal-temperature protective solution is applied as a protective solution for storing and transporting the oral liquid of the pigs and the poultry at normal temperature, the oral liquid of the pigs and the poultry and the normal-temperature protective solution are directly mixed in equal volume, and then the mixture can be stored or transported at normal temperature.

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a normal-temperature protection solution as well as a preparation method and application thereof.

Background

The pig oral liquid is a mixed liquid consisting of saliva and serum exudate in the pig oral cavity. Saliva is a mixed liquid secreted by large and small salivary glands, is colorless, tasteless and approximately neutral (pH 6.6-7.1), wherein the water accounts for about 99 percent, and the rest components are mainly mucin, amylase, lysozyme, lipase, proline-rich glycoprotein and the like; serum exudate is the liquid that enters the oral cavity through the oral mucosa and gingival sulcus and capillaries on the oral mucosa. The oral liquid has wide biological functions, and the oral liquid sample can be used for antibody detection and PCR detection of antigens, can replace a serum sample to a certain extent, and provides a new technical means for monitoring the infectious diseases of swinery.

Pig oral fluid samples are widely applied to detection of pathogens by a PCR method of a plurality of epidemic diseases or determination of antibodies by an Elisa method and the like so as to evaluate real-time health dynamics of swinery. Kosher (Corthier) first reported collecting test swine oral fluid infected with swine fever virus to detect swine antibodies, and then many scholars successively reported using the swine oral fluid to detect antibodies or antigens of swine infectious diseases such as swine colibacillosis, infectious pleuropneumonia, swine transmissible gastroenteritis, swine reproductive and respiratory syndrome, foot and mouth disease, swine pseudorabies, influenza a, swine campylobacter jejuni, streptococcosis, etc., to confirm the infection status of a herd of pigs. The oral liquid of the pig is used as a pathogenic sample to analyze and detect various diseases of the swinery, and provides necessary guarantee for the diagnosis and control of the diseases of the pig.

The swine oral liquid can be used for early diagnosis, and viremia of certain swine infectious diseases exists in the swine oral liquid before the infected swine has clinical symptoms, so the swine oral liquid can be used as a pathogen of infectious diseases such as foot-and-mouth disease, porcine pseudorabies and the like in a latent stage or a subclinical stage for detection, and the infection condition is disclosed early. Similarly, some bacterial infectious diseases, such as gastric ulcer caused by spirochete, usually have lesions observed after necropsy, and can also be diagnosed in advance by collecting oral liquid of pigs for PCR detection, so as to effectively prevent and control the infectious diseases.

Since the advent of the Polymerase Chain Reaction (PCR) technology in 1985, the PCR technology has been widely used in many fields such as medicine and molecular biology due to its high sensitivity, strong specificity and rapid detection speed. The accuracy of the PCR result depends greatly on the quality of the nucleic acid of the sample to be tested. However, in the process of collecting, transporting and storing the sample for PCR detection, the quality of the detected nucleic acid is affected by human and objective factors, such that the quality of the detected nucleic acid is affected by nucleic acid degradation, and false negative of the PCR detection result is caused, so the storage and transportation conditions after the sample collection are particularly important.

Currently, most of the existing methods for preserving samples mainly comprise direct low-temperature preservation after sample collection, such as: dry ice, liquid nitrogen, ice bags and the like must be kept at low temperatures for storage and transportation.

The method for preserving at low temperature has great limitation, namely, the low-temperature preservation cost is high; secondly, the limitation that the sustainable low-temperature condition of the sample is guaranteed for the sample needing to be transported for a long time or a long distance is embodied, such as: the liquid nitrogen is adopted for preservation, and the requirement of special equipment of a liquid nitrogen tank is met; volatilizing the dry ice; melting of ice bags, etc. If the freshly collected sample cannot be immediately stored at low temperature, the nucleic acid molecules may be degraded, and other factors may change the state of the nucleic acid; or improper storage in the transportation process can bring operation errors to subsequent experiments and influence the final experiment results.

In addition, although there are some sample protective solutions at normal temperature, such as the chinese patent application CN105145545A, "nucleic acid protective solution for long-term storage and transportation of tissue samples at normal temperature", this patent application relates to a nucleic acid protective solution and its application, and in particular relates to a nucleic acid protective solution and its application in storage and transportation of tissue samples at normal temperature. Although the protective solution disclosed in this patent is also used for protecting nucleic acid in a sample, it is mainly directed to a tissue sample and is only suitable for normal-temperature storage and transportation of neuroblastoma tissue in humans and mammals other than humans. In addition, in addition to the tissue sample protective solution described in the above patent, some other nucleic acid DNA sample protective solutions are easy to delaminate after use, which results in a decrease in DNA recovery rate and affects the accuracy of the detection result; or the used protective solution has poor protective effect on the sample at normal temperature, and cannot fully protect the nucleic acid DNA of the sample.

Disclosure of Invention

The purpose of the invention is as follows: the present invention has been made in view of the above problems occurring in the prior art, and it is a first object of the present invention to disclose a protective solution for ordinary temperature, which can effectively protect clinical samples of DNA pathogens at ordinary temperature. The second purpose of the invention is to disclose a preparation method of the normal temperature protection solution. The third purpose of the invention is to disclose the application of the normal temperature protective solution.

The protective solution for storing and transporting DNA pathogen samples at normal temperature can be provided, a large number of collected DNA pathogen samples can be quickly and simply treated, and can be directly and uniformly mixed with the protective solution of the samples in an equal volume and then stored or transported at normal temperature, so that the collected samples can be effectively prevented from being polluted and corrupted, the storage time of the DNA pathogen samples can be prolonged better, and the samples can be conveniently stored and transported; the preparation method of the DNA pathogen sample protection solution is simple and rapid, is suitable for treating various DNA pathogen samples, and is simple to operate and convenient to store.

The technical scheme is as follows: an ambient temperature protective solution comprising:

1.0 to 1.5g of sodium chloride, 0.03 to 0.06g of potassium chloride, 0.01 to 0.03g of calcium chloride, 0.1 to 0.6ml of Tween-20, 2 to 8Mol of guanidine hydrochloride, 10 to 50mMol of Tris-HCl and 50 to 100ml of sterile water.

Further, the normal temperature is 4-37 ℃.

A preparation method of a normal-temperature protection solution comprises the following specific implementation steps:

(1) respectively adding 1.0-1.5 g of sodium chloride, 0.03-0.06 g of potassium chloride and 0.01-0.03 g of calcium chloride into 50-100 ml of sterile water, fully dissolving and uniformly mixing to prepare a dissolving solution;

(2) and (2) sequentially adding 0.1-0.6 ml of Tween-20, 2-8 Mol of guanidine hydrochloride and 10-50 mMol of Tris-HCl into the dissolved solution obtained in the step (1), and uniformly mixing to obtain the normal-temperature protection solution.

Further, the normal temperature is 4-37 ℃.

The normal-temperature protective solution is applied as a protective solution for storing and transporting DNA pathogen samples at normal temperature.

Further, the DNA pathogen sample is oral liquid of pigs and poultry. The normal-temperature protection solution disclosed by the invention can be stabilized at normal temperature, and the degradation of total DNA is effectively avoided, so that the integrity of DNA molecules is protected.

Further, when the normal-temperature protection solution is applied as a protection solution for storing and transporting the oral liquid of the pigs and the poultry at the normal temperature, the oral liquid of the pigs and the poultry and the normal-temperature protection solution are directly mixed in equal volume, and then the mixture can be stored or transported at the normal temperature.

Has the advantages that: the invention discloses a normal temperature protective solution and a preparation method and application thereof, and the normal temperature protective solution has the following beneficial effects:

1. the normal-temperature protective solution can effectively prevent the collected sample from being polluted and corrupted, better prolongs the storage time of the DNA pathogen sample and is convenient for the storage and transportation of the sample;

2. the preparation method of the normal-temperature protection solution is simple and rapid;

3. the normal temperature protection solution is suitable for treating the DNA pathogen sample of the oral liquid of the pigs and the poultry, and has simple operation and convenient preservation.

Drawings

FIG. 1 is a detection diagram of a normal temperature protective solution and a sample mixed solution, and PBS and the sample mixed solution which are respectively preserved for 0d, 1d, 3d and 7d under the condition of 4 ℃;

FIG. 2 is a detection diagram of a normal temperature protective solution and a sample mixed solution, and PBS and the sample mixed solution which are respectively preserved for 0d, 1d, 3d and 7d under the condition of 27 ℃;

FIG. 3 is a detection diagram of the mixed solution of the normal temperature protective solution and the sample and the mixed solution of the PBS and the sample after being respectively preserved for 0d, 1d, 3d and 7d at 37 ℃;

wherein: od indicates that sampling was performed as an initial control immediately after formulation was completed.

The specific implementation mode is as follows:

the following describes in detail specific embodiments of the present invention.

The present invention will now be further described by describing in detail preferred embodiments of the DNA pathogen sample preservation solution according to the present invention. The present invention is further described below with reference to examples, but is not limited to the scope of the present invention. The experimental reagents used in the following examples are commercially available without specific reference.