阅读说明：本技术 一种呼吸道病毒核酸采样试剂盒及其应用 (Respiratory virus nucleic acid sampling kit and application thereof ) 是由 李刚 尹天武 汪珂 于 2020-07-23 设计创作，主要内容包括：本发明提供一种呼吸道病毒核酸采样试剂盒及其应用,该试剂盒包括鼻喷剂和样本保存液,鼻喷剂按照重量百分数的组成包括：0.9％-3％的氯化钠,0.1％-0.5％的丙酮酸钠,0.01％-0.1％的丙酮酸,0.01％-0.1％的吐温80,0.01％-0.1％的单硬脂酸甘油酯,余量为无菌水；所述样本保存液按照重量百分数的组成包括：0.08-0.12％的葡萄糖,0.02-0.03％的氯化钙,0.03-0.05％的氯化钾,0.01-0.015％的磷酸氢二钠,0.01-0.015％的异亮氨酸,0.01-0.015％的亮氨酸,0.01-0.015％的盐酸赖氨酸,余量为生理盐水。本发明可以有效提高呼吸道病毒核酸检出率。(The invention provides a respiratory virus nucleic acid sampling kit and application thereof, wherein the kit comprises a nasal spray and a sample preservation solution, and the nasal spray comprises the following components in percentage by weight: 0.9 to 3 percent of sodium chloride, 0.1 to 0.5 percent of sodium pyruvate, 0.01 to 0.1 percent of pyruvic acid, 0.01 to 0.1 percent of Tween 80, 0.01 to 0.1 percent of glycerin monostearate and the balance of sterile water; the sample preservation solution comprises the following components in percentage by weight: 0.08-0.12% of glucose, 0.02-0.03% of calcium chloride, 0.03-0.05% of potassium chloride, 0.01-0.015% of disodium hydrogen phosphate, 0.01-0.015% of isoleucine, 0.01-0.015% of leucine, 0.01-0.015% of lysine hydrochloride and the balance of physiological saline. The invention can effectively improve the detection rate of the nucleic acid of the respiratory virus.)

1. A sampling kit for detecting respiratory virus nucleic acid, which is characterized in that: the nasal spray comprises a nasal spray and a first sample preservation solution, wherein the nasal spray comprises the following components in percentage by weight: 0.9 to 3 percent of sodium chloride, 0.1 to 0.5 percent of sodium pyruvate, 0.01 to 0.1 percent of pyruvic acid, 0.01 to 0.1 percent of Tween 80, 0.01 to 0.1 percent of glycerin monostearate and the balance of sterile water; the sample preservation solution I comprises the following components in percentage by weight: 0.08-0.12% of glucose, 0.02-0.03% of calcium chloride, 0.03-0.05% of potassium chloride, 0.01-0.015% of disodium hydrogen phosphate, 0.01-0.015% of isoleucine, 0.01-0.015% of leucine, 0.01-0.015% of lysine hydrochloride and the balance of sterile physiological saline.

2. A sampling kit for detecting respiratory virus nucleic acid, which is characterized in that: the nasal spray comprises a nasal spray and a sample preservation solution II, wherein the nasal spray comprises the following components in percentage by weight: 0.9 to 3 percent of sodium chloride, 0.1 to 0.5 percent of sodium pyruvate, 0.01 to 0.1 percent of pyruvic acid, 0.01 to 0.1 percent of Tween 80, 0.01 to 0.1 percent of glycerin monostearate and the balance of sterile water; the second sample preservation solution comprises ethanol, guanidine isothiocyanate, DTT, ethylene diamine tetraacetic acid, glycogen and sterile water, wherein the volume fraction of the ethanol in the second sample preservation solution is 50% -80%, the concentration of the guanidine isothiocyanate is 0.2-2M, the concentration of the DTT is 1-10mM, the concentration of the ethylene diamine tetraacetic acid is 1-10mM, and the concentration of the glycogen is 10-100 mu g/ml.

3. The sampling kit for detecting a respiratory virus nucleic acid according to claim 1 or 2, characterized in that: the preparation method of the nasal spray comprises the following steps: the nasal spray comprises the ingredients according to the weight percentage, and is prepared by dissolving sodium chloride, sodium pyruvate, pyruvic acid, tween 80 and glyceryl monostearate in sterile water.

4. The sampling kit for detecting a respiratory virus nucleic acid according to claim 1, characterized in that: the preparation method of the first sample preservation solution comprises the following steps: the sample preservation solution is prepared by dissolving glucose, calcium chloride, potassium chloride, disodium hydrogen phosphate, isoleucine, leucine and lysine hydrochloride in sterile physiological saline according to the weight percentage of the sample preservation solution.

5. The sampling kit for detecting a respiratory virus nucleic acid according to claim 2, characterized in that: the preparation method of the second sample preservation solution comprises the following steps: and (3) preparing the ingredients according to the concentration of the components contained in the sample preservation solution II, and dissolving ethanol, guanidinium isothiocyanate, DTT, ethylene diamine tetraacetic acid and glycogen into sterile water to obtain the product.

6. A sampling method for detecting a nucleic acid of a respiratory virus, comprising: comprising the use of a sampling kit according to any one of claims 1 to 5.

7. The sampling method for detecting a nucleic acid of a respiratory virus according to claim 6, wherein: the sampling method specifically comprises the following steps: continuously spraying the nasal spray from the nose until the nasal secretion is driven to flow to the oropharynx; and (3) spitting the mixed liquid of the oropharynx part into the first sample preservation liquid or the second sample preservation liquid for preservation.

8. The sample for detecting respiratory virus nucleic acid according to claim 7The method is characterized in that: when the mixed liquid of the oropharynx part is preserved by adopting the sample preservation liquid, the method further comprises the following steps: placing the sample-containing preservation solution at 37 deg.C and CO₂And culturing for 20-30 h in an incubator with the concentration of 5%.

9. Use of the sampling kit according to any one of claims 1 to 5, or the sampling method according to any one of claims 6 to 8, for preserving respiratory viral nucleic acids.

10. Use of the sampling kit according to any one of claims 1 to 5, or the sampling method according to any one of claims 6 to 8, for aiding the detection of respiratory virus nucleic acids.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a respiratory virus nucleic acid sampling kit and application thereof.

Background

Respiratory viral infections are the most common mode of infection causing large-scale public health incidents. The new crown epidemic situation continuously brings huge disasters to the world. According to the diagnosis and treatment scheme for novel coronavirus pneumonia published by the national health committee, virus nucleic acid detection is the main diagnosis and treatment standard.

However, the detection rate of the nucleic acid of the new coronavirus is only 30% -50%, which means that 50-70% of infected persons are missed, and these false negative infected persons become a mobile infection source and are a great hidden danger of epidemic prevention and control. The conventional nasopharyngeal swab and oropharyngeal swab sampling methods have insufficient sample collection amount, and are key factors causing missed detection.

Disclosure of Invention

In view of the above, it is necessary to provide a sampling kit for detecting nucleic acid of respiratory virus, which can rapidly and simply increase the sampling amount of virus, and the application thereof, in view of the problems in the prior art. The technical scheme of the invention is as follows:

the invention provides a sampling kit for detecting respiratory virus nucleic acid, which comprises a nasal spray and a first sample preservation solution, wherein the nasal spray comprises the following components in percentage by weight: 0.9 to 3 percent of sodium chloride, 0.1 to 0.5 percent of sodium pyruvate, 0.01 to 0.1 percent of pyruvic acid, 0.01 to 0.1 percent of Tween 80, 0.01 to 0.1 percent of glycerin monostearate and the balance of sterile water; the sample preservation solution I comprises the following components in percentage by weight: 0.08-0.12% of glucose, 0.02-0.03% of calcium chloride, 0.03-0.05% of potassium chloride, 0.01-0.015% of disodium hydrogen phosphate, 0.01-0.015% of isoleucine, 0.01-0.015% of leucine, 0.01-0.015% of lysine hydrochloride and the balance of sterile physiological saline.

Further, the preparation method of the nasal spray comprises the following steps: the nasal spray comprises the ingredients according to the weight percentage, and is prepared by dissolving sodium chloride, sodium pyruvate, pyruvic acid, tween 80 and glyceryl monostearate in sterile water.

Further, the preparation method of the first sample preservation solution comprises the following steps: the sample preservation solution is prepared by dissolving glucose, calcium chloride, potassium chloride, disodium hydrogen phosphate, isoleucine, leucine and lysine hydrochloride in sterile physiological saline according to the weight percentage of the sample preservation solution.

In a second aspect, the invention also provides a sampling kit for detecting the nucleic acid of the respiratory virus, which comprises a nasal spray and a second sample preservation solution, wherein the nasal spray comprises the following components in percentage by weight: 0.9 to 3 percent of sodium chloride, 0.1 to 0.5 percent of sodium pyruvate, 0.01 to 0.1 percent of pyruvic acid, 0.01 to 0.1 percent of Tween 80, 0.01 to 0.1 percent of glycerin monostearate and the balance of sterile water; the second sample preservation solution comprises ethanol, guanidine isothiocyanate, DTT, ethylene diamine tetraacetic acid, glycogen and sterile water, wherein the volume fraction of the ethanol in the second sample preservation solution is 50% -80%, the concentration of the guanidine isothiocyanate is 0.2-2M, the concentration of the DTT is 1-10mM, the concentration of the ethylene diamine tetraacetic acid is 1-10mM, and the concentration of the glycogen is 10-100 mu g/ml.

Further, the preparation method of the nasal spray comprises the following steps: the nasal spray comprises the ingredients according to the weight percentage, and is prepared by dissolving sodium chloride, sodium pyruvate, pyruvic acid, tween 80 and glyceryl monostearate in sterile water.

Further, the preparation method of the second sample preservation solution comprises the following steps: and (3) preparing the ingredients according to the concentration of the components contained in the sample preservation solution II, and dissolving ethanol, guanidinium isothiocyanate, DTT, ethylene diamine tetraacetic acid and glycogen into sterile water to obtain the product.

In a third aspect, the present invention also provides a sampling method for detecting respiratory virus nucleic acid, which comprises using one of the two sampling kits described above.

Further, the sampling method specifically includes: continuously spraying the nasal spray from the nose until the nasal secretion is driven to flow to the oropharynx; and (3) spitting the mixed liquid of the oropharynx part into the first sample preservation liquid or the second sample preservation liquid for preservation.

Further, when the mixed liquid of the oropharynx part is preserved by using the sample preservation liquid, the method further comprises the following steps: placing the sample-containing preservation solution at 37 deg.C and CO₂And culturing for 20-30 h in an incubator with the concentration of 5%.

In a fourth aspect, the present invention provides the use of the above-described sampling kit and the above-described sampling method for preserving respiratory virus nucleic acid.

In a fifth aspect, the invention provides the use of the above sampling kit and the above sampling method in assisted respiratory virus nucleic acid detection.

The invention has the beneficial effects that:

the nasal spray component can wet the nasal mucosa surface, dilute and liquefy nasal mucus, stimulate the ciliary movement of the nasal mucosa and facilitate the discharge of foreign matters including viruses in the nasal cavity.

Secondly, the sample preservation solution can keep the activity of sample cells, viruses can be amplified in vitro on living cells, and the detection success rate is improved.

And thirdly, the sample preservation solution II can effectively inactivate viruses and completely preserve virus nucleic acids, so that the extraction of the virus nucleic acids is facilitated.

The sampling range of the sampling method of the invention comprises all nasal cavity parts, pharyngeal parts and oral parts, and compared with the sampling of the pharyngeal swab and the nasal swab which are mainstream at present, the sampling range is wider, which is beneficial to improving the virus content of the sampling and further improving the nucleic acid detection rate.

The sampling method is simple, different from the situation that sampling of a nose swab and a throat swab needs operation of professional medical care personnel, a patient can sample independently, on one hand, sampling is more convenient and faster, sampling discomfort of the patient is reduced, on the other hand, operation of the medical care personnel is avoided, and therefore infection risk of the medical care personnel is reduced.

Drawings

FIG. 1 is a nucleic acid amplification curve detected after sampling in example 5 of the present invention using the kit of example 2.

FIG. 2 is a nucleic acid amplification curve detected after sampling with a nasal swab in example 5 of the present invention.

FIG. 3 is a nucleic acid amplification curve detected after sampling in example 11 of the present invention using the kit of example 7.

Detailed Description

In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.