CN115747176B

CN115747176B - Virus sample preservation solution capable of being preserved at normal temperature and inactivating viruses and detection method

Info

Publication number: CN115747176B
Application number: CN202211362154.6A
Authority: CN
Inventors: 韩雪娟; 陈海波
Original assignee: Hantech Medical Device Co ltd
Current assignee: Hantech Medical Device Co ltd
Priority date: 2022-11-02
Filing date: 2022-11-02
Publication date: 2023-10-10
Anticipated expiration: 2042-11-02
Also published as: CN115747176A

Abstract

The invention belongs to the field of virus sample preservation solutions, and particularly relates to a virus sample preservation solution capable of being preserved at normal temperature and inactivating viruses and a detection method. The virus sample preservation solution can ensure that the sample is kept in a stable manner at normal temperature and simultaneously ensures that nucleic acid is complete, the sample is not required to be transported by a cold chain, stored by an ultralow temperature refrigerator or liquid nitrogen, and the sample is kept fast and economically.

Description

Virus sample preservation solution capable of being preserved at normal temperature and inactivating viruses and detection method

Technical Field

The invention belongs to the field of virus sample preservation solutions, and particularly relates to a virus sample preservation solution capable of being preserved at normal temperature and inactivating viruses and a detection method.

Background

Outbreaks of new coronavirus epidemic have become a major public health problem and the burden of disease detection is increasing. Molecular diagnostic products, represented by RT-PCR technology, have become an important detection tool for the diagnosis of novel coronavirus infections. Sample preservation is one of the very critical but easily neglected factors, and sample preservation of a particular need is a blind spot.

"false positives" have a great relationship with sample quality. In clinical testing work, a number of problems arise due to improper preservation of virus samples: the virus activity is greatly affected by temperature, cold chain transportation and preservation are needed, and the titer of the virus can be reduced by repeated freezing and thawing; the novel coronavirus is a single-stranded RNA virus, the virus shell is easy to break, and released nucleic acid is easy to degrade and pollute due to the fact that a sample contains a large amount of enzyme substances, so that the problems of false negative caused by pathogen missed detection and insufficient sensitivity occur.

The conventional common preservation solution can not ensure that the sample is stable and not degraded, and needs to be preserved and transported at low temperature. And there is virus sample and can't inactivate rapidly and cause the sample to cause the secondary to spread and cause the extremely high risk of infection to medical personnel when preserving, transporting and uncapping and detecting.

Disclosure of Invention

The invention aims to solve the problems that the conventional common preservation solution in the prior art cannot ensure that a sample is stable and not degraded and needs low-temperature preservation and transportation. Moreover, the technical problem that the virus sample cannot be quickly inactivated exists, and the virus sample preservation solution capable of being preserved at normal temperature and inactivating the virus and the detection method are provided.

The technical scheme adopted by the invention is as follows:

the invention provides a virus sample preservation solution capable of preserving at normal temperature and inactivating viruses, which comprises, by weight, 40-80 parts of guanidine isothiocyanate or guanidine hydrochloride, 2-8 parts of sodium citrate or sodium bicarbonate, 0.2-3 parts of disodium ethylenediamine tetraacetate, 1-2 parts of glycerol and 0.001-0.006 part of allure red or phenol red;

the beneficial effects of the invention are as follows: the virus preservation solution can keep the integrity of virus particles, so that the release, degradation and pollution of RNA are avoided, the effective inactivation of the virus is carried out, the safety of detection personnel is ensured, and the recovery rate of collected RNA is improved.

Aiming at the limitation of preservation solution in the background technology on coronavirus detection preservation, the efficient normal-temperature preservation solution for virus samples is researched and developed, and a series of preservation products for clinical samples are researched and developed based on the same-layer plasma technology by organically combining specific osmotic pressure stabilizer, cell membrane stabilizer, metabolic inhibitor, nuclease inhibitor, anticoagulant and other special components. After the medical staff collects the sample of the suspected patient, the collected sample is put into the preservation solution, and the preservation and transportation can be carried out at normal temperature, so that the temperature is not strictly required, and the preservation time is long. The virus particles keep the complete state shell in the preservation solution without cracking, and meanwhile, the nucleic acid in the sample is ensured not to be degraded, so that the quality of the RNA detection material is not affected, the detection sensitivity is improved, and the problem of false negative caused by nucleic acid degradation is effectively avoided. The preservation solution can quickly inactivate viruses possibly existing in the sample, can effectively inhibit the growth of infectious agents, cut off infectious sources and block the infectious paths, reduces the exposure risk of medical workers to the greatest extent, and provides the most basic and firm guarantee for epidemic prevention and control.

On the basis of the technical scheme, the invention can be improved as follows.

Further, the virus sample preservation solution includes, in parts by weight, 70 parts of guanidine isothiocyanate, 4.3 parts of sodium bicarbonate, 0.5 parts of disodium edetate, 0.8 parts of glycerin, and 0.002 parts of allure red.

The further scheme has the beneficial effects that the reagent used in the formula is convenient to purchase, the price is proper, the formula is relatively simple, and the preparation is convenient. The production of enterprises is facilitated, the cost is reduced, and the operation of operators is facilitated. Nuclease inhibitor based on guanidine isothiocyanate, osmotic stabilizer based on sodium bicarbonate, unique chelating property based on multiple valence bonds in low-toxicity disodium ethylenediamine tetraacetate to replace common anticoagulant EDTA-3K, allure red indicator and antifreeze glycerol are added. The synergistic effect of these components can better play a role in protection.

Further, the pH of the virus sample preservation solution was 7.1.

The further scheme has the beneficial effects that the proper pH value can fully excite the activity of the inactivating component and the degradation preventing component to reach the highest, so that the intact stability of the nucleic acid is realized while the virus protein is effectively inactivated.

The invention also provides a preparation method of the virus sample preservation solution capable of preserving at normal temperature and inactivating viruses, which comprises the following steps:

s1, mixing 40-80 parts of guanidine isothiocyanate or guanidine hydrochloride, 2-8 parts of sodium citrate or sodium bicarbonate, 0.2-3 parts of disodium ethylenediamine tetraacetate, 1-2 parts of glycerol and 0.001-0.006 part of alluring red or phenol red to obtain mixed components;

s2, adding pure water into the mixed components obtained in the step S1, and stirring at normal temperature to obtain mixed liquid, wherein the volume of the pure water is 55% -80% of that of the mixed components;

s3, adding 1mol/L NaOH solution into the mixed solution obtained in the step S2, and adjusting the pH to 6.2-7.8 to obtain a virus sample preservation solution.

The preparation method has the beneficial effects that the preparation method can simply and efficiently produce the virus sample preservation solution with stable quality.

In another aspect, the present invention provides a method for detecting Ct value of a virus sample preservation solution capable of preserving at normal temperature and inactivating viruses as described above, comprising the steps of:

A. adding a virus sample to be tested into the virus sample preservation solution according to any one of claims 1 to 3, marking, taking the preservation solution from a marked tube, and automatically extracting RNA according to the step of the Siami Tianlong nucleic acid extraction kit;

B. after the automatic procedure in the step A is finished, transferring the eluent into a non-nuclease centrifuge tube, preserving at-80 ℃, and marking to obtain nucleic acid of a sample to be detected;

C. and B, performing fluorescent quantitative PCR detection on the nucleic acid to be detected in the step B, and setting the circulation conditions as follows:

after the setting is completed, a file is saved, and a program is operated to obtain a Ct value.

The method has the beneficial effects that the Ct value of the virus sample preservation solution can be stably and accurately measured by the detection method.

In another aspect, the invention provides a nucleic acid detection kit, which comprises the virus sample preservation solution capable of preserving at normal temperature and inactivating viruses.

The nucleic acid detection kit containing the preservation solution has the beneficial effects that the application range of the nucleic acid detection kit containing the preservation solution is wide. Including serum, plasma, pharyngeal swabs, nasal swabs, sputum, stool, urine, and the like. The method is suitable for sampling, transporting and preserving the oropharynx of different virus samples such as respiratory tract and enterovirus of coronavirus, influenza, avian influenza, hand-foot-mouth, swine influenza and the like, and is also suitable for collecting other virus, chlamydia, mycoplasma and ureaplasma urealyticum samples. The stored sample can be used for clinical experiments such as nucleic acid extraction or separation and purification. The virus sample is widely applied to medical clinic, public health emergency, entry and exit inspection, quarantine, food inspection, animal husbandry, aquaculture and other relevant scientific research institutions.

Drawings

FIG. 1 is a box plot of Ct values for ratios 1 to 4 in example 2 of the present invention;

FIG. 2 is a box plot of Ct values for ratio 1, ratio 5, and ratio 6 in example 2 of the present invention;

Detailed Description

The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.

Example 1,

The method for detecting the Ct value in the virus preservation solution comprises the following specific steps:

1) Mu.l of the virus sample was added to a tube containing 3ml of virus stock and labeled.

2) 200. Mu.L of stock solution was taken from the labeled tube and RNA was extracted on-line automatically according to the procedure of the Siamitraz nucleic acid extraction kit, set up below.

3) After the automatic procedure is finished, transferring the eluent in the 6 th or 12 th column into a clean nuclease-free centrifuge tube, preserving at-80 ℃ and marking to obtain the nucleic acid of the sample to be detected.

4) The method for detecting the nucleic acid to be detected by fluorescence quantitative PCR comprises the following specific steps of

a) Reagent preparation (reagent preparation area)

NC (ORF 1 ab/N) PCR reaction liquid A, NC (ORF 1 ab/N) PCR reaction liquid B was taken out of the kit, melted at room temperature, mixed by shaking, centrifuged at 8000rpm for several seconds, and used.

Taking a PCR reaction tube (sample to be detected), an NC (ORF 1 ab/N) negative quality control product and an NC (ORF 1 ab/N) positive quality control product

The single-person amplification system is prepared as follows:

NC (ORF 1 ab/N) PCR reaction solution A	NC (ORF 1 ab/N) PCR reaction liquid B	Amplification system
			17μl	3μl	20μl

After the components are fully mixed, the mixture is centrifuged for a short time, and after the liquid on the tube wall is fully centrifuged to the bottom of the tube, 20 mu l of the amplification system is packaged into PCR tubes.

b) Sample application (sample preparation area)

And respectively adding 5 mu l of treated negative quality control substances, 5 mu l of sample nucleic acid to be detected and 5 mu l of treated positive quality control substances into the PCR reaction tube, tightly covering a tube cover, centrifuging at 8000rpm for a plurality of seconds, and transferring to an amplification detection area.

c) The reaction tube was placed in the instrument sample well and tested using an ABI 7500 instrument.

(1) Opening a setup window, setting a negative quality control (NTC), a positive quality control (positive quality control) and an unknown sample (Unkowm) according to the corresponding sequence of the samples, and setting sample names in a column of a sample name; the probe detection mode is set as follows:

Reponer1:FAMQuencherl:NONE；Reponer2:VIC,Quencher2:NONE:Report er3:Cy5,Quencher3:NONE:Passive Reference:NONE

(2) The instrument window is opened and the loop conditions are set as follows:

after the setting is completed, the file is saved and the program is run.

EXAMPLE 2,

The following 6 virus sample preservation solutions with different proportions are prepared, and the specific steps are as follows:

a mixture ratio of 1,

The virus sample preservation solution capable of being preserved at normal temperature and inactivating viruses comprises, by weight, 62g/L guanidine isothiocyanate, 5.5g/L sodium bicarbonate, 1.2g/L disodium ethylenediamine tetraacetate, 0.6g/L glycerol and 0.002g/L alluring red;

the pH of the virus sample preservation solution was 7.1.

The preparation method comprises the following steps:

s1, mixing 62g/L of guanidine isothiocyanate, 5.5g/L of sodium bicarbonate, 1.2g/L of disodium ethylenediamine tetraacetate, 0.6g/L of glycerol and 0.002g/L of alluring red to obtain mixed components;

s2, adding pure water into the mixed components obtained in the step S1, and stirring at normal temperature to obtain mixed liquid, wherein the volume of the pure water is 70% of that of the mixed components;

s3, adding 1mol/L NaOH solution into the mixed solution obtained in the step S2, and adjusting the pH to 7.1 to obtain a virus sample preservation solution.

Proportioning 2,

The difference with the mixture ratio 1 is that the guanidine isothiocyanate is 70g/L, the sodium bicarbonate is 4.3g/L, the disodium edetate is 0.5g/L, and the glycerol is 0.8g/L.

Proportioning 3,

The difference with the mixture ratio 1 is that the guanidine isothiocyanate is 40g/L, the sodium bicarbonate is 2.1g/L, the disodium edetate is 2.8g/L, the glycerol is 1.4g/L, and the alluring red is 0.005g/L.

Proportioning 4,

The difference with the mixture ratio 1 is that guanidine isothiocyanate is 80g/L, sodium bicarbonate is 7.7g/L, disodium ethylenediamine tetraacetate is 2.1g/L, glycerol is 1.8g/L, and alluring red is 0.004g/L.

Proportioning 5,

The difference with the mixture ratio 1 is that guanidine isothiocyanate is 62g/L, sodium bicarbonate is 5.5g/L, disodium ethylenediamine tetraacetate is 1.2g/L, glycerol is 0.6g/L, and dithiothreitol is added in an amount of 0.05g/L.

Proportioning 6,

The difference with the mixture ratio 1 is that guanidine isothiocyanate is 62g/L, sodium bicarbonate is 5.5g/L, disodium ethylenediamine tetraacetate is 1.2g/L, glycerol is 0.6g/L, and dithiothreitol is added to the mixture 0.75g/L.

In order to compare and prove the effects of the virus preservation solutions of different contents and proportions of proportions 1 to 6 in example 1, ct values were detected in particular according to the method of example 1.

The specific results are as follows:

taking the preservation solution with the mixture ratio of 1 and numbers of 1-1 to 1-5, and analogically taking Jin Weizhi biological synthesis COVID-19 pseudovirus as a sample, detecting whether the Ct value of the ORF is in a normal reference value interval or not after the preservation of the preservation solution with different mixture ratios for preserving the preserved sample for 24 hours, wherein the normal Ct value is 18.00-36.00. The results obtained by measuring the mixture ratios 1 to 4 according to the method of example 1 are shown in the following table 1, and a box diagram is prepared as shown in fig. 1:

TABLE 1

Proportion 1	Ct value	Proportion 2	Ct value	Proportion 3	Ct value	Proportion 4	Ct value
								1-1	29.96	2-1	29.02	3-1	30.96	4-1	31.22
1-2	28.41	2-2	28.96	3-2	29.77	4-2	32.43
								1-3	28.26	2-3	28.78	3-3	29.33	4-3	29.62
1-4	29.50	2-4	28.98	3-4	28.23	4-4	28.05
								1-5	30.01	2-5	28.54	3-5	31.13	4-5	30.10

The prepared box diagram is shown in fig. 1, analysis shows that the measured Ct values of the virus samples stored in the 4 kinds of ratio preservation solutions are in a normal reference interval, but the ratio 2 is more stable, the preservation effect is better, and the improvement of the greater preservation stability of the ratio 2 relative to other ratios 1, 3 and 4 is realized through the change of the ratio.

The results of the tests of the mixture ratio 1, the mixture ratio 5 and the mixture ratio 6 according to the method of example 1 are shown in the following table 2, and the box diagram is shown in fig. 2:

TABLE 2

Proportion 1	Ct value	Proportion 5	Ct value	Proportion 6	Ct value
						1-1	29.96	5-1	35.24	6-1	30.96
1-2	28.41	5-2	26.93	6-2	27.15
						1-3	28.26	5-3	30.69	6-3	29.33
1-4	29.50	5-4	33.21	6-4	34.88
						1-5	30.01	5-5	27.15	6-5	26.43

The invention is characterized in that the common preservation solution component dithiothreitol is added, the fluctuation range of Ct value is large, the data is unstable, and the clinical judgment is not facilitated. The advantages of the mixture ratio of the invention are proved to have synergistic effect, and the storage life can be prolonged well. Importantly, dithiothreitol belongs to nuclease inhibitor, and is unfavorable for long-term preservation of preservation solution after being added, which proves that the invention has unexpected technical effect.

To further illustrate the effect of the preservation solution of the present invention, the following experiments were performed:

1. preservation fluid expiration date experiment

Taking a novel coronavirus pseudovirus as a sample, and detecting whether the Ct values of the ORF gene and the N gene of the sample are in a normal reference value interval (18.00-36.00). (Jin Weizhi biosynthesis of COVID-19 pseudovirus, detection was performed using the novel coronavirus 2019-nCoV nucleic acid detection kit of the da' an gene (fluorescent PCR method).

1.1, 30 preservation solutions of two batches of the mixture ratio 1 in example 2 were taken, and the content of each preservation solution was 3ml. The samples were stored under different conditions and were distributed as shown in Table 3 below:

TABLE 3 Table 3

Batch of	Quantity (branch)	Numbering device	Storage temperature (. Degree. C.)
				1	15	#1	Laboratory room temperature
2	15	#2	Laboratory room temperature
				1	15	#3	35
2	15	#4	35

Wherein, the virus dilution gradient is 1:100;

1.2, respectively taking one sample from the 4 groups of samples every month, diluting and preserving the samples for 24 hours, and performing fluorescence quantitative PCR. Specific detection methods are as in example 1.

The test results are shown in Table 4 below, taken for 12 months in total:

TABLE 4 Table 4

Therefore, the invention can be stored for a long time under normal temperature environment.

2. Virus preservation and amplification efficiency comparison experiment

Three groups of commercially available virus preservation solutions are taken, the pH value of the virus preservation solutions is 6.8, and the content components are as follows: 24g/L guanidine isothiocyanate, 3.2g/L N-acetyl-L cysteine, 1.5g/L polyethylene glycol neo-phenyl ether, 2.6g/L EDTA, 0.13g/L sodium bicarbonate and 0.001g/L phenol red.

The method comprises the steps of taking Jin Weizhi biosynthesis COVID-19 pseudovirus as a test sample, detecting the Ct value of the amplification condition of the ORF gene, and respectively taking three groups of sample preservation solutions of the virus in the proportion 1 in the embodiment 2 and the three groups of commercially available viruses according to the method of the embodiment 1 to detect the Ct value, wherein the experimental results are shown in the following table 5, and the proportion of the preservation solutions is equal to that of the proportion 1 in the embodiment 2.

TABLE 5

According to the data, the preservation performance of the preservation solution is not greatly different from that of the comparison preservation solution in the first 24 hours, but the data of 48 hours and 72 hours show that the preservation solution Ct is stable, but the comparison preservation solution has large change, which indicates that the effect of the comparison preservation solution for preserving the virus sample for a long time is poor.

3. Optimal pH validation

The components of the virus sample preservation solution of the mixture ratio 1 in the example 2 are taken, different pH values are set, 3ml of the preservation solution of the same formula and different pH values are taken, and Ct values are measured according to the method of the example 1. Two replicates were run for each pH, and specific assay data and pH are shown in table 6 below:

TABLE 6

As can be seen from the data in Table 6, the pH values in the above intervals meet the requirements, but the values at 7.1 are the most stable, and the Ct value change of the pH data at both sides of 7.1 is obvious relative to 7.1.

In conclusion, the virus sample preservation solution capable of being preserved at normal temperature and inactivating viruses has good innovation and market prospect, and can make an excellent contribution to virus detection.

In the description of the present specification, the terms "one embodiment," "some embodiments," "particular embodiments," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. The preparation method of the virus sample preservation solution capable of preserving at normal temperature and inactivating viruses is characterized by comprising the following steps of:

s1, mixing 70 parts of guanidine isothiocyanate, 4.3 parts of sodium bicarbonate, 0.5 part of disodium ethylenediamine tetraacetate, 0.8 part of glycerol and 0.002 part of alluring red to obtain a mixed component;

2. A virus sample preservation solution prepared by the method for preparing a virus sample preservation solution capable of preserving at normal temperature and inactivating viruses according to claim 1.

3. A nucleic acid detection kit, characterized in that the kit comprises the virus sample preservation solution which can be preserved at normal temperature and can inactivate viruses as claimed in claim 2.