CN111676216A - Extraction-free inactivated virus sample nucleic acid preservation solution and preparation method and application thereof - Google Patents
Extraction-free inactivated virus sample nucleic acid preservation solution and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a nucleic acid preservation solution for a non-extraction inactivated virus sample, and a preparation method and application thereof. The preservation solution is safe and nontoxic, is simple to use, does not need cold chain preservation, can be transported and preserved at normal temperature for 3 days, does not need further inactivation treatment, is provided with a virus cracking component, a nuclease inhibiting component and a buffer system component, the virus cracking component can inactivate and crack viruses to release nucleic acid, the nuclease inhibiting component can inhibit nuclease activity and effectively avoid nucleic acid degradation, and the nucleic acid preservation solution can be used for directly detecting nucleic acid without further nucleic acid extraction and purification operation, and is particularly suitable for sample nucleic acid collection of epidemic viruses with high infectivity.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to an inactivated virus sample nucleic acid preservation solution and a preparation method and application thereof.
Background
A virus is a special organism without cellular structure, consisting of a protein coat and internal genetic material. The virus is small in individual and simple in structure, and only contains one genetic material (DNA or RNA). The virus has no metabolic mechanism, no enzyme system, can not survive independently, can only be parasitized in host cells, completes life activities depending on substances and energy in the host cells, performs replication, transcription and translation according to genetic information contained in the virus, generates a new generation virus, and realizes replication type proliferation. The new generation virus after replication and proliferation can crack the original host cell and infect the new host cell for the next round of replication and proliferation. Viral infections are extremely harmful, for example the novel coronavirus (SARS-CoV-2), a new type of coronavirus not previously found in humans, and can cause acute infectious pneumonia (COVID-19), the initial symptoms of the infected are fever, fatigue and dry cough, the subsequent breathing difficulty gradually appears, and the severe patients can develop acute respiratory distress syndrome or septic shock, and even die.
The most effective measure for preventing and controlling viral infection is early diagnosis and early treatment, nucleic acid detection is a main method for quickly diagnosing viral infection, the acquisition of a viral nucleic acid sample is a precondition for nucleic acid detection, and the accuracy of a nucleic acid detection result is often determined by the capability of keeping the stability of nucleic acid in a viral sample. However, the nucleic acid of the virus sample is easily degraded at room temperature and needs to be stored below the freezing temperature to keep stability, but still can be degraded to a certain extent along with the lapse of time, which not only increases the cost of sample transportation and storage, but also limits the possibility and distance between a sample collection point and a diagnostic experiment, and directly influences the accuracy of the experimental detection result.
The existing virus sample collection and storage treatment method depends on cold chain transportation, meanwhile, the storage time is mostly within 48 hours based on the consideration of virus activity, and nucleic acid detection can be carried out after nucleic acid is extracted from the virus samples. In these methods, the virus-preserving solution is usually an isotonic saline solution or a phosphate buffer solution, and can preserve the activity of the virus in a short time, but cannot inhibit the degradation of nucleic acid. Based on the high infectivity of some viruses and the requirement of the existing nucleic acid gene detection, virus samples all need high-temperature inactivation treatment, and related treatment has the risk of infecting experimenters. However, existing studies indicate that treatment of high-temperature inactivated virus can significantly reduce the amount of detectable viral nucleic acid (Zhang Qin et al, 2020), and meanwhile, the subsequent steps of viral nucleic acid extraction are complicated, nucleic acid purification reagents are required to be consumed, and the detection time and cost are increased.
Therefore, there is a need to develop a nucleic acid preservation solution for a non-extraction inactivated virus sample, which can directly inactivate viruses, inhibit the activity of nuclease, stably preserve viral nucleic acids, and directly detect nucleic acids without nucleic acid extraction.
Disclosure of Invention
In view of the above, the invention provides a non-extraction inactivated virus sample nucleic acid preservation solution, a preparation method and an application thereof, the preservation solution does not need a cold chain, can be transported and stored at normal temperature for 3 days, does not need inactivation treatment, can directly inactivate viruses, can directly carry out nucleic acid detection without further nucleic acid extraction and purification operation, and is suitable for nucleic acid collection of epidemic virus samples.
In order to achieve the above object, the invention provides a nucleic acid preservation solution for a hands-free inactivated virus sample, which comprises lithium dodecyl sulfate, N-lauroyl sarcosine, Tween-20, bovine serum albumin, tris (2-carboxyethyl) phosphine hydrochloride, vanadyl riboside complex and sodium citrate.
In the nucleic acid preservation solution for the non-extraction inactivated virus sample, the lithium dodecyl sulfate and the N-lauroyl sarcosine are anionic surfactants, the Tween-20 is a nonionic surfactant, and the advantages of the three are complementary, so that the virus coat protein is cracked, and the nucleic acid is released into the solution; bovine serum albumin, tris (2-carboxyethyl) phosphine hydrochloride and the vanadyl riboside complex can inhibit the activity of nuclease, and the combination of the bovine serum albumin, the tris (2-carboxyethyl) phosphine hydrochloride and the vanadyl riboside complex can further inhibit the activity of the nuclease; the sodium citrate acts as a chelating agent and also provides a suitable pH environment. The components are cooperated to realize the effect of storing the nucleic acid of the virus sample, which can carry out nucleic acid detection without cold chain transportation, further inactivation treatment and further nucleic acid extraction and purification.
In one embodiment of the invention, the extraction-free inactivated virus sample nucleic acid preservation solution comprises 1-150mM of lithium dodecyl sulfate, 1-150mM of N-lauroyl sarcosine, 1-15% (V/V) of Tween-205, 0.1-2mM of bovine serum albumin, 1-10mM of tris (2-carboxyethyl) phosphine hydrochloride, 1-10mM of vanadyl riboside complex and 5-200 mM of sodium citrate.
In a preferred embodiment of the invention, the nucleic acid preservation solution for the extraction-free inactivated virus sample comprises 10-100mM of lithium dodecyl sulfate, 10-100mM of N-lauroyl sarcosine, 10-12% (V/V) of Tween-208, 0.5-1.5mM of bovine serum albumin, 2-8mM of tris (2-carboxyethyl) phosphine hydrochloride, 2-8mM of vanadyl riboside complex and 10-100mM of sodium citrate.
In an embodiment of the invention, the pH value of the nucleic acid preservation solution for the extraction-free inactivated virus sample is adjusted by citric acid.
In an embodiment of the invention, the pH value of the nucleic acid preservation solution for the extraction-free inactivated virus sample is 6 to 8.
In one embodiment of the present invention, the virus sample is a DNA virus, an RNA virus, or a phage.
In a preferred embodiment of the present invention, the virus sample is an RNA virus.
The second aspect of the invention provides a method for preserving nucleic acid of a non-extraction inactivated virus sample, which comprises the following steps: collecting a virus sample, placing the virus sample in a preserving fluid which is pre-filled with the extraction-free inactivated virus sample nucleic acid according to the first aspect of the invention, and preserving at room temperature to obtain the extraction-free inactivated virus sample nucleic acid.
In an embodiment of the present invention, the time period for the viral sample nucleic acid preservation solution to stably preserve the viral sample nucleic acid at room temperature is 3 days.
The third aspect of the invention provides a method for detecting nucleic acid of a non-extraction inactivated virus sample, which comprises the following steps: the virus sample nucleic acid preservation solution preserved by the preservation method according to the second aspect of the present invention is directly transferred to perform nucleic acid detection.
In a fourth aspect, the present invention provides a nucleic acid preservation solution for a sample of a non-extracted inactivated virus according to the first aspect of the present invention, a method for preserving a nucleic acid of a sample of a non-extracted inactivated virus according to the second aspect of the present invention, or a method for detecting a nucleic acid of a sample of a non-extracted inactivated virus according to the third aspect of the present invention, for use in preparing a reagent for detecting a nucleic acid of a virus.
The non-extraction inactivated virus sample nucleic acid preservation solution provided by the invention is safe and non-toxic, is simple to use, does not need cold chain preservation, can be transported and preserved at normal temperature for 3 days, does not need further inactivation treatment, is configured with a virus cracking component, a nuclease inhibiting component and a buffer system component, can inactivate and crack viruses to release nucleic acid, can inhibit the activity of the nuclease inhibiting component, and effectively avoids nucleic acid degradation, can be used for directly detecting nucleic acid without further nucleic acid extraction and purification operation, and is particularly suitable for sample nucleic acid collection of epidemic viruses with high infectivity.
Drawings
FIG. 1 is a QPCR amplification result graph of porcine PEDV coronavirus gene under the condition of nucleic acid extraction of a virus sample preserved by a nucleic acid preservation solution of an extraction-free inactivated virus sample according to an embodiment of the invention;
FIG. 2 is a QPCR amplification result graph of porcine PEDV coronavirus genes under the condition of avoiding nucleic acid extraction of virus samples preserved by Hanks liquid provided by the embodiment of the invention;
FIG. 3 is a QPCR amplification result diagram of porcine PEDV coronavirus genes after nucleic acid extraction of a virus sample stored in the nucleic acid preservation solution of the hands-free inactivated virus sample according to the embodiment of the invention;
FIG. 4 is a QPCR amplification result diagram of porcine PEDV coronavirus genes after nucleic acid extraction of virus samples preserved in Hanks solution provided by the embodiment of the invention.
Detailed Description
In order to more clearly understand the technical features, objects, and effects of the present invention, specific embodiments of the present invention will now be described in detail. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the examples of the present invention, unless otherwise specified, reagents and consumables used therein are commercially available.
The nucleic acid preservation solution for the non-extraction inactivated virus sample provided by the invention comprises 1-150mM of lithium dodecyl sulfate, 1-150mM of N-lauroyl sarcosine, 1-15% (V/V) of Tween-205, 0.1-2mM of bovine serum albumin, 1-10mM of tris (2-carboxyethyl) phosphine hydrochloride, 1-10mM of vanadyl riboside complex and 5mM-200mM of sodium citrate.
In the nucleic acid preservation solution for the non-extraction inactivated virus sample, the lithium dodecyl sulfate and the N-lauroyl sarcosine are anionic surfactants, the Tween-20 is a nonionic surfactant, and the advantages of the three are complementary, so that the virus coat protein is cracked, and the nucleic acid is released into the solution; bovine serum albumin, tris (2-carboxyethyl) phosphine hydrochloride and the vanadyl riboside complex can inhibit the activity of nuclease, and the combination of the bovine serum albumin, the tris (2-carboxyethyl) phosphine hydrochloride and the vanadyl riboside complex can further inhibit the activity of the nuclease; the sodium citrate acts as a chelating agent and also provides a suitable pH environment. In addition, in order to realize the purpose of realizing nucleic acid detection without taking hands, the invention preferably selects Tween-20 which has a promoting effect on nuclease detection reaction as a main cracking component and selects bovine serum albumin as a main component of a nuclease inhibitor during component selection, and the components are cooperated to realize the effect of storing the nucleic acid of the virus sample which can carry out nucleic acid detection without cold chain transportation, further inactivation treatment and further nucleic acid extraction and purification.
EXAMPLE 1 preparation of nucleic acid preservation solution for hands-free inactivated Virus sample
1) Preparing an extraction-free inactivated virus sample nucleic acid preservation solution according to the following formula:
| preservative fluid component | Final | Formulation | 1 | Formulation 2 | Formulation 3 | |
Formulation 5 |
| Dodecyl lithium sulfate | 1- |
1 | 30 | 75 | 100 | 150 | |
| N-lauroyl sarcosine | 1- |
1 | 30 | 75 | 100 | 150 | |
| Tween-20 | 5-15％(V/V) | 5 | 8 | 10 | 13 | 15 | |
| Bovine serum albumin | 0.1-2mM | 0.1 | 0.5 | 1 | 1.5 | 2 | |
| Tris (2-carboxyethyl) phosphine hydrochloride | 1- |
1 | 3 | 5 | 8 | 10 | |
| Vanadyl riboside complexes | 1- |
1 | 3 | 5 | 8 | 10 | |
| Citric acid sodium salt | 5mM-200mM | 5 | 50 | 100 | 150 | 200 |
2) And adjusting the pH value of the preservation solution to 6-8 by using citric acid to obtain the extraction-free inactivated virus sample nucleic acid preservation solution.
Example 2 verification of preservation Effect of nucleic acid preservation solution for hands-free inactivated Virus sample
The preservation effect of the preservation solution prepared according to the formula 3 in the example 1 on the nucleic acid of the virus sample is compared with that of the Hanks solution which is widely used in the market at present:
virus samples to be verified: porcine PEDV coronavirus (epidemic diarrhea live vaccine AJ1102-R, purchased from Wuhan family pre-organism) with virus content of more than or equal to 1013TCID50/mL。
The verification scheme comprises the following steps:
scheme one
1) Putting 200 mu L of live virus vaccine in the preservation solution provided by the invention, setting 3 independent samples, simultaneously taking buccal swab samples of the cheek parts on the left side and the right side of 3 healthy volunteers, putting the buccal swab samples in the preservation solution samples, and fully and uniformly mixing;
2) storing in 35 deg.C incubator at day 0 (within 2 hr after sample treatment), day 1 and day 3, respectively, collecting 5 μ L of the storage solution, reverse transcribing into cDNA, and selecting Takara
Viral PEDV gene in the storage solution under the condition of nucleic acid extraction was detected by qPCR using the Universal U + Probe MasterMix V2(Q513-02/03) kit.
Scheme two
1) Putting 200 mu L of live virus vaccine into Hanks liquid, setting 3 independent samples, simultaneously taking oral swab samples of the cheek parts on the left side and the right side of 3 healthy volunteers, putting the oral swab samples into a preservation liquid sample, and fully and uniformly mixing;
2) storing in a refrigerator at 4 deg.C, reverse transcribing 5 μ L of the stock solution to cDNA on days 0 (within 2 hours after sample treatment), 1 and 3, respectively, and selecting Takara
Viral PEDV gene in Hanks solution under the condition of nucleic acid extraction was detected by qPCR using Universal U + Probe Master Mix V2(Q513-02/03) kit.
Scheme three
1) Putting 200 mu L of live virus vaccine in the preservation solution provided by the invention, setting 3 independent samples, simultaneously taking buccal swab samples of the cheek parts on the left side and the right side of 3 healthy volunteers, putting the buccal swab samples in the preservation solution samples, and fully and uniformly mixing;
2) storing in 35 deg.C incubator at day 0 (within 2 hr after sample treatment), day 1 and day 3 respectively, collecting 140 μ L of the storage solution for viral RNA extraction, eluting RNA with volume of 20 μ L, collecting 2 μ L, reverse transcribing into cDNA, and selecting Takara
Viral PEDV gene in the preservation solution after nucleic acid extraction was detected by qPCR using the Universal U + Probe Master Mix V2(Q513-02/03) kit.
Scheme four
1) Putting 200 mu L of live virus vaccine into Hanks liquid, setting 3 independent samples, simultaneously taking oral swab samples of the cheek parts on the left side and the right side of 3 healthy volunteers, putting the oral swab samples into a preservation liquid sample, and fully and uniformly mixing;
2) storing in a refrigerator at 4 deg.C, collecting 140 μ L of preservation solution for virus RNA extraction on day 0 (within 2 hr after sample treatment), day 1 and day 3, respectively, eluting RNA with volume of 20 μ L, collecting 2 μ L, reverse transcribing into cDNA, and selecting Takara
Viral PEDV gene in Hanks solution after nucleic acid extraction was detected by qPCR using Universal U + Probe Master Mix V2(Q513-02/03) kit.
The detection results are as follows:
as a result, the porcine PEDV coronavirus gene cannot be detected in the virus sample stored in the Hanks solution under the condition of no nucleic acid extraction, and the details are shown in FIG. 2, which shows that no virus nucleic acid is released into the solution in the Hanks solution of the virus sample; the fourth scheme is that the virus sample preserved in Hanks liquid can detect the porcine PEDV coronavirus gene after nucleic acid extraction, but the CT value of the virus sample is positively correlated with the preservation time, and detailed figure 4 shows that the virus sample preserved in Hanks liquid can be degraded even if the virus sample is preserved in the Hanks liquid at 4 ℃ by refrigeration; the first scheme is that the virus sample preserved for 3 days at 35 ℃ prepared by the formula 3 can detect the porcine PEDV coronavirus gene under the condition of no nucleic acid extraction, and is shown in detail in figure 1, and the CT value detected for 3 days is relatively stable, which indicates that the nucleic acid in the virus sample is released into the solution, and the nucleic acid in the preservation solution is not obviously degraded even under the condition of 3 days preserved at 35 ℃, and the third scheme is that the virus sample preserved for 3 days at 35 ℃ prepared by the formula 3 can detect the porcine PEDV coronavirus gene after the nucleic acid extraction, is shown in figure 3, and the CT value detected for 3 days is slightly smaller than the CT value detected for 3 days in the first scheme, which indicates that the preservation solution prepared by the formula 3 can obtain the nucleic acid detection effect similar to that obtained after traditional nucleic acid extraction and purification under the condition of no nucleic acid extraction.
In conclusion, the invention provides a non-extraction inactivated virus sample nucleic acid preservation solution which is safe and non-toxic, simple to use, free of cold chain preservation, capable of transporting and preserving virus sample nucleic acid at normal temperature for 3 days, free of further inactivation treatment, capable of inactivating and cracking virus to release nucleic acid, capable of inhibiting nuclease activity, effectively avoiding nucleic acid degradation, capable of directly detecting nucleic acid without further nucleic acid extraction and purification operation, and particularly suitable for sample nucleic acid collection of epidemic viruses with high infectivity.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. A nucleic acid preservation solution for a non-extraction inactivated virus sample is characterized by comprising lithium dodecyl sulfate, N-lauroyl sarcosine, Tween-20, bovine serum albumin, tris (2-carboxyethyl) phosphine hydrochloride, a vanadyl riboside compound and sodium citrate.
2. The fluid for preserving nucleic acid from a sampling-free inactivated virus sample according to claim 1, which comprises 1 to 150mM of lithium dodecyl sulfate, 1 to 150mM of N-lauroylsarcosine, 205 to 15% (V/V) of Tween, 0.1 to 2mM of bovine serum albumin, 1 to 10mM of tris (2-carboxyethyl) phosphine hydrochloride, 1 to 10mM of a oxovanadium riboside complex, and 5 to 200mM of sodium citrate.
3. The preservation solution for nucleic acid of a non-extraction inactivated virus sample according to claim 1, wherein the pH value of the preservation solution for nucleic acid of a non-extraction inactivated virus sample is adjusted by citric acid.
4. The preservation solution for nucleic acid of an extraction-free inactivated virus sample according to claim 1, wherein the pH of the preservation solution for nucleic acid of an extraction-free inactivated virus sample is 6 to 8.
5. A method for preserving nucleic acid of a non-extraction inactivated virus sample is characterized by comprising the following steps: collecting a virus sample, putting the virus sample in a preserving fluid preloaded with the extraction-free inactivated virus sample nucleic acid of claim 1, and preserving at room temperature to obtain the extraction-free inactivated virus sample nucleic acid.
6. The method for preserving a nucleic acid in a sample of a hands-free inactivated virus according to claim 5, wherein the time period for which the nucleic acid in the sample of a virus is stable when the sample of a virus is preserved in the solution for preserving a nucleic acid in a sample of a virus at room temperature is 3 days.
7. A method for detecting nucleic acid in a sample of a non-extraction inactivated virus, characterized in that a nucleic acid is detected by directly removing the nucleic acid preservation solution of the virus sample preserved by the preservation method according to claim 5.
8. Use of the fluid for preserving a nucleic acid from a sample of a non-extracted inactivated virus according to claim 1, the method for preserving a nucleic acid from a sample of a non-extracted inactivated virus according to claim 5, or the method for detecting a nucleic acid from a sample of a non-extracted inactivated virus according to claim 8 for preparing a reagent for detecting a nucleic acid from a virus.
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