CN111183973A

CN111183973A - Saliva or oral swab preserving fluid and preparation method and application thereof

Info

Publication number: CN111183973A
Application number: CN202010063435.6A
Authority: CN
Inventors: 梁玉姣; 耿彪; 曹健荣
Original assignee: Denogen Beijing Bio Sci&tech Co ltd
Current assignee: Denogen Beijing Bio Sci&tech Co ltd
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2020-05-22

Abstract

The invention relates to the technical field of biology, and particularly provides a saliva or oral swab preserving fluid and a preparation method and application thereof. The preservation solution provided by the invention comprises: 10-50mmol/L Tris-HCl, 0.2-1mol/L urea, 0.3-1w/v SDS, 2-8w/v EDTA-Na 21-8 mmol/L sodium alginate and 3-8w/v L-carnitine, and the pH is 7-10. In the invention, the components of the preservation solution can be used for preserving the saliva or oral swab sample for a long time through the effective proportioning and the synergistic effect, and particularly, the good preservation of the sample can be realized in a high-temperature environment with the temperature of more than 35 ℃. The preservation solution enables the application of the genomic DNA extracted from saliva or oral swabs to be wider, and reduces the environment and operation requirements, thereby reducing the operation cost and being beneficial to the subsequent PCR test operation.

Description

Saliva or oral swab preserving fluid and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to a saliva or oral swab preserving fluid and a preparation method and application thereof.

Background

The genome DNA sample extracted from human tissue and body fluid is widely applied in the fields of gene engineering, molecular biology research and the like. Genomic DNA commonly used in PCR reactions and other tests is mainly extracted from anticoagulated blood, but after long-term preservation of anticoagulated blood, blood clots are formed, which brings inconvenience to extraction. And extract DNA through the blood sampling still need professional personnel to draw blood, some people have the psychology of contradicting to drawing blood, and this mode has the shortcoming that can't gather the sample at any time in a large number.

Saliva and oral cavity genome DNA extraction is a method for obtaining genome DNA without harm and pain and with simple operation compared with the mode of collecting genome DNA by anticoagulation, and the method can obtain complete genome DNA even if the saliva and oral cavity genome DNA is stored for a long time at room temperature, and is gradually adopted in recent years.

99% of saliva in human body is water, contains electrolytes such as sodium, potassium, calcium, magnesium, chlorine, bicarbonate radical, phosphate radical and the like, and organic substances such as mucopolysaccharide, glycoprotein, amylase, tongue esterase, antibacterial enzyme, proline-rich protein and the like, and simultaneously contains various bacteria and oral exfoliative cells. It is known that DNA is susceptible to degradation in high temperature environments, and the prior art does not provide an effective preservation agent for preserving DNA in high temperature environments. Therefore, how to ensure the integrity of the genomic DNA in a high-temperature environment for a long time becomes a key point of saliva and oral swab preserving fluid.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first object of the present invention is to provide a saliva or buccal swab preserving solution which solves the technical problem of the prior art that the preserving solution cannot preserve saliva or buccal swabs in a high temperature environment for a long time, thereby ensuring that genomic DNA is not degraded.

A second object of the present invention is to provide a method for producing a saliva or buccal swab preservative solution, which is simple and easy to handle, and which is low in cost.

The third object of the present invention is to provide the use of the above-mentioned preservative fluid for extracting genomic DNA from saliva or buccal swab.

The fourth object of the present invention is to provide the use of the above-mentioned preservative fluid for preserving saliva or a mouth swab.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a saliva or buccal swab preservative solution comprising: 10-50mmol/L Tris-HCl, 0.2-1mol/L urea, 0.3-1w/v SDS, 2-8w/v EDTA-Na 21-8 mmol/L sodium alginate and 3-8w/v L-carnitine, and the pH of the preservation solution is 7-10.

Further, comprising: 15-45mmol/L Tris-HCl, 0.3-0.9mol/L urea, 0.3-0.8w/v SDS, 3-7w/v EDTA-Na 21-6 mmol/L sodium alginate and 3-7w/v L-carnitine, and the pH of the preservation solution is 7-9.

Further, comprising: Tris-HCl 20-40mmol/L, urea 0.3-0.7mol/L, SDS0.4-0.7 w/v%, EDTA-Na 22-5 mmol/L, sodium alginate 4-6 w/v% and L-carnitine 3-5 w/v%, and the pH of the preservation solution is 7-8.

Further, comprising: 30mmol/L Tris-HCl, 0.6mol/L urea, 0.5w/v SDS, 23mmol/L EDTA-Na, 5w/v sodium alginate and 4w/v L-carnitine, and the pH of the preservation solution is 7.5.

The preparation method of the preserving fluid comprises the steps of uniformly mixing Tris-HCl, urea, SDS, EDTA-Na2, sodium alginate and L-carnitine according to the proportion by using water, and adjusting the pH value to obtain the preserving fluid.

Further, the method also comprises a step of sterilization after the pH is adjusted.

The application of the preservation solution in extracting genome DNA from saliva or oral swabs.

The application of the above-mentioned preserving fluid in preserving saliva or oral swab is disclosed.

Further, the preservation temperature is-80 ℃ to 50 ℃.

Further, the saliva is human saliva.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a saliva or oral swab preserving fluid, which comprises: 10-50mmol/L Tris-HCl, 0.2-1mol/L urea, 0.3-1w/v SDS, 2-8w/v EDTA-Na 21-8 mmol/L sodium alginate and 3-8w/v L-carnitine, and the pH of the preservation solution is 7-10. Wherein urea and SDS act as denaturants to inhibit or reduce the growth of microorganisms; SDS and sodium alginate are used as bacteriostatic agents, can kill or reduce the microorganisms in saliva or oral swabs, and sodium alginate is used as a natural bacteriostatic agent, can effectively inhibit the breeding of fungi and bacteria, and prolongs the storage time; EDTA-Na2 as a chelating agent can chelate the activator of nuclease such as magnesium ions and the like, and inhibit the degradation of nuclease to DNA; the L-carnitine can effectively protect the activity of cells obtained from saliva or oral swabs and prolong the storage time; meanwhile, Tris-HCl is used as a buffer reagent, so that the preservation solution is relatively stable, and the pH environment in a certain range is favorable for preserving the sample. In the invention, the components of the preservation solution and the synergistic effect of the components jointly form a relatively stable preservation environment, so that the saliva or buccal swab sample can be preserved for a long time, and particularly, the good preservation of the sample can be realized in a high-temperature environment of more than 35 ℃. The preservation solution enables the application of the genomic DNA extracted from saliva or oral swabs to be wider, and reduces the environment and operation requirements, thereby reducing the operation cost and being beneficial to the subsequent PCR test.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an electrophoretogram of DNA genome extracted after saliva samples in test example 1 of the present invention are stored at room temperature for different time periods using the storage solution of example 1, M represents Trans 15K DNA marker, and 1-8 represent 8 different human samples, respectively;

FIG. 2 is an electrophoretogram of DNA genome extracted after oral swab samples in test example 1 of the present invention were stored at room temperature for different periods of time using the storage solution of example 1, where M represents Trans 15K DNA marker, and 1-8 represent 8 different human samples, respectively;

FIG. 3 is an electrophoretogram of DNA genome extracted from saliva samples stored at high temperature of 50 ℃ for 7 days in different storage solutions in test example 2 of the present invention, wherein M represents Trans 15K DNA Maker, 1 is the sample stored in example 1, and 2-11 are the samples stored in comparative examples 1-10;

FIG. 4 is an electrophoretogram of DNA genome extracted from saliva samples stored at 50 ℃ for 21 days in different storage solutions in test example 2 of the present invention, wherein M represents Trans 15K DNA Maker, 1 is the sample stored in example 1, and 2-11 are the samples stored in comparative examples 1-10;

FIG. 5 is an electrophoretogram of DNA genome extracted from oral swab samples stored at high temperature of 50 ℃ for 7 days in different storage solutions in test example 2 of the present invention, wherein M represents Trans 15K DNA Maker, 1 is the sample stored in example 1, and 2-11 are the samples stored in comparative examples 1-10;

FIG. 6 is an electrophoretogram of DNA genome extracted from oral swab samples stored at high temperature of 50 ℃ for 21 days in different storage solutions in test example 2 of the present invention, wherein M represents Trans 15K DNA Maker, 1 is the sample stored in example 1, and 2-11 are the samples stored in comparative examples 1-10;

FIG. 7 shows the amplification results of genomic DNA extracted from saliva samples stored at room temperature for 7 days in test example 3 of the present invention using the storage solution of example 1, where M represents GeneRuler High Range DNA Ladder, and 1 and 2 represent 2 different human samples, respectively;

FIG. 8 shows the results of amplification of genomic DNA extracted from saliva samples stored at room temperature for 21 days in test example 3 of the present invention using the storage solution of example 1, where M represents GeneRuler High Range DNA Ladder, and 1 and 2 represent 2 different human samples, respectively;

FIG. 9 shows the amplification results of genomic DNA extracted from oral swab specimens in test example 3 of the present invention after storage at room temperature for 7 days using the storage solution of example 1, wherein M represents GeneRuler High Range DNA Ladder, and 1 and 2 represent 2 different human samples, respectively;

FIG. 10 is a graph showing the results of amplification of genomic DNA extracted from an oral swab specimen in test example 3 of the present invention after storage at room temperature for 21 days using the storage solution of example 1, wherein M represents GeneRuler High Range DNA Ladder, and 1 and 2 represent 2 different human samples, respectively;

FIG. 11 is the results of genomic DNA amplification product 1 extracted from saliva samples in test example 3 of the present invention stored at 50 ℃ for 7 days using the storage solutions of example 1 and comparative examples 1 to 10, M representing GeneRuler High Range DNA Ladder, 1 representing the sample stored in example 1, and 2 to 11 representing the samples stored in comparative examples 1 to 10;

FIG. 12 is the results of genomic DNA amplification product 2 extracted from saliva samples in test example 3 of the present invention stored at 50 ℃ for 7 days using the storage solutions of example 1 and comparative examples 1 to 10, wherein M represents GeneRuler High Range DNA Ladder, 1 represents the sample stored in example 1, and 2 to 11 are the samples stored in comparative examples 1 to 10;

FIG. 13 is the results of genomic DNA amplification products 3 extracted from saliva samples in test example 3 of the present invention stored at 50 ℃ for 7 days using the storage solutions of example 1 and comparative examples 1 to 10, M representing GeneRuler High Range DNA Ladder, 1 representing the samples stored in example 1, and 2 to 11 representing the samples stored in comparative examples 1 to 10;

FIG. 14 is the results of genomic DNA amplification product 1 extracted from a saliva sample in test example 3 of the present invention stored at 50 ℃ for 21 days using the storage solutions of example 1 and comparative examples 1 to 10, M representing GeneRuler High Range DNA Ladder, 1 representing the sample stored in example 1, and 2 to 11 representing the samples stored in comparative examples 1 to 10;

FIG. 15 is the results of genomic DNA amplification product 2 extracted from a saliva sample in test example 3 of the present invention stored at 50 ℃ for 21 days using the storage solutions of example 1 and comparative examples 1 to 10, wherein M represents GeneRuler High Range DNA Ladder, 1 represents the sample stored in example 1, and 2 to 11 are the samples stored in comparative examples 1 to 10;

FIG. 16 is the results of genomic DNA amplification products 3 extracted from saliva samples in test example 3 of the present invention stored at 50 ℃ for 21 days using the storage solutions of example 1 and comparative examples 1 to 10, M representing GeneRuler High Range DNA Ladder, 1 representing the samples stored in example 1, and 2 to 11 representing the samples stored in comparative examples 1 to 10;

FIG. 17 is a graph showing the results of preservation of the oral swab specimens for 7 days at 50 ℃ for the genomic DNA amplification product 1 extracted in the preservation solution of example 1 and the preservation solutions of comparative examples 1 to 10 in test example 3 of the present invention, wherein M represents a GeneRuler High Range DNAdder, 1 represents the specimens preserved in example 1, and 2 to 11 represent the specimens preserved in comparative examples 1 to 10;

FIG. 18 is a graph showing the results of preservation of the oral swab specimens for 7 days at 50 ℃ for the genomic DNA amplification product 2 extracted in the preservation solution of example 1 and the preservation solutions of comparative examples 1 to 10 in test example 3 of the present invention, wherein M represents a GeneRuler High Range DNAdder, 1 represents the specimens preserved in example 1, and 2 to 11 represent the specimens preserved in comparative examples 1 to 10;

FIG. 19 is the results of genomic DNA amplification products 3 extracted from oral swab specimens in test example 3 of the present invention by storing the specimens at 50 ℃ for 7 days using the storage solution of example 1 and the storage solutions of comparative examples 1 to 10, wherein M represents GeneRuler High Range DNAader, 1 represents the specimens stored in example 1, and 2 to 11 represent the specimens stored in comparative examples 1 to 10;

FIG. 20 is a graph showing the results of preservation of the oral swab specimens for 21 days at 50 ℃ for the genomic DNA amplification product 1 extracted in the preservation solution of example 1 and the preservation solutions of comparative examples 1 to 10 in test example 3 of the present invention, wherein M represents a GeneRuler High Range DNAdder, 1 represents the specimens preserved in example 1, and 2 to 11 represent the specimens preserved in comparative examples 1 to 10;

FIG. 21 is a graph showing the results of preservation of the oral swab specimens for 21 days at 50 ℃ for the genomic DNA amplification product 2 extracted in the preservation solution of example 1 and the preservation solutions of comparative examples 1 to 10 in test example 3 of the present invention, wherein M represents GeneRuler High Range DNAdder, 1 represents the specimens preserved in example 1, and 2 to 11 represent the specimens preserved in comparative examples 1 to 10;

FIG. 22 is a graph showing the results of preservation of the oral swab specimens for 21 days at 50 ℃ for the genomic DNA amplification product 3 extracted in the preservation solution of example 1 and the preservation solutions of comparative examples 1 to 10 in test example 3 of the present invention, wherein M represents a GeneRuler High Range DNAdder, 1 represents the specimens preserved in example 1, and 2 to 11 represent the specimens preserved in comparative examples 1 to 10.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

In the invention, the components of the preservation solution jointly form a relatively stable preservation environment through effective proportioning and synergistic action, so that the saliva or buccal swab sample can be preserved for a long time, and particularly, the good preservation of the sample can be realized in a high-temperature environment of more than 35 ℃. The preservation solution enables the application of the genomic DNA extracted from saliva or oral swabs to be wider, and reduces the environment and operation requirements, thereby reducing the operation cost and being beneficial to the subsequent PCR test operation.

Urea and SDS act as denaturants to inhibit or reduce the growth of microorganisms; SDS and sodium alginate are used as bacteriostatic agents, can kill or reduce the microorganisms in saliva or oral swabs, and sodium alginate is used as a natural bacteriostatic agent, can effectively inhibit the breeding of fungi and bacteria, and prolongs the storage time; EDTA-Na2 as a chelating agent can chelate the activator of nuclease such as magnesium ions and the like, and inhibit the degradation of nuclease to DNA; the L-carnitine can effectively protect the activity of cells obtained from saliva or oral swabs and prolong the storage time; meanwhile, Tris-HCl is used as a buffer reagent, so that the preservation solution is relatively stable, and the pH environment in a certain range is favorable for preserving the sample.

In the present invention, "w/v%" means the mass g of a substance contained in 100mL of a storage solution, and for example, "0.3 to 1 w/v% SDS" means that 0.3 to 1g of SDS is contained in 100mL of a storage solution; the expression "sodium alginate 2-8 w/v%" means that each 100mL of the preservation solution contains 2-8g of sodium alginate; the 'L-carnitine content of 3-8 w/v%' means that each 100mL of preservation solution contains 3-8g of L-carnitine.

The content of Tris-HCl (Tris-hydroxymethyl-aminomethane hydrochloride) may be, but is not limited to, 10mmol/L, 20mmol/L, 30mmol/L, 40mmol/L or 50 mmol/L; the content of urea can be but is not limited to 0.2mol/L, 0.3mol/L, 0.4mol/L, 0.5mol/L, 0.6mol/L, 0.7mol/L, 0.8mol/L, 0.9mol/L or 1 mol/L; the content of SDS (sodium dodecyl sulfate) may be, but is not limited to, 0.3 w/v%, 0.4 w/v%, 0.5 w/v%, 0.6 w/v%, 0.7 w/v%, 0.8 w/v%, 0.9 w/v%, or 1 w/v%; the content of EDTA-Na2 can be but is not limited to 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L or 8 mmol/L; the content of sodium alginate can be but is not limited to 2 w/v%, 3 w/v%, 4 w/v%, 5 w/v%, 6 w/v%, 7 w/v% or 8 w/v%; the content of L-carnitine can be, but is not limited to, 3 w/v%, 4 w/v%, 5 w/v%, 6 w/v%, 7 w/v%, or 8 w/v%. Preservation solution pH may be, but is not limited to, 7, 8, 9, or 10.

In some embodiments, the preservation solution comprises: 15-45mmol/L Tris-HCl, 0.3-0.9mol/L urea, 0.3-0.8w/v SDS, 3-7w/v EDTA-Na 21-6 mmol/L sodium alginate and 3-7w/v L-carnitine, and the pH of the preservation solution is 7-9.

In a preferred embodiment, the preservation solution comprises: Tris-HCl 20-40mmol/L, urea 0.3-0.7mol/L, SDS0.4-0.7 w/v%, EDTA-Na 22-5 mmol/L, sodium alginate 4-6 w/v% and L-carnitine 3-5 w/v%, and the pH of the preservation solution is 7-8.

In a more preferred embodiment, the preservation solution comprises: 30mmol/L Tris-HCl, 0.6mol/L urea, 0.5w/v SDS0.5, 23mmol/L EDTA-Na, 5w/v sodium alginate and 4w/v L-carnitine, and the pH of the preservation solution is 7.5.

Through further optimization of the proportion of the components in the preservation solution, the preservation solution has more stable performance and better preservation effect.

The invention also provides a preparation method of the preserving fluid, which comprises the steps of uniformly mixing Tris-HCl, urea, SDS, EDTA-Na2, sodium alginate and L-carnitine according to the proportion by using water, and adjusting the pH value to obtain the preserving fluid.

In a preferred embodiment, the step of adjusting the pH further comprises sterilization. The sterilization may preferably be performed by filtration sterilization with a filter membrane.

The invention also protects the application of the preservation solution in extracting genome DNA from saliva or oral swabs. For saliva or oral swabs which are sampled, samples which cannot be used for immediately extracting genome DNA can be preserved by the preservation solution provided by the application, so that the later extraction is facilitated.

The invention also protects the application of the preservation solution in the preservation of saliva or oral swabs. Wherein the preservation temperature can be-80-50 ℃. Further preferably 35 to 50 ℃. The preservation solution provided by the invention is suitable for preserving saliva or oral swab samples in a high-temperature environment, and has a good effect.

In a preferred embodiment, the saliva is human saliva.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1

A saliva or oral swab preserving fluid comprises the following components and contents: 30mmol/L Tris-HCl, 0.6mol/L urea, 0.5w/v SDS, 23mmol/L EDTA-Na, 5w/v sodium alginate and 4w/v L-carnitine.

The preparation method of the saliva and oral swab preservative fluid of the embodiment is as follows:

30.06g of urea, 5g of SDS, 50g of sodium alginate and 40g of L-carnitine are respectively weighed and dissolved in 800ml of sterile water, then 30ml of 1M Tris-HCl and 6ml of 0.5M EDTA-Na2 are added, after being uniformly stirred by a glass rod, the pH value is adjusted to 7.5 by dilute hydrochloric acid, and then the volume is fixed to 1000 ml. Then the mixture is filtered by a disposable filter membrane and placed in a blue-cap bottle.

Example 2

A preservation solution for saliva or oral swabs comprises Tris-HCl 10mmol/L, urea 1mol/L, SDS0.3 w/v%, EDTA-Na 28 mmol/L, sodium alginate 2 w/v% and L-carnitine 8 w/v%, and the pH of the preservation solution is 7.

Example 3

A preservation solution for saliva or oral swabs comprises Tris-HCl 50mmol/L, urea 0.2mol/L, SDS1 w/v%, EDTA-Na 21 mmol/L, sodium alginate 8 w/v% and L-carnitine 3 w/v%, and the pH of the preservation solution is 10.

Example 4

A preserving fluid for saliva or oral swabs comprises Tris-HCl 15mmol/L, urea 0.9mol/L, SDS0.3w/v%, EDTA-Na 26 mmol/L, sodium alginate 3 w/v% and L-carnitine 7 w/v%, and the pH of the preserving fluid is 7.

Example 5

A preserving fluid for saliva or oral swabs comprises Tris-HCl 45mmol/L, urea 0.3mol/L, SDS0.8w/v%, EDTA-Na 21 mmol/L, sodium alginate 7 w/v% and L-carnitine 3 w/v%, and the pH of the preserving fluid is 9.

Example 6

A saliva or buccal swab preserving fluid comprises Tris-HCl 20mmol/L, urea 0.7mol/L, SDS0.4w/v%, EDTA-Na 25 mmol/L, sodium alginate 4 w/v% and L-carnitine 5 w/v%, and the pH of the preserving fluid is 7.

Example 7

A preservation solution for saliva or oral swab comprises Tris-HCl 40mmol/L, urea 0.3mol/L, SDS0.7 w/v%, EDTA-Na 22 mmol/L, sodium alginate 6 w/v% and L-carnitine 3 w/v%, and has pH of 8.

Comparative example 1

A preserving fluid for saliva or oral swab comprises Tris-HCl 5mmol/L, urea 0.15mol/L, SDS0.2w/v%, EDTA-Na20.5mmol/L, sodium alginate 1 w/v% and L-carnitine 2 w/v%, and the pH of the preserving fluid is 7.

Comparative example 2

A saliva or oral swab preserving fluid comprises Tris-HCl 60mmol/L, urea 1.5mol/L, SDS1.5 w/v%, EDTA-Na 210 mmol/L, sodium alginate 10 w/v% and L-carnitine 10 w/v%, and the pH of the preserving fluid is 8.

Comparative example 3

A saliva or buccal swab preservative solution is based on the preservative solution in example 1, and lacks 0.6mol/L urea.

Comparative example 4

A saliva or buccal swab preservative solution, based on the preservative solution of example 1, lacks 0.5 w/v% SDS.

Comparative example 5

A saliva or buccal swab preservative solution is based on the preservative solution in example 1, and lacks 3mmol/L EDTA-Na 2.

Comparative example 6

A saliva or buccal swab preservative solution, based on the preservative solution of example 1, lacks 5 w/v% sodium alginate.

Comparative example 7

A saliva or buccal swab preservative solution, based on the preservative solution of example 1, lacks 4 w/v% L-carnitine.

Comparative example 8

A saliva preservation solution comprises the following components and contents: 0.67mol/L of lithium chloride, 33mmol/L of Tris-HCl, 0.67mol/L of urea, 0.6% (w/v) of SDS, 3.3mmol/L of EDTA, 30% (v/v) of ethanol and 7.5 of system pH.

Comparative example 9

A saliva or buccal swab preservative solution has a pH of 5 based on the preservative solution of example 1.

Comparative example 10

A saliva or buccal swab preservative solution has a pH value of 11 based on the preservative solution of example 1.

Test example 1

Methods of saliva and buccal swab sample collection and preservation:

the requirements before sampling are as follows: gargling 30min before sampling to remove food residues and residual microorganisms, and no diet, smoking or large amount of drinking water is needed within 30min after gargling so as not to influence the recovery quantity of exfoliated cells.

Collecting a saliva sample: before starting spitting, the cheek was released, and after spitting, the saliva was mixed with the storage solution in example 1 at a ratio of 1: 1, reversing the proportion of the total amount of the saliva, collecting 3ml of saliva for each person, collecting 8 persons in total, fully mixing the saliva with a preservation solution, placing the mixture at room temperature for preservation, and extracting DNA in the sample in 0 day, 7 days and 21 days respectively.

Oral swab sample collection: a sterile medical cotton swab was prepared, and the swab was taken out by repeatedly rubbing the inside of the mouth and the left and right cheeks 15 times, and placed in 400. mu.l of the storage solution of example 1 to sufficiently soak the swab. 3 samples are collected from each person, 8 samples are collected, the samples are placed at room temperature for storage, and DNA in the samples is extracted in 0 day, 7 days and 21 days respectively.

The extraction method of the saliva sample DNA comprises the following steps:

adding 500. mu.l of LB and 25. mu.l of pk into 500. mu.l of the mixed sample of saliva and preservation solution, and performing water bath at 56 ℃ for 20 min;

after the supernatant is removed by magnetic attraction, 750 mu l Binding Buffer and 30 mu lMB are added and mixed evenly for 10 min;

after the supernatant was removed by magnetic attraction, 500. mu.l of WB1 was added and washed once;

after the supernatant was magnetically aspirated, 500. mu.l of WB2 was added and washed twice;

after the supernatant is removed by magnetic attraction, 500 mul of WB3 is added for washing once, and 50 mul of EB56 ℃ water bath is added for 10 min;

place the centrifuge tube on a magnetic rack and transfer the liquid to a new centrifuge tube.

Electrophoresis detection is performed on DNA extracted from saliva at 0 day, 7 days and 21 days, and the results are shown in FIG. 1, wherein M is marker, and lanes 1-8 are saliva samples of 8 persons respectively.

The method for extracting the DNA of the oral swab sample comprises the following steps:

adding 400 μ l LB and 20 μ l PK into 400 μ l oral swab sample, and water bathing at 56 deg.C for 20 min;

then taking the liquid out to a new centrifuge tube, adding 600 mu lBD and 30 mu lMB, and uniformly mixing for 10 min;

Electrophoresis detection of DNA extracted from the buccal swab at 0 day, 7 days and 21 days is shown in FIG. 2, wherein M is marker, and lanes 1-8 are saliva samples of 8 persons, respectively.

From the above results, it can be seen that the saliva sample is stored at room temperature for 7 days, and the DNA extracted from 21 days is clear in band and not degraded compared with the DNA extracted from 0 day; the oral swab samples were stored at room temperature for 7 days, 21 days and no degradation, with clear bands compared to the DNA extracted on day 0. Therefore, the preservation solution of the present invention can be used for the preservation of saliva and buccal swabs at room temperature.

In addition, the same tests were carried out on the preservation solutions of examples 2 to 7 according to the above test procedures, and tests show that the preservation solution provided by the present invention has a good effect on the preservation of saliva samples or buccal swab samples at room temperature.

Test example 2

Methods of saliva and buccal swab sample collection and preservation:

collecting a saliva sample: the saliva sample was collected and DNA was extracted according to the method of the saliva sample collection in test example 1. 16.5ml of saliva of 1 person is collected, mixed evenly and centrifuged, and then divided into 1.5ml tubes, 11 tubes in total, and the mixture is mixed with the preservation solution of the example 1 and the comparative examples 1-10 according to the proportion of 1: 1, mixing, placing at 50 ℃ for storage after mixing, and extracting DNA in the sample respectively for 7 days and 21 days.

Oral swab sample collection: DNA was collected and extracted according to the method for collecting oral swab specimens described in test example 1. 22 oral swabs of 1 person were collected, divided into 11 groups, and placed in the storage solutions of example 1 and comparative examples 1 to 10, respectively, and stored at 50 ℃ at the same time, and DNAs in the samples were extracted for 7 days and 21 days, respectively.

Through detection, in saliva samples, DNA can not be degraded in 7 days in the sample of the preservation solution of example 1, and the samples preserved in the preservation solutions of comparative examples 1-10 are degraded to different degrees in 7 days; the samples in the comparative example degraded more severely after 21 days and were in a dispersed state.

In example 1 and comparative examples 1 to 10, as shown in FIG. 3 (stored for 7 days) and FIG. 4 (stored for 21 days), M is maker, 1 is the sample of example 1, and 2 to 11 are the samples of comparative examples 1 to 10.

In the oral swab sample, the DNA can not be degraded in 7 days in the storage solution sample in the example 1, and the samples stored in the storage solutions of the comparative examples 1-10 are degraded to different degrees in 7 days; the samples in the comparative example degraded more severely after 21 days and were in a dispersed state.

In example 1 and comparative examples 1 to 10, as shown in FIG. 5 (stored for 7 days) and FIG. 6 (stored for 21 days), M is maker, 1 is the sample of example 1, and 2 to 11 are the samples of comparative examples 1 to 10.

The results show that the preservation solution provided by the invention can well protect the integrity of the genome DNA after being stored at the high temperature of 50 ℃ for 21 days, and the components and the formula of the preservation solution provided by the invention are more excellent, so that the genome DNA can be protected at the high temperature for a long time.

Test example 3

This test example was conducted to examine the effect of PCR amplification of a target fragment of one fragment (NC-000012.12) of chromosome 12 GRCh38.p13 using the extracted genomic DNA obtained from Takara as a PCR reagent, the storage solutions used in examples 1 and comparative examples 1 to 10 as the storage solution, and two room temperature samples stored for 7 days and 21 days obtained in test example 1 and 50 ℃ samples stored for 7 days and 21 days obtained in test example 2 as the genomic DNA. And (3) carrying out an experiment by taking the DNA as an amplification template to verify whether the extracted genome DNA can be subjected to subsequent PCR.

Product 1 Gene primer (purchased from Shanghai)

F：5’-AGCCTCGGTCTGTTTTCCAG-3’(SEQ ID NO.1)；

R：5’-TGTTCAGGTGCATGACATTTTG-3’(SEQ ID NO.2)；

Product 1 length: 48638 bp;

product 2 Gene primers (purchased from Shanghai)

F：5’-AACAAAGGGACCACCGATCC-3’(SEQ ID NO.3)；

R：5’-CATCAAACCCCACATCATGGC-3’(SEQ ID NO.4)；

Product 2 length: 39570 bp;

product 3 Gene primer (purchased from Shanghai)

F：5’-CAGCCCGAGGTCTTCTGCAA-3’(SEQ ID NO.5)；

R：5’-GTATAACTTGAGATCTGGCC-3’(SEQ ID NO.6)；

Product 3 length: 31290 bp;

and (3) PCR reaction system:

the results of genomic DNA of saliva samples, two room temperature samples stored for 7 days and 21 days are shown in FIGS. 7 and 8, where M is marker, and 1 and 2 represent 2 different human samples.

The results of storing the genomic DNA of saliva samples at 50 ℃ for 7 days and 21 days are shown in FIGS. 11 to 16, in which M is marker, 1 represents the results of the genomic amplification products 1 to 3 extracted from the samples stored in the preservation solution of example 1, and 2 to 11 represent the results of the genomic amplification products 1 to 3 extracted from the preservation solution of comparative examples 1 to 10.

The results of genomic DNA of the buccal swab samples, two room temperature samples stored for 7 days and 21 days, are shown in FIGS. 9 and 10, where M is marker, and 1 and 2 represent 2 different human samples.

The results of storing the genomic DNA of two samples at 50 ℃ for 7 days and 21 days in the buccal swab specimen are shown in FIGS. 17 to 22, in which M is maker, 1 represents the results of the extracted genomic amplification products 1 to 3 of the specimen stored in the preservative solution of example 1, and 2 to 11 represent the results of the extracted genomic amplification products 1 to 3 of the preservative solution of comparative examples 1 to 10.

From the above results, it can be seen that 3 target products were amplified in 21 days at normal temperature and high temperature in the saliva and buccal swab samples stored in example 1, while 3 target products were amplified in 7 days in the samples stored in comparative examples 1 to 10, and 3 target products were not amplified in 21 days, indicating that the stored genome was fragmented and the subsequent PCR experiment could not be performed.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Denorjeyi (Beijing) Biotechnology Ltd

<120> saliva or oral swab preserving fluid and preparation method and application thereof

<160>6

<170>PatentIn version 3.5

<210>1

<211>20

<212>DNA

<213> Artificial sequence

<400>1

agcctcggtc tgttttccag 20

<210>2

<211>22

<212>DNA

<213> Artificial sequence

<400>2

tgttcaggtg catgacattt tg 22

<210>3

<211>19

<212>DNA

<213> Artificial sequence

<400>3

aacaaaggga ccaccgatcc 20

<210>4

<211>21

<212>DNA

<213> Artificial sequence

<400>4

catcaaaccc cacatcatgg c 21

<210>5

<211>20

<212>DNA

<213> Artificial sequence

<400>5

cagcccgagg tcttctgcaa 20

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<400>6

gtataacttg agatctggcc 20

Claims

1. A saliva or buccal swab preservative fluid, comprising: 10-50mmol/L Tris-HCl, 0.2-1mol/L urea, 0.3-1w/v SDS, 2-8w/v EDTA-Na 21-8 mmol/L sodium alginate and 3-8w/v L-carnitine, and the pH of the preservation solution is 7-10.

2. The preservation fluid according to claim 1, characterized by comprising: 15-45mmol/L Tris-HCl, 0.3-0.9mol/L urea, 0.3-0.8w/v SDS, 3-7w/v EDTA-Na 21-6 mmol/L sodium alginate and 3-7w/v L-carnitine, and the pH of the preservation solution is 7-9.

3. The preservation fluid according to claim 1, characterized by comprising: Tris-HCl 20-40mmol/L, urea 0.3-0.7mol/L, SDS0.4-0.7 w/v%, EDTA-Na 22-5 mmol/L, sodium alginate 4-6 w/v% and L-carnitine 3-5 w/v%, and the pH of the preservation solution is 7-8.

4. The preservation fluid according to claim 1, characterized by comprising: 30mmol/L Tris-HCl, 0.6mol/L urea, 0.5w/v SDS, 23mmol/L EDTA-Na, 5w/v sodium alginate and 4w/v L-carnitine, and the pH of the preservation solution is 7.5.

5. The method for preparing a preserving fluid as claimed in any one of claims 1 to 4, wherein Tris-HCl, urea, SDS, EDTA-Na2, sodium alginate and L-carnitine are mixed with water in a ratio, and the pH is adjusted to obtain the preserving fluid.

6. The method of claim 5, further comprising the step of sterilizing after adjusting the pH.

7. Use of a preservation solution according to any of claims 1 to 4 for extracting genomic DNA from saliva or buccal swabs.

8. Use of a preservation solution according to any of claims 1-4 for preserving saliva or buccal swabs.

9. Use according to claim 8, wherein the temperature of storage is-80 ℃ to 50 ℃.

10. Use according to claim 8, wherein the saliva is human saliva.