CN111378719A - Reagent compositions and methods for preserving nucleic acid integrity in human saliva - Google Patents
Reagent compositions and methods for preserving nucleic acid integrity in human saliva Download PDFInfo
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Abstract
The invention provides a reagent composition for preserving the integrity of nucleic acids in human saliva, and applications and methods thereof. The reagent composition comprises urea, lauryl sodium sulfate, trans-1, CDTA, Tris-HCl, sodium ascorbate, propyl p-hydroxybenzoate, diazoalkyl imidazole urea, ethanol, sucrose and proteinase K. The reagent composition is uniformly mixed with human saliva and then stored at normal temperature. The reagent composition and the preservation method can inhibit the activity of nuclease and avoid the nuclease from being polluted by external microorganisms or being oxidized by the external microorganisms, thereby ensuring the purity and the integrity of genome DNA, having long preservation time and wide applicable preservation temperature and ensuring the stability of saliva samples in the transportation process among various collection places.
Description
Technical Field
The present invention relates to the field of biotechnology, and more particularly to reagent compositions and methods for preserving the integrity of nucleic acids in human saliva.
Background
At present, molecular biology detection technology is increasingly used in various fields such as forensics, military, human medicine and scientific research. For example, in human health, after DNA in blood, other body fluids or cells of a detector is extracted and a nucleic acid sequence is amplified, a target sequence or a susceptible site of the detector is detected, and relevant gene information is collected, people can know the gene information of the person through reading known information, and the risk of the disease of the body is predicted, so that the living environment and habit of the person are improved, and the disease is avoided or delayed. In the field of forensic identification, the identity of the deceased can be identified by extracting the DNA of the family.
The body fluids currently used for extracting genomic DNA are usually derived from blood, but the use of blood as a source of detection has many disadvantages. First, since DNA in blood is mainly derived from white blood cells, and a large amount of red blood cells containing no DNA are present in blood, it is necessary to remove the influence of red blood cells when extracting DNA having a high purity. Second, the collection of blood requires a trained professional to operate. In addition, blood collection is an invasive procedure, often causing some discomfort and pain to the test subject, and furthermore, the blood collection process needs to avoid infection by some blood-borne pathogens and bacteria, and the collected blood must be refrigerated or DNA extracted as quickly as possible. Compared with the saliva, the saliva is easily obtained body fluid for a human body, and is more convenient to collect. Pure saliva does not contain human DNA, but since oral epithelial cells are constantly metabolized, there is cell shedding at every moment, and thus DNA is available through saliva.
Saliva collection has the following distinct advantages over blood collection: 1, no stabbing pain and no harm to sampling, thereby avoiding unnecessary pain to a tester; 2, the operation is simple and convenient, and the collection personnel can sample and send the samples by themselves without professional training; 3, the application is wide, some people are not suitable for blood sampling, such as the old, children and the like, and other people resist blood sampling, such as patients with blood sickness and mental diseases; 4, the cost of consumables such as an injector, an anticoagulation tube and the like is saved; 5, the transportation is convenient, and if the transportation is carried out in a large range and a long distance, the saliva is obviously more suitable to be transferred between a laboratory and each collection place.
The purity and integrity of DNA used for gene detection are ensured firstly, and if the DNA is damaged by breakage, degradation and the like, subsequent experiments are certainly and directly influenced, and the accuracy of an analysis result is further influenced; organic matters, bacteria and the like contained in saliva can also have certain influence on the integrity of cell DNA; in addition, saliva preservation solution is used as a preservation medium for saliva DNA, and is also at risk of oxidation and microbial contamination. The microorganism can use DNA as a nutrient, even secrete enzymes for promoting the accelerated degradation of the DNA, damage the integrity of the DNA and further greatly reduce the reliability of subsequent experimental data. Therefore, the saliva is well preserved, and the saliva is about important for being used as a source of genome DNA in gene detection. Most saliva preservation solutions in the market at present cannot well ensure the purity and integrity of saliva DNA, and the better saliva preservation solutions in the market cannot preserve the saliva DNA for a long time. Therefore, the research and development of the human saliva sample preservation solution with good preservation effect, convenient preservation and long preservation time has very important significance.
Disclosure of Invention
To overcome the disadvantages of the prior art, the present invention aims to provide a DNA composition for preserving human body fluids such as saliva and a method for preparing the same. The present invention relates to a reagent composition for extracting nucleic acid from a sample of human body fluid, such as saliva, which comprises a denaturant, a detergent, a metal ion chelating agent, a reducing agent, a bacteriostatic agent, a buffering agent and a protease, so that the nucleic acid in the sample can be stably stored for at least 24 months at room temperature and can be directly used for downstream experiments such as polymerase chain reaction, gene detection and the like without other treatments. Specifically, the composition comprises urea, sodium dodecyl sulfate, trans-1, 2-cyclohexanediaminetetraacetic acid (CDTA), Tris-HCl, sodium ascorbate, propyl p-hydroxybenzoate, diazoalkylimidazolium urea, ethanol, sucrose and proteinase K. More specifically, in order to achieve the above object, the present invention adopts a technical solution comprising: a reagent composition for preserving the integrity of nucleic acids in human saliva is provided, comprising a denaturant, a detergent
Further, Tris-HCl, urea, SDS, CDTA, ethanol, NaAc, propyl paraben, diazoalkylimidazolium urea, sodium ascorbate, sucrose and proteinase K.
Further, the method comprises 1-10mmol/L of CDTA; 10% -50% of ethanol; NaAc0.1-1 mol/L; 1-5mg/mL propyl p-hydroxybenzoate; 1-5mg/mL diazoalkyl imidazole urea; 0.01-0.5mol/L of sodium ascorbate; 1 to 10 percent of cane sugar.
Further, Tris-HCl with pH of 6.5-8.5 is contained in 0.05-0.5 mol/L; 0.1-1mol/L of urea; 0.1% -1% of SDS; 1-10mmol/L of CDTA; 10% -50% of ethanol; NaAc0.1-1 mol/L; 1-5mg/mL propyl p-hydroxybenzoate; 1-5mg/mL diazoalkyl imidazole urea; 0.01-0.5mol/L of sodium ascorbate; 1% -10% of sucrose; proteinase K10ug/mL, system pH 7-9.
Further, Tris-HCl 0.2mol/L with PH8.0 is included; 0.45mol/L of urea; 0.6% of SDS; CDTA3.6mmol/L; 40% of ethanol; 2mg/mL propyl p-hydroxybenzoate; diazoalkyl imidazole urea 2 mg/mL; sodium acetate 0.67 mol/L; 0.05mol/L of sodium ascorbate; 4% of sucrose; 10ug/mL of protease K; the system pH was 8.0.
The invention also provides application of the reagent composition in preserving the integrity of nucleic acid in human saliva.
The invention also provides a method for preserving the integrity of nucleic acids in human saliva, comprising providing the reagent composition and mixing the reagent composition with a human saliva sample.
Further, the reagent composition is mixed with the human saliva sample uniformly according to the ratio of 1: 1.
Further, the mixture is uniformly mixed and stored at normal temperature.
The CDTA introduced into the human saliva preservation composition provided by the invention is a powerful metal ion chelating agent, can chelate divalent metal cations such as Mg, Ga and the like contained in saliva per se, inhibit the activity of nuclease and prevent DNA from being degraded, and inhibit the occurrence of redox reaction based on metal ions; the introduced sodium acetate can precipitate part of DNA released by cell lysis to prevent suspension fracture, and can provide a weak alkaline environment for ensuring the stability of the DNA; the introduced propyl p-hydroxybenzoate and diazo alkyl imidazole urea can better inhibit the growth of microbial colonies without damaging DNA, and the propyl p-hydroxybenzoate and diazo alkyl imidazole urea are used in a matching way to form a very broad-spectrum antibacterial system, so that the propyl p-hydroxybenzoate and diazo alkyl imidazole urea can effectively inhibit bacteria and sterilize, prevent the pollution of microorganisms and the damage to DNA, and prolong the storage time of a sample; the introduced ethanol can not only destroy the structure of the protein and play the role of a denaturant, but also inhibit the growth of microorganisms; the introduced cane sugar can play a role in increasing the viscosity of a system and maintaining proper osmotic pressure to prevent DNA from being damaged by mechanical force; the most important point is that a strong reducing agent sodium ascorbate is introduced, is an endogenous free radical scavenger and can play an anti-oxidation role, and the sodium ascorbate can generate a color change reaction in an aqueous solution along with the prolonging of time, and gradually changes from colorless to yellow so as to indicate the preservation time of the preservation solution. In conclusion, the components are introduced and matched according to the set proportion, so that the problems that DNA in a saliva sample of a human body is not broken and incomplete or low in DNA purity due to DNA breakage and pollution caused by enzymolysis or oxidation of nuclease and other factors generated by external conditions are solved, and the saliva sample can be stably stored for a long time under appropriate conditions such as pH, system viscosity and the like, so that the saliva sample can be better stored for a longer time.
The composition for preserving the human saliva is used for preserving the human saliva sample, can inhibit the activity of nuclease, and avoids the contamination or the self-oxidation of the nuclease by external microorganisms, thereby ensuring the purity and the integrity of genome DNA, having long preservation time and wide applicable preservation temperature, and being capable of ensuring the stability of the saliva sample in the transportation process among various collection places.
Drawings
FIG. 1 is an agarose gel electrophoresis image of genomic DNA extracted from saliva samples of human saliva preservation compositions prepared in example 1, stored at room temperature for various periods of time, wherein: m: HindIII Marker; 1: standing at room temperature for 3 d; 2: standing at room temperature for 7 d; 3: standing at room temperature for 14 d; 4: standing at room temperature for 1 month; 5: standing at room temperature for 3 months; 6: standing at room temperature for 6 months; 7: standing at room temperature for 12 months; 8: standing at room temperature for 18 months; 9: standing at room temperature for 24 months
FIG. 2 is an agarose gel electrophoresis image of genomic DNA extracted from saliva samples after storage of the composition for human saliva storage prepared in example 1 at 37 ℃ for various times, in which: m: HindIII Marker; 1: standing at 37 ℃ for 3 d; 2: standing at 37 ℃ for 7 d; 3: standing at 37 ℃ for 14 d; 4: standing at 37 deg.C for 1 month; 5: standing at 37 deg.C for 2 months; 6: standing at 37 deg.C for 3 months; 7: standing at 37 deg.C for 6 months; 8: standing at 37 deg.C for 9 months; 9: standing at 37 deg.C for 12 months.
FIG. 3 is an agarose gel electrophoresis image of genomic DNA extracted from a saliva sample obtained after storing the composition for human saliva storage prepared in example 1 at a high temperature of 45 ℃ for various times, in which: m: HindIII Marker; 1: standing at 45 ℃ for 14 d; 2: standing at 45 deg.C for 1 month; 3: standing at 45 deg.C for 3 months; 4: standing at 45 deg.C for 6 months; 5: standing at 45 deg.C for 9 months; 6: standing at 45 deg.C for 12 months.
FIG. 4 is an agarose gel electrophoresis image of genomic DNA extracted from a saliva sample obtained after shaking and mixing the composition for preserving human saliva prepared in example 1 on a vortex mixer for various times, wherein: m: HindIII Marker; 1: vortex and shake for 1 day; 2: vortex and shake for 3 days; 3: vortex and shake for 5 days; 4: vortex and shake for 7 days; 5: vortex and shake for 9 days; 6: vortex for 12 days; 7: vortex for 15 days.
Detailed Description
Example 1
The composition for preserving human saliva comprises the following components in percentage by weight: Tris-HCl 0.2mol/L at pH 8.0; 0.45mol/L of urea; 0.6% of SDS; CDTA3.6 mmol/L; 40% of ethanol; 2mg/mL propyl p-hydroxybenzoate; diazoalkyl imidazole urea 2 mg/mL; sodium acetate 0.67 mol/L; 0.05mol/L of sodium ascorbate; 4% of sucrose; 10ug/mL of protease K; the system pH was 8.0.
Test of preservation Effect of the above-mentioned composition for preserving saliva of human body:
the subjects cleaned their mouth 30 minutes before the saliva samples were collected, kept clean, and did not eat, smoke, and chew their gums. Before starting to collect saliva, the cheek was relaxed and gently massaged with fingers for 15-30 seconds to produce saliva. Spit the saliva into the saliva collection tube gently to avoid excessive foam as much as possible. After collecting about 2ml of saliva, the saliva was mixed with 2ml of the composition for preserving human saliva of example 1 by inversion, and then stored under the following preservation conditions for use.
1. Stability test of saliva samples preserved with the composition for preserving human saliva after standing at room temperature for various periods of time
The saliva samples preserved by the composition for preserving human saliva are respectively placed at room temperature for 3 days, 7 days, 14 days, 1 month, 3 months, 6 months, 12 months, 18 months and 24 months, and then the genomic DNAs of the saliva samples obtained by the kit for extracting saliva genome by the Koning column method are respectively numbered 1-9 for standby.
Agarose gel electrophoresis: each of the saliva samples DNA2ul was applied to 1% agarose gel, labeled with HingIIImarker, and electrophoresed. After electrophoresis, the gel image system was used to take pictures, see in particular FIG. 1.
And (3) detecting concentration and purity: the saliva sample DNA2ul was collected and tested for concentration and purity by Nano-100, as shown in Table 1.
Table 1: and (3) carrying out DNA detection on saliva samples placed at room temperature for different times:
| sample numbering | Concentration (ng/ul) | A260/A280 |
| 1 | 46.3 | 1.86 |
| 2 | 45.6 | 1.84 |
| 3 | 48.2 | 1.85 |
| 4 | 42.8 | 1.84 |
| 5 | 38.8 | 1.83 |
| 6 | 42.1 | 1.83 |
| 7 | 39.7 | 1.82 |
| 8 | 42.6 | 1.85 |
| 9 | 46.1 | 1.83 |
To summarize: as shown in FIG. 1 and Table 1, the saliva sample is placed at room temperature for 3 days until 24 months, the DNA is not degraded, and the electrophoresis result shows that the integrity of the genome DNA is good.
2. Stability test of saliva samples preserved with the composition for preserving human saliva after standing at 37 ℃ for various periods of time
The saliva samples preserved by the composition for preserving human saliva are respectively placed in an environment of 37 ℃ for 3 days, 7 days, 14 days, 1 month, 2 months, 3 months, 6 months, 9 months and 12 months, and then the genomic DNA of each saliva sample obtained by using a kit for extracting saliva genome by a Koning column method is respectively numbered 1-9 for standby.
Agarose gel electrophoresis: each of the saliva samples DNA2ul was applied to 1% agarose gel, labeled with HingIIImarker, and electrophoresed. After electrophoresis, the gel image was taken with a gel imaging system, see fig. 2.
And (3) detecting concentration and purity: the saliva sample DNA2ul was collected and tested for concentration and purity by Nano-100, as shown in Table 2.
Table 2: DNA detection of saliva samples placed at 37 ℃ for different times:
| sample numbering | Concentration (ng/ul) | A260/A280 |
| 1 | 44.2 | 1.81 |
| 2 | 44.8 | 1.84 |
| 3 | 45.9 | 1.86 |
| 4 | 46.6 | 1.82 |
| 5 | 45.1 | 1.88 |
| 6 | 43.8 | 1.83 |
| 7 | 42.6 | 1.89 |
| 8 | 45.1 | 1.86 |
| 9 | 44.7 | 1.82 |
To summarize: as shown in FIG. 2 and Table 2, when the saliva sample is placed in a 37 ℃ environment for 3 days until 12 months, the DNA is not degraded, and the electrophoresis result shows that the integrity of the genome DNA is good.
3. Stability test of saliva samples preserved with the composition for preserving human saliva after standing at 45 ℃ for various periods of time
The saliva samples preserved by the composition for preserving human saliva are respectively placed under the environment of 45 ℃ for 14 days, 1 month, 3 months, 6 months, 9 months and 12 months, and then the genomic DNA of each saliva sample obtained by a Koning column method saliva genomic extraction kit is respectively numbered 1-6 for standby.
Agarose gel electrophoresis: each of the saliva samples DNA2ul was applied to 1% agarose gel, labeled with HingIIImarker, and electrophoresed. After electrophoresis, the gel image was taken with a gel imaging system, see FIG. 3.
And (3) detecting concentration and purity: the saliva sample DNA2ul was collected and tested for concentration and purity by Nano-100, as shown in Table 3.
Table 3: and (3) carrying out DNA detection on saliva samples placed at room temperature for different times:
| sample numbering | Concentration (ng/ul) | A260/ |
| 1 | 40.5 | 1.88 |
| 2 | 43.9 | 1.82 |
| 3 | 46.7 | 1.86 |
| 4 | 42.8 | 1.84 |
| 5 | 39.8 | 1.86 |
| 6 | 43.7 | 1.83 |
To summarize: as shown in FIG. 3 and Table 3, when the saliva sample is left at 45 ℃ for 14 days until 12 months, the DNA is not degraded, and the electrophoresis result shows that the integrity of the genome DNA is good.
4. Stability test of saliva samples preserved with the composition for preserving human saliva after shaking and mixing the saliva samples on a vortex mixer for different time
The saliva samples preserved by the composition for preserving human saliva are respectively shaken and mixed on a vortex mixer for 1 day, 3 days, 5 days, 7 days, 9 days, 12 days and 15 days, and then the genomic DNA of each saliva sample obtained by a Koning column method saliva genomic extraction kit is respectively numbered for 1-7 for standby.
Agarose gel electrophoresis: each of the saliva samples DNA2ul was applied to 1% agarose gel, labeled with HingIIImarker, and electrophoresed. After electrophoresis, the gel image was taken with a gel imaging system, see FIG. 4.
And (3) detecting concentration and purity: the saliva samples, DNA2ul, were collected and tested for concentration and purity using Nano-100, see Table 4.
Table 4: and (3) carrying out DNA detection on saliva samples placed at room temperature for different times:
to summarize: as shown in FIG. 4 and Table 4, the saliva sample is shaken and mixed on a vortex mixer for 1 day until 15 days, the DNA is not degraded, and the electrophoresis result shows that the integrity of the genome DNA is good.
Relevant saliva preservation and genome DNA extraction detection experiments show that the composition for preserving human saliva can stably preserve saliva samples for more than two years at room temperature; the saliva sample can be stably preserved for more than one year under the condition of high temperature (37 ℃ and 45 ℃); can be stably stored for more than 7 days under the condition of violent shaking.
Claims (10)
1. A reagent composition for preserving the integrity of nucleic acids in human saliva comprising a denaturant, a detergent, a metal ion chelator, a reducing agent, a bacteriostatic agent, and a buffer and a protease.
2. The reagent composition of claim 1, comprising Tris-HCl, urea, SDS, CDTA, ethanol, NaAc, propyl paraben, diazoalkylimidazolium urea, sodium ascorbate, sucrose, and proteinase K.
3. The reagent composition of claim 2, comprising CDTA 1-10 mmol/L; 10% -50% of ethanol; NaAc0.1-1 mol/L; 1-5mg/mL propyl p-hydroxybenzoate; 1-5mg/mL diazoalkyl imidazole urea; 0.01-0.5mol/L of sodium ascorbate; 1 to 10 percent of cane sugar.
4. The reagent composition of claim 2, comprising Tris-hci 0.05-0.5mol/L at PH 6.5-8.5; 0.1-1mol/L of urea; 0.1% -1% of SDS; 1-10mmol/L of CDTA; 10% -50% of ethanol; NaAc0.1-1 mol/L; 1-5mg/mL propyl p-hydroxybenzoate; 1-5mg/mL diazoalkyl imidazole urea; 0.01-0.5mol/L of sodium ascorbate; 1% -10% of sucrose; proteinase K10ug/mL, system PH 7-9.
5. The reagent composition of claim 4, comprising Tris-HCl 0.2mol/L at PH 8.0; 0.45mol/L of urea; 0.6% of SDS; CDTA3.6 mmol/L; 40% of ethanol; 2mg/mL propyl p-hydroxybenzoate; diazoalkyl imidazole urea 2 mg/mL; sodium acetate 0.67 mol/L; 0.05mol/L of sodium ascorbate; 4% of sucrose; 10ug/mL of protease K; the system pH was 8.0.
6. Use of a reagent composition according to one of claims 1 to 5 for preserving the integrity of nucleic acids in human saliva.
7. A method for preserving the integrity of nucleic acids in human saliva, comprising providing a reagent composition according to any one of claims 1 to 3; and mixing the reagent composition with a human saliva sample.
8. The method of claim 7, wherein the reagent composition is mixed with the human saliva sample at a 1:1 ratio.
9. The method of claim 8, wherein the mixture is stored at ambient temperature after being mixed uniformly.
10. The method of claim 9, wherein the storage is performed at ambient temperature for 24 months.
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| CN112662661A (en) * | 2020-12-30 | 2021-04-16 | 华南理工大学 | Genome DNA room temperature preservation card and manufacturing method and application thereof |
| CN112760318A (en) * | 2020-12-30 | 2021-05-07 | 苏州白垩纪生物科技有限公司 | Reagent composition for stabilizing nucleic acid molecules and application thereof |
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