CN111440790A - Method for simultaneously extracting DNA and RNA of salivary spots - Google Patents

Abstract

The invention discloses a method for simultaneously extracting DNA and RNA from saliva stains. The present invention provides a method for synchronously extracting DNA and RNA from on-site saliva stains. 1) Immerse the on-site saliva stains with carriers in a reaction system containing DTT and cell dissociation buffer, incubate the reaction, and obtain Reaction product; 2) removing the carrier in the reaction product, collecting the reaction product of removing the carrier, and then passing the reaction product of removing the carrier through the DNA adsorption column, collecting the adsorption column that is adsorbed with DNA and the column liquid containing RNA 3) Purify the described column liquid containing RNA and the described adsorption column adsorbed with DNA respectively to obtain RNA and DNA; this method can be used for nucleic acid extraction of common on-site inspection materials such as oral swabs, cigarette butts, water cup wipes, etc. , the obtained DNA and RNA samples obtained ideal detection results in downstream STR typing and fluorescence quantitative PCR detection.

Description

Simultaneous extraction of DNA and RNA from saliva blots

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for simultaneously extracting DNA and RNA from saliva stains.

Background technique

Saliva stains are one of the most common types of biological evidence at the scene of various crimes, including oral swab samples, cigarette butts, tableware wipes, bite marks and other traces of on-site inspection materials that may adhere to oral epithelial cells. It contains nucleic acid information such as DNA and RNA. With the development of DNA testing technology, compared with other biological evidences such as exfoliated cells, saliva stains are easier to obtain DNA STR typing with comparative value through DNA analysis, which plays an irreplaceable role in case investigation and suspect investigation. important role. At the same time, on the basis of DNA STR typing test, the tissue origin of saliva stains and other on-site biological samples can be inferred by technical means such as RNA analysis, which can provide accurate information for the comprehensive analysis of the biological evidence involved and the reconstruction of the crime process. Enhance the evidential value of the biological evidence involved in the case.

However, under the current criminal form and the development trend of criminal technology, trace and old biological evidence has become the main object of inspection and research in the field of forensic science, and plays an important role in the practice of handling cases. Due to the low content of nucleic acid and the high degree of nucleic acid damage, it is difficult to extract nucleic acid samples that meet both DNA STR test and RNA analysis at the same time by applying existing technical methods for trace and old biological evidence. In the practice of handling cases, often due to technical limitations, a more urgent analysis content has to be selected in DNA and RNA analysis, and a lot of important information is lost. Therefore, there is an urgent need for a simultaneous extraction method of DNA and RNA that can be applied to saliva stains in the field.

The study found that in the fields of basic medicine and biological research, there have been relatively well-developed kits or methods for simultaneous extraction of DNA and RNA. However, these methods are only suitable for a large number of clean culture samples such as cells and tissues, and it is difficult to obtain DNA and RNA nucleic acid samples that meet the needs of downstream detection when applied to on-site biological samples with complex traces, inhibitors or impurities. The practical requirements for simultaneous extraction and analysis of DNA and RNA from trace saliva stains at the scene of criminal technology in a crime scene.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for the simultaneous extraction of DNA and RNA from live saliva blots.

The method provided by the present invention comprises the following steps:

1) soaking the spot saliva stain with the carrier in the reaction system containing DTT and cell dissociation buffer, incubating the reaction to obtain the reaction product;

2) remove the carrier in the reaction product, collect the reaction product of removing the carrier, and then pass the reaction product of removing the carrier through the DNA adsorption column (in the embodiment of the present invention, be specifically AllPrep DNA/RNA Mini Kit kit; DNeasy Spin Column in the middle), collect the adsorption column adsorbed with DNA and the column liquid containing RNA;

3) purifying the column-passing liquid containing RNA and the adsorption column adsorbing DNA respectively to obtain RNA and DNA;

The purification of the RNA-containing column liquid is carried out according to a method comprising the following steps:

3)-1, flow through the RNA adsorption column (in the embodiment of the present invention, be specifically the RNeasy Spin Column in the AllPrep DNA/RNA Mini Kit test kit) of the described column-crossing liquid containing RNA, obtain the adsorption that is adsorbed with RNA column;

3)-2, use RNA elution buffer (in the embodiment of the present invention, specifically adopt RNase-free water as RNA elution buffer) to flow through the described adsorption column with RNA, room temperature (18-25 ℃) ) stand for 5-10min, centrifuge (centrifugal force 16200g centrifugation for 1min) to collect the first column liquid to obtain RNA;

The DNA purification is carried out according to a method comprising the following steps:

3)-a, use DNA elution buffer (EB Buffer or water or other DNA elution buffer can be used as DNA elution buffer, which can be the one in the AllPrep DNA/RNA Mini Kit, or Can be prepared or purchased), flow through the adsorption column with DNA adsorbed, stand at 60-65°C for 8-15min (the standstill time may be 10min in particular), and collect the first column liquid by centrifugation to obtain DNA. .

In the above-mentioned method, in the method for purifying the liquid through the column containing RNA, between steps 3)-1 and 3)-2, the following steps are further included: washing the adsorption column adsorbed with RNA, and obtaining the adsorption column after washing. RNA adsorption column.

Washing is specifically divided into the following:

①Add 700μl Buffer RW1 (washing) to the adsorption column RNeasy Spin Column with RNA, and centrifuge at 13000rpm for 15s;

② Add 500 μl BufferRPE (washing) to the adsorption column RNeasy Spin Column with RNA adsorbed after the previous step, and centrifuge at 13000rpm for 15s;

Note: An appropriate volume of absolute ethanol should be added to Buffer RPE before use.

③ Add 500 μl BufferRPE (washing) to the adsorption column RNeasy Spin Column with RNA adsorbed in the upper step, and centrifuge at 13000 rpm for 2 min;

④ Centrifuge at 13000rpm for 1min, repeat twice to ensure that no liquid remains on the RNeasy Spin Column;

⑤ Transfer the RNA-adsorbed adsorption column treated in the previous step to a new RNase-free and DNase-free clean 2mL EP tube, and centrifuge at 13000rpm for 1min;

In the above method, in the method for purifying the RNA-containing column liquid, in step 3)-1, before flowing through the RNA adsorption column, the following steps are also included: adding to the RNA-containing column liquid, etc. The volume percentage of the volume is 70%-80% of the ethanol aqueous solution to obtain a mixed solution.

In the above method, in the method for purifying the RNA-containing column liquid, in 3)-2, after the first time passing the column liquid is collected, the first time passing the column liquid is passed through the The adsorption column with the RNA adsorbed is allowed to stand for 5-10 min at room temperature, and the second column passing liquid is collected by centrifugation; the first passing column liquid and the second passing column liquid are combined to obtain RNA.

In the DNA purification method, before the DNA elution buffer is used to flow through the DNA-adsorbed adsorption column, the following steps are further included: washing the DNA-adsorbed adsorption column to obtain the washed DNA-adsorbed adsorption column. adsorption column.

In the above method,

Washing is as follows:

① Add 500 μl BufferAW1 (washing) to the DNA-adsorbed adsorption column DNeasy Spin Column obtained in the above 2), and centrifuge at 13000 rpm for 15 s;

Note: An appropriate volume of absolute ethanol should be added to Buffer AW1 before use.

② Add 500 μl BufferAW2 (washing) to the DNA-adsorbed adsorption column DNeasy Spin Column treated in the previous step, and centrifuge at 13000 rpm for 2 min;

③ Transfer the DNA-adsorbed DNeasy Spin Column treated in the previous step to a new RNase-free and DNase-free clean 2mL EP tube, and centrifuge at 13000rpm for 1min;

④ Centrifuge at 13,000 rpm for 1 min and repeat twice to ensure that no liquid remains on the DNeasy Spin Column;

In the above-mentioned method, in the method for DNA purification, in 3)-a, after the first time passing the column liquid is collected, the first time passing the column liquid is then passed through the washing and adsorbed thereon. DNA adsorption column, stand at 60-65° C. for 8-15 min, and centrifuge to collect the second-pass column liquid; combine the first-pass column liquid and the second-pass column liquid to obtain DNA.

In the above method, the concentration of the DTT in the reaction system is 1-2 mM; the concentration of the DTT in the reaction system is specifically 1.6 mM. In the embodiment of the present invention, the reaction system containing DTT and cell dissociation buffer is to mix DTT and cell dissociation buffer to obtain a reaction system containing DTT and cell dissociation buffer; in the embodiment of the present invention , the cell dissociation buffer is Buffer RLT in the AllPrep DNA/RNA Mini Kit.

In the above method, in step 1), the reaction conditions are incubation at room temperature for more than or equal to 1.5 hours; the above reaction conditions are specifically incubation at 18-25° C. for 2.5 hours.

In the above method, the RNA elution buffer is RNase-free water.

In the above method, the on-site saliva stains with the carrier are traces of on-site inspection materials such as oral swabs, cigarette butts or water cup wipes.

In the above-mentioned method step 2), the above-mentioned removal of the carrier in the reaction product, the collection and removal of the carrier reaction product is carried out according to the following method: from the reaction product, use RNase-free, DNase-free clean ophthalmic tweezers to pick up the carrier, and place the RNase-free tweezers on the carrier. , In a DNase-free disposable syringe, press the plunger of the syringe to squeeze out all the residual liquid on the carrier, recover the residual liquid on the carrier, and recycle the residual liquid into the original centrifuge tube to remove the carrier from the reaction product. The external liquid is mixed to obtain the reaction product (liquid) from which the carrier is removed;

Before passing the carrier-removed reaction product through the DNA adsorption column, the following steps are also included: centrifuge the carrier-removed reaction product at 13,000 rpm for 2 min (to remove common dust, soil and other small particle impurities on the carrier of the on-site inspection material), take Centrifuge the supernatant;

The above collection is to pass the centrifugation supernatant through the DNA adsorption column, repeat the centrifugation twice at 13000 rpm for 30 s, until there is no liquid in the adsorption column, and collect the adsorption column adsorbed with DNA and the column liquid containing RNA.

The above-mentioned DNA adsorption column may specifically be the AllPrep DNASpin Column adsorption column in the AllPrep DNA/RNA Mini Kit.

The DNA and RNA synchronous extraction method established in this study can be used for nucleic acid extraction of common field samples such as oral swabs, cigarette butts, and water cup swabs. The DNA and RNA samples obtained can be obtained in downstream STR typing and fluorescence quantitative PCR detection. Ideal detection results, simple operation, stable results, in line with actual case handling requirements, can be used for on-site DNA and RNA analysis and detection of saliva plaques.

The establishment of this method is based on the optimization of the method of the finished kit. The kit, which is only suitable for a large number of clean samples such as cells and tissues, is applied to common cases such as oral swabs, cigarette butts, water cup swabs and other common cases with trace amounts and complex inhibitors. The method of simultaneous extraction of DNA and RNA from saliva stains of on-site inspection materials provides the possibility for the simultaneous analysis of DNA and RNA of on-site inspection materials, in order to play a role in the inspection of biological evidence involved in the case, and to provide information for case investigation, characterization of suspects and reconstruction of the crime process. More comprehensive information will further enhance the evidence value of saliva stains on site. However, the cost of the finished kit is high. In the future promotion and application, the extraction method with lower cost should be further developed, and its application effect in blood, exfoliated cells and other samples should be investigated according to actual needs.

The method advantages of the present invention are as follows:

1. Carrier (cell) treatment: In the digestion and lysis of the carrier, the operation steps different from the cell and tissue suspension are adopted. The oral epithelial cells attached to the cotton swab and the cigarette butt together with the carrier are directly immersed in the lysing solution Buffer RLT. At the same time, in order to avoid the loss of cells or free nucleic acid fragments due to cell fragmentation, no other treatment is performed before this, and only the carrier is cut into pieces to form fragments of suitable size, forming a large surface area to facilitate the penetration of the lysate. resulting in higher digestion efficiency;

2. Digestion and cleavage: instead of using β-ME (2-mercaptoethanol) as a reducing agent to destroy protein disulfide bonds, DTT (dithiothreitol), which is less toxic and more reducing, is used to destroy protein disulfide bonds. At the same time, it prevents DNA from forming dimers, which is beneficial to dissociate oral epithelial cells from the complex carrier surface, and can also destroy cells more efficiently in the presence of impurities or even inhibitors, so as to obtain nucleic acid samples , which is also more conducive to obtaining satisfactory test results in subsequent tests;

3. Nucleic acid purification: After the nucleic acid purification step is completed, increase the number of centrifugations and remove the purification buffer to avoid the subsequent nucleic acid elution due to the presence of the purification buffer. De-efficiency, while avoiding the adverse effects of the buffer system on nucleic acid detection;

4. Nucleic acid elution conditions: ①RNA: After adding the eluent, add the elution step of standing at room temperature for 10 minutes at room temperature. Properly extending the elution time can make the RNA more fully dissolved in the eluent, It will not cause RNA degradation due to long standing time; ②DNA: increase the elution temperature to 65°C during elution, and at the same time extend the incubation time to ten minutes, and increase the elution temperature, which can reduce the binding force between DNA and spin column, Increasing the solubility of DNA and prolonging the elution time is conducive to the full dissolution of DNA and the improvement of elution efficiency; the change of elution conditions can improve the extraction rate of nucleic acid, and obtain nucleic acid to a greater extent in limited trace samples, so that in subsequent detections A more realistic response to the sample situation.

5. The synchronous extraction method of DNA and RNA established in this study can be used for nucleic acid extraction of common on-site samples such as oral swabs, cigarette butts, and water cup swabs. The DNA and RNA samples obtained are used in downstream STR typing and fluorescence quantitative PCR detection. All of them obtained relatively ideal detection results, with simple operation and stable results, which met the actual case handling requirements, and could be used for DNA and RNA analysis and detection of saliva plaques on site. The establishment of this method is based on the method optimization of the finished kit, relying on the spin column system of the kit, changing the key steps such as carrier treatment, cell lysis, nucleic acid elution, etc., and will be applied to the DNA and RNA nucleic acid extraction methods of basic research. Introduce forensic nucleic acid testing, and apply kits that are only suitable for large, clean, cultured samples such as cells and tissues to on-site biological samples with trace amounts and complex inhibitors at the scene of the case, for the simultaneous analysis of DNA and RNA of biological samples involved in the case on the scene of the case. It is possible to further investigate the application effect of this method in on-site biological samples such as blood and exfoliated cells.

Description of drawings

Figure 1 shows the results of STR typing of oral swabs extracted by the instruction method.

Figure 2 shows the results of STR typing of cigarette butt samples extracted by the manual method.

Figure 3 shows the results of STR typing of water cup wipes extracted by the instruction method.

Figure 4 shows the results of STR typing of oral swabs extracted by the optimized method.

Figure 5 shows the results of STR typing of cigarette butt samples extracted by the optimized method.

Figure 6 shows the results of STR typing of water cup wipes extracted by the optimized method.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The instruments and reagents used in the following examples are as follows:

1) The equipment required for the experiment is as follows:

2) Main reagents:

AllPrep DNA/RNA Mini Kit QIAGEN, Germany

GlobalFiler ^TM PCR Amplification Kit Thermo, USA

Embodiment 1, saliva stain DNA and RNA synchronous extraction method

1. Simultaneous extraction of DNA and RNA from saliva blots

1. Preparation of experimental samples (the following three are saliva stains at the scene of common cases in criminal technical work practice, and are also the most important biological evidence at the scene of the case):

(1) Oral swab: A cotton swab rolls to wipe the side wall of the oral cavity of the person to be tested.

(2) Cigarette butts: samples of fresh cigarette butts.

(3) Water cup wipes: Wipe the saliva stains at the mouth of the water cup with a dry and wet method.

2. Comparative example: DNA and RNA extracted by AllPrep DNA/RNA Mini Kit

Use the existing AllPrep DNA/RNA Mini Kit to extract the DNA and RNA of the above-mentioned sample 1. The instructions are as follows:

1) Synchronous lysis to obtain DNA and RNA

(1) Cut an appropriate amount of the carrier (the swab part of a cotton swab or the outer layer of the cigarette butt filter) into pieces of suitable size, put it in a clean 1.5mL centrifuge tube without RNase and DNase, add 600μl Buffer RLT and 6μl β -ME;

(2) Use RNase-free and DNase-free ophthalmic tweezers to pick up the carrier, put it in a RNase-free, DNase-free disposable syringe, and press the plunger of the syringe to squeeze out all the residual liquid on the carrier and recover it to the original centrifuge tube in order to remove the carrier;

(3) Centrifuge at 13,000 rpm for 2 min (to remove common dust, soil and other small particles of impurities on the carrier of the field test material), take the centrifugal supernatant and pass it through the AllPrep DNA Spin Column adsorption column (13,000 rpm, 30 s, repeat the centrifugation 2 times, until the adsorption column No liquid), put the adsorption column in a clean 2mL EP tube without RNase and DNase, and store it at 4°C (AllPrep DNASpin Column adsorption column is used for DNA purification, and the column liquid is used for RNA purification);

2) Purified RNA:

①Add an equal volume of 70% ethanol to the liquid passing through the column, take 700μl and centrifuge it through the RNeasy Spin Column (13000rpm, 15s), repeat the operation until all the liquid is transferred;

②Add 700μl Buffer RW1 to RNeasy Spin Column and centrifuge at 13000rpm for 15s;

③Add 500μl Buffer RPE to the RNeasy Spin Column and centrifuge at 13000rpm for 15s;

Note: An appropriate volume of absolute ethanol should be added to Buffer RPE before use.

④Add 500μl Buffer RPE to the RNeasy Spin Column and centrifuge at 13000rpm for 2min;

Note: Repeat the procedure to ensure that no liquid remains on the RNeasy Spin Column.

⑤ Transfer the RNeasy Spin Column to a new RNase-free, DNase-free clean 2mL EP tube, and centrifuge at 13000rpm for 1min;

⑥Put the RNeasy Spin Column in a clean 1.5mL centrifuge tube without RNase and DNase, add 35μl of RNase-free water, centrifuge at 13000rpm for 1min, and collect the supernatant as RNA;

⑦The obtained RNA was stored at -80℃ for later use.

Purified DNA:

①Add 500μl Buffer AW1 to DNeasy Spin Column, and centrifuge at 13000rpm for 15s;

Note: An appropriate volume of absolute ethanol should be added to Buffer AW1 before use.

②Add 500μl Buffer AW2 to the RNeasy Spin Column and centrifuge at 13000rpm for 2min;

③ Transfer the RNeasy Spin Column to a new RNase-free and DNase-free clean 2mL EP tube, and centrifuge at 13000rpm for 1min;

Note: Repeat the procedure to ensure that no liquid remains on the DNeasy Spin Column.

④Put the DNeasy Spin Column in a clean 1.5mL centrifuge tube without RNase and DNase, add 100μl EB Buffer, let stand at room temperature for 1min, and centrifuge at 13000rpm for 1min; repeat the operation twice;

⑤ Store the obtained DNA at -80°C for later use.

3. The synchronous extraction method of DNA and RNA from saliva stains of the present invention

Based on the description of the existing AllPrep DNA/RNA Mini Kit, this study optimized the steps of lysis, adsorption, and elution, and finally formed a method for simultaneous extraction of DNA and RNA that can be used for on-site samples and trace nucleic acid samples. The reagents and materials used are the components of the AllPrep DNA/RNA Mini Kit unless otherwise specified.

1) Synchronous lysis to obtain DNA and RNA

(1) Cut an appropriate amount of the carrier (the wiping part of a cotton swab or the outer layer of the cigarette butt filter) into pieces of suitable size, put them in a clean 1.5mL centrifuge tube without RNase and DNase, and add 600μl Buffer RLT (solution). to separate the cells) and 20 μl DTT (destroy the membrane to lyse the cells, the final concentration in the reaction system is 1.6 mM), fully shake and mix, and incubate the reaction at room temperature (25 degrees) for 2.5 hours to obtain the reaction product;

(2) Use RNase-free, DNase-free clean ophthalmic tweezers to pick up the carrier from the reaction product, put it in a RNase-free, DNase-free disposable syringe, press the plunger of the syringe to squeeze out all the remaining liquid on the carrier, and recover the carrier The residual liquid on the surface is recovered, and the residual liquid is recovered into the original centrifuge tube, and it is mixed with the liquid in the reaction product except the carrier, to obtain the reaction product with the carrier removed;

(3) the reaction product of removing the carrier is centrifuged at 13000rpm for 2min (removing small particles of impurities such as dust and soil that are common on the on-site inspection material carrier), and the centrifugal supernatant is taken through the AllPrep DNA Spin Column adsorption column (13000rpm, 30s, Repeat the centrifugation twice until there is no liquid in the adsorption column), and collect the RNA-containing liquid and the adsorption column adsorbed with DNA.

AllPrep DNA Spin Column adsorption column is used for DNA purification, and the column liquid is used for RNA purification.

Place the adsorption column in a clean 2mL EP tube without RNase and DNase, and store at 4°C.

2) Purified RNA:

①Add an equal volume of 70% ethanol aqueous solution to the RNA-containing column liquid obtained in 1) above (to remove other components such as proteins and fatty acids, and to help nucleic acids to bind to the RNA adsorption column), take 700 μl of centrifugation through RNeasy SpinColumn (RNA adsorption column, 13000rpm, 15s), repeat the operation until all the liquids are transferred to obtain an adsorption column with RNA adsorbed;

②Add 700μl Buffer RW1 (washing) to the adsorption column RNeasy Spin Column adsorbed with RNA, and centrifuge at 13000rpm for 15s;

③ Add 500 μl BufferRPE (wash) to the RNeasy Spin Column adsorbed on RNA after the previous step, and centrifuge at 13,000 rpm for 15 s;

Note: An appropriate volume of absolute ethanol should be added to Buffer RPE before use.

④ Add 500 μl BufferRPE (washing) to the adsorption column RNeasy Spin Column with RNA adsorbed after the above step, and centrifuge at 13000 rpm for 2 min;

⑤ Centrifuge at 13,000 rpm for 1 min and repeat twice to ensure that no liquid remains on the RNeasy Spin Column;

⑥ Transfer the RNA-adsorbed adsorption column treated in the previous step to a new RNase-free and DNase-free clean 2mL EP tube, and centrifuge at 13000rpm for 1min;

⑦Put the RNA-adsorbed adsorption column treated in the previous step into a clean 1.5mL centrifuge tube without RNase and DNase, add 35μl RNase-free water, stand at room temperature (25°C) for 10min, and centrifuge at 13000rpm (16200g) for 1min , collect the first centrifugation column fluid;

Then pass the first centrifugation through the column liquid through the adsorption column with RNA adsorbed in this step again, let stand at room temperature for 10min, centrifuge at 13000rpm (centrifugal force 16200g) for 1min, and collect the second centrifugation through the column liquid;

Combine the first pass and the second pass to obtain RNA.

⑧ Store the obtained RNA at -80℃ for later use.

Purified DNA:

① Add 500 μl BufferAW1 (washing) to the DNA-adsorbed adsorption column DNeasy Spin Column obtained in 1) above, and centrifuge at 13,000 rpm for 15 s;

Note: An appropriate volume of absolute ethanol should be added to Buffer AW1 before use.

② Add 500 μl BufferAW2 (washing) to the DNA-adsorbed adsorption column DNeasy Spin Column treated in the previous step, and centrifuge at 13000 rpm for 2 min;

③ Transfer the DNA-adsorbed DNeasy Spin Column treated in the previous step to a new RNase-free and DNase-free clean 2mL EP tube, and centrifuge at 13000rpm for 1min;

④ Centrifuge at 13,000 rpm for 1 min and repeat twice to ensure that no liquid remains on the DNeasy Spin Column;

⑤Put the DNA-adsorbed DNeasy Spin Column after the previous step in a clean 1.5mL centrifuge tube without RNase and DNase, add 100μl EB Buffer (provided with the kit), stand at 65°C for 10min, and centrifuge at 13000rpm for 1min , collect the first centrifugation column fluid;

Then pass the first centrifugation through the column liquid again through the adsorption column with the DNA adsorbed in this step, stand at 65°C for 10min, centrifuge at 13000rpm (centrifugal force 16200g) for 1min, and collect the second centrifugation through the column liquid;

Combine the first and second passes to obtain DNA.

⑥ Store the obtained DNA at -80℃ for later use.

2. Detection

1. Nucleic acid concentration and OD value

The DNA concentration and OD value were measured by Nanodrop2000 spectrophotometer, respectively.

The DNA concentration and OD _260/280 obtained by extraction are shown in Table 1 and Table 2. Compared with the optimized method, the OD _260/280 of the DNA extracted by the kit method are all greater than 2, indicating that the RNA removal is incomplete and the DNA extraction quality is low. .

Table 1 is the DNA concentration and OD value extracted by the comparative example (kit detection)

Table 2 is the DNA concentration and OD value extracted by the method of the present invention

2. DNA STR typing

The DNA extracted by the above two different methods was amplified by GlobalFiler ^™ PCR Amplification Kit, sequenced by ABI3500xl sequencer, and DNA STR typing was performed.

Part of the results of oral swabs, cigarette butts and water cup swabs extracted from the kit instructions are shown in Figures 1-3. It can be seen that no valid STR typing has been obtained.

The partial results of all DNA extracted by the method of the present invention are shown in Figures 4-6. It can be seen that the DNA samples have obtained correct STR typing results, and the peaks are clear, sharp and balanced, achieving DNA STR detection and comparison. Require.

The results of oral swabs, cigarette butts and water cup swabs extracted according to the kit instructions and the method of the present invention are summarized in Table 3. The kit method only detected 2-4 loci in the oral swab samples, and there was no comparison. For value, no other samples were detected. All the loci are detected in all samples by the method of the invention, the STR typing results are correct, the peak map is clear, sharp and balanced, and the DNA STR detection and comparison requirements are met.

Table 3 DNA STR typing results of each group of samples

3. Detection of β-actin mRNA and 18S rRNA signal in RNA by reverse transcription fluorescence quantitative PCR

1) Reverse transcription to obtain cDNA

Using Invitrogen ^™ SuperScript ^™ III First-Strand Synthesis SuperMix forqRT-PCR kit (Thermo Fisher, USA, 2×RT Reaction Mix in this kit contains RandomPrimers, Oligo dT Primers) The RNA obtained above was reverse transcribed to synthesize cDNA, details as follows:

(1) Reaction system (20 μl):

(2) Reaction procedure:

After the reaction, the reaction products were collected to obtain cDNA.

Take an appropriate amount of cDNA template and carry out PCR amplification according to the following reaction system and reaction procedure, and each sample is amplified twice in parallel.

2) Fluorescence quantitative PCR

principle:

Gene Expression Assays系列中的18S GE Assays和ACTB GE Assays(Thermo Fisher美国)。PCR试剂为

FastAdvanced Master Mix(Thermo Fisher美国)。反应在Step One定量PCR仪上进行。Using the TaqMan probe method, the probes and primers are

18S GE Assays and ACTB GE Assays in the Gene Expression Assays series (Thermo Fisher USA). PCR reagents are

FastAdvanced Master Mix (Thermo Fisher USA). The reaction was performed on a Step One quantitative PCR machine.

(1) PCR reaction system (20 μl):

(2) PCR amplification procedure:

(3) Data processing and analysis:

After the PCR reaction, the CT value of each reaction was obtained, and the average CT value and the ratio of the CT values of the two genes expressed in each sample were calculated. SPSS software was used for statistical analysis.

The detection results of 18S rRNA and β-actin mRNA signals are shown in Table 4 and Table 5. The 18S rRNA and β-actin mRNA Ct values of the RNA samples extracted according to the kit instructions were close to the threshold, and many samples did not obtain β-actin mRNA detection signals (no variance statistics were performed in the group in which they belonged), which failed to meet the detection requirements. In the RNA samples obtained after the method optimization, the detection results of 18S rRNA and β-actin mRNA signals were more reliable.

Table 4 Comparative example (kit detection) Ct values of 18S rRNA and β-actin mRNA extracted from RNA samples

Table 5 Ct value of RNA sample 18S rRNA and β-actin mRNA extracted by the method of the present invention

Claims (10)

1. A method for synchronously extracting DNA and RNA from a spot saliva stain comprises the following steps:

1) soaking the spot saliva spots with the carriers in a reaction system containing DTT and cell dissociation buffer solution, and incubating for reaction to obtain reaction products;

2) removing the carrier in the reaction product, collecting the reaction product with the carrier removed, passing the reaction product with the carrier removed through a DNA adsorption column, and collecting the adsorption column adsorbed with DNA and the column passing liquid containing RNA;

3) purifying the column passing liquid containing RNA and the adsorption column adsorbed with DNA respectively to obtain RNA and DNA;

the purification of the RNA-containing column-passing liquid is carried out according to a method comprising the following steps:

3) -1, flowing the RNA-containing column-passing liquid through an RNA adsorption column to obtain the adsorption column adsorbed with RNA;

3) 2, flowing RNA elution buffer solution through the adsorption column adsorbed with RNA, standing at room temperature for 5-10min, and centrifuging to collect first column passing solution to obtain RNA;

the DNA purification is carried out according to a method comprising the following steps:

3) and a, using a DNA elution buffer solution to flow through the adsorption column adsorbed with the DNA, standing for 8-15min at 60-65 ℃, and centrifugally collecting the first column passing solution to obtain the DNA.

2. The method of claim 1, wherein:

the method for purifying the RNA-containing column-passing liquid further comprises the following steps between the steps 3) -1 and 3) -2: and washing the adsorption column adsorbed with the RNA to obtain the washed adsorption column adsorbed with the RNA.

3. The method of claim 2, wherein:

in the method for purifying the RNA-containing column-passing liquid, the method further comprises the following steps before flowing through the RNA adsorption column in the step 3) -1: and adding an equal volume of 70-80% ethanol aqueous solution into the RNA-containing column-passing liquid to obtain a mixed solution.

4. A method according to claim 2 or 3, characterized in that:

in the method for purifying the column passing liquid containing the RNA, in 3) -2, after the first column passing liquid is collected, the first column passing liquid flows through the adsorption column adsorbed with the RNA again, stands for 5-10min at room temperature, and is centrifuged to collect the second column passing liquid; and combining the first column solution and the second column solution to obtain RNA.

5. The method according to any one of claims 1-4, wherein:

in the method for purifying DNA, before passing through the adsorption column with adsorbed DNA with DNA elution buffer, the following steps are included: and washing the adsorption column adsorbed with the DNA to obtain the adsorption column adsorbed with the DNA after washing.

6. The method according to any one of claims 1-5, wherein:

in the method for purifying the DNA, in 3) -a, after the first column-passing liquid is collected, the first column-passing liquid flows through the adsorption column which is adsorbed with the DNA after washing, stands for 8-15min at 60-65 ℃, and is centrifuged to collect the second column-passing liquid; and combining the first column solution and the second column solution to obtain DNA.

7. The method according to any one of claims 1-6, wherein: the concentration of the DTT in the reaction system is 1-2 mM.

8. The method according to any one of claims 1-7, wherein: in the step 1), the reaction condition is that the incubation is carried out for more than or equal to 1.5 hours at room temperature.

9. The method according to any one of claims 1-8, wherein: the RNA elution buffer is RNase-free water.

10. The method according to any one of claims 1-9, wherein:

the on-site salivary spot with carrier is a mouth swab, a cigarette butt or a cup wipe.

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