A blood pathogen nucleic acid extraction kit and its usage method

Abstract

The present invention provides a nucleic acid extraction kit for blood. The nucleic acid extraction kit for blood includes a pre-lysis solution and a variety of extraction solutions, and at least one of the extraction solutions contains 0.5% to 3% octyl glucoside neopentyl glycol. With the wide application of nucleic acid detection, nucleic acid extraction technology has also developed rapidly. However, in the existing pathogen nucleic acid extraction kits on the market, there are the following drawbacks: 1. The sample pretreatment operation is cumbersome: the whole blood sample needs to be pretreated; 2. Low sensitivity and other reasons are likely to cause missed detections; 3. Incomplete nucleic acid extraction causes quantitative deviation and cannot well reflect the true concentration of the sample. By adjusting the components of the extraction solution and adding octyl glucoside neopentyl glycol (OGNG) to the extraction solution, the present invention improves the extraction efficiency, and the extracted nucleic acid has higher content and purity, showing higher detection sensitivity in the PCR reaction.

Description

Technical Field

[0001] The present invention belongs to the field of molecular diagnostic DNA extraction, and particularly relates to a kit for rapidly extracting blood pathogen DNA and human DNA and a using method thereof. Background Art

[0002] Existing methods for extracting nucleic acid (DNA) from blood mainly include phenol extraction method, alkaline lysis method, CTAB extraction method, boiling method, centrifugal column extraction method, glass bead adsorption method and micro-nano magnetic bead extraction method. Currently, the main methods available for blood virus extraction are adsorption column method and magnetic bead method. The centrifugal column nucleic acid extraction method has obvious advantages in the nucleic acid purity obtained finally compared with traditional methods due to the specific adsorption of nucleic acid and repeated washing of impurities. And the magnetic bead method nucleic acid extraction technology has become the mainstream method in the industry because of its high automation degree, large sample throughput, good nucleic acid concentration and purity, especially high safety. The mainstream domestic nucleic acid extraction manufacturers have fully automatic nucleic acid extraction systems using the magnetic bead method principle, which do not require centrifugation and are automated in operation, can reduce personnel contact, and use deep well plate consumables to avoid cross-contamination.

[0003] With the development of clinical molecular diagnosis, in some fields of infectious disease screening, after the virus nucleic acid extraction and purification are identified by PCR amplification, the detection results can be used as the basis for clinical reference. However, due to the existence of exogenous and endogenous nucleases and the relatively stable enzyme activity, etc., it is relatively difficult to extract and purify high-quality virus nucleic acid from peripheral blood. Due to the continuous development of molecular biology technology, the detection of pathogen infection by molecular biology technology has become an important and indispensable tool in disease diagnosis. In clinical pathogen detection, the application of molecular biology technology can improve the accuracy of detection results and play an extremely important role in the rapid detection of diseases.

[0004] With the wide application of nucleic acid detection, nucleic acid extraction technology is also developing rapidly. However, in the existing pathogen nucleic acid extraction kits on the market currently, the kits have the following drawbacks: 1. The sample pretreatment operation is cumbersome: the whole blood sample needs to be pretreated; 2. Low sensitivity and other reasons are likely to cause missed detection; 3. Incomplete nucleic acid extraction causes quantitative deviation and cannot well reflect the true concentration of the sample. Summary of the Invention

[0005] The present invention provides a kit for extracting nucleic acid from blood and a using method thereof to improve the extraction efficiency of nucleic acid.

[0006] The present invention provides a nucleic acid extraction kit for blood. The nucleic acid extraction kit for blood includes a pre-lysis solution and a variety of extraction solutions. At least one of the extraction solutions contains octyl glucoside neopentyl glycol with a volume ratio of 0.5% to 3%. Preferably, the volume ratio of octyl glucoside neopentyl glycol is 0.8% to 2.5%; more preferably, the volume ratio of octyl glucoside neopentyl glycol is 1% to 2%. For example, in the present invention, extraction solutions with a volume ratio of octyl glucoside neopentyl glycol of 0.5%, 0.7%, 0.9%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0% can all efficiently extract nucleic acids from blood.

[0007] Further, the pre-lysis solution includes 5 to 15 mmol / L SDS and 0.2 mmol to 1 mmol / L calcium chloride.

[0008] Further, the extraction solutions include a first extraction solution and a second extraction solution. The second extraction solution contains octyl glucoside neopentyl glycol with a volume ratio of 0.5% to 3% and 0.2 to 0.7 mg / mL magnetic beads.

[0009] Still further, the first extraction solution includes 1 to 5 mol / L guanidine hydrochloride, 10 to 200 mmol / L TrisHCl, 1 to 5 mol / L guanidine thiocyanate, 1 to 50 mmol / L EDTA, and 0.01 to 0.5 mmol / L dithiothreitol.

[0010] Still further, the second extraction solution includes 0.5 mol / L to 1 mol / L NaCl, isopropanol with a volume ratio of 15% - 30%, octyl glucoside neopentyl glycol with a volume ratio of 0.5% to 3%, and 0.2 to 0.7 mg / mL magnetic beads.

[0011] Further, the nucleic acid extraction kit for blood further includes a washing solution and an elution solution.

[0012] Still further, the nucleic acid extraction kit for blood further includes a first washing solution, a second washing solution, and an elution solution.

[0013] Still further, the first washing solution includes isopropanol with a volume ratio of 10% to 20%, NP 40 with a volume ratio of 0.1% to 5%, 0.5 to 2 mol / L guanidine thiocyanate, 1 to 50 mmol / L EDTA, and 0.01 to 0.5 mmol / L dithiothreitol; the second washing solution includes 10 to 200 mmol / L Tris HCl and absolute ethanol, and the elution solution includes 10 to 200 mmol / L Tris - HCl and 1 to 50 mmol / L EDTA.

[0014] Further, the nucleic acid extraction kit for blood also includes Proteinase K.

[0015] On the other hand, the present invention also discloses a method for using the nucleic acid extraction kit for blood according to any one of the above, in the process of use, 0.5-1.5 volumes of the extraction solution containing octyl glucoside neopentyl glycol are used for 1 volume of the sample.

[0016] Further, the nucleic acid extraction kit for blood includes a pre-lysis solution, a first extraction solution, a second extraction solution, a first washing solution, a second washing solution, and an elution solution. The method of use includes the following steps:

[0017] S1. Take 1 volume of the sample, add 0.15-0.4 volume of the lysis solution, add 0.05-0.15 volume of Proteinase K, and shake.

[0018] S2. Add 2-5 volumes of the first extraction solution, shake, centrifuge, and let stand at 30-60 °C.

[0019] S3. Add 0.5-1.5 volumes of the second extraction solution, shake, centrifuge, and let stand; wherein, the second extraction solution contains octyl glucoside neopentyl glycol and magnetic beads with a volume ratio of 0.5%-3%.

[0020] S4. Perform magnetic separation to remove the waste liquid.

[0021] S5. Add 2-6 volumes of the first washing solution, shake, perform magnetic separation, and remove the waste liquid.

[0022] S6. Add 2-6 volumes of the second washing solution, shake, perform magnetic separation, and remove the waste liquid.

[0023] S7. Repeat S6.

[0024] S8. Centrifuge, perform magnetic separation, and remove the waste liquid; let stand.

[0025] S9. Add 0.2-0.8 volumes of the elution solution, shake and mix well, elute the magnetic beads, and let stand; perform magnetic separation again to complete the extraction process.

[0026] Compared with the prior art, the present invention improves the extraction efficiency by adjusting the components of the extraction solution and adding octyl glucoside neopentyl glycol (OGNG) to the extraction solution. The extracted nucleic acid has a higher content and purity, and shows higher detection sensitivity in the PCR reaction. Description of the Drawings

[0027] Figure 1 It is the detection result of extracting the Epstein-Barr virus sample from whole blood in Example 1 of the present invention.

[0028] Figure 2Detection results of the whole blood EBV virus sample extracted in Comparative Example 1 of the present invention;

[0029] Figure 3 Detection results of the whole blood EBV virus sample extracted in Comparative Example 2 of the present invention. Detailed implementation manners

[0030] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0031] In the embodiment of the present invention, in order to improve the extraction efficiency of nucleic acids in blood, a nucleic acid extraction or purification kit based on the magnetic bead method for blood is developed. This extraction reagent is based on the principle of the magnetic bead method, has a high extraction efficiency, and can obtain high-quality nucleic acids in whole blood without pretreatment. At the same time, this extraction reagent can also be adapted to semi-automatic extraction instruments, and adopts a pre-packaged production process, which shortens the overall extraction time and improves the efficiency.

[0032] The solution provided by the embodiment of the present invention is as follows:

[0033] The nucleic acid extraction kit includes a pre-lysis solution and a variety of extraction solutions, and at least one of the extraction solutions contains octyl glucoside neopentyl glycol with a volume ratio of 0.5% to 3%.

[0034] Among them, the nucleic acid extraction kit can be used to extract viral DNA or human DNA. In the embodiment of the present invention, the EBV virus is taken as an example specifically. In addition, octyl glucoside neopentyl glycol (OGNG) is added to the extraction solution in the embodiment of the present invention, which improves the extraction efficiency, and the content and purity of the extracted nucleic acids are higher, showing higher detection sensitivity in the PCR reaction.

[0035] Further, the pre-lysis solution includes: 5 - 15 mmol / L SDS, 0.2 mmol - 1 mmol / L calcium chloride.

[0036] Further, the extraction solution includes a first extraction solution and a second extraction solution. The second extraction solution contains octyl glucoside neopentyl glycol with a volume ratio of 0.5% to 3% and 0.2 - 0.7 mg / mL magnetic beads.

[0037] Even further, the first extraction solution includes 1 - 5 mol / L guanidine hydrochloride, 10 - 200 mmol / L TrisHCl, 1 - 5 mol / L guanidine thiocyanate, 1 - 50 mmol / L EDTA, 0.01 - 0.5 mmol / L dithiothreitol.

[0038] Further, the second extraction solution comprises 0.5 mol / L to 1 mol / L of NaCl, isopropanol with a volume ratio of 15% - 30%, octyl glucoside neopentyl glycol with a volume ratio of 0.5% - 3%, and magnetic beads at 0.2 - 0.7 mg / mL.

[0039] Further, the nucleic acid extraction kit from blood further comprises a first washing solution, a second washing solution, and an elution solution.

[0040] Further, the first washing solution comprises: isopropanol with a volume ratio of 10% - 20%, NP 40 with a volume ratio of 0.1% - 5%, guanidine thiocyanate at 0.5 - 2 mol / L, EDTA at 1 - 50 mmol / L, and dithiothreitol at 0.01 - 0.5 mmol / L; the second washing solution comprises: 10 - 200 mmol / L of Tris HCl and absolute ethanol, and the elution solution comprises: 10 - 200 mmol / L of Tris - HCl and 1 - 50 mmol / L of EDTA.

[0041] It should be noted that the above - mentioned compositions of the first washing solution, the second washing solution, and the elution solution are preferred solutions in the embodiments of the present invention. However, for those skilled in the art, other known washing solutions and elution solutions can also be used for substitution. The dosage of absolute ethanol can be specifically set according to the experience of those in this industry.

[0042] Further, the nucleic acid extraction kit from blood further comprises proteinase K.

[0043] The using method of the nucleic acid extraction kit from blood according to the present invention comprises the following steps:

[0044] S1. Take 1 volume of the sample, add 0.15 - 0.4 volume of the lysis solution, add 0.05 - 0.15 volume of proteinase K, and shake.

[0045] Among them, in the embodiment of the present invention, the sample is directly subjected to lysis treatment without pre - treatment operation, further simplifying the extraction process;

[0046] S2. Add 2 - 5 volumes of the first extraction solution, shake, centrifuge, and let stand at 30 - 60 °C.

[0047] S3. Add 0.5 - 1.5 volumes of the second extraction solution, shake for 1 min, perform instantaneous centrifugation, and let stand at room temperature for 5 min.

[0048] S4. Perform magnetic separation to remove the waste liquid.

[0049] S5. Add 2 - 6 volumes of the first washing solution, shake for 30 s, perform magnetic separation for 2 min, and remove the waste liquid.

[0050] S6. Add 2 - 6 volumes of the second washing solution, shake for 30 s, perform magnetic separation for 2 min, and remove the waste liquid;

[0051] S7. Repeat S6;

[0052] S8. Centrifuge, perform magnetic separation, and remove the waste liquid; Let it stand for 5 min;

[0053] S9. Add 0.2 - 0.8 volumes of the elution solution, shake and mix evenly, elute the magnetic beads, and let it stand for 10 min; Perform magnetic separation again for 3 min to complete the extraction process.

[0054] To specifically illustrate the solutions and effects of the embodiments of the present invention, Example 1, Comparative Example 1, and Comparative Example 2 are provided for description. The components of the kits of Example 1, Comparative Example 1, and Comparative Example 2 are as follows:

[0055]

[0056]

[0057] I. The following operating steps are adopted for Example 1 and Comparative Examples 1 - 2 of the present invention:

[0058] S1 Randomly select 10 EBV whole blood samples, after shaking and mixing evenly, take 200 μL of the sample and add it to a 1.5 mL centrifuge tube, add 45 μL of the pre - lysis solution PL, add 20 μL of proteinase K, and shake for 10 s;

[0059] S2 Add 700 μL of the first extraction solution, shake for 1 min, perform instantaneous centrifugation, and let it stand at 60 °C for 12 min.

[0060] S3 Add 200 μL of the second extraction solution, shake for 1 min, perform instantaneous centrifugation, and let it stand at room temperature for 5 min;

[0061] S4 Place the centrifuge tube on the magnetic separator, after 2 min, slowly aspirate and discard the waste liquid (*note not to touch the magnetic beads adsorbed on the inner wall of the tube);

[0062] S5 Add 800 μL of the first washing solution, shake for 30 s, place the centrifuge tube on the magnetic separator, after 2 min, slowly aspirate and discard the waste liquid (*note not to touch the magnetic beads adsorbed on the inner wall of the tube);

[0063] S6 Add 800 μL of the second washing solution, shake for 30 s, place the centrifuge tube on the magnetic separator, after 2 min, slowly aspirate and discard the waste liquid (*note not to touch the magnetic beads adsorbed on the inner wall of the tube);

[0064] S7 Repeat the above steps once;

[0065] S8 Instant centrifugation: Place the centrifuge tube on the magnetic separator, carefully aspirate and discard the liquid at the bottom of the tube, and let it stand at room temperature for 5 min (*Note: Residual ethanol will have an adverse effect on subsequent experiments, and it is necessary to ensure that the ethanol has fully evaporated; at the same time, do not dry for too long to avoid difficulty in eluting the magnetic beads from the wall).

[0066] S9 Add 100 μL of elution buffer, mix well by shaking for 30 s, elute the magnetic beads on the centrifuge tube wall to the bottom of the tube, and let it stand at room temperature for 10 min (it is also possible to choose to stand at 56 °C for 10 min, which can improve the nucleic acid elution efficiency of some complex samples); Place the centrifuge tube on the magnetic separator again and magnetically separate for 3 min, then transfer the eluted nucleic acid to a new 1.5 mL centrifuge tube to complete nucleic acid extraction.

[0067] II. Detection of the nucleic acids obtained in Example 1 of the present invention and Comparative Examples 1-2 is as follows:

[0068] Take 10 μL of the extracted nucleic acid and detect it using an Epstein-Barr virus nucleic acid detection kit (PCR-fluorescent probe method).

[0069] III. Test results

[0070] 1. Comparison of the content and purity of DNA in the elution buffer:

[0071] Extract whole blood samples using the extraction reagents in Example 1 of the present invention and Comparative Examples 1-2. After extraction, use a micro ultraviolet spectrophotometer to measure the content and purity of DNA in the elution buffer, as shown in Table 1 specifically

[0072] Extraction reagent OD260 / 280 OD260 / 280 Concentration (ng / μL) Example 1 1.8 1.9 126 Comparative Example 1 1.5 0.8 20 Comparative Example 2 1.6 0.9 50

[0073] 2. Comparison of nucleic acid detection results:

[0074] Extract whole blood Epstein-Barr virus samples (400 copies / mL) in Example 1 of the present invention and Comparative Examples 1-2, and the nucleic acid detection results are as follows (see specifically Figures 1 to 3 shown, the dark line in the figure is the EBV-DNA gene and the light line is the internal standard detection);

[0075] Sensitivity test of whole blood EBV sample at 400 copies / mL

[0076] Example 1 Comparative Example 1 Comparative Example 2 Ct value 33 37 37 Detection rate 100％ 80％ 100％

[0077] It can be seen that in Example 1 of the present invention, by using octyl glucoside neopentyl glycol, EBV samples as low as 400 copies / mL can be extracted and accurately detected. The time for the automated instrument to extract 96 samples is 35 minutes. Compared with Comparative Examples 1-2, in Example 1 of the present invention, OGNG is added to the extraction solution, which can more effectively extract EBV virus, the Ct value of the amplification curve is appropriate, and the detection rate and Ct value comparison are both good, fully reflecting its characteristics of high sensitivity and simple operation, and is particularly suitable for the detection and screening of blood pathogens, and can quickly and accurately obtain the detection results (in Example 1 of the present invention, when the cycle is 39 times, the Rn value is close to that of the internal standard, while in Comparative Example 1 and Comparative Example 2, 45 cycles are required to have a higher Rn value, and the Rn value of Example 1 of the present invention at 45 cycles is much higher than that of Comparative Example 2 at 45 cycles).

[0078] 3. Testing of serum, plasma and whole blood EBV samples

[0079] The EBV samples in serum, whole blood and plasma were tested with the kit of Example 1, and the results are as follows

[0080]

[0081] It can be seen that the kit of Example 1 of the present invention is applicable to plasma, serum, whole blood samples, etc., and has the advantage of wide application range. Combined with the specific usage method, it can effectively extract and detect low-concentration EBV virus, with high sensitivity and good detection efficiency.

[0082] In summary, the embodiments of the present invention have the following advantages

[0083] 1. Starting from simplifying the operation steps, the kit of the embodiment of the present invention does not require pretreatment and can be manually extracted or semi-automatic nucleic acid extraction instrument can be used;

[0084] 2. The kit of the embodiment of the present invention is applicable to nucleic acid extraction of whole blood, plasma, serum and other samples;

[0085] 3. The embodiment of the present invention also adopts pre-packaged nucleic acid extraction reagents that can be applicable to the above various sample types, which can be matched with nucleic acid extraction equipment. After actual testing, through the semi-automatic instrument extraction scheme, 48 or 96 samples can be extracted within 35 minutes. Compared with the traditional schemes (Comparative Example 1, Comparative Example 2), the nucleic acid extraction efficiency and report sending time are greatly improved, the detection throughput is increased, and the cost is further reduced, and it is simple and efficient;

[0086] 4. The nucleic acid extraction yield is high, and the obtained nucleic acid purity is good, thereby improving the detection sensitivity, reducing the quantitative deviation caused by missed detection and incomplete nucleic acid extraction, and thus well reflecting the true concentration of the sample.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that after reading the specification of this application, they can still modify the specific implementation manners of the present invention or make equivalent substitutions, but these modifications or changes do not depart from the protection scope of the pending claims of this invention application.

Claims

1. A nucleic acid extraction kit for blood, characterized in that, The nucleic acid extraction kit for blood includes a pre-lysis solution, a first extraction solution, and a second extraction solution; The pre-lysis solution includes 5 - 15 mmol / L SDS and 0.2 - 1 mmol / L calcium chloride; The first extraction solution includes 1 - 5 mol / L guanidine hydrochloride, 10 - 200 mmol / L Tris HCl, 1 - 5 mol / L guanidine thiocyanate, 1 - 50 mmol / L EDTA, and 0.01 - 0.5 mmol / L dithiothreitol; The second extraction solution includes 0.5 - 1 mol / L NaCl, isopropanol with a volume ratio of 15% - 30%, octyl glucoside neopentyl glycol with a volume ratio of 0.5% - 3%, and 0.2 - 0.7 mg / mL magnetic beads.

2. The nucleic acid extraction kit for blood according to claim 1, wherein The nucleic acid extraction kit for blood further includes a washing solution and an elution solution.

3. The nucleic acid extraction kit for blood according to claim 2, wherein The washing solution includes a first washing solution and a second washing solution. Among them, the first washing solution includes isopropanol with a volume ratio of 10% - 20%, NP 40 with a volume ratio of 0.1% - 5%, 0.5 - 2 mol / L guanidine thiocyanate, 1 - 50 mmol / L EDTA, and 0.01 - 0.5 mmol / L dithiothreitol; the second washing solution includes 10 - 200 mmol / L Tris HCl and absolute ethanol, and the elution solution includes 10 - 200 mmol / L Tris - HCl and 1 - 50 mmol / L EDTA.

4. The nucleic acid extraction kit from blood according to claim 1, characterized in that, The nucleic acid extraction kit for blood further includes proteinase K.

5. A method for using the nucleic acid extraction kit for blood according to any one of claims 1 to 4, characterized in that, During use, 1 volume of the sample uses 0.5 - 1.5 volumes of the extraction solution containing octyl glucoside neopentyl glycol.

6. The method for using the nucleic acid extraction kit in blood according to claim 5, wherein, The nucleic acid extraction kit for blood includes a pre-lysis solution, a first extraction solution, a second extraction solution, a first washing solution, a second washing solution, and an elution solution. The usage method includes the following steps: S1. Take 1 volume of the sample, add 0.15 - 0.4 volume of the lysis solution, add 0.05 - 0.15 volume of proteinase K, and shake; S2. Add 2 - 5 volumes of the first extraction solution, shake, centrifuge, and let stand at 30 - 60 °C; S3. Add 0.5 - 1.5 volumes of the second extraction solution, shake, centrifuge, and let stand; among them, the second extraction solution contains octyl glucoside neopentyl glycol with a volume ratio of 0.5% - 3% and magnetic beads; S4. Perform magnetic separation to remove the waste liquid; S5. Add 2 - 6 volumes of the first washing solution, shake, perform magnetic separation, and remove the waste liquid; S6. Add 2 - 6 volumes of the second washing solution, shake, perform magnetic separation, and remove the waste liquid; S7. Repeat S6; S8. Centrifuge, perform magnetic separation, and remove the waste liquid; let stand; S9. Add 0.2 - 0.8 volume of the elution solution, shake and mix evenly, elute the magnetic beads, and let stand; perform magnetic separation again to complete the extraction process.

Images