A method for rapid extraction of nucleic acids from fresh brain tissue

Abstract

The present application relates to the technical field of biological detection, and provides a nucleic acid rapid extraction method of fresh brain tissue, wherein the obtained fresh brain tissue is cut into small pieces, then is added with a lysis buffer for lysis, and then is ground for 20-40s and centrifuged; after 98 DEG C incubation for several minutes, an extraction buffer is added and mixed uniformly, and is directly used for nucleic acid genetic information detection. Using the method of the present application, only 8 minutes are needed to complete the detection, and the clinical requirement of rapid molecular typing detection during brain tumor surgery can be met. In combination with a rapid fluorescent quantitative PCR system, the PCR amplification and data analysis process can be completed in 20 minutes, and the whole detection process is controlled within half an hour from obtaining the tissue sample to obtaining the detection report of the patient. The requirement of rapid molecular typing detection during brain tumor surgery in clinic can be met.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection technology, and relates to a nucleic acid rapid extraction method suitable for fresh brain tissue in a surgical process. BACKGROUND

[0002] Glioma is a tumor originating from brain supporting cells, and is one of the most common primary brain tumors in adults and children, and the incidence increases with age. Glioma surgery is an important treatment for glioma. Since glial cells are one of the most abundant cell types in brain tissue, supporting the normal function of neurons and maintaining homeostasis in the nervous system, glioma surgery is a complex and delicate process that requires superb skill and precision. Too little resection may leave tumor tissue and lead to recurrence; too much resection may lead to increased patient sequelae, and the longer the surgery time, the greater the risk of complications such as bleeding, infection and swelling.

[0003] In clinical practice, when performing intracranial surgery on brain glioma patients, rapid acquisition of tumor tissue genomic DNA (gDNA) and detection of tumor genetic information characteristics can help guide precise tumor resection: if it is a malignant tumor, the tumor tissue can be removed as much as possible to improve the survival period of the patient; if it is a benign or better prognosis tumor, the brain tumor can be conservatively resected to minimize the risk of sequelae 【1】 . Accurate molecular typing can assist clinicians in minimizing the risk of tumor tissue residue, minimizing sequelae, and ultimately improving patient outcomes 【2】 .

[0004] For safety reasons, brain glioma craniotomy surgery time needs to be strictly controlled, and the time for molecular typing detection during surgery needs to be controlled within 30 minutes. Therefore, for intracranial surgery on brain glioma patients, it is very important to quickly obtain brain tissue DNA. The DNA extraction time is usually required to be controlled within 10 minutes, and then combined with a rapid detection instrument, it is possible to achieve the requirement of completing molecular detection in 30 minutes in clinical practice.

[0005] Brain tissue and other tissue organs are different, and contain very high levels of lipids, so the conventional method based on protease and RNase digestion, phenol chloroform extraction and alcohol precipitation often has low yield. In order to overcome this problem, the usual method is to add SDS removing agent, and then high temperature treatment for more than 30 minutes to improve DNA yield 【3】 . But also because of this, the brain tissue DNA extraction time is longer than the conventional tissue organs.

[0006] At present, the methods applied to fresh tissue DNA extraction mainly include three kinds: solution extraction method, magnetic bead extraction method and column extraction method. The extraction time ranges from several tens of minutes to several hours, plus the subsequent PCR amplification and data analysis time, which far exceeds the clinical requirements. The fat content in brain tissue is relatively high, the extraction difficulty is greater, the required time is longer, and the stability of some reagents for extracting brain tissue is also lower, so it is urgent to develop a new fast and stable fresh brain tissue extraction method. SUMMARY

[0007] The present application is directed to the above problems, based on the technical defects in the prior art that the DNA extraction processing time of fresh glioma tissue is too long to meet the needs of typing detection during surgery, a simple and fast brain tissue DNA extraction method is provided, which can complete the sample preparation work before PCR within 8 minutes. Combined with the use of fast qPCR system, PCR amplification and data analysis process can be completed within 20 minutes, from getting the tissue sample to the patient getting the test report, the whole detection process is controlled within half an hour. Meet the needs of rapid molecular typing detection during brain tumor surgery in clinic.

[0008] In order to achieve the above purpose, the specific technical scheme adopted by the present application is as follows:

[0009] The steps of the nucleic acid rapid extraction method of fresh brain tissue provided by the present application are as follows: after the obtained fresh brain tissue is cut into small pieces and added with lysis buffer for lysis, it is ground for 20-40s and simply centrifuged, after 98℃ incubation for several minutes, extraction buffer is added and mixed uniformly, and it is directly used for nucleic acid genetic information detection.

[0010] The preferred conditions of each step are as follows:

[0011] (1) The fresh brain tissue is cut into small pieces with the size of 2-3mm x 2-3mm x 2-3mm.

[0012] (2) The lysis buffer is composed of 10-1000mmol / L NaOH, 50-500mmol / L NaCl and 0.1%-10% SDS. SDS is a surfactant with strong lysis ability, which also has the effect of promoting cell solubilization; NaCl is an electrolyte for coordinating the ion balance inside and outside the cell membrane; NaOH makes the cell membrane change from lipid bilayer structure to vesiculation, so as to lyse the cell and release the cell DNA.

[0013] (3) After lysis, the brain tissue is ground with a plastic grinding rod for 30s, and the centrifugation conditions are as follows: 800rpm, 20s.

[0014] (4) Incubation condition after centrifugation is 98℃, 5 minutes. The purpose of incubation is to lyse cells and release DNA in cells. Too high temperature will cause DNA degradation, affecting detection. Too low temperature will cause insufficient lysis, affecting detection sensitivity.

[0015] (5) The extraction buffer is composed of Tris-HCl 10-1000mmol / L, pH 6.0-9.0, EDTA 0.1-10mmol / L, and sodium deoxycholate 0.1-10%.

[0016] Tris-HCl ensures stable pH value during nucleic acid extraction;

[0017] EDTA is a chelating agent, which has the following effects: (a) EDTA forms stable complex with metal ions, such as calcium and magnesium in cell lysis solution, so as to dissociate cell membrane and cell organs; by complexing with metal ions in nucleases, the degradation of nucleic acids is reduced, and the integrity of nucleic acids in cells is protected; (b) the osmotic pressure of cell lysis solution is adjusted, so as to be closer to the intracellular environment, which is conducive to maintaining the integrity of intracellular molecules and preventing nucleic acids from being damaged; (c) the activity of some enzymes, such as DNA polymerase, is improved, so as to better meet the subsequent experimental requirements.

[0018] Sodium deoxycholate is an anionic detergent, which can effectively destroy the interaction between many proteins and help to separate cell nuclei.

[0019] The present application has a very obvious advantage in sample processing time. The conventional kit needs sample preparation time of dozens of minutes to several hours, and the present application can complete the sample preparation in only 8 minutes. Figure 2 The present application can meet the clinical demand for rapid molecular typing detection during brain tumor surgery. In combination with a rapid fluorescent quantitative PCR system, PCR amplification and data analysis can be completed in 20 minutes. From obtaining the tissue sample to obtaining the test report of the patient, the whole detection process is controlled within half an hour, which meets the demand for rapid molecular typing detection during brain tumor surgery.

[0020] The nucleic acids of the same quality of fresh brain tissue are extracted by the present application and Qiagen kit respectively, the nucleic acid extraction products obtained by the two methods are compared, and then the detection of nucleic acid genetic information is carried out. The results of fluorescent quantitative PCR (qPCR) show that the Ct values obtained by the two methods are very close, with a difference of less than 0.5 Figure 3 , and good repeatability Figure 4 .

[0021] The beneficial guarantee and effect of the present application are as follows:

[0022] The nucleic acid extraction method of the present application has two advantages: (1) Compared with the magnetic bead extraction method and the column extraction method, the difference is that the final DNA solution must be pure. The present application optimizes the related reagent formula and experimental process. The DNA suspension obtained after tissue lysis can be directly used for nucleic acid genetic information detection, such as fluorescent quantitative PCR (qPCR), and the experimental process is more simple and convenient; (2) Compared with the magnetic bead extraction method and the column extraction method, the operation time is more than half an hour. The operation time of the method of the present application can be shortened to within 10 minutes, which perfectly matches the demand for rapid nucleic acid detection of brain tissue during brain glioma clinical surgery.

[0023] Therefore, under the premise of reducing operation steps and shortening operation time, the nucleic acid extraction product obtained by the method of the present application can achieve the same detection effect as the commercialized kit using other methods, and thus better matches the demand for rapid nucleic acid detection of brain tissue during brain glioma clinical surgery. BRIEF DESCRIPTION OF DRAWINGS

[0024] The present disclosure will be further described below in conjunction with the drawings, wherein these displays are only for illustrating the embodiments of the present disclosure, and are not for limiting the scope of the present disclosure.

[0025] Figure 1 The sample processing flowchart of the present application is shown.

[0026] Figure 2 The processing time of the present application, the traditional extraction method and the Qiagen kit extraction are shown.

[0027] Figure 3 The qPCR detection results of the method of the present application and the Qiagen kit are shown.

[0028] Figure 4 The repeatability experiment results of the present application are shown. DETAILED DESCRIPTION

[0029] The following examples and experimental examples further illustrate the present application and should not be construed as limiting the present application. The examples do not include detailed descriptions of conventional methods, such as PCR methods, methods for constructing vectors and plasmids, methods for inserting genes encoding proteins into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 ndCold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present application. Throughout the description and claims of this specification, terms mean their art-recognized meanings as follows unless the context clearly dictates otherwise. Preferably, the most preferred methods and materials are described herein.

[0031] Example 1: Rapid extraction of nucleic acid from fresh brain tissue according to the present application

[0032] According to the present application, the steps of the method for rapid extraction of nucleic acid from fresh brain tissue are as follows: Figure 1

[0033] (1) The obtained fresh brain tissue is cut into small pieces of rice size, specifically 2-3 mm x 2-3 mm x 2-3 mm in size;

[0034] (2) After cutting into small pieces, place them in a 1.5 mL centrifuge tube and add lysis buffer for lysis. The lysis buffer is composed of 10-1000 mmol / L NaOH, 50-500 mmol / L NaCl, and 0.1%-10% SDS.

[0035] (3) Grind the brain tissue with a plastic grinding rod for about 30 s, and then centrifuge at 800 rpm for 20 s. Incubate at 98°C for 5 min;

[0036] (4) Add extraction buffer and mix well, and directly use for detection of nucleic acid genetic information, such as fluorescence quantitative PCR (qPCR). The extraction buffer is composed of Tris-HCl 10-1000 mmol / L at pH 6.0-9.0, EDTA 0.1-10 mmol / L, and Sodium deoxycholate 0.1-10%.

[0037] Comparative Example 1: Traditional nucleic acid extraction method

[0038] (1) Take 0.1 g (0.5 cm 3 ) of fresh brain tissue, add 1 ml of cell lysis solution, and homogenize the tissue until no tissue pieces are visible, about 10 min. Transfer into a 1.5 ml centrifuge tube, add 20 μl of 500 μg / ml proteinase K, and water-bath at 65°C for 30 min. Centrifuge at 12000 rpm for 5 min, and transfer the supernatant into another centrifuge tube;

[0039] (2) Add an equal amount of phenol-chloroform (phenol: chloroform: isopropyl alcohol = 25:24:1), mix well by oscillation, and centrifuge at 12000 rpm for 5 min; ​

[0040] (3) Take the supernatant to another tube, add 1 / 2 volume of 7.5 mol / L ammonium acetate, add 2 volumes of anhydrous ethanol, mix well, precipitate at room temperature for 10 min, centrifuge at 12000 rpm for 15 min;

[0041] (4) Carefully pour off the supernatant, and invert the centrifuge tube on a paper towel to remove residual drops adhering to the tube wall;

[0042] (5) Wash the precipitate with 1 ml of 70% ethanol once, centrifuge at 12000 rpm for 5 min;

[0043] (6) Carefully pour off the supernatant, and invert the centrifuge tube on a paper towel to remove residual drops adhering to the tube wall, dry at room temperature for about 5 min;

[0044] (7) Redissolve the precipitate with 200 ul of TE, and then store at 4°C or -20°C for standby use.

[0045] Comparative Example 2: QIAgen method

[0046] (1) Take 10 mg of brain tissue, cut into pieces and put into a 1.5 ml centrifuge tube, add 180 ul of Buffer ATL, 20 ul of proteinase K, mix well, and incubate at 56°C until completely dissolved, about 40 min;

[0047] (2) Add 200 ul of Buffer AL, mix well, and incubate at 56°C for 10 min;

[0048] (3) Add 200 ul of ethanol, mix well;

[0049] (4) Transfer the solution into a DNeasy Mini spin column, and put into a 2 ml collection tube, centrifuge at 8000 rpm for 1 min.

[0050] (5) Put the centrifugal column into a new 2 ml collection tube, add 500 ul of Buffer AW1, centrifuge at 8000 rpm for 1 min;

[0051] (6) Put the centrifugal column into a new 2 ml collection tube, add 500 ul of Buffer AW2, centrifuge at 14000 rpm for 3 min;

[0052] (7) Put the centrifugal column into a new 1.5 ml centrifuge tube, add 200 ul of Buffer AE, centrifuge at 8000 rpm for 1 min, and store the DNA in the centrifuge tube for standby use.

[0053] The DNA extracted by the above three extraction methods is detected by conventional qPCR.

[0054] Figure 2 The processing time of the three extraction methods is shown, and the processing time of the present application, QIAgen method and traditional extraction method is 8min, 60min and 90min respectively; Figure 3 The qPCR detection results of the DNA extracted by the present application and the QIAgen method are shown, and the obtained Ct values are very close, with a difference of <0.5; Figure 4 The repeatability experiment results of the extraction method of the present application are shown, and there is no obvious difference between the Ct values of different batches of DNA.

[0055] The reference involved in the background art of the present application:

[0056] 1.Goodenberger ML,Jenkins RB(December 2012)."Genetics of adult glioma".Cancer Genetics.205(12):613-21.

[0057] 2.Holdhoff,M;Guner,G;Rodriguez,FJ;Hicks,JL;Zheng,Q;Forman,MS;Ye,X;Grossman,SA;Meeker,AK;Heaphy,CM;Eberhart,CG;De Marzo,AM;Arav-Boger,R(15June 2017)."Absence of Cytomegalovirus in Glioblastoma and Other High-grade Gliomas by Real-time PCR,Immunohistochemistry,and In Situ Hybridization".Clinical Cancer Research.23(12):3150-3157.

[0058] 3.John Saldanha,Alison Gannicliffe,Ruth F.Itzhaki,An improved method for preparing DNA from human brain,Journal of Neuroscience Methods,Volume 11,Issue 4,1984,Pages275-279

[0059] The preferred embodiments of the present application have been disclosed with the above particularity, but the application is not limited to the embodiments disclosed, and variations and modifications can be made by those skilled in the art without deviating from the spirit of the application, and such variations and modifications are intended to be included within the scope of the application as defined in the following claims.

Claims

1. A method for rapid extraction of nucleic acids from fresh brain tissue, characterized by, In combination with the fluorescent quantitative PCR, the method comprises the following steps: after the obtained fresh brain tissue is cut into small pieces with a size of 2-3mm*2-3mm*2-3mm, the small pieces are added into a lysis buffer for lysis, then the small pieces are ground for 30s and centrifuged, after 98℃ incubation for 5 minutes, an extraction buffer is added and mixed uniformly, and the mixture is directly used for detection of nucleic acid genetic information, The lysis buffer is composed of 10-1000mmol / L NaOH, 50-500mmol / L NaCl and 0.1%-10% SDS; The centrifugal condition is as follows: 800rpm, 20s; The extraction buffer is composed of 10-1000mmol / L Tris-HCl with a pH of 6.0-9.0, 0.1-10mmol / L EDTA and 0.1-10% sodium deoxycholate.

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