阅读说明：本技术 一种高通量柑橘黄龙病叶脉dna的提取方法 (Method for extracting DNA (deoxyribonucleic acid) of veins of citrus Huanglongbing with high flux ) 是由 刘福平 廖惠红 陈东奎 黄宏明 王茜 黄其椿 汪妮娜 张兰 施平丽 于 2020-05-27 设计创作，主要内容包括：本发明涉及分子生物学技术领域,特别涉及一种高通量柑橘黄龙病叶脉DNA的提取方法,本发明利用机器研磨柑橘叶脉,在同样的时间内磨样数量大大增加,同时利用分液器,采用用试剂盒直接提取DNA,简单方便,加快柑橘黄龙病检测的效率,且确保检测结果准确,进而能大规模地开展柑橘黄龙病检测工作,为促进柑橘产业健康可持续发展提供技术服务。(The invention relates to the technical field of molecular biology, in particular to a method for extracting DNA (deoxyribonucleic acid) of veins of citrus Huanglongbing with high flux.)

1. A method for extracting high-throughput citrus Huanglongbing vein DNA is characterized by comprising the following steps:

(1) weighing 200mg of leaf vein sample of leaf base, and cutting up;

(2) placing the cut leaf vein sample at the basal part of the leaf into a 2ml round-bottom centrifuge tube A, and placing two steel balls with the diameter of 3mm into the centrifuge tube A;

(3) placing the centrifuge tube A into a hole of a grinding box of a grinding machine, immersing the grinding box into a foam box which is filled with liquid nitrogen, soaking the liquid nitrogen, rapidly placing the liquid nitrogen into the grinding machine for grinding, and placing the liquid nitrogen into a refrigerator at the temperature of-70 ℃ for batch extraction after grinding;

(4) adding the material obtained in the step (3) into FP 1400 ul in the plant rapid extraction kit, and shaking once;

(5) adding 6u L RNaseA into the plant rapid extraction kit treated in the step (4), carrying out vortex oscillation for 1min, and then standing at normal temperature for 10 min;

(6) adding 130ul of buffer solution FP2 into the plant rapid extraction kit treated in the step (4), fully and uniformly mixing, and carrying out vortex oscillation for 1 min; centrifuging at 12000rpm for 8min, and transferring the supernatant to a new centrifuge tube B; centrifuging at 12000rpm for 5min, and transferring the obtained supernatant to centrifuge tube B;

(7) adding isopropanol with volume 0.7 times of that of the supernatant into the centrifuge tube B by using a liquid separator, fully and uniformly mixing, and centrifuging for 2min at the rotating speed of 12000 rpm;

(8) adding 600ul of ethanol with volume fraction of 70% into the centrifuge tube B treated in the step (7), shaking up, centrifuging at 12000rpm for 2min, then discarding the supernatant, and repeating the above operation once;

(9) opening the cover and inverting, standing at room temperature, and completely airing residual ethanol; adding 60ul of elution buffer TE into a liquid separator, reversing and uniformly mixing for several times to help dissolve, and thus obtaining the dissolved citrus Huanglongbing vein DNA.

2. The method for extracting the vein DNA of the citrus Huanglongbing disease with high throughput according to claim 1, wherein the step (3) further comprises storing the ground finished product in a refrigerator at-70 ℃.

3. The method for extracting the vein DNA of the citrus Huanglongbing disease with high throughput according to claim 1, wherein the specific modes of soaking in liquid nitrogen and grinding in the step (3) are as follows: soaking liquid nitrogen for 50-70s, grinding in a grinder for 25-35s, soaking liquid nitrogen for 50-70s, and grinding for 25-35s at a frequency of 45-60 Hz.

4. The method for extracting the vein DNA of the citrus Huanglongbing disease with high throughput according to claim 1, wherein the specific modes of soaking in liquid nitrogen and grinding in the step (3) are as follows: soaking in liquid nitrogen for 60s, and grinding in a grinder for 60s, wherein the grinding frequency is 55 Hz.

5. The method for extracting the vein DNA of the citrus Huanglongbing disease according to the claim 1, wherein the plant rapid extraction kit in the step (4) comprises a buffer solution FP1 of 400u L.

6. The method for extracting high-throughput citrus Huanglongbing vein DNA according to claim 1, wherein the concentration of RNaseA added in the step (5) is 10 mg/ml.

7. The method for extracting the vein DNA of the citrus Huanglongbing disease with high flux according to claim 3, wherein the specific modes of soaking in liquid nitrogen and grinding are as follows: soaking liquid nitrogen for 60s, then putting into a grinder to grind for 30s, soaking liquid nitrogen for 60s, and finally grinding for 30s, wherein the grinding frequency is 60 Hz.

[ technical field ] A method for producing a semiconductor device

The invention belongs to the technical field of molecular biology, and particularly relates to a method for extracting high-flux citrus Huanglongbing vein DNA.

[ background of the invention ]

The Citrus yellow shoot disease (Citrus Huanganglingbin, H L B) is one of the most destructive diseases in the world Citrus production, after plants are infected with the yellow shoot disease, the tree vigor is reduced, the yield of the Citrus is reduced, the quality of the Citrus is reduced, and the Citrus yellow shoot withers within 3-5 years, and the Citrus yellow shoot disease has wide occurrence area and huge loss.

At present, no effective prevention and control agent for citrus huanglongbing belongs to controllable and untreatable diseases, and the comprehensive prevention and control measures are to plant disease-free seedlings, strictly prevent citrus psyllids, felled trees, planting isolation belts and the like. In order to reduce the loss, the Guangxi nationwide promulgated a local regulation, namely the prevention and control regulation of citrus yellow shoot disease in the Guangxi Zhuang autonomous region, and guided and encouraged measures are taken; the citrus grower and the citrus seedling breeder are main citrus greening disease prevention and control bodies; establishing a monitoring and early warning mechanism, and definitely identifying a detection program; perfecting the quarantine supervision mechanism of citrus seedlings; the removal of the citrus greening disease plants by the county-level people government or the township people government is an emergency treatment measure which must be taken in response to natural disasters of the citrus greening disease. Whether the nursery stock is quarantined, the sick tree is dug out, the monitoring and early warning are carried out, and the infection source is controlled, the sick tree is diagnosed firstly.

The citrus huanglongbing disease is diagnosed mainly according to field symptoms for a long time, typical symptoms of the citrus huanglongbing disease comprise yellow tips, mottled yellowed leaves and red noses, but different varieties have different expression symptoms and complex field expression symptoms. At present, only when mottled leaves and red naseberry appear, the field can be used as the basis for determining that citrus trees are infected with yellow shoot, but the two symptoms are not easy to appear in the early stage of some citrus varieties, fruit drop occurs in some varieties, leaves are normal, and young trees of some varieties only have yellow tips, so that the PCR detection technology is a technology for accurately diagnosing whether the citrus trees are infected with the yellow shoot, and plays an important role in quarantine, monitoring, early warning, prevention and control diagnosis.

However, the most accumulated pathogenic bacteria of citrus yellow dragon disease are veins, and the method is different from other detection methods, and only needs to detect leaves by other detection methods. The blade is easy to grind the sample, the mechanical grinding effect is good, large-scale monitoring is convenient to develop, and industrial service is well provided. The citrus leaf vein is required to be cut and milled for detecting citrus yellow dragon disease, the citrus leaf vein is difficult to mill than leaves, especially old leaf veins, and the veins of oranges and grapefruits are large, and the milling time is very long, so that the detection speed is greatly reduced, the detection time is long, and the large-scale and high-efficiency detection work is not facilitated. The inventor also refers to the method of grinding citrus veins by a machine, but the method has the problems of no grinding, poor extraction effect, DNA degradation and the like.

[ summary of the invention ]

In view of the above, there is a need for a method for extracting citrus Huanglongbing vein DNA with high throughput, which uses a machine to grind citrus veins, and the number of ground samples is greatly increased in the same time.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for extracting high-throughput citrus Huanglongbing vein DNA comprises the following steps:

(1) weighing 200mg of leaf vein sample of leaf base, and cutting up;

(2) placing the cut leaf vein sample at the basal part of the leaf into a 2ml round-bottom centrifuge tube A, and placing two steel balls with the diameter of 3mm into the centrifuge tube A;

(3) placing the centrifuge tube A into a hole of a grinding box of a grinding machine, immersing the grinding box into a foam box which is filled with liquid nitrogen, soaking the liquid nitrogen, rapidly placing the liquid nitrogen into the grinding machine for grinding, and placing the liquid nitrogen into a refrigerator at the temperature of-70 ℃ for batch extraction after grinding;

(4) adding the material obtained in the step (3) into the plant rapid extraction kit, and shaking once;

(5) adding 6u L RNaseA into the plant rapid extraction kit treated in the step (4), carrying out vortex oscillation for 1min, and then standing at normal temperature for 10 min;

(6) adding 130ul of buffer solution FP2 into the plant rapid extraction kit treated in the step (4), fully and uniformly mixing, and carrying out vortex oscillation for 1 min; centrifuging at 12000rpm for 8min, and transferring the supernatant to a new centrifuge tube B; centrifuging at 12000rpm for 5min, and transferring the obtained supernatant to centrifuge tube B;

(7) adding isopropanol with volume 0.7 times of that of the supernatant into the centrifuge tube B by using a liquid separator, fully and uniformly mixing, and centrifuging for 2min at the rotating speed of 12000 rpm;

(8) adding 600ul of ethanol with volume fraction of 70% into the centrifuge tube B treated in the step (7), shaking up, centrifuging at 12000rpm for 2min, then discarding the supernatant, and repeating the above operation once;

(9) opening the cover and inverting, standing at room temperature, and completely airing residual ethanol; adding 60ul of elution buffer TE into a liquid separator, reversing and uniformly mixing for several times to help dissolve, and thus obtaining the dissolved citrus Huanglongbing vein DNA.

In the invention, the step (3) further comprises the step of preserving the ground finished product in a refrigerator at-70 ℃.

In the invention, further, the specific modes of soaking in liquid nitrogen and grinding in the step (3) are as follows: soaking liquid nitrogen for 50-70s, grinding in a grinder for 25-35s, soaking liquid nitrogen for 50-70s, and grinding for 25-35s at a frequency of 45-60 Hz.

In the invention, further, the specific modes of bubbling liquid nitrogen and grinding are as follows: soaking in liquid nitrogen for 60s, grinding in a grinder for 30s, soaking in liquid nitrogen for 60s, and grinding for 30s at a frequency of 60 Hz.

In the invention, further, the specific modes of soaking in liquid nitrogen and grinding in the step (3) are as follows: soaking in liquid nitrogen for 60s, and grinding in a grinder for 60s, wherein the grinding frequency is 55 Hz.

In the invention, further, the plant rapid extraction kit in the step (4) comprises a buffer solution FP1 of 400u L.

In the present invention, further, the RNaseA added in the step (5) has a concentration of 10 mg/ml.

The detection method comprises the following steps: 6 samples of P1, P4, C2, C6, Y3 and Y5 are picked from hand-ground samples, DNA is extracted after the grinding by the method, the extraction effect of the DNA is detected on 1% agarose gel electrophoresis, then 16SrDNA specific primers fD1/fD2 and OI1/OI2 of Asian species of the yellow dragon disease are used for performing Nested-PCR amplification, and the amplification result is detected on 1% agarose gel electrophoresis to see whether the samples are infected with the yellow dragon disease or not.

The invention has the following beneficial effects:

the invention utilizes a machine to grind citrus veins, greatly increases the number of ground samples in the same time, can ensure the grinding degree of the samples, and accelerates the cell lysis of the huanglongbing pathogen by putting steel balls into a centrifugal tube for grinding, thereby effectively improving the DNA yield of the huanglongbing pathogen. In addition, the applicant also researches the influence of the grinding frequency and time on DNA extraction, optimizes extraction parameters, and simultaneously utilizes a liquid separator and a kit to directly extract DNA, so that the method is simple and convenient, the efficiency of citrus greening disease detection is accelerated, the accuracy of the detection result is ensured, further citrus greening disease detection work can be carried out on a large scale, and technical service is provided for promoting the healthy and sustainable development of the citrus industry.

[ description of the drawings ]

FIG. 1 is a diagram showing the result of DNA agarose gel electrophoresis detection by a conventional liquid nitrogen hand milling method;

FIG. 2 is a diagram showing the results of electrophoretic detection of Nested-PCR products of DNA extracted by a conventional liquid nitrogen hand milling method;

FIG. 3 is a diagram showing the results of DNA agarose gel electrophoresis detection by the method of the present application;

FIG. 4 is a graph showing the results of electrophoretic detection of Nested-PCR products of DNA extracted by grinding using the method of the present application;

FIG. 5 is a graph showing the results of DNA agarose gel electrophoresis performed in the manner described in the first set of experiments in the second set of experiments;

FIG. 6 is a graph showing the results of DNA agarose gel electrophoresis performed in the second set of protocols in experiment two;

FIG. 7 is a graph showing the results of DNA agarose gel electrophoresis performed in the manner described in the third group of experiment two;

FIG. 8 is a graph showing the results of DNA agarose gel electrophoresis performed in the manner described in the fourth group of experiments in the second group;

FIG. 9 is a diagram showing the result of DNA agarose gel electrophoresis performed in the manner described in the fifth group of experiments;

FIG. 10 is a graph showing the result of DNA agarose gel electrophoresis performed in the manner described in the sixth group of experiments III;

FIG. 11 is a diagram showing the result of DNA agarose gel electrophoresis performed in the manner described in the seventh group of experiments III;

FIG. 12 is a diagram showing the result of DNA agarose gel electrophoresis performed in the manner described in the eighth group of experiments;

FIG. 13 is a graph showing the results of electrophoretic detection of Nested-PCR products of 9-16 bubbles in FIG. 10 in liquid nitrogen 60s, ground 60s, and ground extracted DNA at 55 Hz.

[ detailed description ] embodiments

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The grinder used in the present application was (Shanghai Jingxin industry development Co., Ltd., Multi-sample tissue grinder-48), the Rapid extraction kit was (TIANGEN DNA quick Plant System Rapid Plant genomic DNA extraction System (non-centrifugation column type))