CN111500678A - Method for extracting DNA (deoxyribonucleic acid) of veins of citrus Huanglongbing with high flux - Google Patents
Method for extracting DNA (deoxyribonucleic acid) of veins of citrus Huanglongbing with high flux Download PDFInfo
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Abstract
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.
Description
[ 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))
Example 1:
the embodiment provides a method for extracting vein DNA of citrus Huanglongbing with high throughput, which 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) putting the centrifugal tube A into a grinding box hole of a grinding machine, soaking in liquid nitrogen for 50s, then putting the centrifugal tube A into the grinding machine for grinding for 25s, soaking in liquid nitrogen for 50s, and finally grinding for 25-35 s; storing the ground finished product in a refrigerator at the temperature of-70 ℃; the above-mentioned grinding is grinding at a frequency of 45 Hz;
(4) adding the material obtained in the step (3) into the FP 1400 u L rapid plant extraction kit;
(5) adding 6u L RNaseA into the plant rapid extraction kit treated in the step (4), carrying out vortex oscillation for 1min, and then placing at normal temperature for 10min, wherein the concentration of RNaseA is 10 mg/ml;
(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.
Example 2:
the embodiment provides a method for extracting vein DNA of citrus Huanglongbing with high throughput, which 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) putting the centrifuge tube A into a grinding plate box of a grinding machine, soaking liquid nitrogen for 60s, then putting the centrifuge tube A into the grinding machine for grinding for 30s, soaking liquid nitrogen for 60s, and finally grinding for 30 s; storing the ground finished product in a refrigerator at the temperature of-70 ℃; the above-mentioned grinding is grinding at a frequency of 60 Hz;
(4) adding the material obtained in the step (3) into the FP 1400 u L rapid plant extraction kit;
(5) adding 6u L RNaseA into the plant rapid extraction kit treated in the step (4), carrying out vortex oscillation for 1min, and then placing at normal temperature for 10min, wherein the concentration of RNaseA is 10 mg/ml;
(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.
Example 3
The embodiment provides a method for extracting vein DNA of citrus Huanglongbing with high throughput, which 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) putting the centrifugal tube A into a grinding box hole of a grinding machine, soaking in liquid nitrogen for 70s, then putting into the grinding machine for grinding for 35s, soaking in liquid nitrogen for 70s, and finally grinding for 35 s; storing the ground finished product in a refrigerator at the temperature of-70 ℃; the above-mentioned grinding is grinding at a frequency of 60 Hz;
(4) adding the material obtained in the step (3) into the FP 1400 u L rapid plant extraction kit;
(5) adding 6u L RNaseA into the plant rapid extraction kit treated in the step (4), carrying out vortex oscillation for 1min, and then placing at normal temperature for 10min, wherein the concentration of RNaseA is 10 mg/ml;
(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.
Example 4
The embodiment provides a method for extracting vein DNA of citrus Huanglongbing with high throughput, which 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) putting the centrifuge tube A into a grinding box hole of a grinding machine, soaking liquid nitrogen for 60s, and then putting the centrifuge tube A into the grinding machine for grinding for 60 s; storing the ground finished product in a refrigerator at the temperature of-70 ℃; the above-mentioned grinding is grinding at a frequency of 55 Hz;
(4) adding the material obtained in the step (3) into the FP 1400 u L rapid plant extraction kit;
(5) adding 6u L RNaseA into the plant rapid extraction kit treated in the step (4), carrying out vortex oscillation for 1min, and then placing at normal temperature for 10min, wherein the concentration of RNaseA is 10 mg/ml;
(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.
Test and results
Test one:
samples of ponkan P1, P2 and P3 with patchy leaves and samples of ponkan P4, P5 and P6 with normal leaves are respectively adopted; samples of Hongjiang orange C1, C2 and C3 with mottled leaves, and samples of Hongjiang orange C4, C5 and C6 with normal leaves; samples of Shatian pomelo Y1, Y2 and Y3 with mottled leaves, and samples of Shatian pomelo Y4, Y5 and Y6 with normal leaves; a total of 18 samples. The conventional liquid nitrogen hand milling method is adopted, and then DNA is extracted, 48 samples take 2 days.
As shown in FIG. 1, it is a picture of DNA running gel of the hand-milled sample, wherein M is Mark; 1: P1; 2: P2; 3: P3; 4: P4; 5: P5; 6: P6; 7: C1; 8: C2; 9: C3; 10: C4; 11: C5; 12: C6; 13: Y1; 14: Y2; 15: Y3; 16: Y4; 17: Y5; and 18: Y6. As shown in FIG. 2, it is a photograph of glue-running fruit of Nested-PCR product, in which M is Mark; 1: P1; 2: P2; 3: P3; 4: P4; 5: P5; 6: P6; 7: C1; 8: C2; 9: C3; 10: C4; 11: C5; 12: C6; 13: Y1; 14: Y2; 15: Y3; 16: Y4; 17: Y5; and 18: Y6.
6 samples of P1, P4, C2, C6, Y3 and Y5 were selected from the hand-milled samples, and the DNA extraction took 0.5 day after milling by the method of the present invention.
FIG. 3 is a picture of a DNA running gel fruit after the grinding according to the method of example 2 and the extraction of DNA; as shown in FIG. 4, it is a photograph of the glue running fruit of the Nested-PCR product.
And (4) conclusion: machine grinds time saving than hand mill, if 48 samples, just can practice thrift 1.5 days time, if extract in batches, the time of saving is just more, and it is more high-efficient to compare, and the Nested-PCR product of the DNA of extracting runs the glue effect and shows, and the testing result is unanimous with the hand mill testing result, and the end of the yellow dragon disease fungus also can detect. The citrus greening disease vein DNA extracted by the method can be completely used for Nested-PCR detection.
And (2) test II:
to illustrate the practical value of some of the parameters (bubble liquid nitrogen time, milling time) in the present application, the applicant set the following comparison groups (first to fourth groups) and observed the extraction of DNA at parameters not within the scope of protection of the present application:
a first group: liquid nitrogen does not bubble, the grinding time is 1min, and other modes are strictly carried out according to the embodiment 2; the picture of DNA running gel is shown in FIG. 5; wherein M is Mark; 1: P1; 2: P2; 3: P3; 4: P4; 5: P5; 6: P6; 7: C1; 8: C2; 9: C3; 10: C4; 11: C5; 12: C6; 13: Y1; 14: Y2; 15: Y3; 16: Y4; 17: Y5; and 18: Y6. As can be seen from FIG. 5, the DNA extracted by the first group of methods has high enzyme activity, more degradation and poor extraction effect due to the absence of liquid nitrogen low-temperature quick freezing.
Second group: soaking in liquid nitrogen for 30s, grinding for 1min, and performing other modes strictly according to the embodiment 2; the picture of DNA running gel is shown in FIG. 6; wherein M is Mark; 1: P1; 2: P2; 3: P3; 4: P4; 5: P5; 6: P6; 7: C1; 8: C2; 9: C3; 10: C4; 11: C5; 12: C6; 13: Y1; 14: Y2; 15: Y3; 16: Y4; 17: Y5; and 18: Y6. As can be seen from FIG. 6, the DNAs extracted by the second group were degraded because the time for bubbling liquid nitrogen was not long enough, and some of the DNAs were degraded, resulting in non-uniform extraction amount and poor extraction purity.
Third group: soaking in liquid nitrogen for 3min, grinding for 1min, and performing other modes strictly according to the embodiment 2; the picture of DNA running gel is shown in FIG. 7; wherein M is Mark; 1: P1; 2: P2; 3: P3; 4: P4; 5: P5; 6: P6; 7: C1; 8: C2; 9: C3; 10: C4; 11: C5; 12: C6; 13: Y1; 14: Y2; 15: Y3; 16: Y4; 17: Y5; and 18: Y6. As can be seen from FIG. 7, the DNA extracted by the third group method had a poor extraction effect because the vein was hardened due to the long time of soaking in liquid nitrogen during grinding, and only a small amount of ground DNA was extracted during grinding.
And a fourth group: soaking in liquid nitrogen for 4min, grinding for 1min, and performing other modes strictly according to the embodiment 2; the picture of DNA running gel is shown in FIG. 8; wherein M is Mark; 1: P1; 2: P2; 3: P3; 4: P4; 5: P5; 6: P6; 7: C1; 8: C2; 9: C3; 10: C4; 11: C5; 12: C6; 13: Y1; 14: Y2; 15: Y3; 16: Y4; 17: Y5; and 18: Y6. As can be seen from FIG. 8, the DNA extracted by the fourth group method has too hard veins due to too long time for soaking in liquid nitrogen, which increases the grinding difficulty, and results in insufficient grinding of the sample, and the basic extraction is not successful.
And (3) test III:
after the applicant finds out that the time for soaking liquid nitrogen is 60s, the liquid nitrogen is ground for 30s, then the liquid nitrogen is soaked for 60s, the liquid nitrogen is ground for 30s, the grinding frequency is 60Hz, and the extracted DNA has a good effect, the applicant further finds out the above steps. Since the grinding time frequency is too high, tube breakage occurs, and then the liquid nitrogen soaking grinding is repeated, so that the operation is increased. Therefore, we have designed the following experiment again.
The samples taken were mottled and healthy leaves of different varieties of diseased trees with known results. (iii) patchy leaves of ponkan orange trees (gb1, gb2.. gtb 12), normal upper tree leaves (gz1, gz2.. gtz 12); red river orange diseased tree mottled leaves (cb1, cb2.... cnb 12), normal treetop leaves (cz1, cz2.. cna. cz 12); mottled leaves of shatian pomelo diseased trees (yb1, yb2.... yb12), normal foliage leaves on trees (yz1, yz2.... yz 12); mottled leaves of honey pomelo diseased trees (mb1, mb2.... mb12), and leaves of normal trees (mz1, mz2.... mz 12).
In FIG. 9, M is Mark; gb 1; 2: gz 1; cb 1; 4: cz 1; 5: yb 1; 6: yz 1; mb 1; 8: mz 1; gb 2; gz 2; cb 2; 12: cz 2; 13: yb 2; 14: yz 2; 15: mb 2; 16: mz 2; gb 3; 18: gz 3; cb 3; 20: cz 3; 21: yb 3; 22: yz 3; 23: mb 3; 24: mz 3. Wherein, 1-8 detection bands with bubble liquid nitrogen for 30s, grinding for 30s and frequency of 55 Hz; the 9-16 strips are detection strips which are soaked in liquid nitrogen for 30s, ground for 60s and have the frequency of 55 Hz; the 17-24 bands are detection band types of bubble liquid nitrogen for 30s, grinding for 90s and frequency of 55 Hz. (set as the fifth group)
In FIG. 10, M is Mark; gb 4; 2: gz 4; cb 4; 4: cz 4; 5: yb 4; 6: yz 4; mb 4; 8: mz 4; gb 5; gz 5; cb 5; 12: cz 5; 13: yb 5; 14: yz 5; 15: mb 5; 16: mz 5; gb 6; 18: gz 6; cb 6; 20: cz 6; 21: yb 6; 22: yz 6; 23: mb 6; 24: mz 6. Wherein, 1-8 detection bands with bubble liquid nitrogen for 60s, grinding for 30s and frequency of 55 Hz; the 9-16 strips are detection strips which are soaked in liquid nitrogen for 60s, ground for 60s and have the frequency of 55 Hz; the 17-24 strips are detection strips with 60s of bubble liquid nitrogen, 90s of grinding and 55Hz of frequency. (set as the sixth group)
In FIG. 11, M is Mark; gb 7; 2: gz 7; cb 7; 4: cz 7; 5: yb 7; 6: yz 7; mb 7; 8: mz 7; gb 8; gz 8; cb 8; 12: cz 8; 13: yb 8; 14: yz 8; 15: mb 8; 16: mz 8; gb 9; 18: gz 9; cb 9; 20: cz 9; 21: yb 9; 22: yz 9; 23: mb 9; 24: mz 9; wherein, 1-8 detection bands with bubble liquid nitrogen for 30s, grinding for 30s and frequency of 50Hz are formed; the 9-16 strips are detection strips which are soaked in liquid nitrogen for 30s, ground for 60s and have the frequency of 50 Hz; the 17-24 bands are detection band types of bubble liquid nitrogen for 30s, grinding for 90s and frequency of 50 Hz. (set as the seventh group)
In FIG. 12, M is Mark; gb 10; 2: gz 10; cb 10; 4: cz 10; 5: yb 10; 6: yz 10; mb 10; 8: mz 10; gb 11; gz 11; cb 11; 12: cz 11; 13: yb 11; 14: yz 11; 15: mb 11; 16: mz 11; gb 12; 18: gz 12; cb 12; 20: cz 12; 21: yb 12; 22: yz 12; 23: mb 12; 24: mz 12; wherein, 1-8 detection bands with bubble liquid nitrogen for 30s, grinding for 30s and frequency of 45 Hz; the 9-16 strips are detection strips which are soaked in liquid nitrogen for 30s, ground for 60s and have the frequency of 45 Hz; the 17-24 bands are detection band types of bubble liquid nitrogen for 30s, grinding for 90s and frequency of 45 Hz. (set as the eighth group)
As can be seen from the above FIG. 9-FIG. 12, the bands 9-16 in FIG. 10 bubble liquid nitrogen 60s, grind the extracted DNA band with the frequency of 55Hz brightest, and the Nested-PCR product run gel results are shown in FIG. 13, M is Mark; negative control; a positive control; gb 5; 2: gz 4; cb 5; 4: cz 5; 5: yb 5; 6: yz 5; mb 5; 8: mz 5. As can be seen from FIG. 13 (gel running result of Nested-PCR product), the detection result of normal leaf is negative, the detection result of mottled leaf is positive, and the detection result is consistent with the selected sample.
And (4) conclusion: through DNA agarose electrophoresis gel running, the DNA bands extracted by soaking liquid nitrogen for 60s, grinding for 60s and extracting at the frequency of 55Hz are the clearest and the best effect is found. According to the calculation, one sample grinding machine can grind about 2000 samples according to the work calculation of 7 hours a day. The sample grinding efficiency is greatly improved.
The above examples merely represent some embodiments of the present invention, which are described in more detail and in more detail, but are not to be construed as limiting the scope of the invention.
Claims (7)
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.
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