CN112481386A - Normal-temperature rapid PCR (polymerase chain reaction) kit for detecting nucleic acid of pine wood nematode and detection method - Google Patents
Normal-temperature rapid PCR (polymerase chain reaction) kit for detecting nucleic acid of pine wood nematode and detection method Download PDFInfo
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Abstract
The invention discloses a detection method of a normal-temperature rapid PCR kit for detecting nucleic acid of pine wood nematodes, which comprises Q-PCR freeze-dried powder, complex solution and a nucleic acid extraction reagent, and belongs to the technical field of biological detection. The invention designs a specific primer probe aiming at the pine wood nematode, detects the pine wood nematode by a Q-PCR method, has the sensitivity of detecting 100 copies in each reaction system, and has no significant difference in detection results. The detection result is sensitive and specific, and compared with the traditional PCR, the method is simpler and more efficient to operate. Meanwhile, a normal-temperature detection kit is prepared, can be transported and stored at normal temperature, is more beneficial to the high-efficiency operation of the pine wood nematode disease prevention and control department, and meets the 'early discovery, early report, early removal and early removal' pine wood nematode disease prevention and control requirements. The pine wood nematode disease detection and quarantine method can be used for various levels of pine wood nematode disease detection and quarantine units, research institutions and social service units, can complete pine wood nematode disease detection, quarantine and identification work in laboratories, can also realize field detection and quarantine operation outdoors, and is particularly suitable for basic pine wood nematode disease detection and quarantine departments.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a normal-temperature rapid PCR (polymerase chain reaction) kit and a detection method for pine wood nematode nucleic acid detection.
Background
Pine wood nematode disease, also known as pine wilt disease, is a devastating forest disease caused by pine wood nematodes, is the most dangerous and devastating disease in the global forest ecosystem, and has been listed as the target of forest plant quarantine in China. In 38 years of invasion into China, the pine trees are diffused to 18 provinces (districts and cities) and 672 county-level administrative districts, more than 6 hundred million pine trees are fatally killed, pine forest resources, natural landscapes and ecological environments in China are seriously damaged, serious economic and ecological losses are caused, and the situation is very severe.
At present, China has performed great-effect work on the aspect of the detection and identification method of the pine wilt disease, and great progress is made from the traditional detection of modern molecular detection technology. In the eighties and ninety years of the 20 th century, Chinese scholars mainly rely on means such as symptom inspection, gummosis method, chemical method and chromogenic reaction to judge the morbidity of hosts. The method is simple and easy to implement, does not need complex instruments and equipment, and is widely applied to scientific research and basic production. From the beginning of the 21 st century, along with the development and application of molecular biology technology, the detection and quarantine technology of the pine wood nematode has been developed rapidly. Novel detection tools such as RAPD and PCR based on biochemical technology, bioelectricity technology and bioinformatics technology are applied, and novel methods and novel technologies such as rapid detection kit technology, immunodiagnosis technology and trapping and collecting tube capture diagnosis technology are developed. Compared with the traditional method, the development and the application of the new technology greatly improve the quarantine efficiency and the accuracy. However, many inspection and quarantine methods are limited by conditions and technologies or are still imperfect, so that a certain distance is left for wide popularization and application. At present, a portable and portable means which is simple and convenient to operate, fast and efficient and can be used for field detection is lacked in the market to deal with increasingly serious bursaphelenchus xylophilus disasters.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides the normal-temperature rapid PCR kit and the detection method for detecting the nucleic acid of the pine wood nematode.
The technical scheme adopted by the invention is as follows: the pine wood nematode nucleic acid detection normal temperature type rapid PCR kit comprises Q-PCR freeze-dried powder, a complex solution and a nucleic acid extraction reagent, wherein the Q-PCR freeze-dried powder comprises 1-2U of amplification enzyme, 100-500nM of primer, 100-500nM of probe, 20-30mM of KCl and 10mM of MgSO 42, the complex solution comprises 10-20mM of Tris-HCl, the nucleic acid extraction reagent comprises 50-200mM of Tris-HCl, 10-30mM of EDTA, 500mM of NaCl200-5 and 1-3% of SDS, and the sequences of the primer and the probe are as follows:
and (3) primer F: CCTATGACACATTTATTCGTGCTCGTC
And (3) primer R: CGGCAAGCAGCTGGCTG
And (3) probe P: FAM- -GGAACGACGCGAATCGAACCG- -BHQ 1.
Preferably, the primer F and the primer R in the Q-PCR freeze-dried powder of the pine wood nematode nucleic acid detection normal-temperature rapid PCR kit are used at the concentration of 200nM, and the probe P is used at the concentration of 200 nM.
Preferably, the using concentration of Taq DNA polymerase in the Q-PCR freeze-dried powder of the pine wood nematode nucleic acid detection normal-temperature rapid PCR kit is 5U.
Preferably, Q-PCR freeze-dried powder of the pine wood nematode nucleic acid detection normal-temperature rapid PCR kit contains KCl 20mM and MgSO45 mM.
Preferably, the re-solution of the pine wood nematode nucleic acid detection normal temperature type rapid PCR kit contains Tris-HCl 20 mM.
Preferably, the nucleic acid extraction reagent of the pine wood nematode nucleic acid detection normal temperature type rapid PCR kit comprises 100mM Tris-HCl, 25mM EDTA, 500mM NaCl and 1% SDS.
The invention also provides a detection method of the normal-temperature rapid PCR kit for detecting the nucleic acid of the pine wood nematode, which comprises the following steps:
(1) extracting the genome DNA of the pine wood nematode from the wood chip sample and the nematode liquid sample of the pine wood nematode by using the nucleic acid extraction reagent;
(2) adding the pine wood nematode genome DNA extracted in the step (1) as an amplification template into the pine wood nematode nucleic acid detection normal-temperature rapid PCR kit, and setting a reaction program for amplification detection;
(3) performing fluorescence interpretation on the amplification detection result obtained in the step (2) by using a fluorescence detection device.
Preferably, the bursaphelenchus xylophilus genomic DNA obtained in step (1) is obtained by the following steps:
and (2) taking about 100mg of epidemic wood chip sample into a 1.5mL Eppendorf tube, adding 1mL of nucleic acid extraction reagent, centrifuging at 10,000rpm for 30 seconds, and treating at 95 ℃ for 5-20 minutes to obtain lysate, namely the bursaphelenchus xylophilus genome DNA amplification template.
Preferably, the bursaphelenchus xylophilus genomic DNA obtained in step (1) is obtained by the following steps:
and (3) taking 1mL of nematode liquid sample into a 1.5mL Eppendorf tube, centrifuging at 10,000rpm for 1 minute, removing the supernatant, adding 100uL of nucleic acid extraction reagent, and treating at 95 ℃ for 5-20 minutes to obtain lysate, namely the pine wood nematode genome DNA amplification template.
Preferably, the step (2) is specifically:
adding 5ul of the pine wood nematode DNA amplification template prepared in the step (1) into 20ul of the normal temperature type rapid PCR kit for pine wood nematode nucleic acid detection, wherein the reaction process comprises the following steps: at 95 ℃ for 3 min; 95 ℃ for 10 s; 60 ℃ for 20 s; at 40 cycles, the FAM channel detects fluorescence.
Preferably, in the step (3), the excitation wavelength 497nm and the detection wavelength 520nm are required for the fluorescence detection device, and the Ct value for positive determination of the fluorescence detection result is 42.
Preferably, the pine wood nematode nucleic acid detection normal temperature type rapid PCR kit is used for single or more pine wood nematodes.
After adopting the structure, the invention has the following beneficial effects: the invention relates to a normal-temperature rapid PCR kit for detecting nucleic acid of pine wood nematode and a detection method. The pine wood nematode detection and quarantine work can be carried out in a laboratory, and the field rapid detection and quarantine work can also be carried out outdoors. The detection time of the normal-temperature rapid PCR kit for detecting the pine wood nematode is finished within 40 minutes, the operation is simple, the practicability is high, and the pine wood nematode detection, quarantine and identification work of all levels of pine wood nematode disease detection and quarantine units, research institutions and social service units in laboratories and outdoors can be conveniently carried out; the normal-temperature rapid PCR kit for detecting the pine wood nematode nucleic acid has high detection sensitivity, and can realize the detection work of single or more pine wood nematode samples. The nucleic acid detection normal-temperature rapid PCR kit for the pine wood nematodes has good detection specificity, and can distinguish the pine wood nematodes, the pseudopine wood nematodes and other nematodes; the detection method of the normal-temperature rapid PCR kit for detecting the nucleic acid of the pine wood nematode has the advantages of simple sample treatment mode, short time, no more than 10 minutes in the whole sample treatment process, and economic and efficient sample treatment equipment.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
FIG. 1 is a graph showing the results of fluorescence detection of a single sample of Bursaphelenchus xylophilus by the normal temperature type rapid PCR kit for nucleic acid detection of Bursaphelenchus xylophilus of example 2;
FIG. 2 is a graph showing the results of fluorescence detection of a sample of a Bursaphelenchus xylophilus isolate and a sample of a Bursaphelenchus pseudoxylophilus isolate by the normal temperature type rapid PCR kit for detecting nucleic acid of Bursaphelenchus xylophilus in example 3;
FIG. 3 is a graph showing the result of fluorescence detection of a wood chip sample by the normal temperature type rapid PCR kit for nucleic acid detection of Bursaphelenchus xylophilus of example 4;
FIG. 4 is a graph showing the results of fluorescence detection of a sample of nematode fluid of different concentrations by the normal temperature type rapid PCR kit for nucleic acid detection of Bursaphelenchus xylophilus of example 5.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Example 1 construction of a fluorescent colorimetric kit for DNA detection of Bursaphelenchus xylophilus
1. Raw material preparation
Pine wood nematode wood chip samples, pine wood nematode separating medium samples, pseudopine wood nematode separating medium samples and other more than twenty kinds of nematode samples used in the experiment are all provided by a forest pest control station of national forestry and grassland bureau. Taq DNA polymerase, reaction Buffer, amplification primers, probe sequence and other chemical reagents were supplied by Nanjing Naddy high-tech Co.
2. Design and Synthesis of amplification primers
According to the specific comparison of the pine wood nematode and pine wood nematode simulating gene sequences in GenBank, selecting a conserved sequence region as an amplification region, and designing a specific amplification primer by utilizing Oligo7.0 primer design software and a BLAST software program, wherein the sequences of the primer and the probe are respectively as follows:
and (3) primer F: CCTATGACACATTTATTCGTGCTCGTC
And (3) primer R: CGGCAAGCAGCTGGCTG
And (3) probe P: FAM- -GGAACGACGCGAATCGAACCG- -BHQ1
3. Preparation of genome DNA template of pine wood nematode
(1) Preparation of genome DNA template of pine wood nematode infected wood chip sample
About 100mg of wood chip samples of the epidemic trees are taken to be put into a 1.5mL Eppendorf tube, 1mL of nucleic acid extraction reagent (containing Tris-HCl100mM, EDTA25mM, NaCl 500mM and SDS 1%) is added, the mixture is centrifuged at 10,000rpm for 30 seconds and is treated at 95 ℃ for 10 minutes, and the obtained lysate is the amplification template of the genome DNA of the bursaphelenchus xylophilus.
(2) Preparation of genome DNA template of pine wood nematode separating medium sample
Taking 1mL of nematode liquid sample into a 1.5mL Eppendorf tube, centrifuging at 10,000rpm for 1 minute, discarding the supernatant, adding 100uL of nucleic acid extraction reagent (containing Tris-HCl100mM, EDTA25mM, NaCl 500mM and SDS 1%), processing at 95 ℃ for 10 minutes, and obtaining lysate which is the bursaphelenchus xylophilus genome DNA amplification template.
It should be noted that the sample lysis time is optimized, one sample is divided into four parts, the sample is lysed at 95 ℃ for 2 minutes, 5 minutes, 10 minutes and 15 minutes respectively, the lysis efficiency is evaluated according to the fluorescence amplification Ct value, and the optimal lysis time is finally determined to be 10 minutes.
4. Optimization of amplification systems
The normal-temperature rapid Q-PCR kit for the pine wood nematode comprises an amplification system in 25 ul:
Q-PCR freeze-dried powder 1 hole
Compound solution 20ul
Genomic DNA template 5ul
It should be noted that the concentration of each component in the reaction system is optimized, specifically:
(1) adding the treated bursaphelenchus xylophilus genome DNA template into a Q-PCR freeze-dried powder system prepared by MgSO4(2mM, 5mM and 10mM) with different concentrations, judging the optimal concentration according to the Ct value, and finally determining the optimal concentration of MgSO4 to be 5 mM.
(2) Adding the processed bursaphelenchus xylophilus genome DNA template into a Q-PCR freeze-dried powder system prepared by amplification enzymes (2U, 5U and 10U) with different concentrations, judging the dosage of the enzymes according to Ct values, and finally determining the optimal amplification enzyme dosage to be 5U.
5. Amplification reaction procedure
The normal-temperature rapid Q-PCR kit for the pine wood nematode comprises the following reaction procedures: at 95 ℃ for 3 min; 95 ℃ for 10 s; 60 ℃ for 20 s; at 40 cycles, the FAM channel detects fluorescence. The excitation wavelength 497nm and the detection wavelength 520nm of the fluorescence detection equipment, and the positive judgment Ct value of the fluorescence detection result is 42.
The reaction temperature of the reaction program is optimized, the processed bursaphelenchus xylophilus genome DNA template is added into an amplification system, the reaction temperature is respectively set to 59 ℃, 60 ℃ and 61 ℃, the optimal reaction temperature is judged according to the Ct value, and the optimal reaction temperature is finally determined to be 60 ℃.
6. Determination of results
The excitation wavelength 497nm and the detection wavelength 520nm of the fluorescence detection equipment, and the positive judgment Ct value of the fluorescence detection result is 42.
Example 2 verification of sensitivity of nucleic acid detection Normal temperature type Rapid PCR kit for Bursaphelenchus xylophilus
Diluting the nematode liquid sample with water, picking a single nematode sample by means of a microscope, preparing a genome DNA template by using an optimized sample processing program, and adding an optimized kit amplification system, wherein the detection result is shown in figure 1, the figure is a fluorescence result, 1 and 2 are single nematode sample detection results, 3 is a negative control result, and 4 is a positive control result. As can be seen from FIG. 1, the detection results of single nematode samples are all positive, and the single nematode can be detected by the kit sensitivity.
Example 3 specificity verification of nucleic acid detection Normal temperature type Rapid PCR kit for Bursaphelenchus xylophilus
The method comprises the steps of using a pine wood nematode separating medium sample and a pseudopine wood nematode separating medium sample, using an optimized sample processing program to prepare a genome DNA template, adding an optimized kit amplification system, and obtaining a detection result as shown in figure 2, wherein the figure is a fluorescence result, 1 is a pine wood nematode separating medium sample detection result, 2 is a pseudopine wood nematode separating medium sample detection result, 3 is a negative control result, and 4 is a positive control result. As can be seen from FIG. 2, the result No. 1 is positive, and the result No. 2 is negative, indicating that the kit has good detection specificity, and can distinguish the bursaphelenchus xylophilus sample from the bursaphelenchus xylophilus sample.
Example 4 detection and verification of pine wood nematode nucleic acid detection Normal temperature type fast PCR kit on wood chip sample
Two pine wood nematode infected wood chip samples are used, an optimized sample processing program is used for preparing a genome DNA template, an optimized kit amplification system is added, the detection result is shown in figure 3, the figure is a fluorescence result, 1 and 2 are infected wood chip sample detection results, 3 is a negative control result, and 4 is a positive control result. As can be seen from FIG. 3, the detection results of samples No. 1 and No. 2 are both positive, which indicates that the kit can accurately detect different wood chip samples.
Example 5 detection and verification of nucleic acid detection Normal temperature type fast PCR kit for pine wood nematode
And (3) diluting the nematode liquid sample by four concentration gradients to prepare four nematode liquid samples, preparing a genome DNA template by using an optimized sample processing program, and adding an optimized kit amplification system, wherein the detection result is shown in figure 4, the figure is a fluorescence result, 1, 2, 3 and 4 are nematode liquid sample detection results, 5 is a negative control result, and 6 is a positive control result. As can be seen from FIG. 4, the detection results of samples No. 1, 2, 3 and 4 are all positive, and the kit can accurately detect nematode fluid samples with different concentrations.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. Pine wood nematode nucleic acid detects quick PCR kit of normal atmospheric temperature type, its characterized in that: comprises Q-PCR freeze-dried powder, double solution and nucleic acid extraction reagent, wherein the Q-PCR freeze-dried powder comprises 1-2U of amplification enzyme, 100-500nM of primer, 100-500nM of probe, 20-30mM of KCl and 10mM of MgSO 42, the double solution comprises 10-20mM of Tris-HCl, and the nucleic acid extraction reagent comprises 50-200mM of Tris-HCl, 10-30mM of EDTA, 200-500mM of NaCl and 1-3% of SDS, and the sequences of the primer and the probe are as follows:
and (3) primer F: CCTATGACACATTTATTCGTGCTCGTC
And (3) primer R: CGGCAAGCAGCTGGCTG
And (3) probe P: FAM- -GGAACGACGCGAATCGAACCG- -BHQ 1.
2. The detection method of the normal temperature type rapid PCR kit for detecting the nucleic acid of the pine wood nematode is characterized in that: the method comprises the following steps:
(1) extracting the genome DNA of the pine wood nematode from the wood chip sample and the nematode liquid sample of the pine wood nematode by using the nucleic acid extraction reagent;
(2) adding the pine wood nematode genome DNA extracted in the step (1) as an amplification template into the pine wood nematode nucleic acid detection normal-temperature rapid PCR kit, and setting a reaction program for amplification detection;
(3) performing fluorescence interpretation on the amplification detection result obtained in the step (2) by using a fluorescence detection device.
3. The detection method of the normal temperature type rapid PCR kit for detecting the nucleic acid of the pine wood nematode as claimed in claim 2, which is characterized in that: the bursaphelenchus xylophilus genome DNA obtained in the step (1) is obtained through the following steps:
and (2) taking about 100mg of epidemic wood chip sample into a 1.5mL Eppendorf tube, adding 1mL of nucleic acid extraction reagent, centrifuging at 10,000rpm for 30 seconds, and treating at 95 ℃ for 5-20 minutes to obtain lysate, namely the bursaphelenchus xylophilus genome DNA amplification template.
4. The detection method of the pine wood nematode nucleic acid detection normal temperature type rapid PCR kit according to claim 2, characterized in that the pine wood nematode genome DNA of the step (1) is obtained by the following steps:
and (3) taking 1mL of nematode liquid sample into a 1.5mL Eppendorf tube, centrifuging at 10,000rpm for 1 minute, removing the supernatant, adding 100uL of nucleic acid extraction reagent, and treating at 95 ℃ for 5-20 minutes to obtain lysate, namely the pine wood nematode genome DNA amplification template.
5. The detection method of the normal temperature type rapid PCR kit for detecting the nucleic acid of the pine wood nematode as claimed in claim 2, which is characterized in that: the step (2) is specifically as follows:
adding 5ul of the pine wood nematode DNA amplification template prepared in the step (1) into 20ul of the normal temperature type rapid PCR kit for pine wood nematode nucleic acid detection, wherein the reaction process comprises the following steps: at 95 ℃ for 3 min; 95 ℃ for 10 s; 60 ℃ for 20 s; at 40 cycles, the FAM channel detects fluorescence.
6. The detection method of the normal temperature type rapid PCR kit for detecting the nucleic acid of the pine wood nematode as claimed in claim 2, which is characterized in that: in the step (3), the fluorescence detection device needs to have an excitation wavelength of 497nm and a detection wavelength of 520 nm.
7. The application of the normal temperature type rapid PCR kit for detecting the nucleic acid of the pine wood nematode is characterized in that: the nucleic acid detection normal-temperature rapid PCR kit for the pine wood nematodes is used for detecting single or more pine wood nematodes.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115786536A (en) * | 2022-11-03 | 2023-03-14 | 北京林业大学 | Nucleic acid detection system for pine wood nematode and nucleic acid detection method of pine wood nematode |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101041851A (en) * | 2006-03-24 | 2007-09-26 | 叶建仁 | Special primer pair for pine beam thread insect PCR detection |
| CN102925545A (en) * | 2012-07-06 | 2013-02-13 | 天津出入境检验检疫局动植物与食品检测中心 | New Bursaphelenchus xylophilus detection kit and detection method thereof |
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- 2020-11-19 CN CN202011304249.3A patent/CN112481386A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101041851A (en) * | 2006-03-24 | 2007-09-26 | 叶建仁 | Special primer pair for pine beam thread insect PCR detection |
| CN102925545A (en) * | 2012-07-06 | 2013-02-13 | 天津出入境检验检疫局动植物与食品检测中心 | New Bursaphelenchus xylophilus detection kit and detection method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115786536A (en) * | 2022-11-03 | 2023-03-14 | 北京林业大学 | Nucleic acid detection system for pine wood nematode and nucleic acid detection method of pine wood nematode |
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