CN113186263A - Real-time fluorescence detection method of Taqman probe for rapidly detecting tea thrips - Google Patents
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Abstract
The invention discloses a Taqman probe real-time fluorescence detection method for rapidly detecting tea yellow thrips, which is characterized in that the sequence of the ITS2 gene of tea yellow thrips on GenBank is compared and analyzed with the sequence of the ITS2 gene of Thripidae of thrips, a plurality of sets of primer premier 5.0 software are designed, and 1 set of primers and probes are screened by experiments, wherein the method comprises the following steps: the upstream primer, the downstream primer and the primer probe are 3 pieces in total. The invention has the beneficial effects that: a method for identifying the tea thrips is established by designing a specific primer and a TaqMan probe of a real-time fluorescent quantitative PCR based on a tea thrips rDNA ITS2 gene sequence, has important significance for early monitoring and port rapid quarantine of the tea thrips, and improves the detection efficiency.
Description
Technical Field
The invention relates to the technical field of tea thrips detection, in particular to a Taqman probe real-time fluorescence detection method for rapidly detecting tea thrips.
Background
Scirtothrips horsala belonging to the genus Thysanoptera, Thripidae of the family thrips, Scirtothrips durothripes. Originally originated in india, has spread rapidly to a global distribution in the last two decades, being a globally important invasive pest. The host range is very wide, and recorded hosts comprise more than 100 plants of 40 families, which are mainly harmful to young leaves and young fruits of the plants and cause poor growth and even death of the plants when the plants are serious; in addition, the tea yellow thistle horse can transmit eight plant viruses of tomato spotted wilt virus genus Tospovir, equiaxed unstable ringspot virus group Ilarvirus and bean golden mosaic virus Begomovirus for indirect damage. Due to the harm of tea yellow thrips, the yield loss caused by the production of Indian hot pepper can reach 61-74%, and the production of mango, Japanese orange and grape and Chinese tea in Malaysia poses serious threats. At present, EPPO, Canada, New Zealand and other areas and countries are listed in quarantine pest lists, and the quarantine risk of the EPPO, Canada, New Zealand and other areas and countries has great influence on global trade. Therefore, the establishment of the method capable of accurately and rapidly identifying the frankliniella cha is urgent and necessary for rapidly passing through the border between the imported and exported plants and plant products, protecting the agricultural and ecological safety of China, promoting the export of agricultural products and early monitoring of field prevention and control so as to prevent and control in time.
At present, the detection and identification technologies of tea thrips comprise two main technologies: one is morphological characterization, which is the most common method for insect identification, but it has some limitations for the identification of theaflavin. Firstly, the tea thrips has small body size, the length of adults is 0.8-1mm, the morphological characteristics distinguished from other similar thrips are few and not obvious, a glass slide must be made for microscopic examination, and the requirement on the flaking technology is very high, so the requirement on the specialty of an identifier is very high; secondly, the classification of thrips generally takes the external form of adults as the basis for identification, and in the incomplete maturation stage (such as eggs and nymphs), the identification is often difficult to distinguish due to unstable form or unobvious characteristics. At present, the tea yellow thistle which is found in an immature period is usually required to be raised to adults for identification, so that not only is time consumed, but also the thrips raising method is not mature, and the rate of successfully raising the tea yellow thistle to adults is low under the condition that the number of found insects is not large. The other is a molecular detection method, and the currently reported methods comprise specific PCR detection based on rDNA ITS2 gene sequences, DNA barcode detection based on mitochondrial CO1 genes and the like, the method can accurately identify the Thesium japonicum in different growth stages, but has long detection time, and the fussy detection process comprises fussy steps of nucleic acid extraction, 㧡 acid amplification, gel imaging and the like and needs professional technicians to operate, so that the detection cost is increased, the application range of the method is limited, and the requirements of rapid customs clearance at the port cannot be well met.
The real-time fluorescent quantitative PCR (polymerase chain reaction) technology is a technology which adds a fluorescent group in the PCR reaction process and monitors the generated PCR product in real time by accumulating the accumulation of a fluorescent signal so as to judge whether a target exists. Compared with the conventional PCR, the method has the advantages of simpler operation, accurate quantification, real-time monitoring, less pollution and the like. Fluorescent probes and fluorescent dyes are 2 types of fluorescent groups commonly adopted by the technology, wherein the fluorescent probes have stronger specificity than the fluorescent dyes, the TaqMan probes are the most commonly used fluorescent probes, and the TaqMan probes are widely used in the fields of food, medicine, agriculture and the like at present, and have better effects in the detection and identification of various thrips although few reports are reported in the detection and identification of insects, for example, Wuxia et al adopts the TaqMan real-time fluorescent quantitative PCR technology to carry out the specificity identification on the Frankliniella occidentalis, and can quantitatively detect the copy number of different insect-state target DNA fragments of the Frankliniella occidentalis; the Thrips palmi was well distinguished from 23 other Thrips using TaqMan real-time fluorescent quantitative PCR technology. At present, no report that the technology is applied to the detection and identification of the tea yellow thistle is found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a specific primer and a TaqMan probe for designing real-time fluorescence quantitative PCR based on the gene sequence of the cynara scolymus rDNA ITS2, establishes a method for identifying the cynara scolymus more sensitively, specifically and quickly, and has important significance for early monitoring and rapid port quarantine of the cynara scolymus.
The purpose of the invention is realized by the following technical scheme: based on the sequence of the Thesium japonicum ITS2 gene on GenBank (GenBank accession number: AB 063343.1), by comparison analysis with the ITS2 gene sequence of Thripidae of other thrips, a plurality of sets of primers and probes were designed and experimentally screened by using primer premier 5.0 software, wherein 1 set of primers and probes comprises: the upstream primer, the downstream primer and the primer probe are 3 in total, the specific sequence is shown in the following table 1,
| name | Base sequence (5 '-3') |
| Upstream primer Upestream primer (SdiF) | TGACCAAACTCAACGACCAGAC |
| Downstream primer Downstem primer (SdIR) | ATCCAAGCGAACGAGACAAG |
| Primer Probe (SdIP) | CGAACGCCGAGCCCACACT |
TABLE 1
The primer for rapidly detecting the tea cynara scolymus comprises the following steps:
first primer synthesis selection a primer is synthesized by primer synthesis company in the profession of primer synthesis selection.
Extracting the DNA of the sample: the extraction of total DNA of the single-headed thrips adopts a commercial kit method. Using Takara Universal 9765 kit, a single-headed thrips was placed on a glass slide on which 20. mu.L of LBuffer GL lysate was dropped, and the insects were punctured with a miniature insect needle to release body fluids. The slides were then washed 3-4 times with Buffer GL for a total dose of 300. mu.L. The rest steps are extracted according to the kit instruction. The total DNA is extracted and stored in a refrigerator at the temperature of 20 ℃ below zero for standby.
The real-time fluorescent quantitative PCR reaction: quantitative PCR amplification reactions were performed on a fluorescent quantitative PCR instrument (BIO-RAD CFX 96) using a 96-well optical plate. A25. mu.L reaction system included PCR Master mix (2X) 12.5. mu.L, forward primer (10. mu.M) 0.5. mu.L, reverse primer (10. mu.M) 0.5. mu. L, TaqMan probe (10. mu.M) 1. mu. L, DNA template 2. mu.L, the remainder being made up with ddH 2O. Water was used as a negative control and the plasmid DNA as a positive control in the experiment. The amplification procedure is specifically: the initial step is 95 ℃ for 30s, then the denaturation at 95 ℃ is carried out for 5s, the annealing and extension at 60 ℃ is carried out for 30s (the fluorescent signal is collected), and the amplification is carried out for 40 cycles.
Example 1: establishment of a Standard Curve
Under the optimal reaction condition, plasmid DNA with 102-107 times dilution gradient is used as a template to carry out real-time fluorescence quantitative PCR reaction to establish a standard curve. The standard graph (fig. 1) shows that: the copy number of the plasmid DNA and the Ct value have a good linear relation, the linear equation is Y = -3.107X + 40.878, the Delta Rn value is increased along with the increase of the concentration of the standard substance, the slope of the standard curve is 3.107, the amplification efficiency is more than 100%, and the determination coefficient is 0.9963.
Example 2: specificity verification
The results of the real-time fluorescent quantitative PCR reaction on 5 thrips in the tea garden under the optimal reaction conditions show that (see figure 2): only three different geographical populations of thrips thea and positive control amplified fluorescent signals were detected, and no amplified fluorescent signal was seen for the other thrips DNA samples tested and the negative control. The experiment shows that the designed SdiF/SdiR and SdiP primers and probes have stronger specificity to the tea yellow thrips and stronger specificity to the tea yellow thrips of three geographical populations, and can better distinguish the tea yellow thrips from other thrips in a tea garden.
Example 3: sensitivity verification
The real-time fluorescent quantitative PCR detection is carried out on the individual DNA of the single-head tea yellow thistle at different development stages, and the result shows that (shown in figure 3) the method can not only detect the imagoes, but also detect the nymphs, the pupae and the eggs, the amplification curves show the same trend, the Ct values are all smaller than 35, the Ct values are respectively the imagoes < the pupae < the nymph < the egg from small to large, and the content of the single-egg target DNA fragment is 1729.00 copy number/muL.
The invention has the following advantages:
1. the upstream primer, the downstream primer and the primer probe of the invention, and the specific primer and the TaqMan probe of the real-time fluorescent quantitative PCR designed based on the gene sequence of the rDNA ITS2 of the tea yellow thistle establish a method for identifying the tea yellow thistle more sensitively, specifically and rapidly, and have important significance for early monitoring and rapid quarantine of the tea yellow thistle.
2. The invention relates to a method for rapidly identifying the thrips tea through establishing a method capable of accurately and rapidly identifying the rapid clearance of imported and exported plants and plant products by comparing and analyzing an ITS2 gene sequence of the thrips tea on GenBank with an ITS2 gene sequence of Thripidae in thrips, using primer premier 5.0 software to design a plurality of sets, and experimentally screening 1 set of primers and probes, thereby protecting the agricultural and ecological safety of China, promoting the export of agricultural products, and early monitoring of field prevention and control so as to prevent and treat in time.
3. The DNA extraction of the invention: extracting total DNA of the single-headed thrips by adopting a commercially available kit method; placing single-headed thrips on a glass slide on which 20 mu LBuffer GL lysate is dropped by adopting a Takara universal 9765 kit of Baozoia, and puncturing insects by using a miniature insect needle to release body fluid; then washing the slide 3-4 times by Buffer GL, the total dosage is 300 mu L; extracting the rest steps according to the kit instruction; the extracted total DNA is stored in a refrigerator at the temperature of 20 ℃ below zero for standby, the method identifies the morphological characteristics of the tea yellow thistle, and the detection efficiency and the speed are high.
Drawings
FIG. 1 is a schematic diagram of a DNA fragment standard curve of thrips Camellia in the present invention;
FIG. 2 is a schematic diagram of the specificity test of the fluorescent primers and probes of the present invention;
FIG. 3 is a diagram showing the sensitivity test of the fluorescent primers and probes of the present invention.
Detailed Description
The invention will be further described with reference to the accompanying drawings, without limiting the scope of the invention to the following:
as shown in FIGS. 1-3, a Taqman probe real-time fluorescence detection method for rapidly detecting tea thrips comprises the following steps of comparing and analyzing an ITS2 gene sequence of Thripidae of other thrips by using an ITS 5.0 software according to an ITS2 gene sequence (GenBank accession number: AB 063343.1) of tea thrips on GenBank, designing a plurality of sets of primers and experimentally screening 1 set of primers and probes, wherein the primers and probes comprise: the upstream primer, the downstream primer and the primer probe are 3 in total, the specific sequence is shown in the following table 1,
| name | Base sequence (5 '-3') |
| Upstream primer Upestream primer (SdiF) | TGACCAAACTCAACGACCAGAC |
| Downstream primer Downstem primer (SdIR) | ATCCAAGCGAACGAGACAAG |
| Primer Probe (SdIP) | CGAACGCCGAGCCCACACT |
TABLE 1
The primer for rapidly detecting the tea cynara scolymus comprises the following steps:
first primer synthesis selection a primer is synthesized by primer synthesis company in the profession of primer synthesis selection.
And extracting total DNA of the single-headed thrips by using a commercially available kit method. Using Takara Universal 9765 kit, a single-headed thrips was placed on a glass slide on which 20. mu.L of LBuffer GL lysate was dropped, and the insects were punctured with a miniature insect needle to release body fluids. The slides were then washed 3-4 times with Buffer GL for a total dose of 300. mu.L. The rest steps are extracted according to the kit instruction. The total DNA is extracted and stored in a refrigerator at the temperature of 20 ℃ below zero for standby.
Performing real-time fluorescent quantitative PCR reaction on a fluorescent quantitative PCR instrument (BIO-RAD CFX 96) by using a 96-hole optical plate to perform quantitative PCR amplification reaction. A25. mu.L reaction system included PCR Master mix (2X) 12.5. mu.L, forward primer (10. mu.M) 0.5. mu.L, reverse primer (10. mu.M) 0.5. mu. L, TaqMan probe (10. mu.M) 1. mu. L, DNA template 2. mu.L, the remainder being made up with ddH 2O. Water was used as a negative control and the plasmid DNA as a positive control in the experiment. The amplification procedure is specifically: the initial step is 95 ℃ for 30s, then the denaturation at 95 ℃ is carried out for 5s, the annealing and extension at 60 ℃ is carried out for 30s (the fluorescent signal is collected), and the amplification is carried out for 40 cycles.
Example 1: establishment of a Standard Curve
Under the optimal reaction condition, plasmid DNA with 102-107 times dilution gradient is used as a template to carry out real-time fluorescence quantitative PCR reaction to establish a standard curve. The standard graph (fig. 1) shows that: the copy number of the plasmid DNA and the Ct value have a good linear relation, the linear equation is Y = -3.107X + 40.878, the Delta Rn value is increased along with the increase of the concentration of the standard substance, the slope of the standard curve is 3.107, the amplification efficiency is more than 100%, and the determination coefficient is 0.9963.
Example 2: specificity verification
The results of the real-time fluorescent quantitative PCR reaction on 5 thrips in the tea garden under the optimal reaction conditions show that (see figure 2): only three different geographical populations of thrips thea and positive control amplified fluorescent signals were detected, and no amplified fluorescent signal was seen for the other thrips DNA samples tested and the negative control. The experiment shows that the designed SdiF/SdiR and SdiP primers and probes have stronger specificity to the tea yellow thrips and stronger specificity to the tea yellow thrips of three geographical populations, and can better distinguish the tea yellow thrips from other thrips in a tea garden.
Example 3: sensitivity verification
The real-time fluorescent quantitative PCR detection is carried out on the individual DNA of the single-head tea yellow thistle at different development stages, and the result shows that (shown in figure 3) the method can not only detect the imagoes, but also detect the nymphs, the pupae and the eggs, the amplification curves show the same trend, the Ct values are all smaller than 35, the Ct values are respectively the imagoes < the pupae < the nymph < the egg from small to large, and the content of the single-egg target DNA fragment is 1729.00 copy number/muL.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A Taqman primer group for rapidly detecting tea thrips, which is characterized in that: consists of the following primers:
an upstream primer: TGACCAAACTCAACGACCAGAC, respectively;
a downstream primer: ATCCAAGCGAACGAGACAAG, respectively;
primer probe: CGAACGCCGAGCCCACACT are provided.
2. The Taqman primer group for rapidly detecting thrips Camellia according to claim 1, wherein: the primer is prepared by comparing and analyzing an ITS2 gene sequence of tea thistle on GenBank with an ITS2 gene sequence of Thripidae of thrips, a plurality of sets are designed by using primer premier 5.0 software, and 1 set of primer and probe are experimentally screened.
3. The Taqman primer group for rapidly detecting thrips Camellia according to claim 1, wherein: the primer group adopts ITS2 of thrips theae as a target gene.
4. A Taqman probe real-time fluorescence detection method for rapidly detecting tea thrips, which is characterized in that: the method comprises the following steps:
(1) primer synthesis: selecting a professional primer synthesis company to synthesize a primer;
(2) extracting sample DNA: extracting total DNA of the single-headed thrips by adopting a commercially available kit method; placing single-headed thrips on a glass slide on which 20 mu LBuffer GL lysate is dropped by adopting a Takara universal 9765 kit of Baozoia, and puncturing insects by using a miniature insect needle to release body fluid; then washing the slide 3-4 times by Buffer GL, the total dosage is 300 mu L; extracting the rest steps according to the kit instruction; the extracted total DNA is stored in a refrigerator at the temperature of 20 ℃ below zero for standby;
(3) real-time fluorescent quantitative PCR reaction: carrying out quantitative PCR amplification reaction on a 96-well optical plate on a fluorescent quantitative PCR instrument, wherein a 25-mu L reaction system comprises 2 XPCR Master mix 12.5 mu L, 10 mu M upstream primer 0.5 mu L, 10 mu M downstream primer 0.5 mu L, 10 mu M TaqMan probe 1 mu L, DNA template 2 mu L, and the rest is complemented with ddH 2O; water was used as a negative control, and DNA was used as a positive control in the experiment, and the amplification protocol was specifically: the initial step is 95 ℃ and lasts for 30s, and then the denaturation is carried out at 95 ℃ for 5 s; collecting fluorescence signals at 60 ℃, detecting, annealing and extending for 30 s; amplification was performed for 40 cycles.
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| CN110283917A (en) * | 2019-07-24 | 2019-09-27 | 南昌师范高等专科学校 | A kind of the LAMP primer group and method of quick Testing and appraisal Scirothrips dorsalis |
| JP2021073945A (en) * | 2019-11-13 | 2021-05-20 | 国立研究開発法人農業・食品産業技術総合研究機構 | Molecular identification technique for thrips |
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