CN113604577B - Primer, probe, kit and method for detecting cassava mealy bugs based on fluorescent quantitative PCR - Google Patents
Primer, probe, kit and method for detecting cassava mealy bugs based on fluorescent quantitative PCR Download PDFInfo
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Abstract
The invention discloses a primer and a probe for detecting cassava mealy bugs based on fluorescent quantitative PCR, and a kit and a detection method adopting the primer and the probe. The detection primer set comprises an upstream primer PM-F1, a downstream primer PM-R1 and a Probe PM-Probe1; the detection kit comprises a primer, a probe, a template, a positive sample, a negative sample and a premix. The detection method comprises the steps of extracting DNA of a sample to be detected, rapidly detecting the DNA of the sample to be detected by utilizing a fluorescent quantitative PCR technology, and judging whether the sample to be detected is a cassava mealy scale or not according to the real-time fluorescent PCR amplification condition. The invention has the advantages of rapidness, sensitivity, specificity, easy operation and the like, and is convenient for popularization and application.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a primer, a probe, a kit and a method for detecting cassava mealy bugs based on fluorescent quantitative PCR.
Background
Cassava mealybugsPhenacoccusmanihotiMatile-Ferrio belonging to genus Ericerus of the family Lepidoptera (Hemiptera) of the family Lepidoptera (Coccoidea) of the family Lepidoptera (Pseudomonas) of the subfamily Lepidoptera (Phenococcidae)Phenacoccus) Is a world quarantine pest and is also an important quarantine pest in the entrance and exit inspection and quarantine of the port of China at present. The cassava mealy bugs are addicted to cassava, especially sweet cassava, and often develop disasters on the cassava, and the existing researches show that the host plants of the cassava mealy bugs have 27 species and 3 genera in 15 families, including crops, garden plants, weeds, fruit trees and the like. The cassava mealybugs are native in the middle of south america, are an important pest on local cassava, and are then spread along with host plants and products thereof, and are found in western africa, middle africa and eastern africa, thus causing great loss to the africa cassava industry. The cassava mealybugs are highly suitable for most areas of provinces (autonomous areas) such as Guangdong, guangxi, hainan and the like in China, and are moderately suitable for some areas such as Fujian, jiangxi, guizhou, yunnan and the like in Chongqing, sichuan and the like.
Therefore, the rapid detection technology of the cassava mealy bugs is developed as soon as possible, the monitoring and detection of the cassava mealy bugs are enhanced, and the method is a necessary premise for guaranteeing the healthy development of the cassava, vegetable and flower industries and the fruit and cotton industries in China. At present, the cassava mealy bugs are aimed atPhenacoccusmanihotiThe detection scheme of (1) is that common PCR detection is carried out on the cassava mealy gecko moth by using a specific SS-COI primer, and the publication number is CN104293970A; the common PCR detection method is required to be used for electrophoresis detection, the time consumption is long, the real-time fluorescence quantitative PCR technology is not required to be used for electrophoresis detection, time and labor are saved, the specificity is high, the sensitivity is high, and the like, but the technology is not specific to the cassava mealybugs at presentP.manihotiReal-time fluorescent quantitative PCR detection method of (2)A kit. Therefore, providing a real-time fluorescent quantitative PCR detection method for specifically detecting tapioca becomes a problem to be solved by those skilled in the art.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem solved by the invention is to provide a primer and a probe for detecting the cassava mealy bugs based on fluorescent quantitative PCR.
The technical problem solved by the invention is also to provide a kit for detecting the cassava mealy bugs based on fluorescent quantitative PCR.
The technical problem solved by the invention is also to provide a method for detecting the cassava mealy bugs based on fluorescent quantitative PCR.
In order to solve the technical problems, the invention is realized by adopting the following technical scheme:
in one aspect, the invention provides a primer and a probe for detecting cassava mealy bugs based on fluorescent quantitative PCR, which comprises an upstream primer PM-F1, a downstream primer PM-R1 and a specific probe PM-probe, wherein the nucleotide sequences of the primers are respectively shown as follows:
PM-F1: 5’-ACCTTTGATGATTTCTTCTTCTG-3’；
PM-R1：5’-TGAAGTTTAAAGTAATGAAATTTTG-3’；
PM-Probel:5'-ATTTTAGATTTTGATTACTACTTCCTTCTT-3' wherein the 5 '-end-labeled fluorophore is FAM and the 3' -end-labeled quencher is BHQ1.
On the other hand, the invention also discloses a kit for detecting the cassava mealy bugs based on fluorescent quantitative PCR, which comprises the detection primer and the probe as described in claim 1 or 2.
Preferably, the kit for detecting the cassava mealy bugs based on fluorescence quantitative PCR further comprises part or all of the following components: (1) a template; (2) a positive control and a negative control; (3) PCR premix.
Preferably, the positive control is recombinant plasmid and the negative control is ddH 2 O, the PCR premix is Takara Premix Ex Tag produced by Dalianbao biological company.
The invention also discloses a method for detecting the cassava mealy bugs based on fluorescent quantitative PCR, which comprises the following steps:
1) Extracting DNA of a sample to be detected;
2) Preparing a fluorescent quantitative PCR reaction system: adding the DNA of the sample to be detected extracted in the step 1) into a fluorescent quantitative PCR reaction system, wherein the fluorescent quantitative PCR reaction system contains the primer and the probe according to the claim 1 or 2;
3) Standard plasmid construction: performing conventional PCR amplification on the cassava mealy scale DNA by using a primer pair PM-F1/PM-R1; the obtained target fragment is connected with PUC57, and then the connection product is transformed to obtain a colony transformed with plasmid; selecting a monoclonal colony, and inoculating the monoclonal colony into a culture solution for culture; extracting positive recombinant plasmid by using a plasmid preparation kit for PCR identification and sending to Hangzhou qingke organism Co-Ltd for sequencing verification; after the plasmid identification is correct, measuring the plasmid concentration by using a spectrophotometer, and taking the plasmid concentration as a gene standard substance of the method after quantification;
4) Fluorescent quantitative PCR amplification: setting positive control and negative control, and reacting the prepared fluorescent quantitative PCR reaction system on a fluorescent quantitative PCR amplification instrument, wherein the reaction procedure is as follows: pre-denaturation at 95℃for 5min; then deforming for 10sec at 95 ℃, annealing for 30sec at 58 ℃, cycling for 40 times, storing a file after the reaction is finished, and opening analysis software;
5) And (3) judging results: and judging the detection result by observing the amplification condition of the fluorescent quantitative PCR.
Preferably, the fluorescent quantitative PCR reaction system is a 20. Mu.L reaction system containing 10. Mu.M upstream and downstream primers of 0.4. Mu.L each, ROX Reference Dye II of 0.5. Mu.L, DNA template of 2. Mu.L, 10. Mu.MTaqMan probe 0.8. Mu.L, add ddH 2 O to 20. Mu.L.
The invention utilizes the real-time fluorescence quantitative PCR technology and is based on the mitochondria of the cassava mealy bugsCOIGenes, design specific primersTaqMan probe, and the kit and method for fast and accurately detecting the cassava mealy bugs are established, and can be used in the form of kit in the fields of port, cassava production base, organic vegetable and organic fruit production base, fresh cut flower production base, cotton planting area, vegetable and ornamental plantAnd the fruit tree seedlings/plants are promoted in the transportation.
The invention can rapidly and accurately realize the cassava mealybugsP. manihotiWith the genus GeckoPhenacoccusGenus LecanitisDysmicoccusIdentification of insects, avoiding confusion that may result from morphological identification; compared with the conventional PCR, the real-time fluorescence quantitative PCR has the advantages that the time consumption is greatly shortened, the detection result can be obtained only by 1h, meanwhile, the specificity is higher, the sensitivity is higher, the minimum DNA concentration of detection can reach 10fg/uL, the result can be directly observed, and the pollution caused in the operation process can be effectively avoided. The invention relates to a cassava mealybugsP. manihotiPrimer, probe and kit for real-time fluorescent quantitative PCR detection, and can rapidly and accurately detect cassava mealy bugsP. manihotiAnd (5) detecting.
Drawings
FIG. 1 is a schematic diagram showing the positions of the primer PM-F1/PM-R1 and the Probe PM-Probe1 on the mtDNA of the Gecko in cassava;
FIG. 2 is based onTaqA standard curve of real-time fluorescence PCR detection of the man probe;
FIG. 3 is a species-specific assay for fluorescent primers and probes, in which, numeral 1: plasmid DNA;2:P. manihoti；3-8：P.solani,Phenacoccussp.,P. solenopsis,P.aceris,D.neobrevipesand a negative control;
FIG. 4 illustrates a cassava mealy scalePhenacoccusmanihotiThe sensitivity of the fluorescent primer and probe used in the fluorescent quantitative detection system is shown in the figure, wherein a-g respectively represent 1.0ng/uL,100pg/uL,10pg/uL,1.0pg/uL,100fg/uL and 10fg/uL.
Detailed Description
The invention is further illustrated, but not limited, by the following examples.
According to the current research results, we have chosen to ensure the specificity of the primers designed
Cassava mealybugsPhenacoccusmanihoti) Short-range lycoris mealy bugs
（P.solani) Radix seu radix Kadsurae HeteroclitaePhenacoccussp.. Hibiscus sabdariffa L.var.sinensisP. solenopsis) Acer ginnala Makino is a kind of Chinese medicineP.aceris) And the outer edge is planted with new pineapple gray mealy bugsDysmicoccusneobrevipes) Based on cassava mealybugsP. manihotiMitochondrial genomeCOIAfter the Primer PM-F1/PM-R1 is designed by using the software Primer5 for the gene sequence, the Probe PM-Probe1 is designed by using the software Primer Exprss according to the amplified specific fragment, so that the cassava meadow can be specifically amplifiedP. manihotiBoth primers and probes were synthesized by the Hangzhou department of Optimago.
Example 1: DNA extraction
The six kinds of adults are used for DNA extraction, the insect bodies are put into a 1.5ml centrifuge tube, added with liquid nitrogen and ground into powder, and DNeasy three Tissue Kit of Germany QIAGEN company is used for extracting DNA, and specific steps are carried out by referring to the Kit instruction book.
Example 2: target sequence selection
For cassava mealybugsPhenacoccusmanihotiIn mtDNA of (2)COIAnd analyzing the gene sequence, and selecting a sequence fragment with higher mutation rate as an amplified target fragment. Multiple groups of real-time fluorescent quantitative PCR primers and probes are designed, and a group of PCR primers for detecting the cassava mealybugs is finally determined through preliminary screening of experimentsP. manihotiFluorescent quantitative PCR primers and probes of (a).
Example 3: specific primer and probe design
For the group, designing an upstream primer PM-F1, a downstream primer PM-R1, a specific Probe PM-Probe1,
PM-F1: 5’-ACCTTTGATGATTTCTTCTTCTG-3’；
the length of the amplified product of PM-R1:5'-TGAAGTTTAAAGTAATGAAATTTTG-3', PM-F1/PM-R1 is 179bp;
PM-Probe1:5'-ATTTTAGATTTTGATTACTACTTCCTTCTT-3' the 5 'end of the probe is labeled with a FAM reporter fluorophore and the 3' end is labeled with a BHQ1 quencher.
The schematic diagrams of the positions of the primer PM-F1/PM-R1 and the Probe PM-Probe1 on the mtDNA of the cassava mealy bugs are shown in figure 1.
Example 4: standard plasmid construction
Performing conventional PCR amplification on the cassava mealy scale DNA by using a primer pair PM-F1/PM-R1; the obtained target fragment is connected with PUC57, and then the connection product is transformed to obtain a colony transformed with plasmid; selecting a monoclonal colony, and inoculating the monoclonal colony into a culture solution for culture; extracting positive recombinant plasmid by using a plasmid preparation kit for PCR identification and sending to Hangzhou qingke organism Co-Ltd for sequencing verification; after the plasmid identification is correct, the plasmid concentration is measured by a spectrophotometer, and the plasmid is quantified to be used as a gene standard product of the method.
Example 5: real-time fluorescent PCR detection
The real-time fluorescent quantitative PCR amplification system is 20 mu L:2×TaqMan probe Real-time PCR Master Mixture. Mu.L (Dalianbao biological Co Takara Premix Ex Tag), upstream and downstream primers (10. Mu.M) 0.4. Mu.L, ROX Reference Dye II 0.5. Mu.L, DNA template 2. Mu.L, respectively,Taqman probe (10. Mu.M) 0.8. Mu.L, add ddH 2 O to 20. Mu.L. Real-Time fluorescent PCR amplification was performed on a CFX Opus 96 Real-Time PCR System quantitative PCR amplification instrument. Opening CFX Opus 96 Real-Time PCR System Software, setting PCR reaction conditions, and performing a two-step amplification reaction procedure, wherein the pre-denaturation is carried out at 95 ℃ for 5min; then deformed at 95℃for 10sec, annealed at 58℃for 30sec, and cycled 40 times. Clicking for operation, performing PCR reaction, finishing the reaction about 1h, saving the file, and opening analysis software.
Example 6: standard Curve establishment
The recombinant plasmid is used as a standard template, the empty plasmid is used as a negative control, the recombinant plasmid is subjected to 10-time decreasing gradient dilution, and 2 mu L of each concentration is used as a template for real-time fluorescence quantitative PCR detection. The analysis results show that the higher the template concentration,C t the lower the value, the parallel test results show that the DNA concentration (x) is equal toC t (Y) has a correlation (x is in ng/ul) of the formula Y= -3.084x+12.148, see FIG. 2, with a high correlation coefficient, in particularR 2 =0.998 (more than 0.98), meets the requirement of real-time fluorescence quantitative PCR detection, and can be used for quantitative rapid detection analysis of cassava mealybugs.
Example 7: species-specific assays for fluorescent primers and probes
6 kinds of mealy scale DNA are used as templates and heavyThe group plasmid is positive control and ddH 2 O was used as a negative control, and was performedTaqAnd (5) carrying out man qRT-PCR specific detection. The detection result shows that only the cassava mealy bugs and the recombinant plasmid send out obvious fluorescent signals, obvious amplification curves are generated, as shown in figure 3, and other 5 mealy bugs and negative controls are not amplified, so that the primers and probes designed by the method have better specificity. The detection system can be used for detection, identification and analysis of the cassava mealy bugs.
Example 8: sensitive detection of fluorescent primers and probes
Purification of recombinant plasmid at each dilutionTaqThe results of the manqRT-PCR detection show that the DNA concentration is 1.0ng/uL,100pg/uL,10pg/uL,1.0pg/uL,100fg/uL and 10fg/uL, and the DNA concentration lower than 10fg/uL shows negative amplification without increase of fluorescent signal. It can be seen that the establishmentTaqA minimum of 10fg/uL was detected by manqRT-PCR, as shown in FIG. 4.
Claims (6)
1. The primer and the probe for detecting the cassava mealy bugs based on fluorescent quantitative PCR are characterized by comprising an upstream primer PM-F1 and a downstream primer PM-R1, wherein the nucleotide sequences of the primers are respectively shown as follows:
PM-F1: 5’-ACCTTTGATGATTTCTTCTTCTG-3’；
PM-R1：5’-TGAAGTTTAAAGTAATGAAATTTTG-3’；
the length of the amplification product of the PMF1/PMR1 is 179bp;
the probe is PM-Probe, and the nucleotide sequence is:
5'-ATTTTAGATTTTGATTACTACTTCCTTCTT-3' the 5 '-end-labeled fluorophore is FAM and the 3' -end-labeled quencher is BHQ1.
2. A kit for detecting cassava mealy bugs based on fluorescence quantitative PCR, which is characterized in that the kit comprises the detection primer and the probe as described in claim 1.
3. The kit for fluorescence quantitative PCR detection of casserole according to claim 2, further comprising part or all of the following components: (1) a template; (2) a positive control and a negative control; (3) PCR premix.
4. The kit for fluorescence quantitative PCR detection of Gecko according to claim 3, wherein the positive control is recombinant plasmid and the negative control is ddH 2 O, the PCR premix is Takara Premix Ex Tag produced by Dalianbao biological company.
5. The method for detecting the cassava mealy bugs based on fluorescent quantitative PCR is characterized by comprising the following steps of:
1) Extracting DNA of a sample to be detected;
2) Preparing a fluorescent quantitative PCR reaction system: adding the DNA of the sample to be detected extracted in the step 1) into a fluorescent quantitative PCR reaction system, wherein the fluorescent quantitative PCR reaction system contains the primer and the probe according to the claim 1 or 2;
3) Standard plasmid construction: performing conventional PCR amplification on the cassava mealy scale DNA by using a primer pair PM-F1/PM-R1; the obtained target fragment is connected with PUC57, and then the connection product is transformed to obtain a colony transformed with plasmid; selecting a monoclonal colony, and inoculating the monoclonal colony into a culture solution for culture; extracting positive recombinant plasmid by using a plasmid preparation kit for PCR identification and sending to Hangzhou qingke organism Co-Ltd for sequencing verification; after the plasmid identification is correct, measuring the plasmid concentration by using a spectrophotometer, and taking the plasmid concentration as a gene standard substance of the method after quantification;
4) Fluorescent quantitative PCR amplification: setting positive control and negative control, and reacting the prepared fluorescent quantitative PCR reaction system on a fluorescent quantitative PCR amplification instrument, wherein the reaction procedure is as follows: pre-denaturation at 95℃for 5min; then deforming for 10sec at 95 ℃, annealing for 30sec at 58 ℃, cycling for 40 times, storing a file after the reaction is finished, and opening analysis software;
5) And (3) judging results: and judging the detection result by observing the amplification condition of the fluorescent quantitative PCR.
6. A fluorescent-based assay as defined in claim 5A quantitative PCR method for detecting the cassava mealy bugs is characterized in that a fluorescent quantitative PCR reaction system is a 20 mu L reaction system which contains 0.4 mu L of 10 mu M upstream and downstream primers, 0.5 mu L of ROX Reference Dye II, 2 mu L of DNA template and 10 mu M TaqMan probe 0.8. Mu.L, add ddH 2 O to 20. Mu.L.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168142A (en) * | 2011-03-02 | 2011-08-31 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Primer, probe and detection method for real-time fluorescent polymerase chain reaction (PCR) of Planococcus minor |
| CN104293970A (en) * | 2014-10-30 | 2015-01-21 | 中国农业科学院植物保护研究所 | Phenacoccus manihoti matile-ferrero specificity SS-COI primer pair as well as method and kit for quick PCR detection |
| CN107653328A (en) * | 2017-10-13 | 2018-02-02 | 中国农业科学院植物保护研究所 | The beauteously specific SS COI detection primers and detection method and kit of continuous mealybug |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168142A (en) * | 2011-03-02 | 2011-08-31 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Primer, probe and detection method for real-time fluorescent polymerase chain reaction (PCR) of Planococcus minor |
| CN104293970A (en) * | 2014-10-30 | 2015-01-21 | 中国农业科学院植物保护研究所 | Phenacoccus manihoti matile-ferrero specificity SS-COI primer pair as well as method and kit for quick PCR detection |
| CN107653328A (en) * | 2017-10-13 | 2018-02-02 | 中国农业科学院植物保护研究所 | The beauteously specific SS COI detection primers and detection method and kit of continuous mealybug |
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