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Primers, probe, kit and method for detecting lycoris radiata mealybug based on fluorescent quantitative PCR

A technology for quantification of Lycoccus lycoccus and fluorescence, which is applied in the field of molecular biology, can solve the problems of gel product pollution, lack of specificity, cross-contamination, etc., and achieve the effects of avoiding confusion, strong specificity, and avoiding pollution

Pending Publication Date: 2021-08-06
ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the inspection scheme for P. amaryllis is to use specific SS-COI primers to perform ordinary PCR detection on P. amaryllis moths, the publication number is CN104745576A. This species is prone to cross-contamination by using ordinary PCR detection methods and has specificity. What is lacking; electrophoresis detection is required, which takes a long time, and the gel product pollutes the environment; while real-time fluorescent quantitative PCR technology does not require electrophoresis detection, saves time and effort, and has strong specificity, high sensitivity, and good repeatability. There is no real-time fluorescent quantitative PCR detection method for Lycopa lycoccus

Method used

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  • Primers, probe, kit and method for detecting lycoris radiata mealybug based on fluorescent quantitative PCR
  • Primers, probe, kit and method for detecting lycoris radiata mealybug based on fluorescent quantitative PCR
  • Primers, probe, kit and method for detecting lycoris radiata mealybug based on fluorescent quantitative PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: DNA Extraction

[0031] The above six kinds of adults were used for DNA extraction. The cotton mealybug was put into a mortar, and liquid nitrogen was added to grind it into a powder. DNA extraction was performed using the DNeasy® Tissue Kit from QIAGEN, Germany. For specific steps, refer to the kit instructions.

Embodiment 2

[0032] Embodiment 2: target sequence selection

[0033] mtDNA of P. amaryllis COI The sequence of the gene is analyzed, and the sequence fragment with a higher mutation rate is selected as the target fragment for amplification. Several sets of real-time fluorescent quantitative PCR primers and probes were designed. After preliminary screening, a set of fluorescent quantitative PCR primers and probes for detecting P. amaryllis was finally determined.

Embodiment 3

[0034] Example 3: Specific Primer and Probe Design

[0035] like figure 1 As shown, for the group, design the upstream primer PsF1, the downstream primer PsR1, and the specific probe as PsProbe,

[0036] PsF1:5'-TAGATTCTGACTTCTTTTTACCTTCT-3',

[0037] PsR1: 5'-TTAAGGTAATAAAATTTTGATT-3', the length of the amplified product of PsF1 / PsR1 is 125bp;

[0038] PsProbe: 5'-ATACAGGTTGAACTTTATATCCTCCTT-3', the 5' end of the probe is labeled with a FAM reporter fluorescent group, and the 3' end is labeled with a BHQ1 quencher group.

[0039]The probe PsProbe is an oligonucleotide probe with a fluorophore attached to the 5' end of the probe and a quencher at the 3' end. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the primers and P. amaryllis mtDNA complete the thermal cycle of high temperature denaturation, low temperature annealing and extension, and follow the polymerase chain reaction T...

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Abstract

The invention discloses primers and a probe for detecting lycoris radiata mealybug based on fluorescent quantitative PCR, and a kit and a detection method adopting the primers and the probe. A detection primer group comprises an upstream primer PsF1, a downstream primer PsR1 and a probe PsProbe; and the detection kit comprises the primers, the probe, a template, a positive sample, a negative sample and a premixed solution. The detection method comprises the following steps of: extracting DNA of a sample to be detected, quickly detecting the DNA of the sample to be detected by using a fluorescent quantitative PCR technology, and judging whether the sample to be detected is the lycoris radiata mealybug according to the amplification condition of the fluorescent quantitative PCR. The primers, the probe, the kit and the method have the advantages of quickness, sensitivity, specificity, easiness in operation and the like, and are convenient to popularize and apply.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer, a probe, a kit and a method for detecting Pycoccus amaryllis based on fluorescent quantitative PCR. Background technique [0002] Amaryllis spp. Phenacoccussolani Ferris, belonging to the order Hemiptera (Hemiptera) Coccoidea (Coccoidea) mealybug family (Pseudococcidae) cotton mealybug subfamily (Phenacocccinae) cotton mealybug genus Phenacoccus , is a new pest that has not been reported in mainland my country and was discovered in July-September 2008. It is also an important quarantine pest in the entry-exit inspection and quarantine of ports in my country. The host range of P. amaryllis is very wide. Existing studies have shown that its host plants have more than 30 families, covering vegetables, medicinal materials, ornamental plants and tobacco. P. amaryllis is an important and potentially dangerous pest of lettuce, eggplant, potato and oth...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11
CPCC12Q1/6888C12Q1/6851C12Q2531/113C12Q2545/113C12Q2561/101
Inventor 吴志毅陈鹏程黄芳程帆方文渊田红伟唐慧骥党志浩吴颖刘鹏娟任琰蔡军
Owner ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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