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Method for detecting potato virus Y by using digital PCR and special complete set of reagents thereof

A potato and virus technology, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve the problems of long detection period, low sensitivity, cumbersome operation steps, etc.

Inactive Publication Date: 2017-06-20
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Potato virus Y is the most harmful to potato, which can lead to potato degeneration, reduce potato yield, serious yield reduction can reach more than 80%, or even extinction
[0003] At present, the methods for detecting Potato virus Y mainly include enzyme-linked immunosorbent assay (ELISA), electron microscope technology and RT-PCR technology. false positive result

Method used

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  • Method for detecting potato virus Y by using digital PCR and special complete set of reagents thereof
  • Method for detecting potato virus Y by using digital PCR and special complete set of reagents thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1, the preparation of the complete set of reagents that detect potato virus Y

[0083] The set of reagents for detecting Potato virus Y consists of primer pair X1 and probe PVY-p. Primer pair X1 consists of primer PVY-f: 5'-CCAAACCCGAACAAAGGAAAG-3' (sequence 1 in the sequence listing) and primer PVY-r: 5'-GGACGTGATAGCCTTGATTCTC-3' (sequence 2 in the sequence listing). The probe PVY-p is: 5'-FAM-AATGCAGGCACATCTGGGACACATACTGT-TAMRA-3' (sequence 3 in the sequence listing). The 5' end of the probe PVY-p has a FAM fluorescent label, and the 3' end has a TAMRA fluorescent label.

[0084] In the complete set of reagents for detecting potato virus Y, primer PVY-f, primer PVY-r and probe PVY-p are separately packaged. In the complete set of reagents, the molar ratio of primer PVY-f, primer PVY-r and probe PVY-p is 2:2:1.

[0085] Primer PVY-f, primer PVY-r and probe PVY-p were synthesized.

Embodiment 2

[0086] Embodiment 2, the establishment of the method utilizing digital PCR to detect potato virus Y

[0087] 1. Extraction of total RNA from the sample to be tested

[0088] The total RNA of the sample to be tested was extracted with a plant total RNA extraction kit to obtain the total RNA of the sample to be tested.

[0089] 2. Obtaining the cDNA of the sample to be tested

[0090] Take the total RNA of the sample to be tested, and perform reverse transcription using the primer PVY-r synthesized in Example 1 according to the steps in the instructions of the reverse transcription kit to obtain the cDNA of the sample to be tested.

[0091] 3. Digital PCR amplification reaction

[0092] (1) Preparation of reaction system

[0093]Reaction system A is 20 μL, consisting of 10 μL 2×SuperMix, 1 μL primer PVY-f, 1 μL primer PVY-r, 0.5 μL probe PVY-p, 2 μL cDNA of the sample to be tested and 5.5 μL nuclease-free water; in the initial system , the concentration of primer PVY-f was 0...

Embodiment 3

[0104] Embodiment 3, specific detection

[0105] According to the method of Example 2, specific detection was carried out. The samples to be tested were 0.1g potato leaves infected with PVM, 0.1g potato leaves infected with PVX, 0.1g potato leaves infected with PVY, 0.1g tobacco leaves infected with TMV or 0.1g cucumber leaves infected with CMV. In the cDNA of 2 μL samples to be tested, the DNA content was 580 ng / μL.

[0106] See the experimental results figure 1 (both A05 and B05 are PVY, C05 is PVX, D05 is PVM, E05 is TMV, F05 is CMV, G05 is a negative control, and H05 is a blank control). The results showed that only the potato leaves infected with PVY produced positive droplets, and the other samples to be tested, the negative control and the blank control did not produce positive droplets (the fluorescence threshold limit for judging as positive droplets at this time was 0, and those below 0 The droplet instrument is judged as negative, and the droplet instrument is ju...

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Abstract

The invention discloses a method for detecting potato virus Y by using digital PCR and a special complete set of reagents thereof. The complete set of reagents is prepared from a primer pair X1 and a probe PVY-p, wherein the primer X1 consists of a primer PVY-f and a primer PVY-r; the primer PVY-f is a single-chain DNA molecule expressed by a sequence 1 in a sequence table; the primer PVY-r is a single-chain DNA molecule expressed by a sequence 2 in the sequence table; the sequence of the probe PVY-p is a sequence 3 in the sequence table; an FAM fluorescent mark is arranged at a 5'end of the probe PVY-p; a TAMRA fluorescent mark is arranged at a 3'end of the probe PVY-p. Experiments show that the method for detecting the potato virus Y by using the digital PCR is high in specificity and high in sensitivity, is applicable to detection of latent diseases and the PVY in samples with low virus content, and has an important application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting potato virus Y by digital PCR and a set of special reagents thereof. Background technique [0002] Potatoes are the most grown and eaten crop in the world and the fourth largest food crop in the global economy after rice, wheat and corn. Potatoes are one of the high-yield crops in the world, and China's potato planting area ranks first in the world. In recent years, my country's potato industry has developed rapidly, and potatoes account for an important proportion of my country's national economic growth. However, the overall production level of potatoes in my country at this stage is lower than the world's average level. The main reason is the impact of potato viruses. Potato virus Y (PVY) is a potato virus, a representative species of the family Potyviridae and the genus Potyvirus, and is distributed in all potato-growing regions. Potato virus Y is the mos...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 张永江黄洁芳朱鹏宇付伟王晨光
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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