Chip for screening avsunviroid viroid and application of chip

A virus-like, avocado-like technology, applied in the field of chips for screening avocado sunspot viroids, can solve the problems of low throughput and inability to detect virus-like viruses, and achieve good results

Inactive Publication Date: 2012-10-10
CHINESE ACAD OF INSPECTION & QUARANTINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing technologies such as biological assays, forward and reverse polyacrylamide gel electrophoresis, and RT-PCR have low throughput, and can only detect one type of virus in a sample at a time, and they are specific detections, which cannot detect large variations and Detection of viroids with many mutation sites

Method used

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  • Chip for screening avsunviroid viroid and application of chip
  • Chip for screening avsunviroid viroid and application of chip
  • Chip for screening avsunviroid viroid and application of chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the preparation of the chip of screening avocado sunspot viroid genus viroid

[0040] 1. Design of genus-level highly compatible oligonucleotide probes

[0041] Download the whole genome and nucleic acid sequence of Viroids from the database of National Center for Biotechnology Information (NCBI) and the International Committee on Virology (ICTV); remove more than 90% of the nucleic acid sequences with 95% similarity to other sequences; use 5 Bases are used as intervals to continuously extract all 40mer nucleic acid sequences; 40%≤GC content≤60%, single base content≤50%, number of consecutive repeated bases≤4, and no hairpin structure of more than 6 bases is Nucleic acid sequences were screened for standards, and homology comparisons were performed in the NCBI database to ensure the specificity of the probes obtained.

[0042] According to the above principles, five probes (probe 1-probe 5) of viroids of the avocado sunspot viroid genus were designed, the...

Embodiment 2

[0047] Embodiment 2, the application of the chip of screening avocado sunspot viroid genus viroid

[0048] 1. Chip detection samples for screening avocado sunspot viroids

[0049] 1. Extraction of total RNA from samples used for detection

[0050]1) Take 0.1 g of avocado leaves infected with Avocado sunblotch viroid (Latin name of Avocado sunblotch viroid, purchased from American type culture collection, referred to as ATCC, PV-663), and grind it into powder with liquid nitrogen Finally, the total RNA of the sample was extracted according to the instructions of the Nanometer Magnetic Beads Plant Virus RNA Extraction Kit (Wuhan Wawatiana Technology Development Co., Ltd., catalog number: EX1011), and stored at -20°C for future use.

[0051] 2. Sample labeling and hybridization

[0052] The total RNA obtained above was reverse transcribed to obtain cDNA.

[0053] PCR amplification, that is, add 2 μL of cDNA product, 0.5 μL of upstream primer (final concentration of 0.5 mmol / L)...

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Abstract

The invention discloses a chip for screening the avsunviroid viroid and application of the chip. A probe group identifying or assisting in identifying the avsunviroid viroid consists of 5 probes, and the nucleotide sequences of the 5 probes are the sequences from 1 to 5 in a sequence table respectively. The invention also provides a gene chip for screening the avsunviroid viroid, which is obtained by fixing the probe group on the surface of the chip base. The experiment proves that the nucleotide sequence of the avsunviroid viroid is analyzed by a bioinformatics method, the compatible probes of the group are designed, and the standard viroid sample verification result indicates that a probe effect is good. The chip for screening the avsunviroid viroid disclosed by the invention can be applied to the quarantine identification of the avsunviroid viroid.

Description

technical field [0001] The invention relates to the technical fields of bioinformatics and bioquarantine identification, in particular to a chip for screening avocado sunspot viroids and its application. Background technique [0002] Avocado sunblotch viroid (Avsunviroid) belongs to the avocado sunblotch viroid family (Avsunviridae), currently only includes avocado sunblotch viroid (ASBVd) one species. This type of virus infects avocado (Persea Americana), causing yellow, white or pink spots on the fruit, and sometimes sunken crater-shaped lesions can be formed on the fruit. The lesions can be yellow, green and dark red, and the affected The fruit cannot be listed, causing economic losses. This type of virus is a quarantine pest in the "List of Imported Plant Quarantine Pests of the People's Republic of China" released in 2007, and its host plants and their products need to be quarantined according to law. [0003] With the increase of trade in plants and their products at...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C40B40/06
Inventor 张永江朱水芳辛言言李桂芬赵文军李明福魏梅生
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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