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Lily virus gene chip and preparation method thereof as well as method for detecting lily viruses using lily virus gene chip

A virus gene and gene chip technology, applied in the field of microbial detection, can solve the problems of long time, difficulty in detecting a large number of samples, cumbersome operation, etc., and achieve the effect of short operation time, stable reproducibility and strong sensitivity

Inactive Publication Date: 2015-03-25
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The practical application of these technologies has cumbersome purification processes and takes a long time; some cumbersome operations are prone to contamination and false positives; the advantages of electron microscope identification of plant viruses are fast, simple and direct, and can directly observe the virus morphology and structure And the ultrastructural changes of cells caused by the virus infecting the host, but the operation of the electron microscope requires certain skills, and the work is relatively concentrated, and the number of samples is not easy to be too large; and these detection methods have a large workload, long time, and poor sensitivity, making it difficult to detect a large number of samples

Method used

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  • Lily virus gene chip and preparation method thereof as well as method for detecting lily viruses using lily virus gene chip
  • Lily virus gene chip and preparation method thereof as well as method for detecting lily viruses using lily virus gene chip
  • Lily virus gene chip and preparation method thereof as well as method for detecting lily viruses using lily virus gene chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1 Experimental material

[0068] 1. The source of the virus

[0069] Positive samples: tested by ELISA and electron microscopy, the lily samples confirmed to be carrying CMV, LSV, and LMoV were provided by the Tianjin Animal and Plant Quarantine Institute and the Vegetable and Flower Research Center of Beijing Academy of Agriculture and Forestry Sciences.

[0070] Field specimens: Field lily plant specimens were collected from the lily planting base in Zhangsanying Town, Yanqing County, Beijing. They were divided into 4 specimens, numbered: A, B, C, and D; the lily bulbs were imported from the Netherlands, from Tianjin Entry-Exit Inspection and Quarantine Bureau.

[0071] Negative specimens: maize rough dwarf virus (MRDV), wheat yellow dwarf virus (BYDV), hibiscus chlorotic ringspot virus (HCRSV), geminivirus (WTG); luciferase gene plasmid.

[0072] 2. Reagents

[0073] Trizol RNA extraction kit (purchased from GIBCO, USA); TaqDNA polymerase (purchased fro...

Embodiment 2

[0076] Example 2 Preparation of gene chip

[0077] 1. Design probe

[0078] According to the sequence homology of CMV, LSV, and LMoV strains in the GeneBank database (CMV is subgroup I), alternative probes were designed using Mprobe software for the conserved sequence of the virus coat protein (CP) gene and the negative control rat brain gene , by GeneBank Blast sequence homology comparison, the specific oligonucleotide probe sequences of the CP genes of the three viruses were obtained. Online Blast analysis designed 3 pairs of primers and 6 specific oligonucleotide probes, as shown in Tables 1 and 2.

[0079] Table 1 Probe sequences

[0080]

[0081]

[0082] Table 2 Primer sequences

[0083]

[0084] 2. Synthetic primers and probes

[0085] Primers (CMV F1, CMV R1; LSV F2, LSV R2; LMoV F3, LMoV R3), probes (CMV P1, CMV P2; LSVP3 , LSV P4; LMoV P5, LMoV P6), the specific steps are as follows:

[0086] After the synthesis was completed, deprotection / cleavage treat...

Embodiment 3

[0099] Example 3 Detection of Lily virus

[0100] 1. Primer design

[0101] According to the homology of coat protein gene sequences of CMV, LSV, and LMoV strains in the GeneBank database, oligonucleotide primers were designed, and the primer sequences are shown in Table 3.

[0102] Table 3 Primer sequences

[0103]

[0104] 2. Extraction of total RNA from lily materials

[0105] For the extraction of total plant RNA, refer to the third edition of Molecular Cloning. details as follows:

[0106] 1) After taking out the collected lily corm sample impregnated with CMV from the ultra-low temperature refrigerator, immediately place it in a precooled mortar, add liquid nitrogen to quickly grind it into powder, and obtain the lily corm powder impregnated with CMV;

[0107] The lily bulb powder dipped in LSV and LMoV was prepared according to the method for preparing lily bulb powder dipped in CMV.

[0108] 2) Add 800 μl Trizol reagent to a 2ml centrifuge tube, take 1 g of the...

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Abstract

The invention discloses a lily virus gene chip and a preparation method thereof as well as a method for detecting lily viruses using the lily virus gene chip. The lily virus gene chip includes a solid phase carrier and an oligonucleotide probe fixed on the carrier, wherein the probe is selected from nucleotide segments corresponding to one or more virus genes of lily symptomless virus (LSV), cucumber mosaic virus (CMV) or lily mottle virus (LMoV). The viruses detected by the lily virus gene chip provided by the invention include three types of viruses which have severe impact and damage on cut lily flowers. The method for detecting lily viruses using the lily virus gene chip, provided by the invention has the advantages that specific primers are used for PCR amplification; amplification products are fluorescence-labeled, with high accuracy; the gene chip is high in sensitivity; a probe with specificity is selected to hybridize with the amplification products, with high specificity; the defect that a solution is false positive when the PCR products are detected through gel electrophoresis can be avoided; the detection is fast; the method is good in stability and low in cost, and is applicable to an accurate detection on a large number of lily samples in short time.

Description

technical field [0001] The invention relates to a plant virus gene chip and a detection method thereof, in particular to a gene chip capable of simultaneously detecting multiple lily viruses and a detection method thereof, belonging to the field of microbial detection. Background technique [0002] Lily (Lilium) is an important economic crop that combines ornamental, edible and medicinal uses, and has become one of the most important cut flowers and ornamental flowers in the world. In recent years, with the growing area of ​​lily planting, long-term asexual reproduction has caused the accumulation of viruses; viral diseases have become more and more harmful to lily, affecting the yield and quality of lily, and causing huge economic losses to the lily planting industry. In the late 19th century, viral diseases nearly drove lily cultivars and varieties to extinction. Due to the different sources of bulbs of lily cultivated in various regions, and most of them lack strict plan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C12Q1/70C12Q1/68
Inventor 王进忠贾慧董金臯郝少东王升启张志勇尚巧霞杨宝东
Owner BEIJING UNIV OF AGRI
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