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Liquid phase chip detection primer of potato spindle tuber viroid, and detection method thereof

A liquid-phase chip detection and potato technology, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., to achieve high throughput, improve detection efficiency, and strong specificity

Active Publication Date: 2014-03-26
哈尔滨海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology has been used in the detection of Plum pox virus (PPV); so far, there has been no report on the application of this technology in the detection of potato spindle tuber viroids at home and abroad.

Method used

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  • Liquid phase chip detection primer of potato spindle tuber viroid, and detection method thereof
  • Liquid phase chip detection primer of potato spindle tuber viroid, and detection method thereof
  • Liquid phase chip detection primer of potato spindle tuber viroid, and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1, Preparation of Potato Spindle Tuber Viroid Liquid Chip Detection Primers, Probes and Kit

[0046] According to the nucleotide sequence of the potato spindle tuber viroid reported by NCBI, primers and probes were designed using Primer Premier5.0 software, and finally a pair of primers and probes with high specificity were screened and artificially synthesized. The sequence is shown in the table 1.

[0047] Table 1 Designed primers and expected target fragment length

[0048]

[0049]

[0050] The 5' end of the primer shown in Sequence 2 above was labeled with Biotin.

[0051] After each of the above primers and probes are packaged independently, they can be used as components of a kit for detecting the potato spindle tuber viroid liquid chip.

Embodiment 2

[0052] Example 2, the application of potato spindle tuber virus-like liquid chip detection primers, probes and kits

[0053] 1. Establishment of a liquid-phase chip detection method for potato spindle tuber viroids

[0054] 1. Detection of cDNA acquisition of samples

[0055] 1) Extraction of detection sample RNA

[0056] The sample RNA was extracted with Trizol Reagent (Catalog No.: 15596-026) from Invitrogen, and the operation was performed according to the instructions.

[0057] 2) cDNA synthesis: Add 1 μL total RNA, 2 μL (10 μmol / L) downstream primer PSTVd-R (no biotin labeling required), 1 μL dNTP Mix (10 mmol / L) and 10 μL DEPC-H to a 0.2 mL reaction tube 2 O; after mixing, heat at 70°C for 10 min, quickly transfer to ice for 5 min, and centrifuge briefly; then add 5 μL of 5× reverse transcription buffer and 0.5 μL RNase inhibitor (40 U / μL) to the reaction tube, 0.5 μL reverse transcriptase (200 U / μL); gently mix, centrifuge briefly; react at 42°C for 1 hour, quench on...

Embodiment 3

[0095] Example 3, Potato spindle tuber virus-like liquid chip detection primers and detection method to detect the actual sample to be tested

[0096] In order to better verify the practical application effect of the primer probe of the present invention, the present invention uses potato (Solanum tuberosum) leaves (sample 1) infected with potato spindle tuber viroid and detects with RT-PCR method to determine that it is not infected by potato spindle Three samples (sample 2, sample 3, sample 4) infected by tuber viroid were tested at the same time.

[0097] The method is the same as Step 1 in Example 2.

[0098] The result shows (table 4), the test result of the sample that is infected is positive, and the sample that has not been infected with potato spindle tuber viroid is negative, thus proves that the method of the present invention can effectively detect whether there is Potato spindle tuber viroid.

[0099] Table 4 liquid chip method detects actual sample results

[...

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Abstract

The invention discloses a liquid phase chip detection primer of potato spindle tuber viroid, and a detection method thereof. The invention also provides a kit used for the liquid phase chip detection or the auxiliary detection of the potato spindle tuber viroid. The kit comprises a primer group and a probe, and the primer is composed of a primer 1 and a primer 2. Experiments prove that a sample can be rapidly detected through the detection method, and only about 4h is spent on a process from sample treatment to result obtaining; and there is no cross reaction of the primers designed in the invention and common plant viruses on potatoes, so the false negative result caused by the non-specific amplification is reduced.

Description

technical field [0001] The invention belongs to the technical field of plant virus detection, and in particular relates to a liquid phase chip detection primer and a detection method for potato spindle tuber viroid. Background technique [0002] Potato spindle tuber viroid (Potato spindle tuber viroid, PSTVd) belongs to Potato spindle tuber viroid family (Pospiviroidae) Potato spindle tuber viroid (Pospiviroid). At present, my country has included this type of virus in the list of plant quarantine pests prohibited from entering the country (see "List of Imported Plant Quarantine Pests of the People's Republic of China"). [0003] Potato spindle tuber virus causes 20%-70% yield loss in potato production. If it is mixed with other potato viruses such as potato leafroll virus and potato virus Y, it will cause more serious yield loss. The virus can also cause spindle deformity and cracking of potato tubers, leading to a severe loss of quality. At present, the only way to preve...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2563/149C12Q2563/131
Inventor 刘洪义张永江张洪祥刘忠梅辛言言朱水芳
Owner 哈尔滨海关技术中心
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