Viroid testing method for fusoid tuber of potato

A detection method and potato technology are applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effects of strong specificity, high sensitivity and short time consumption

Inactive Publication Date: 2003-01-29
NANKAI UNIV +1
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  • Summary
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AI Technical Summary

Problems solved by technology

The current detection methods can no longer meet the needs of future production, and there is an urgent need for rapid and sensitive methods to replace existing technologies

Method used

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Examples

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Comparison scheme
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Embodiment

[0016] Take 1 g of fresh infected potato leaves (Jinyinshu No. 9), add 5 ml of RNA extraction buffer, add a little quartz sand, and grind it into powder in liquid nitrogen. Extract with 2 volumes of water-saturated phenol and 2 volumes of chloroform:isoamyl alcohol (49:1), and ice-bath for 15 min. Centrifuge at 10,000rpm, 4°C for 20min, and absorb the supernatant. Precipitate at -20°C for 30 min with 3 volumes of cold absolute ethanol and 0.25 volumes of NaAC (4M). 12,000rpm, 4°C, centrifuge for 20min. Repeat the above steps 1 time. Rinse twice with 70% ethanol. Air-dry ethanol with appropriate amount of ddH 2 O dissolved.

[0017] Design of PCR primers: According to the complete sequence of PSTVd ​​on GenBank, primers P1 and P2 were designed with computer assistance: P1: 5'-GAAACCTGGAGCGAACTG-3', P2: 5'-CGGTTCCAAGGGCTAAAC-3'.

[0018] PCR amplification: (reaction system 20 μl) take 1 μl of crude potato nucleic acid solution, primer P1 10 Pmol, P2 10 Pmol, 10×Buffer 2 μl...

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Abstract

The present invention is that in PCR amplification system, Taq enzyme is used to reverse-transcribe viroid RNA into cDNA and cDNA amplification is completed with the amplification product being tested through electrophoresis of agarose to judge whether there is viroid or not. The present invention has the advantages of fast test speed of testing decades of samples simultaneously, high sensitivity to nanograme-level viroid RNA, strong specificity of amplification viroid cDNA only and low cost owing to Taq enzyme being greatly cheaper than reverse transcriptase.

Description

Technical field [0001] The invention belongs to the detection method of potato spindle tuber viroid. Background technique [0002] Potato spindle tuber viroid (PSTVd) has no protein coat <Qu Feng, Tian Bo: Acta Virology, 9: 288, 1993>, its detection cannot be detected by the enzyme-linked immunosorbent assay (ELISA) method for detecting common viruses <Zheng Guangyu, Wichai et al.: Acta Virology, 4:150, 1988>. At present, the reciprocating electrophoresis (Return-PAGE) method is generally used to detect <Chen Wei, Tian Bo: Microbiology Bulletin, 1(3):132, 1985>, and its sensitivity and simplicity are slightly insufficient. Polymerase chain reaction (PCR) technology has the advantages of low cost, rapidity, sensitivity, and strong specificity in virus detection <Chen Zhinan et al.: Acta Phytopathology, 27(3): 198, 1997>. PSTVd ​​is an RNA virus. RNA cannot be directly amplified by PCR. First, it must be reverse-transcribed into cDNA and amplified usi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/25C12Q1/68
Inventor 杜荣骞叶明李德森居玉玲李戌彤古瑜罗智敏魏众济
Owner NANKAI UNIV
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