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Method for real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) quantitative detection of virus RNA (ribose nucleic acid) copy number in SRBSV (southern rice black-streaked dwarf virus) plants

A technology for black-streaked dwarf virus and rice black-streaked dwarf, which is applied in the field of agricultural science and can solve the problems of complex operation, high sensitivity, and inability to accurately quantify the number of virus RNA copies.

Inactive Publication Date: 2012-11-21
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the high genome sequence homology between SRBSDV and rice black-streaked dwarf virus (RBSDV), they are very similar in field symptoms, virion morphology, size, and serology, so they are given Diagnosis and identification have caused difficulties, and traditional methods have defects such as time-consuming, complicated operation, and high cost. Molecular methods such as PCR are highly sensitive, but they can only be semi-quantitative or draw a "yes or no" conclusion, and cannot be accurately quantified The copy number of viral RNA has limited the basic research of pathogenesis and SRBSDV

Method used

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  • Method for real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) quantitative detection of virus RNA (ribose nucleic acid) copy number in SRBSV (southern rice black-streaked dwarf virus) plants
  • Method for real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) quantitative detection of virus RNA (ribose nucleic acid) copy number in SRBSV (southern rice black-streaked dwarf virus) plants
  • Method for real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) quantitative detection of virus RNA (ribose nucleic acid) copy number in SRBSV (southern rice black-streaked dwarf virus) plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1, in vitro transcription and purification preparation of RNA standards

[0028] Select the appropriate restriction endonuclease Sal I, add the reagents sequentially according to Table 1, and carry out single enzyme digestion on the positive recombinant plasmid DNA at 37°C for 8 hours.

[0029] Table 1 Restriction enzyme analysis system

[0030] Table1 The system of restriction enzyme analysis

[0031]

[0032] The single-digested product was subjected to 2% agarose gel electrophoresis, and was recovered according to the recovery and purification instructions of AxyPrep DNA Gel Extraction Kit (Axygen) to obtain linear plasmid DNA.

[0033] Using linear plasmid DNA as a template, SRBSDV-S9-F and SRBSDV-S9-R as primers, carry out PCR identification of linear plasmid DNA, conventional PCR system (25μL), amplification conditions: first, pre-denaturation at 95°C for 5min, then Carry out 35 cycles under the following conditions: denaturation at 95°C for 45s, anne...

Embodiment 2

[0050] Embodiment 2, optimization of southern rice black-streaked dwarf virus SYBR Green I-based one-step real time RT-PCR reaction conditions

[0051] Optimization of reaction system and reaction conditions for Southern rice black-streaked dwarf virus SYBR Green I-based one-step real time RT-PCR

[0052] In order to obtain the best reaction system and parameters, follow the kit iScript TM One-Step RT-PCR Kit With SYBR Green (BIO-RAD) requirements, the specific steps are as follows:

[0053] (1) Take a special tube for fluorescent quantitative PCR, add the reagents in Table 3, and mix gently;

[0054] (2) Seal the light-transmitting film and press the film tightly with a scraper;

[0055] Table 3 Reaction system of real-time fluorescent quantitative RT-PCR

[0056] Table3 The system of real time RT-PCR

[0057]

[0058] (3) at Bio-Rad IQ TM 5 The amplification reaction was performed on the Multicolor Real-Time PCR Detection System. First, cDNA was synthesized at 50°C...

Embodiment 3

[0065] Embodiment 3, the establishment of southern rice black-streaked dwarf virus SYBR Green I-based one-step real time RT-PCR standard curve

[0066] According to the copy number calculated in Example 2, standard substance RNA was carried out with Nuclease-free water to carry out 10-fold gradient dilution (2.5 × 10 10 -2.5×10 4 copies / μL), using this group of standard products as a template to optimize the best reaction system and conditions obtained in Example 2 to react, to obtain the respective C T Value, using random software analysis to obtain the amplification curve and standard curve, the logarithmic value of the initial template copy number in the standard curve is the abscissa (x-axis), the corresponding C T The value is the ordinate (y-axis). The results showed that the RNA standard of each dilution gradient had a basic absorbance value measured after the end of the first amplification cycle, and there was no significant change until the end of the fourth cycle. ...

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Abstract

The invention provides a method for SRBSV (southern rice black-streaked dwarf virus) detection on rice plants and accurate quantification of RNA (ribose nucleic acid) copy number. The method can be used for eliminating the interference of the SRBSV, the copy number of the virus RNA in the SRBSV plants is fast obtained, and further, the basis is laid for studying the SRBSV pathogenic mechanism, the molecular biology study and the like.

Description

Technical field: [0001] The invention relates to the detection of southern rice black-streaked dwarf virus on rice plants and the accurate quantification of virus RNA copy number, and belongs to the field of agricultural science and technology. Background technique: [0002] Southern rice black-streaked dwarf virus (SRBSDV) is a proposed new species of Group 2 of the genus Fijivirus in the family Reoviridae. It is mainly transmitted by the white-backed planthopper (Sogatella furcifera) in a persistent and non-oval way, which can infect rice, corn, etc. and cause southern rice black-streaked dwarf and corn rough dwarf. The virion is spherical, and the genome consists of 10 double-stranded RNAs. All rice growth stages can be infected by SRBSDV. The symptoms in the field are mainly dwarfed plants, dark green leaves, milky white and brown tumor-like protrusions on the back of leaves and stems in the early stage, and several nodes in the stem nodes of diseased plants at the join...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 周彤杜琳琳周益军
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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