CN102787176A - 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 - Google Patents

Abstract

The invention provides a method for detecting southern rice black-streaked dwarf virus on rice plants and an accurate quantitative method for RNA copy number. Using this method, the interference of rice black-streaked dwarf disease can be eliminated, and the copy number of viral RNA in the southern rice black-streaked dwarf virus plant sample can be quickly obtained, which will lay a foundation for the pathogenic mechanism and molecular biology research of SRBSDV.

Description

Quantitative detection of virus RNA copy number in southern rice black-streaked dwarf plants by real-time fluorescent quantitative RT-PCR

Technical field:

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. the

Background technique:

Southern rice black-streaked dwarf virus (SRBSDV) is a proposed new species in 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 jointing stage. Anatropic aerial fibrous roots and high-node tillers, hosts include corn and various weeds in addition to rice. It has a great impact on the yield of rice, and severely diseased fields can even lead to no yield. In recent years, the disease has spread and occurred in the lower reaches of the Yangtze River, and it is becoming more and more serious. In 2009, the southern rice black-streaked dwarf virus occurred in an area of 3 million mu nationwide, and it rose rapidly to 19 million mu in 2010. In Hunan Province alone, nearly 10 million mu, 80,000 mu of harvested area, brought huge losses to rice production. There is an urgent need to establish reliable and accurate detection methods. the

At present, the commonly used detection methods for plant viruses include: biological inoculation experiment, serological detection, electron microscope observation and molecular method detection. However, due to the high genome sequence homology between SRBSDV and rice black-streaked dwarf virus (RBSDV), they are very similar in many aspects such as field symptoms, virion morphology, size and serology. 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 restricts the pathogenesis and basic research of SRBSDV. the

Real time RT-PCR is a simple and effective method for detecting gene copy number, which realizes the leap from qualitative to quantitative PCR, can accurately determine the copy number of viral RNA, and has the advantages of less time-consuming, high sensitivity and easy operation , safety and economy advantages. SYBR Green I is a double-stranded DNA dye. The fluorescence intensity is significantly enhanced after binding to double-stranded DNA. Adding this dye to the amplification system can directly detect the amplification product in real time. The dye has no specificity and cannot recognize specific double strands. It does not need special customization for different templates, and it does not need to design probes with good specificity. It has good versatility. Specificity and nonspecificity can be correctly distinguished by measuring the melting curve of the amplified product. After making a standard curve with a standard product of known initial copy number, as long as the cycle threshold (threshold cycle, _CT value) of the unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve. It has the incomparable advantages of conventional PCR technology: strong specificity, template identification is completed by specific hybridization of specific primers or probes with target genes, high accuracy, non-specific products such as dimers are not easy to appear; sensitivity is higher High, using an indicator that can generate a fluorescent signal to display the amount of the amplified product. The fluorescent signal is obtained by embedding a fluorescent dye into double-stranded DNA, or a double-labeled sequence-specific fluorescent probe or energy signal transfer probe, which greatly improves Improve the sensitivity of the detection; the accuracy is good, the _CT value of the exponential phase can be used to accurately quantify the amount of the initial template, and the single-copy gene can also be detected; it is fast and simple, and the amplification and detection are completed in the same tube without opening the cap. Electrophoresis detection greatly reduces the operation, shortens the time, avoids cross-contamination and environmental pollution, and effectively solves the problem of PCR pollution; the degree of automation is high, and the spectrum technology is combined with computer technology. Amplification and detection are completed simultaneously. Through The computer performs real-time detection and the operating system is closed. Real-time fluorescent quantitative PCR technology is being more and more widely used in various fields of scientific research.

Invention content:

The invention provides a method for detecting and accurately quantifying southern rice black-streaked dwarf virus in diseased rice plants, by which the interference of rice black-streaked dwarf virus can be eliminated, and whether rice plants carry southern rice black-streaked dwarf virus can be quickly diagnosed The virus is dwarfed and the copy number of the viral RNA is obtained. the

A kind of rapid detection and quantitative method for southern rice black-streaked dwarf virus in rice plants provided by the present invention is obtained by the following method:

1) According to the S9 nucleotide sequence of Southern Rice Black-streaked Dwarf Virus reported by NCBI, select a relatively conserved region (especially compared with RBSDV) after analysis by software DNAstar, and use the corresponding region on EU523359.1 to design primers through Primer5 , select the best two primers SRBSDV-S9-F and SRBSDV-S9-R;

Reagent(Invitrogen)提取水稻病株总RNA；  2)

Reagent (Invitrogen) extracts the total RNA of rice diseased plants;

3) Use the extracted total RNA of diseased rice plants as a template, SRBSDV-S9-F and SRBSDV-S9-R as primers to obtain the target gene by RT-PCR, recover the SRBSDV S9 target fragment (141bp) by tapping rubber, and connect it to pGEM-T easy to construct a cloning vector, and then transform the ligation product into Escherichia coli (Escherichia coli) competent cell DH5α, screen and identify positive clones, extract and purify the recombinant plasmid, and identify the recombinant plasmid again by RT-PCR and BLAST analysis, so far the recombinant plasmid containing the target Fragments of recombinant plasmids. the

4) Carry out single enzyme digestion on the positive recombinant plasmid DNA with restriction endonuclease Sal I, recover and purify the obtained linear plasmid DNA, use the linear plasmid DNA containing the target fragment as a template, and carry out in vitro according to the instructions of T7Transcription Kit (Fermentas). Transcribe to obtain linear RNA, purify the in vitro transcribed RNA according to the instructions of the in vitro transcription purification kit EZ-10Spin Column5 Minutes RNA Cleanup&Concentration Kit, and obtain RNA standards;

5) Refer to the requirements of the kit iScript ^™ One-Step RT-PCR Kit With SYBR Green (BIO-RAD), use the obtained RNA standard as a template, and set the concentration of primers SRBSDV-S9-F and SRBSDV-S9-R at 5 ×5 matrix format (final concentration of upstream and downstream primers are 100nM, 200nM, 300nM, 400nM and 500nM respectively) for optimization to determine the best reaction system; optimize annealing-extension in the range of 55-65°C to determine the best reaction Conditions, the result data were analyzed by software IQ5 Optical System Software Version 2.0;

6) Calculate the standard RNA copy number, perform 10-fold serial dilution (2.5×10 ¹⁰ -2.5×10 ⁴ copies/μL) with Nuclease-free water, and optimize to obtain SYBR Green I-based one-step real time RT-PCR Perform the amplification reaction with the best reaction system and conditions to obtain the respective C _T values, and use random software analysis to obtain the quantitative curve and standard curve. The logarithmic value of the initial template copy number in the standard curve is the abscissa (x axis), and the corresponding C _{The T} value is the ordinate (y axis);

7) After Real time RT-PCR amplification, from 55°C to 95°C, measure the absorbance value of the amplified product every 0.5°C (80 cycles), use random software to analyze the melting curve, and judge the specificity of the method . the

8) Compare with RT-PCR method to verify the sensitivity of SYBR Green I-based one-step real time RT-PCR. the

The primer sequences provided by the present invention are as follows:

SRBSDV-S9-F GAGACCCACCTCCACTGATT

SRBSDV-S9-R ACGTTTACCACTGCGCCTTC

In the SYBR Green I-based one-step real time RT-PCR reaction system, the concentration of primers SRBSDV-S9-F and SRBSDV-S9-R is 300nM, the amplification effect is the best, and the annealing-extension temperature is 60.0℃. According to the established standard curve, the copy number of SRBSDV RNA in the diseased rice plant samples in the field can be accurately quantified, and the specificity of the method can be determined by analyzing the melting curve of the amplified product. the

The application of a method for detecting and quantifying the RNA copy number of southern rice black-streaked dwarf virus in plants includes: rapid diagnosis and quantification of suspected diseased plants such as rice and corn. the

Using this method, the copy number of SRBSDV RNA in the southern rice black-streaked dwarf virus plant sample can be quickly obtained, which provides support for the pathogenesis of the virus disease and the basic research of SRBSDV. the

Description of drawings:

Figure 1 Primer concentration optimization (1, SRBSDV-S9-F and SRBSDV-S9-R final concentration are 300nM; 2, SRBSDV-S9-F final concentration 400nM and SRBSDV-S9-R final concentration 300nM; 3, SRBSDV-S9 -F final concentration 500 nM and SRBSDV-S9-R final concentration 300 nM). the

Fig. 2 Temperature gradient optimization (1, 56.4°C; 2, 60.0°C; 3, 57.9°C; 4, 55.4°C; 5, 63.1°C; 6, 65.3°C). the

Figure 3: Fluorescence quantification curves of 10-fold serially diluted RNA standards (from left to right are 2.5×10 ¹⁰ , 2.5×10 ⁹ , 2.5×10 ⁸ , 2.5×10 ⁷ , 2.5×10 ⁶ , 2.5×10 ⁵ , 2.5× 10 ⁴ copies/μL of RNA standard).

Fig. 4 Standard curve (2.5×10 ¹⁰ copies/μL-2.5×10 ⁴ copies/μL) obtained by 10-fold serial dilution of RNA standards.

Figure 5 Melting curves of 10-fold serially diluted RNA standards. the

Specific implementation method:

Example 1, in vitro transcription and purification preparation of RNA standard

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

Table 1 Restriction enzyme analysis system

Table1 The system of restriction enzyme analysis

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

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, annealing at 57-60°C for 45s, extension at 72°C for 30s, after the last cycle, incubate at 72°C for 10min, take 2μL PCR product for 2% agarose gel electrophoresis detection . the

Using the linear plasmid DNA containing the target fragment as a template, perform in vitro transcription according to the instructions of T7 Transcription Kit (Fermentas) to obtain linear RNA. the

(1) Add the reagents in order according to the order in Table 2, mix thoroughly, centrifuge slightly, and keep at 37°C for 2 hours to obtain the transcription product;

Table 2 In vitro transcription

Table2 In vitro transcription

(2) Add 2 μL of RNase-free DNase I (2U/μL, Fermentas) to the transcript RNA, mix well, and digest the untranscribed DNA in the transcript at 37°C for 15 min;

(3) Add 2 μL EDTA (50mM), mix well, and inactivate the activity of DNase I at 65°C for 10 minutes. the

Purify the in vitro transcribed RNA according to the instructions of the in vitro transcription purification kit EZ-10 Spin Column5 Minutes RNA Cleanup & Concentration Kit to obtain RNA standards. the

(1) Add 500 μL Solution A to 55 μL RNA solution obtained from in vitro transcription, and mix well;

(2) Transfer the mixture to an RNA preparation tube (placed on a 2mL centrifuge tube), centrifuge at 12,000rpm for 30s at room temperature, and discard the supernatant;

(3) Add 700μL RNA Wash Sloution, centrifuge at 12,000rpm for 30s at room temperature, discard the liquid;

(4) Centrifuge at 10000rpm for 2min at room temperature to remove residual RNAWash Sloution;

(5) Transfer the preparation tube to a new RNase-free centrifuge tube, add 60 μL RNA Elution Buffer, and let stand at room temperature for 2 minutes;

(6) Centrifuge at 12,000rpm for 1min to obtain RNA standards, which are stored at -70°C for later use. the

The concentration and OD value of the RNA standard were measured with an Eppendorf BioPhotometer Plus nucleic acid and protein analyzer, and the copy number was calculated according to the following formula (Santhosh et al., 2007). The standard RNA obtained by transcription was diluted 10-fold with 0.1% DEPC-treated water for sensitivity determination and standard curve establishment. the

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

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

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

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

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

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

Table3 The system of real time RT-PCR

(3) Perform amplification reaction on Bio-Rad IQ ^TM 5 Multicolor Real-Time PCR Detection System. First, cDNA was synthesized at 50°C for 10 minutes, and the activity of reverse transcriptase was inactivated at 95°C for 5 minutes; then 40 PCR cycles were performed: 95°C for 10s, 60°C for 10s (signal was collected at this time); 95°C for 1min, 55°C for 1min, Then perform 80 cycles at 55-95°C, one cycle at an interval of 0.5°C, and one cycle time is 10s;

(4) After the reaction is completed, analyze the data through the software IQ5 Optical System Software Version 2.0. the

The annealing-extension temperature was optimized, and the annealing-extension was optimized in the range of 55-65°C. the

The primer concentration was optimized. Under the same standard RNA template concentration, the upstream and downstream primer concentrations were optimized in a 5×5 matrix (the final concentrations of the upstream and downstream primers were 100nM, 200nM, 300nM, 400nM and 500nM, respectively). the

The amplification curve of the primer concentration optimization experiment is shown in Figure 1. It can be seen that the concentrations of the upstream primer SRBSDV-S9-F are 300nM, 400nM, and 500nM respectively, and the C _T value of the quantitative curve when the concentration of the downstream primer SRBSDV-S9-R is 300nM and The fluorescence value is more appropriate, and the amplification result is the best when the primer concentration is 300nM.

The results of the annealing-extension temperature optimization experiment (Figure 2) show that when the annealing-extension temperature is 56.4°C, 57.9°C and 60.0°C, the C _T values of the quantitative curves are basically the same and smaller, and the time to reach the plateau is earlier and the fluorescence value Relatively high, in view of the fact that the annealing temperature is too low to easily produce non-specific amplification products, so the annealing-extension temperature is 60.0°C is the most suitable.

The optimized 20μL reaction system is: 2×SYBR Green RT-PCR reaction mix 10μL, SRBSDV-S9-F and SRBSDV-S9-R each 0.6μL (10μM), iscript reverse transcriptase for one-step RT-PCR 0.4μL , template RNA 2 μL, and finally make up 20 μL with nuclease-free water. The optimal reaction conditions are reverse transcription at 50°C for 10 minutes; 95°C for 5 minutes to inactivate reverse transcriptase; then enter 40 cycles: 95°C for 10s, 60°C for 30s; then 95°C for 1min, 55°C for 1min, then 55-95°C Increase the temperature by 0.5°C every 10s to obtain the melting curve of the amplification product. the

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

According to the copy number calculated in Example 2, carry out a 10-fold serial dilution of standard RNA with Nuclease-free water (2.5×10 ¹⁰ -2.5×10 ⁴ copies/μL), and use this set of standards as a template to use Example 2 Optimize the best reaction system and conditions for the reaction to obtain their respective C _T values, and use random software to analyze the amplification curve and standard curve. The logarithmic value of the initial template copy number in the standard curve is the abscissa (x-axis), and the corresponding The C _T 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. From the 5th cycle, the absorbance value of 2.5×10 ¹⁰ copies/μL RNA standard began to increase, and the quantitative curve began to rise. After the 7th cycle ( _CT value), the quantitative curve rose rapidly, and reached the peak around the 25th cycle. , and then entered the plateau, until the end of the reaction, the quantitative curve was a typical S-type. The change trend of the quantitative curve of 2.5×10 ⁹ -2.5×10 ⁴ copies/μL RNA standard is the same as this, except that the position of the rising curve (C _T value) will be 3 to 4 cycles later with the decrease of the concentration, and their C _{The T} values were 7, 10.5, 14, 18, 21, 24, and 27 in turn, showing a group of parallel curves with the same slope and equal spacing (as shown in Figure 3).

The standard curve shows (Fig. 4) that the C _T values of all RNA standards are basically on a straight line, and there is no large deviation in any standard. The amplification efficiency of the standard curve in this figure is E=100.2%; the coefficient of determination (R ² ) is greater than 0.98, which is 0.996, the slope is -3.317, and the calculation formula is y=-3.317x+30.659, which can be used to calculate and obtain unknown samples The exact copy number of SRBSDV RNA in .

Embodiment 4, the determination of the melting curve of southern rice black-streaked dwarf virus SYBR Green I-based one-step real time RT-PCR

According to the conditions in Example 2, the melting curve of the amplified product was obtained. Figure 5 shows that the melting temperature Tm of the amplified product is all at 78°C, with a single peak, sharp and narrow peak, indicating that the set primers have good specificity and the amplified product is single , no primer dimers and non-specific amplification products. the

The above implementation does not limit the present invention in any form. the

Claims (1)

1. A method for accurately detecting and quantifying southern rice black-streaked dwarf virus on plants, characterized in that: using the specific primers of SYBR Green I-based one-step real time RT-PCR reaction to extract the rice virus total RNA is used as the template for amplification reaction, and the data is analyzed by software after the reaction is completed. This method can eliminate the interference of rice black-streaked dwarf disease, accurately identify the virus in the southern rice black-streaked dwarf virus plant, and obtain the SRBSDV RNA at the same time. exact copy number. The above "specific primers for SYBR Green I-based one-step real time RT-PCR reaction" refers to the following two primers: SRBSDV-S9-F GAGACCCACCTCCACTGATT SRBSDV-S9-R ACGTTTACCACTGCGCCTTC.

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