CN112430687A - Method for rapidly detecting potato virus based on RT-PCR (reverse transcription-polymerase chain reaction) of filter paper strip capture virus nucleic acid - Google Patents
Method for rapidly detecting potato virus based on RT-PCR (reverse transcription-polymerase chain reaction) of filter paper strip capture virus nucleic acid Download PDFInfo
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Abstract
Aiming at the problem of the complicated RNA extraction step in the current plant virus detection, the inventor establishes a method for capturing virus nucleic acid based on a filter paper strip, the treated filter paper strip is utilized to adsorb the virus nucleic acid in plant tissue liquid obtained after grinding and cracking, the filter paper strip is washed by washing liquid to remove impurities, and then the adsorbed virus nucleic acid is eluted into deionized water or RT-PCR reaction liquid without ribonuclease. Accordingly, the inventor also establishes a corresponding RT-PCR method for rapidly detecting the potato viruses. Researches show that the method can quickly, simply, economically and accurately detect the potato viruses from the potato leaves, and has important significance for large-scale quick detection of the potato viruses.
Description
Technical Field
The invention belongs to the technical field of potato virus detection, and particularly relates to a method for rapidly detecting potato viruses based on RT-PCR (reverse transcription-polymerase chain reaction) of virus nucleic acid captured by filter paper strips.
Background
The potato (Solanum tuberosum L.) is propagated by a vegetative mass, viruses are accumulated year by year along with the propagation of the potato, so that the potato seed degeneration is caused, and researches prove that the potato viruses cause 30 to 50 percent of yield reduction of the potato, and more than 80 percent of yield loss is possibly caused if different viruses are infected in a compound way. The main measure for preventing and treating potato virus diseases is to strictly detect virus-free seed potatoes and replace seedlings in time to avoid virus accumulation. Therefore, the rapid and accurate qualitative or quantitative detection of the potato virus is an important guarantee for the healthy production of the potatoes. More than 40 viruses have been reported to infect potatoes, wherein Potato Virus Y (PVY), Potato Virus S (PVS) and Potato Leaf Roll Virus (PLRV) are indispensable items for Potato virus detection in our country.
The sensitivity and specificity of virus detection from the nucleic acid level are higher than those of serological detection, and the method is particularly suitable for sample detection with high accuracy requirement. The nucleic acid detection method of potato virus mainly comprises PCR, RT-PCR, double-stranded RNA (dsRNA) electrophoresis, loop-mediated isothermal amplification (LAMP) and the like, wherein the RT-PCR is the most widely applied technology for detecting RNA virus at present. The nucleic acid extraction is the link which is most difficult to realize high-throughput operation in the virus detection process, and the traditional CTAB method, SDS method or Trizol method has the problems of long time consumption or high cost and complicated operation steps.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a rapid, simple and economic RT-PCR method for rapidly detecting potato viruses based on the viral nucleic acid captured by filter paper strips, which omits the RNA extraction step with complex operation and can realize high-throughput viral nucleic acid capture and detection.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for capturing virus nucleic acid based on a filter paper strip comprises the steps of adsorbing virus nucleic acid in plant tissue liquid obtained after grinding and cracking by utilizing the treated filter paper strip, washing the filter paper strip by using a washing solution to remove impurities, and eluting the adsorbed virus nucleic acid into deionized water or RT-PCR reaction liquid without ribonuclease (RNase-free); the treatment comprises soaking filter paper strip in DEPC water at 37 deg.C for 1 hr, draining, wrapping with kraft paper, sterilizing at high temperature and high pressure, and oven drying.
The lysis solution used for lysis contained 800mM guanidine hydrochloride, 0.5% polyethylene glycol octylphenyl ether (Triton X-100), 1% Tween-20 and 50mM Tris-HCl (pH 8.0); the wash contained 0.1% Tween-20 and 10mM Tris-HCl (pH 8.0).
The plant tissue fluid is prepared by the following operations: quickly freezing plant tissue in 2mL centrifuge tube with liquid nitrogen, grinding with tissue grinder at 55Hz for 40 s to obtain powder, and adding 1mL lysate to lyse plant cells.
A method for rapidly detecting potato viruses based on RT-PCR of capturing virus nucleic acid by filter paper strips is characterized in that the virus nucleic acid in potatoes is captured by the method, and then RT-PCR detection is carried out; the primer group used for RT-PCR detection comprises four pairs of PCR primers respectively aiming at three potato viruses of PVY, PVS and PLRV and a potato reference gene Actin, and the primers respectively have base sequences of sequence tables SEQ.ID.NO.1 to SEQ.ID.NO. 8.
The method for rapidly detecting the potato viruses by the RT-PCR is operated according to the following steps:
(1) preparation of RT-PCR reaction solution
Taking 2 XFastKing One Step RT-PCR MasterMix 25 muL in a FastKing One Step RT-PCR kit, adding upstream and downstream specific primers (10 muM) into a PCR tube of RNase-free respectively by 1.25 muL, and supplementing RNase-free deionized water to 50 muL;
(2) method for capturing nucleic acid by using filter paper strip
Placing 0.1g of potato tissue sample to be detected in a 2mL freezing and grinding tube containing 2-3 RNase-free steel balls or zirconium beads with the diameter of 3mm, quickly freezing by liquid nitrogen, placing the potato tissue sample into a grinding instrument, and grinding for 40 seconds under the condition of 55 Hz; then adding 1mL of prepared lysate into the centrifuge tube, fully and uniformly mixing, dipping and washing the filter paper strip in the lysate for 3 times, then placing the filter paper strip in 1mL of washing solution for three times, and then dipping and washing the filter paper strip in an RNase-free PCR tube containing 50 muL of RT-PCR reaction solution or RNase-free deionized water for 3 times for elution;
(3) RT-PCR reaction procedure
30min at 42 ℃; pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, 35 cycles; and finally, supplementary extension is carried out for 5min at 72 ℃.
Aiming at the problem of the complicated RNA extraction step in the current plant virus detection, the inventor establishes a method for capturing virus nucleic acid based on a filter paper strip, the treated filter paper strip is utilized to adsorb the virus nucleic acid in plant tissue liquid obtained after grinding and cracking, the filter paper strip is washed by washing liquid to remove impurities, and then the adsorbed virus nucleic acid is eluted into deionized water or RT-PCR reaction liquid without ribonuclease. The method utilizes the filter paper strip to rapidly capture the virus nucleic acid in the specific buffer solution, thereby saving the complicated RNA extraction step, further saving the reagent cost required by RNA extraction, saving time and labor, and being widely applied to the rapid detection of various plant viruses. Accordingly, the inventor also establishes a corresponding RT-PCR rapid detection method for potato viruses, captures viral nucleic acids in potatoes by using the method, and then carries out RT-PCR detection; the primer group used for RT-PCR detection comprises four pairs of PCR primers respectively aiming at three potato viruses of PVY, PVS and PLRV and a potato reference gene Actin (used as a positive control of RT-PCR reaction and used for evaluating the amount of total nucleic acid of potatoes in a reaction system). Researches show that the method can quickly, simply, economically and accurately detect the potato viruses from the potato leaves, and has important significance for large-scale quick detection of the potato viruses.
Drawings
FIG. 1 is a flow chart of the method for capturing viral nucleic acid based on a filter paper strip according to the present invention.
FIG. 2 is an alignment chart of the genome sequence of PVY showing the regions (sequence-conserved regions) where the PVY detection primers PVY-F and PVY-R are located.
FIG. 3 is an alignment chart of the genome sequence of PVS showing the region (sequence-conserved region) where the PVS detection primers PVS-F and PVS-R are located.
FIG. 4 is an alignment chart of the genomic sequence of PLRV showing the region (sequence conserved region) where PLRV detection primers PLRV-F and PLRV-R are located.
FIG. 5 is an electrophoretogram for detecting potato virus, in which: lane M is DNA molecular weight Standard (GeneRuler)TM1kb plus DNA Ladder, Marker); lane 1 nucleic acid capture and amplification of Actin gene fragments with healthy potato plant samples; lane 2 nucleic acid capture and amplification of an Actin gene fragment with PVY infected potato plant samples; lane 3 nucleic acid capture and amplification of PVY fragments with healthy potato plant samples; lane 4 nucleic acid capture and amplification of PVY fragments with PVY infected potato plant samples; lane 5 nucleic acid capture and amplification of PVS fragments with healthy potato plant samples; lane 6 nucleic acid capture and amplification of PVS fragments with PVS infected potato plant samples; lane 7 nucleic acid capture and amplification of PLRV fragments with healthy potato plant samples; lane 8 is nucleic acid capture and amplification of PLRV fragments with PLRV infected potato plant samples.
Detailed Description
First, test materials
A filter paper strip: the DEPC water treated and dried Whatman No.1 filter paper (or other qualitative filter paper) is cut into filter paper strips of appropriate size, and one half of the filter paper strips are soaked in molten wax to produce a hydrophobic region which is convenient to hold and has waterproof function.
Test reagents and drugs: the FastKing one-step RT-PCR kit is purchased from Tiangen Biochemical technology (Beijing) Co., Ltd, the primers are synthesized by Shanghai Biotechnology engineering Co., Ltd, and other medicines are domestic analytical pure.
The instrument equipment comprises: a multi-sample tissue grinding instrument (tissue grinder-96, Shanghai Jingxin), a gene amplification instrument (life ECO, Hangzhou Bori), a Junyi electrophoresis instrument (JY300C, Beijing Junyi), a Gel imaging system (Gel Doc 1000, BIO-RAD), an electronic analytical balance, an oven and the like.
Test potato samples: healthy potato samples stored in the laboratory and samples infected with three potato viruses (PVY, PVS, PLRV).
Second, test method
1. Design and Synthesis of primers
Based on the conserved region sequences of PVY, PVS and PLRV genomes reported in the literature and on the GenBank website, the Primer 6.0 Primer design software was used to design the Specific primers (Gene Specific Primer, GSP) for these 3 viruses and a potato reference Gene, Actin, respectively (see Table 1). The primer binding regions for the 3 viruses are shown in FIGS. 2, 3 and 4.
TABLE 1 primer set for RT-PCR detection of potato virus of the present invention
Note: y ═ C, T; k ═ G, T; m ═ A, C
Preparation of RT-PCR reaction solution
2 XFastKing One Step RT-PCR MasterMix 25. mu.L in FastKing One Step RT-PCR kit is taken to be put in the PCR tube of RNase-free, 1.25. mu.L of each of the upstream and downstream specific primers (10. mu.M) is added, RNase-free deionized water is complemented to 50. mu.L, and the mixture is put on ice for standby.
3. Capture of potato virus nucleic acid by filter paper strip
As shown in FIG. 1, 0.1g of potato leaf to be tested was put into a 2mL cryo-grinding tube containing 2-3 grinding beads (steel balls or zirconium beads) with a diameter of 3mm, frozen by liquid nitrogen, and placed in a grinder (MP)
-24 homogeneous disruptor) for cell disruption (55Hz, 40 seconds), removing 1mL of lysis buffer (800mM guanidine hydrochloride, 50mM pH 8.0Tris, 0.5% Triton x 100, 1% Tween-20), mixing well, dipping the filter paper strip into the crude extract of the previous step three times to bind the nucleic acids, and then washing three times in a wash solution (0.1% Tween-20, 10mM Tris, pH 8.0). Then, the mixture was dipped and washed 3 times in an RNase-free PCR tube containing 50. mu.L of RT-PCR reaction solution or RNase-free deionized water to elute.
3.RT-PCR
Placing the PCR tube filled with the reaction solution containing the nucleic acid on a PCR instrument, wherein the RT-PCR reaction procedure comprises the following steps: 30min at 42 ℃; pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, 35 cycles; and finally, supplementary extension is carried out for 5min at 72 ℃.
RT-PCR amplification product detection
And detecting the RT-PCR amplification product by agarose gel electrophoresis, and judging whether the sample to be detected contains the three potato viruses or not according to the existence and the size of the strips.
As shown in FIG. 5, the results show that the virus nucleic acid extraction method of the present invention can effectively detect the three potato viruses by combining with the RT-PCR detection method.
Sequence listing
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GUANGXI ACADEMY OF AGRICULTURAL SCIENCES
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Claims (5)
1. A method for capturing viral nucleic acid based on a filter paper strip, characterized in that: adsorbing virus nucleic acid in plant tissue liquid obtained after grinding and cracking by using the treated filter paper strip, washing the filter paper strip by using a washing solution to remove impurities, and eluting the adsorbed virus nucleic acid into deionized water or RT-PCR reaction liquid without ribonuclease; the filter paper strips are soaked in DEPC water at 37 ℃ for 1 hour, are wrapped by kraft paper after water is drained, and are sterilized at high temperature and high pressure and dried.
2. The filter paper strip-based viral nucleic acid capture method of claim 1, wherein: the lysis solution used for the lysis contains 800mM guanidine hydrochloride, 0.5% polyethylene glycol octyl phenyl ether, 1% Tween-20 and 50mM Tris-HCl pH 8.0; the wash contained 0.1% Tween-20 and 10mM Tris-HCl pH 8.0.
3. The filter paper strip-based viral nucleic acid capture method of claim 1, wherein the plant tissue fluid is prepared by: quickly freezing plant tissue in 2mL centrifuge tube with liquid nitrogen, grinding with tissue grinder at 55Hz for 40 s to obtain powder, and adding 1mL lysate to lyse plant cells.
4. A method for rapidly detecting potato viruses based on RT-PCR (reverse transcription-polymerase chain reaction) for capturing virus nucleic acids by filter paper strips, which is characterized in that the virus nucleic acids in potatoes are captured by the method of claim 1 and then subjected to RT-PCR detection; the primer group used for RT-PCR detection comprises four pairs of PCR primers respectively aiming at three potato viruses of PVY, PVS and PLRV and a potato reference gene Actin, and the primers respectively have base sequences of sequence tables SEQ.ID.NO.1 to SEQ.ID.NO. 8.
5. The RT-PCR rapid detection method of potato virus according to claim 4, characterized by the following steps:
(1) preparation of RT-PCR reaction solution
Taking 2 XFastKing One Step RT-PCR MasterMix 25 muL in a FastKing One Step RT-PCR kit, adding upstream and downstream specific primers 10 muM into an RNase-free PCR tube, respectively 1.25 muL, and supplementing RNase-free deionized water to 50 muL;
(2) method for capturing nucleic acid by using filter paper strip
Placing 0.1g of potato tissue sample to be detected in a 2mL freezing and grinding tube containing 2-3 RNase-free steel balls or zirconium beads with the diameter of 3mm, quickly freezing by liquid nitrogen, placing the potato tissue sample into a grinding instrument, and grinding for 40 seconds under the condition of 55 Hz; then adding 1mL of prepared lysate into the centrifuge tube, fully and uniformly mixing, dipping and washing the filter paper strip in the lysate for 3 times, then placing the filter paper strip in 1mL of washing solution for three times, and then dipping and washing the filter paper strip in an RNase-free PCR tube containing 50 muL of RT-PCR reaction solution or RNase-free deionized water for 3 times for elution;
(3) RT-PCR reaction procedure
30min at 42 ℃; pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, 35 cycles; and finally, supplementary extension is carried out for 5min at 72 ℃.
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