CN102392018A

CN102392018A - Rapid extraction method of single laodelphax striatellus

Info

Publication number: CN102392018A
Application number: CN201110370566XA
Authority: CN
Inventors: 徐秋芳; 周益军; 张晓霞; 程兆榜; 周彤; 季英华
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2011-11-21
Filing date: 2011-11-21
Publication date: 2012-03-28

Abstract

The invention provides a rapid extraction method of a single laodelphax striatellus and belongs to the technical field of agriculture. A routine extraction method of a single laodelphax striatellus RNA employs an RNA extraction reagent like Trizol for extraction, but the method has disadvantages of uneasiness to insect body milling, high cost and long time consumption. The rapid extraction method of the invention employs sterile water to wash laodelphax striatellus insect body; then the insect body is triturated with toothpick; the laodelphax striatellus RNA is dissolved in DEPC water, so as to obtain laodelphax striatellus RNA. Compared with a routine RNA extraction method, the RNA extraction method of the present invention is simply operated and requires no expensive RNA extraction reagent, so as to substantially reduce detection cost and save time. Provided is a rapid, economical and convenient extraction method; and the obtained laodelphax striatellus RNA is suitable for virus detection on one or a plurality of RNAs in laodelphax striatellus body.

Description

The method of a kind of rapid extraction single head small brown rice planthopper RNA

Technical field

This paper invents the method for a kind of rapid extraction single head small brown rice planthopper RNA, belongs to the agricultural cience and farming techniques field.

Background technology

Small brown rice planthopper (Laodelphax striatellus Fall é n) is a kind of important Agricultural pests; Its harm is inhaled rice strain phloem juice except that thorn and is caused that paddy growth is slow, delays of tillering, flat grain increase; The more important thing is that it can propagate various plants virus; Cause the virus disease of paddy rice, corn, wheat etc., the serious harm agriculture prodn.The virus that small brown rice planthopper is propagated is mainly RNA viruses; Comprise rice stripe virus (Rice stripe virus; RBSDV), rice black-streaked dwarf virus (Rice black streaked dwarf virus, RBSDV), wheat rosette stunt virus (Wheat rosette stunt virus, WRSV) etc.In addition, small brown rice planthopper can also be carried a kind of picornavirus---Himetobi Pvirus (HiPV).

Detecting the intravital RNA viruses of small brown rice planthopper quickly and accurately has important practical significance aspects such as work for the prediction of virus disease and disease control, research virus and amboceptor mutually.At present the detection method of RNA viruses mainly contains two big types in the insect body, and one type is based on the method that detects viral nucleic acid, like RT-PCR etc.; Another kind of is to utilize specific antibody to carry out immunodetection.Based on the method that detects viral nucleic acid owing to need not prepare the specific antibody of virus, and have detection sensitivity with the precision height, can receive widely-used to advantages such as one or more viruses detect simultaneously.But no matter be RT-PCR, Real-time PCR, or loop-mediated isothermal amplification method (RT-LAMP), these had an essential step promptly to extract small brown rice planthopper RNA before detecting based on the method that detects viral nucleic acid.The conventional method of extracting small brown rice planthopper RNA is with behind the liquid nitrogen grinding polypide, adds RNA extraction reagent such as Trizol etc. and extracts.The subject matter that this method exists is that the small brown rice planthopper polypide is little, and small brown rice planthopper body length is merely 1.8～4.0mm, grinds the polypide inconvenient operation, and extraction cost is high in addition, wastes time and energy.Commercially available Trizol reagent price is at 6～10 yuan/milliliter.The Trizol that extraction single head small brown rice planthopper RNA needs more than 300 μ L, only extracts reagent and just needs 2～3 yuan at least when also promptly extracting single head small brown rice planthopper RNA, also need buy reagent such as chloroform, Virahol, absolute ethyl alcohol in addition.The time of accomplishing a RNA extraction usually was at least 50 minutes.To above problem, this paper has invented the method for a kind of rapid extraction single head small brown rice planthopper RNA, and this method is easy and simple to handle, does not need extraction reagent such as Trizol, can accomplish leaching process in the 5min, thereby save the detection cost greatly, has shortened detection time.

Summary of the invention

The invention provides the method for a kind of rapid extraction single head small brown rice planthopper RNA.

Concrete operation method of the present invention is: the single head small brown rice planthopper is put into centrifuge tube; The sterilized water that adds 100 μ L; Clean small brown rice planthopper with pipettor piping and druming, add the DEPC water of 25 μ L after the sterile water wash 2～3 times, adopt aseptic toothpick that the single head small brown rice planthopper is smashed to pieces; The centrifugal 1min of 12000g, the gained supernatant is small brown rice planthopper RNA.

The small brown rice planthopper RNA that adopts the present invention to obtain is applicable to the detection of RNA viruses in the small brown rice planthopper body.In aforesaid method, the water that is used to extract RNA is DEPC water, can prevent to extract the degraded of back RNA, and the minimal volumes of DEPC water is 15 μ L.The eccentricity minimum is 8000g, and centrifugation time is minimum to be 1 minute, and eccentricity is high more, the longer effect of then removing impurity of centrifugation time is good more.For the higher virus of small brown rice planthopper in-vivo content, the volume of DEPC water does not influence detected result in 25 μ L～200 μ L scopes.The supernatant that centrifugal back obtains is can not storage period oversize, promptly carries out reverse transcription after preferably centrifugal.

Small brown rice planthopper RNA process for extracting of the present invention has omitted conventional RNA extraction step, directly smashs small brown rice planthopper to pieces with toothpick, and the RNA of small brown rice planthopper is dissolved in the water, promptly obtains the RNA (operation chart is seen Fig. 1) of small brown rice planthopper after centrifugal.This process for extracting need not bought chemical reagent such as Trizol; Only need article cheaply such as DEPC water and toothpick, overcome and detected the high problem of cost, and easy and simple to handle; RNA extraction time is reduced in the 5min more than 50 minutes by what routine was extracted, has significantly improved extraction efficiency.

Description of drawings

Fig. 1 toothpick synoptic diagram of milling

Directly detect RSV synoptic diagram as a result in Fig. 2 single head small brown rice planthopper

Directly detect RBSDV synoptic diagram as a result in Fig. 3 single head small brown rice planthopper

Directly detect HiPV synoptic diagram as a result in Fig. 4 single head small brown rice planthopper

Detect RSV and HiPV synoptic diagram as a result in Fig. 5 single head small brown rice planthopper simultaneously

The practical implementation method

Instance one detects the intravital rice stripe virus of single head small brown rice planthopper (RSV)

With the single head small brown rice planthopper 200 μ L centrifuge tubes of packing into; Add 100 μ L sterile water wash 3 times, smash the centrifugal 1min of 12000g to pieces with the toothpick of sterilization; Get 10 μ L supernatants and carry out reverse transcription, adopt the Auele Specific Primer RSV CP-F/RSVCP-R of RSV virus to carry out the PCR detection.

The concrete operations step of reverse transcription is: in 200 μ L centrifuge tubes, add 10 μ L supernatants and 1 μ L random primer Random6; Take out to place immediately behind 70 ℃ of placement 5min and place 1min on ice; Add 5 * M-MuLV ThermoScript II damping fluid, 3 μ L, dNTPs (10mM/dNTP) 0.5 μ L, RNase inhibitor (20U/ μ L) 0.5 μ L, M-MuLV ThermoScript II (200U/ μ L) 0.5 μ L successively; Place 1h for 42 ℃; 70 ℃ of insulation 5min promptly obtain cDNA, can place-20 ℃ to preserve subsequent use or directly be used for the PCR reaction.PCR method is: the system shown in the table 1 of pressing adds the required amount of each component.The sequence of upstream primer RSV CP-F is: 5 '-ATGGGTACCAACAAGCCAGCCAC-3 ', and the sequence of downstream primer RSV CP-R is 5 '-GTCATCTGCACCTTCTGCCTCGTC-3 ', the target sequence length of amplification is 966bp.Increase by following PCR program after adding each component: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 50s, 58 ℃ of annealing 50s, 72 ℃ of extension 90s, 40 circulations; Last 72 ℃ are extended 10min.PCR finishes after product and carries out agarose gel electrophoresis.Get 8 μ L PCR products through 1% sepharose electrophoresis 20min under 0.5 * tbe buffer liquid and 150V voltage conditions, dyeing 10min in the ethidium bromide (EB) of 0.5 μ g/mL, the dyeing back is observed in gel imaging system.Carry the electrophoretic band that can detect 966bp in the single head small brown rice planthopper of RSV, as shown in Figure 2.

Table 1PCR composition and usage quantity

Instance two detects the intravital rice black-streaked dwarf virus of single head small brown rice planthopper (RBSDV)

Nontoxic small brown rice planthopper was fed 3 days on the sick seedling of RBSDV; Afterwards the single head small brown rice planthopper is packed in the 200 μ L centrifuge tubes; With 100 μ L sterile water wash 3 times, smash the centrifugal 1min of 12000g to pieces with the toothpick of sterilization; Get 10 μ L supernatants and carry out reverse transcription, adopt the Auele Specific Primer RBSDV P10-F of RBSDV and RBSDV P10-R to carry out the RT-PCR detection.

The concrete grammar of reverse transcription is with instance one.PCR method is: the system shown in the table 1 of pressing adds the required amount of each component.Auele Specific Primer with RBSDV increases.The sequence of upstream primer RBSDV P10-F is: 5 '-ATGGCTGACATAAGACTCGA-3 ', and the sequence of downstream primer RBSDV P10-R is 5 '-TCATCTTGTCACTTTGTTTA-3 ', the target sequence length of amplification is 1677bp.Carry out pcr amplification and agarose gel electrophoresis according to the method and the step of instance one after adding each component.Carry the electrophoretic band that can detect 1677bp in the small brown rice planthopper of RBSDV, the small brown rice planthopper of not carrying RBSDV then detects less than this target stripe, and is as shown in Figure 3.

Instance three detects the intravital HiPV virus of small brown rice planthopper

The single head small brown rice planthopper is packed in the 200 μ L centrifuge tubes; With 100 μ L sterile water wash 3 times, smash the centrifugal 1min of 12000g to pieces with the toothpick of sterilization; Get 10 μ L supernatants and carry out reverse transcription, carry out RT-PCR with the Auele Specific Primer HiPV CP-F of HiPV and HiPVCP-R and detect.

The concrete grammar of reverse transcription is with instance one.PCR method is: the system shown in the table 1 of pressing adds the required amount of each component.Auele Specific Primer with HiPV increases.The sequence of upstream primer HiPV CP-F is: 5 '-CTGGACAACATGATATTAGA-3 ', and the sequence of downstream primer HiPV CP-R is 5 '-CTATTTCCCAGTTCCAAG-3 ', the target sequence length of amplification is 678bp.Add the laggard performing PCR reaction of each component.PCR response procedures and agarose gel electrophoresis are with instance one.Carry the electrophoretic band that can detect 678bp in the single head small brown rice planthopper of RBSDV, as shown in Figure 4.

Instance four detects intravital RSV of single head small brown rice planthopper and HiPV simultaneously

The single head small brown rice planthopper is packed in the 200 μ L centrifuge tubes; With 100 μ L sterile water wash 3 times; Toothpick with sterilization is smashed to pieces; The centrifugal 1min of 12000g gets 10 μ L supernatants and carries out reverse transcription, carries out RT-PCR with Auele Specific Primer HiPV CP-F, HiPV CP-R, RSV CP-F and the RSV CP-R of HiPV and RSV and detects.

The concrete grammar of reverse transcription is with instance one.PCR method is: the system shown in the table 2 of pressing adds the required amount of each component.The primer that adds two kinds of viruses in the PCR reaction simultaneously is that primer RSV CP-F, RSV CP-R, HiPV CP-F and HiPV CP-R increase; Primer sequence is with instance one and instance three, and the target sequence length of amplification is difference 966bp (RSV) and 638bp (HiPV).Add the laggard performing PCR reaction of each component.PCR response procedures and agarose gel electrophoresis are with instance one.Carry simultaneously and can detect two electrophoretic bands of size in the single head small brown rice planthopper of RSV and HiPV for 966bp and 638bp; The small brown rice planthopper of only carrying RSV virus only can detect the electrophoretic band of 966bp; The small brown rice planthopper of only carrying HiPV then only can detect the electrophoretic band of 638bp, and the then detection of not carrying these two kinds of viruses is less than corresponding band.Detected result is as shown in Figure 5.

Table 2 detects the PCR system of the sharp HiPV of RSV simultaneously

Claims

1. the technical characterictic of " method of a kind of rapid extraction single head small brown rice planthopper RNA " is not use RNA to extract reagent to extract RNA; The single head small brown rice planthopper is with sterile water wash 2～3 times; Add the DEPC water of at least 15 μ L, smash polypide to pieces, small brown rice planthopper RNA is dissolved in the DEPC water with aseptic toothpick; More than the rotating speed 8000g centrifugal 1 minute, the supernatant that obtains was small brown rice planthopper RNA.