CN103088160B - Method for detecting RT-LAMP of strawberry latent ringspot virus - Google Patents

Abstract

The invention discloses a method for detecting RT-LAMP (Loop-mediated Isothermal Amplification) of strawberry latent ringspot virus. The method comprises a step of extracting a sample RNA, and a step of carrying out RT-LAMP amplification to obtain the detection results. The overall detection reaction does not exceed 2.5 hours, and the LAMP amplification is waterfall type amplification, so that the amplification efficiency is high, and the sensitivity is 100 times as that of the RT-LAMP detection. The complex detectors and the detection process are not needed, the operation is simple, and the real-time detection is carried out on spot, so that the method disclosed by the invention has the advantages of high detection speed, simplicity in operation, easiness in judging the detection results and high sensitivity.

Description

The RT-LAMP detection method of strawberry latent ringspot virus

Technical field

The present invention relates to the detection technique of strawberry latent ringspot virus, be specifically related to the RT-LAMP detection method of strawberry latent ringspot virus.

Background technology

Strawberry latent ringspot virus ( strawberry latent ringspot virus, SLRSV) and be under the jurisdiction of Comoviridae Nepovirus ^[1], be one of important disease on Rosaceae fruit class crop, the important quarantine harmful organisms that Ye Shi State General Administration for Quality Supervision issues.SLRSV can infect seed, plant ball, stem tuber and nursery stock etc., and after virus infection plant, main manifestations is degradation phenomenon under blade chlorisis, deformity, plant dwarfing, production declining and quality, when serious, can cause crushing disaster.The detection method of SLRSV mainly contains plant indicator leaflet grafting, serology detection and molecular Biological Detection etc. at present.The Symptoms more complicated of SLRSV, virus of the same race is on different plant indicators, and symptom variation is larger, and aforesaid method is for the identification and detection length consuming time of viral species, sensitivity is lower.

Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) is as a kind of nucleic acid amplification method of novelty, has equally the advantages such as fast, the simple to operate and sensitivity of detection speed is high; This technology depends on the Bst archaeal dna polymerase that can identify primer and a kind of tool de-rotation function in 6 special regions on target sequence and be waterfall type amplification, amplified target sequence efficiently, fast and specifically under isothermal condition.Because LAMP is waterfall type amplification, therefore its amplification efficiency is very high; Meanwhile, 6～8 specificity sites on LAMP identification target sequence, its specificity is also very strong; LAMP only carries out constant-temperature amplification at 60 DEG C～65 DEG C, as long as water-bath, without special instrument, operates very simple; And the whole detection reaction of LAMP only needs 1 h～2.5 h, sense cycle is very short.

Summary of the invention

Technical problem to be solved by this invention is to provide the RT-LAMP detection method that a kind of detection speed is fast, simple to operate, detected result judges easy and highly sensitive strawberry latent ringspot virus.

The present invention solves the problems of the technologies described above adopted technical scheme: the RT-LAMP detection method of strawberry latent ringspot virus, and its step is as follows:

A, total RNA extract: detect sample and after liquid nitrogen is processed, grind powderedly, extract total RNA with Trizol nucleic acid extracting reagent, obtain the total RNA of sample; Concrete extracting method carries out according to the specification sheets of Trizol nucleic acid extracting reagent;

B, RT-LAMP amplification: the RT-LAMP amplification reaction system of 20 μ L is 2 × reaction buffer, 10 μ L, the Bst archaeal dna polymerase 1.5 μ L of concentration 5 U/ μ L, the AMV RNA polymerase 1 μ L of concentration 5 U/ μ L, the total RNA1 μ of sample L, primers F 3, B3, Loop1, Loop2, FIP and BIP, surplus is ddH ₂o, in this amplification reaction system, the final concentration of primers F 3 and B3 is respectively 0.2 μ M, the final concentration of primer Loop1 and Loop2 is respectively 0.8 μ M, the final concentration of primers F IP and BIP is respectively 1.6 μ M, wherein the nucleotides sequence of primers F 3 is classified gagaatcacc gttactggaa agg as, the nucleotides sequence of primer B3 is classified acagcaaaca gagaaccact acc as, the nucleotides sequence of primer Loop1 is classified gcaaagccca tttccacagt as, the nucleotides sequence of primer Loop2 is classified ggttgctacg tgccaaggtt as, the nucleotides sequence of primers F IP is classified cgataggcat cgctctccct ttttagaggt gatctttaac ctccc as, the nucleotides sequence of primer BIP is classified tactttggtt cgacagtggt ggattttccc attagataac caccataacc a as, reaction conditions is 1～1.5 h that increases in the thermostat water bath of 60～65 DEG C, obtain amplified production,

C, in amplified production, add the fluorescence dye SYBR green I of 0.1 μ L, infect in sample and have strawberry latent ringspot virus if be green, if be in orange red sample without strawberry latent ringspot virus.

Compared with prior art, the invention has the advantages that the RT-LAMP detection method of strawberry latent ringspot virus, by sample, RNA extracts, RT-LAMP amplification, just can obtain detected result, whole detection reaction is no more than 2.5 h, again because LAMP is waterfall type amplification, therefore its amplification efficiency is very high, sensitivity is 100 times that RT-PCR detects, and without complicated detecting instrument and testing process, simple to operate, can on-the-spotly detect in real time, therefore the present invention is that a kind of detection speed is fast, simple to operate, detected result judges the RT-LAMP detection method of easy and highly sensitive strawberry latent ringspot virus.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.

Embodiment 1

1, according to GenBank(http: //www.ncbi.nlm.nih.gov) in the Coat protein gene sequence (AY860979) of listed strawberry latent ringspot virus, adopt Blast program to carry out homology comparison, in the conserved regions of sequence, use Primer Express 3.0 softwares, design obtains F3, B3, FIP, BIP, Loop1 and Loop2 totally 6 primers, identifies respectively 8 sites of coat protein encoding gene, primers F 3, B3, FIP, BIP, Loop1 and Loop2 are synthetic by Shanghai Ying Jun biotech company, the nucleotides sequence of primers F 3 is classified gagaatcacc gttactggaa agg as, the nucleotides sequence of primer B3 is classified acagcaaaca gagaaccact acc as, the nucleotides sequence of primer Loop1 is classified gcaaagccca tttccacagt as, the nucleotides sequence of primer Loop2 is classified ggttgctacg tgccaaggtt as, the nucleotides sequence of primers F IP is classified cgataggcat cgctctccct ttttagaggt gatctttaac ctccc as, the nucleotides sequence of primer BIP is classified tactttggtt cgacagtggt ggattttccc attagataac caccataacc a as,

2, RT-LAMP amplification reaction system is set up: 2 × reaction buffer, 10 μ L, the Bst archaeal dna polymerase 1.5 μ L of concentration 5 U/ μ L, the AMV RNA polymerase 1 μ L of concentration 5 U/ μ L, the total RNA1 μ of sample L, primers F 3 and B3 final concentration are respectively 0.2 μ M, primer Loop1 and Loop2 final concentration are respectively 0.8 μ M, and primers F IP and BIP final concentration are respectively 1.6 μ M, ddH ₂o is supplemented to 20 μ L; Bst archaeal dna polymerase and AMV RNA polymerase are all purchased from Beijing Mei Laibo medical science and technology company limited;

3, reaction condition optimization: strawberry latent ringspot virus standard model (being purchased from Agdia company of the U.S.) is increased with above-mentioned RT-LAMP amplification reaction system, the amplified reaction time is set as respectively: 30min, 60min and 90min, 30 min do not see precipitation as a result, 60min and 90min can see precipitation, amplified production adds the fluorescence dye SYBR green I(of 0.1 μ L purchased from Shanghai bio-engineering corporation), all be green, therefore choose the reaction times of 60～90 min; Amplified reaction bath temperature is set as respectively 60 DEG C, 63 DEG C and 65 DEG C, and result can be seen precipitation, and amplified reaction temperature is preferably lower 60 DEG C;

4, RT-LAMP specific test: with above-mentioned RT-LAMP amplification reaction system respectively to two samples of strawberry latent ringspot virus, sample of nepovirus ( tobacco ringspot virus, TRSV), sample of carnation ringspot virus ( carnation ringspot virus, CRSV), sample of cucumber green mottle virus ( cucumber green mottle mosaic virus, CGMMV), sample of arabis mosaic virus ( arabis mosaic virus, ArMV) and sample of potato V virus ( potato virus VpVV) increase, above-mentioned sample is all purchased from Agdia company of the U.S., reaction times 60 min, and bath temperature is 60 DEG C, two samples of result strawberry latent ringspot virus can be seen precipitation, amplified production adds the fluorescence dye SYBR green I of 0.1 μ L, is all green, and other sample is without precipitation, after SYBR green I dyeing, be orange red, illustrate that RT-LAMP amplification reaction system is special to strawberry latent ringspot virus.

Embodiment 2

1,2 grams of positive Bulbus Lilii leaf samples of strawberry latent ringspot virus (sample is provided by Shanghai inspection and quarantine bureau) grind powdered after approximately 50 milliliters of liquid nitrogen are processed, extract total RNA with Trizol nucleic acid extracting reagent (purchased from Shanghai bio-engineering corporation), obtain the total RNA of sample; Concrete extracting method carries out according to the specification sheets of Trizol nucleic acid extracting reagent;

2, RT-LAMP susceptibility test: respectively the total RNA of sample is carried out to amplified reaction with RT-PCR reaction system and above-mentioned RT-LAMP amplification reaction system, RT-PCR reaction system primer is F3 and B3, test kit is that One-step RNA PCR kit(is purchased from Dalian TaKaRa company), PCR reaction conditions is as follows: 50 DEG C of synthetic 30 min of cDNA, 94 DEG C of denaturation 2 min; 94 DEG C of sex change 30 s, 58 DEG C of renaturation 30 s, 72 DEG C are extended 1 min, 35 circulations; Last 72 DEG C are extended 5 min.Sample total rna concentration is respectively stoste, 10 of stoste ^-1, 10 ^-2, 10 ^-3, 10 ^-4with 10 ^-5diluent, RT-LAMP amplified reaction times 60 min, bath temperature is 60 DEG C, 10 of result stoste and stoste ^-1with 10 ^-3diluent is seen precipitation, and amplified production adds the fluorescence dye SYBR green I of 0.1 μ L, 10 of stoste and stoste ^-1, 10 ^-2, 10 ^-3, 10 ^-4it is green that diluent is all; RT-PCR only has stoste and 10 ^-1diluent has fluorescent reaction, and therefore RT-LAMP amplification sensitivity is the more than 100 times of RT-PCR, also illustrates in Bulbus Lilii leaf and infects and have strawberry latent ringspot virus.

Embodiment 3

Above-mentioned RT-LAMP amplification reaction system detects rose, rose and narcissus leaf sample respectively.Detection method is with embodiment 2, just bath temperature is 65 DEG C, result Leaf of Japanese Rose amplified production has precipitation, after the fluorescence dye SYBR green I of 0.1 μ L, be green, rose leaf amplified production muddiness, is green after the fluorescence dye SYBR green I of 0.1 μ L, narcissus amplified production is without precipitation, after the fluorescence dye SYBR green I of 0.1 μ L, be orange red, illustrate in rose, rose leaf and infect and have strawberry latent ringspot virus, in narcissus leaf, do not infect strawberry latent ringspot virus.

<110> Ningbo Institute of Inspection and Quarantine Science Technology

The RT-LAMP detection method of <120> strawberry latent ringspot virus

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<170> PatentIn version 3.5

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Claims (1)

1. the RT-LAMP detection method of strawberry latent ringspot virus, is characterized in that step is as follows:

A, total RNA extract: detect sample and after liquid nitrogen is processed, grind powderedly, extract total RNA with Trizol nucleic acid extracting reagent, obtain the total RNA of sample; Concrete extracting method carries out according to the specification sheets of Trizol nucleic acid extracting reagent;

B, RT-LAMP amplification: 20 μ L RT-LAMP amplification reaction systems are 2 × reaction buffer, 10 μ L, the Bst archaeal dna polymerase 1.5 μ L of concentration 5 U/ μ L, the AMV RNA polymerase 1 μ L of concentration 5 U/ μ L, the total RNA1 μ of sample L, primers F 3, B3, Loop1, Loop2, FIP and BIP, surplus is ddH ₂o, in this amplification reaction system, the final concentration of primers F 3 and B3 is respectively 0.2 μ M, the final concentration of primer Loop1 and Loop2 is respectively 0.8 μ M, the final concentration of primers F IP and BIP is respectively 1.6 μ M, wherein the nucleotides sequence of primers F 3 is classified gagaatcacc gttactggaa agg as, the nucleotides sequence of primer B3 is classified acagcaaaca gagaaccact acc as, the nucleotides sequence of primer Loop1 is classified gcaaagccca tttccacagt as, the nucleotides sequence of primer Loop2 is classified ggttgctacg tgccaaggtt as, the nucleotides sequence of primers F IP is classified cgataggcat cgctctccct ttttagaggt gatctttaac ctccc as, the nucleotides sequence of primer BIP is classified tactttggtt cgacagtggt ggattttccc attagataac caccataacc a as, reaction conditions is 1～1.5 h that increases in the thermostat water bath of 60～65 DEG C, obtain amplified production,

C, in amplified production, add the fluorescence dye SYBR green I of 0.1 μ L, infect in sample and have strawberry latent ringspot virus if be green, if be in orange red sample without strawberry latent ringspot virus.