CN102732642B - Narcissus latent virus detection kit and detection method thereof - Google Patents
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Abstract
The invention relates to a narcissus latent virus detection kit and its detecting method that are special for narcissus latent virus detection. The kit comprises: an upstream primer, a downstream primer, an RT Buffer, an RNA enzyme inhibitor, a reverse transcriptase, dNTPs, a PCR Buffer, Mgcl2, Taq DNA polymerase, a positive control, a negative control and RNase-free ddH2O. In the invention, specific primers are designed according to a coat protein CP gene sequence of the narcissus latent virus, which is detected by a reverse transcription-polymerase chain reaction (RT-PCR) technology. With the obvious advantages of rapidity, accuracy, sensitivity, strong operability and simple kit preparation method, the kit and method of the invention are not only suitable for ports of China to conduct rapid detection on the narcissus latent virus of entry and exit narcissus, and also can be used for narcissus latent virus detection and diagnosis, epidemic surveillance as well as early warning and prediction, thus having broad application prospects.
Description
Technical field
The present invention relates to a kind of narcissus cryptovirus detection kit and detection method thereof, belong to the Plant Quarantine technical field, be not only applicable to the Check and Examination of Port sanitary authority to the rapid detection of narcissus cryptovirus, also can be applicable to agricultural sector to real-time monitoring and the early-warning and predicting of narcissus cryptovirus.
Background technology
The narcissus cryptovirus (
Narcissus latent virus, NLV) belong to marmor upsilon section (
Potyviridae), three-bristle cudrania orange Tobamovirus (
Macluravirus) member.The virion form is straight line or slight bending wire, the about 650nm * 13nm of size of particles.Viral genome is sense single stranded rna, and total length is not appeared in the newspapers as yet.NLV mainly propagates by juice contact, aphid perishability mode.This virus can infect Amaryllidaceae (
Amaryllidaceae), Iridaceae (
Iridaceae), Amaranthaceae (
Amaranthaceae), Solanaceae (
Solanaceae), Papilionaceae (
Papilionaceae) plant, the natural host of having reported comprise daffodil (
Narcissus pseudonarcissus), iris (
Iris xiphium), gladiolus (
Gladiolus hybrids), the uncommon lily of root (
Nerine sarniensis).NLV usually with other viral mixed infection narcissuses, cause that blade tip produces the yellow streak of chlorisis after infecting narcissus separately, cause during compound infecting the narcissus plant to occur that floral leaf, mottled, yellow, necrosis, flower arrow reduce, fragrance is thin out with other viruses and downgrade symptom such as deformity, cause the narcissus commodity value to reduce significantly, have a strong impact on or directly lose ornamental value.NLV is distributed in the narcissus cultivation area of Britain, Holland, Germany, Italy, Israel, Australia, China.Because narcissus cryptovirus (NLV) can and be planted ball and carry out long-distance communications with the plant that catches an illness; therefore strengthen the research of this virus detection techniques; set up quick, accurate and sensitive detection method for generation and the diffusion of this virus of prevention; protection narcissus production safety is extremely important.
At present, the detection method of narcissus cryptovirus (NLV) comprises that mainly biology is measured, serology detects and Electronic Speculum is identified.The biology measuring method identifies that NLV is by relevant hosts such as inoculation New Zealand spinach, Globeamaranth Flower, elder brother's promise lamb's-quarters, Kidney bean and Cleveland cigarettes, judge according to the symptom that produces then, wherein produce circular local chlorisis spot at the New Zealand spinach blade, symptom shows as local lesion on Globeamaranth Flower and elder brother's promise lamb's-quarters, the irregular chlorisis spot (Xie Lianhui etc. of system then appear on Kidney bean and Cleveland tobacco leaf, Yunnan Prov Agriculture University's journal, 1990,5(1): 17～20).The shortcoming of biology measuring method is to need special Isolation warm house or solarium, and sense cycle is long, and the result is subject to the external factor influence, and detecting reliability is not high.It mainly is to adopt indirect ELISA method (Clark etc., Australasian Plant Pathology, 2000,29:227～229 that serology detects; Xie Lianhui etc., Yunnan Prov Agriculture University's journal, 1990,5(1): 17～20), react to measure by antigen, antibody generation immunologic opsonin, compare with the biology measuring method, this method weak point detection time, specificity and sensitivity obviously improve, but this method needs the antiserum(antisera) of high specificity, good stability, causes detecting cost because Antiserum Preparation is comparatively complicated and improves.In addition, serology detects the viral sample that serological relation is arranged for the low sample of NLV content and other and NLV, may cause omission or false retrieval.The Electronic Speculum evaluation has fast, direct advantage, but needs expensive plant and instrument and the professional who possesses certain operating skill, therefore is difficult to penetration and promotion.In recent years, more and more be applied to the plant virus detection range based on the RT-PCR molecular detecting method of nucleic acid level, this method is compared with above-mentioned detection method, and the time is faster, sensitivity is higher, specificity is stronger, the result is more reliable.But up to the present, very few about the report of narcissus cryptovirus (NLV) molecular detecting method, do not see as yet so far to be specifically applied to the molecular detection kit that NLV detects.
Summary of the invention
The object of the present invention is to provide a kind of narcissus cryptovirus detection kit and detection method thereof, overcome the defective and the deficiency that exist in the prior art, can be to the evaluation of quarantining fast and accurately of narcissus cryptovirus on pass in and out narcissus and the field narcissus.
Technical solution of the present invention is as follows:
(1) a kind of detection kit for the detection of narcissus cryptovirus is characterized in that described test kit comprises:
1) upstream primer: concentration is 10 μ mol/L, and primer sequence is 5 '-CGAACAAAGCAAGCGAACT-3 ';
2) downstream primer: concentration is 10 μ mol/L, and primer sequence is 5 '-AAGATAGGGCCTCTGGTCAAC-3 ';
3）RT Buffer：5×；
4) RNA enzyme inhibition factor: concentration is 40U/ μ L;
5) ThermoScript II: concentration is 200U/ μ L;
6) dNTPs: concentration is 10mmol/L;
7）PCR Buffer：10×；
8) Mgcl
2: concentration is 25mmol/L;
9) Taq archaeal dna polymerase: concentration is 5U/ μ L;
10) positive control sample of narcissus cryptovirus;
11) do not contain the negative control sample of narcissus cryptovirus;
12）RNase-free ddH
2O。
(2) detection method of above-mentioned narcissus cryptovirus detection kit is characterized in that, may further comprise the steps:
1) reverse transcription reaction: the total RNA 2 μ L of adding testing sample, concentration are downstream primer 1 μ L and the RNase-free ddH of 10 μ mol/L in the PCR pipe
2O 5 μ L, 70 ℃ of water-bath 10min, rapid ice bath 5min, add following reagent again: 5 * RT Buffer, 2.5 μ L, concentration are that 10mmol/L dNTPs 1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ L RNA enzyme inhibition factor 0.5 μ L.42 ℃ of water-bath 60min naturally cool to room temperature behind 70 ℃ of water-bath 10min, synthetic cDNA;
2) PCR reaction: get the synthetic cDNA 3 μ L of step 1), adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 * PCR Buffer, 2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that 10 μ mol/L upstream primers, 1 μ L, concentration are 10 μ mol/L downstream primers, 1 μ L, RNase-free ddH
2O 14.5 μ L, making the reaction cumulative volume is 25 μ L; Mixed reaction solution, 94 ℃ of pre-sex change 3min, then 94 ℃ of sex change 45s, 55 ℃ of annealing 50s, 72 ℃ extend 60s, totally 35 circulations so, 72 ℃ are continued to extend 10min after last loop ends, reaction finishes;
3) after pcr amplification product electrophoresis detection: PCR reaction finishes, get PCR product 10 μ L and detect with 1.5% agarose gel electrophoresis, observe and the record experimental result at gel imaging system behind the ethidium bromide staining; Contain the electrophoresis detection result of narcissus cryptovirus sample for bright DNA band occurs at the 829bp place.
The present invention is according to the coat protein CP gene order design Auele Specific Primer of narcissus cryptovirus, through a large amount of screening experiments, optimal upstream primer and downstream primer have been determined, optimum response system and response procedures have been determined by optimization again, detection narcissus cryptovirus that can be quick, accurate, sensitive.Compared to prior art, narcissus cryptovirus detection kit provided by the present invention and detection method beneficial effect thereof are: 1) detection time is fast: compare with traditional biology measuring method, need not narcissus is cultivated, can directly detect each positions such as the kind ball of narcissus, blade, flowers, and whole testing process can be finished in general 6 hours, had saved detection time greatly; 2) high specificity, highly sensitive: compare with the serology detection method, specificity of the present invention is stronger, sensitivity is higher, not only narcissus cryptovirus and other virus-specifics can be made a distinction, and can accurately identify the narcissus cryptovirus of low levels in the narcissus sample; 3) the detection kit making method is simple, use is economical, has good actual application and is worth.
Description of drawings
Fig. 1 is the narcissus cryptovirus detected result of embodiment 2.Wherein 1: positive control; 2: the sample that carries the narcissus cryptovirus; 3: negative control.
Fig. 2 be embodiment 3 specificity checking result.Wherein 1: healthy narcissus sample; 2: narcissus mosaic virus (NMV) sample; 3: daffod bar virus (NYSV) sample; 4: the slow season yellow of narcissus virus (NLSYV) sample; 5: arabis mosaic virus (ArMV) sample; 6: narcissus cryptovirus (NLV) sample.
Embodiment
Below the invention will be further described by specific embodiment.
Embodiment 1:The configuration of narcissus cryptovirus detection kit (10 detection limits)
1) upstream primer: 10 μ mol/L, 1 pipe (30 μ L);
2) downstream primer: 10 μ mol/L, 1 pipe (30 μ L);
3) RT Buffer:5 *, 1 the pipe (30 μ L);
4) RNA enzyme inhibition factor: 40U/ μ L, 1 pipe (5 μ L);
5) ThermoScript II: 200U/ μ L, 1 pipe (5 μ L);
6) dNTPs:10mmol/L, 1 pipe (30 μ L);
7) PCR Buffer:10 *, 1 the pipe (30 μ L);
8) Mgcl
2: 25mmol/L, 1 pipe (30 μ L);
9) Taq archaeal dna polymerase: 5U/ μ L, 1 pipe (5 μ L);
10) positive control sample of narcissus cryptovirus, 1 pipe (20 μ L);
11) do not contain the negative control sample of narcissus cryptovirus, 1 pipe (20 μ L);
12) RNase-free ddH
2O, 1 pipe (1mL).
Embodiment 2:The detection method of narcissus cryptovirus detection kit
The detection method of above-mentioned narcissus cryptovirus detection kit may further comprise the steps:
1) reverse transcription reaction: the total RNA 2 μ L of adding testing sample, concentration are downstream primer 1 μ L and the RNase-free ddH of 10 μ mol/L in the PCR pipe
2O 5 μ L, 70 ℃ of water-bath 10min, rapid ice bath 5min, add following reagent again: 5 * RT Buffer, 2.5 μ L, concentration are that 10mmol/L dNTPs 1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ L RNA enzyme inhibition factor 0.5 μ L.42 ℃ of water-bath 60min naturally cool to room temperature behind 70 ℃ of water-bath 10min, synthetic cDNA;
2) PCR reaction: get the synthetic cDNA 3 μ L of step 1), adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 * PCR Buffer, 2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that 10 μ mol/L upstream primers, 1 μ L, concentration are 10 μ mol/L downstream primers, 1 μ L, RNase-free ddH
2O 14.5 μ L, making the reaction cumulative volume is 25 μ L; Mixed reaction solution, 94 ℃ of pre-sex change 3min, then 94 ℃ of sex change 45s, 55 ℃ of annealing 50s, 72 ℃ extend 60s, totally 35 circulations so, 72 ℃ are continued to extend 10min after last loop ends, reaction finishes;
3) after pcr amplification product electrophoresis detection: PCR reaction finishes, get PCR product 10 μ L and detect with 1.5% agarose gel electrophoresis, observe and the record experimental result at gel imaging system behind the ethidium bromide staining; Contain the DNA band (Fig. 1) of electrophoresis detection result for occurring at the 829bp place becoming clear of narcissus cryptovirus sample, otherwise do not have.
Embodiment 3:The specificity checking of narcissus cryptovirus detection kit
1) extraction of the total RNA of narcissus sample: respectively with carry narcissus cryptovirus (NLV), narcissus mosaic virus (
Narcissus mosaic virus, NMV), daffod bar virus (
Narcissus yellow stripe virus, NYSV), the slow season yellow of narcissus virus (
Narcissus late season yellows virus, NLSYV), arabis mosaic virus (
Arabis mosaic virus, narcissus sample ArMV) is material, respectively gets 0.1g and places mortar, adds 1mL PBST damping fluid and grinds, 4 ℃, the centrifugal 5min of 10000g gets supernatant and supernatant liquor is transferred to rapidly in the 1.5mL centrifuge tube of sterilization, add 1mL TrizoL reagent, behind the thermal agitation, room temperature leaves standstill 5min; 4 ℃, the centrifugal 10min of 12000g gets supernatant; Add chloroform 300 μ L, concuss 15s, room temperature leaves standstill 5min, and 4 ℃, the centrifugal 15min of 12000g gets the upper strata water; Add isopyknic Virahol, put upside down behind the mixing and leave standstill 15min under the room temperature, 4 ℃, the centrifugal 10min of 12000g abandons supernatant; The washing with alcohol that adds 1mL 75% precipitates 2 times, each 4 ° of C, and the centrifugal 3min of 7500g abandons supernatant; After the RNA precipitation drying, with 20 μ L RNase-free ddH
2The O dissolving.
2) reverse transcription reaction: the total RNA 2 μ L of adding testing sample, concentration are downstream primer 1 μ L and the RNase-free ddH of 10 μ mol/L in the PCR pipe
2O 5 μ L, 70 ℃ of water-bath 10min, rapid ice bath 5min, add following reagent again: 5 * RT Buffer, 2.5 μ L, concentration are that 10mmol/L dNTPs 1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ L RNA enzyme inhibition factor 0.5 μ L.42 ℃ of water-bath 60min naturally cool to room temperature behind 70 ℃ of water-bath 10min, synthetic cDNA;
3) PCR reaction: get step 2) synthetic cDNA 3 μ L, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 * PCR Buffer, 2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that 10 μ mol/L upstream primers, 1 μ L, concentration are 10 μ mol/L downstream primers, 1 μ L, RNase-free ddH
2O 14.5 μ L, making the reaction cumulative volume is 25 μ L; Mixed reaction solution, 94 ℃ of pre-sex change 3min, then 94 ℃ of sex change 45s, 55 ℃ of annealing 50s, 72 ℃ extend 60s, totally 35 circulations so, 72 ℃ are continued to extend 10min after last loop ends, reaction finishes;
4) after pcr amplification product electrophoresis detection: PCR reaction finishes, get PCR product 10 μ L and detect with 1.5% agarose gel electrophoresis, observe and record experimental result (Fig. 2) at gel imaging system behind the ethidium bromide staining.From Fig. 2 as seen, only bright DNA band appears in narcissus cryptovirus sample at the 829bp place, and other viral sample and healthy narcissus sample all do not have, and illustrate that test kit of the present invention has good specificity.
Embodiment 4:The detection of narcissus cryptovirus on the inward narcissus of Britain
10 parts in Britain's narcissus sample (sample A-J) that intercept and capture at picked at random China port detects after employing embodiment 3 methods are extracted the total RNA of sample, verifies with Indirect ELISA method simultaneously.As seen from Table 1, have 3 parts to detect narcissus cryptovirus (NLV) in 10 parts of Britain's narcissus samples, recall rate is 30%, and this result conforms to Indirect ELISA detected result.
The table 1 Britain narcissus test result of samples that enters the territory
Annotate :+expression detects NLV;-expression does not detect NLV
Among above-described embodiment 1-4 in the concentration of employed each material and the technical solution of the present invention concentration of each listed material corresponding identical.
＜110〉Inspection ﹠ Quarantine Technology Center of Fujian Entry-Exit Inspection ﹠ Quar
＜120〉narcissus cryptovirus detection kit and detection method thereof
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<170> PatentIn version 3.3
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cgaacaaagc aagcgaact 19
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Claims (2)
1. narcissus cryptovirus detection kit, it is characterized in that described test kit comprises: 1) upstream primer: 10 μ mol/L, primer sequence are 5 '-CGAACAAAGCAAGCGAACT-3 '; 2) downstream primer: 10 μ mol/L, primer sequence are 5 '-AAGATAGGGCCTCTGGTCAAC-3 '; 3) RT Buffer:5 *; 4) RNA enzyme inhibition factor: 40U/ μ L; 5) ThermoScript II: 200U/ μ L; 6) dNTPs:10mmol/L; 7) PCR Buffer:10 *; 8) MgCl
2: 25mmol/L; 9) Taq archaeal dna polymerase: 5U/ μ L; 10) positive control sample of narcissus cryptovirus; 11) do not contain the negative control sample of narcissus cryptovirus; 12) RNase-free ddH
2O.
2. utilize the detection method of the described narcissus cryptovirus of claim 1 detection kit, it is characterized in that, may further comprise the steps:
1) reverse transcription reaction: the total RNA2 μ of adding testing sample L, concentration are downstream primer 1 μ L and the RNase-free ddH of 10 μ mol/L in the PCR pipe
2O5 μ L, 70 ℃ of water-bath 10min, rapid ice bath 5min, add following reagent again: 5 * RT Buffer2.5 μ L, concentration are that 10mmol/L dNTPs1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ L RNA enzyme inhibition factor 0.5 μ L, 42 ℃ of water-bath 60min, naturally cool to room temperature behind 70 ℃ of water-bath 10min, synthetic cDNA;
2) PCR reaction: get the synthetic cDNA3 μ L of step 1), adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs0.5 μ L, 10 * PCR Buffer2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that 10 μ mol/L upstream primers, 1 μ L, concentration are 10 μ mol/L downstream primers, 1 μ L, RNase-free ddH
2O14.5 μ L, making the reaction cumulative volume is 25 μ L; Mixed reaction solution, 94 ℃ of pre-sex change 3min, then 94 ℃ of sex change 45s, 55 ℃ of annealing 50s, 72 ℃ extend 60s, totally 35 circulations so, 72 ℃ are continued to extend 10min after last loop ends, reaction finishes;
3) after pcr amplification product electrophoresis detection: PCR reaction finishes, get PCR product 10 μ L and detect with 1.5% agarose gel electrophoresis, observe and the record experimental result at gel imaging system behind the ethidium bromide staining; Contain the electrophoresis detection result of narcissus cryptovirus sample for bright DNA band occurs at the 829bp place.
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| CN 201210195687 CN102732642B (en) | 2012-05-14 | 2012-06-13 | Narcissus latent virus detection kit and detection method thereof |
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| CN 201210195687 CN102732642B (en) | 2012-05-14 | 2012-06-13 | Narcissus latent virus detection kit and detection method thereof |
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| CN103484567B (en) * | 2013-09-13 | 2014-09-24 | 福建出入境检验检疫局检验检疫技术中心 | Narcissus late season yellows virus detection kit and method |
| CN104120192B (en) * | 2014-06-26 | 2016-03-16 | 福建出入境检验检疫局检验检疫技术中心 | Narcissus degenerates viral RT-Nested PCR detection kit and detection method thereof |
| CN104059999B (en) * | 2014-07-09 | 2016-08-24 | 福建省农业科学院作物研究所 | A kind of napiform root Rhizoma Iridis Tectori Viral diagnosis primer and method |
| CN104988245B (en) * | 2015-07-28 | 2018-05-08 | 中华人民共和国北京出入境检验检疫局 | Detection dahlia hides the RT-qPCR detection kits and oligonucleotides of viroid |
| CN107955841B (en) * | 2017-12-15 | 2021-05-11 | 福建出入境检验检疫局检验检疫技术中心 | Multiplex RT-PCR detection kit for simultaneously detecting seven narcissus RNA viruses and detection method thereof |
| CN107828918B (en) * | 2017-12-15 | 2021-11-16 | 福州海关技术中心 | Dual RT-PCR (reverse transcription-polymerase chain reaction) detection kit for Nihonin latent virus and narcissus common latent virus and detection method thereof |
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