CN102382890A - Pythium splendens braun fluorescence quantitative PCR detection reagent and detection kit and application - Google Patents
Pythium splendens braun fluorescence quantitative PCR detection reagent and detection kit and application Download PDFInfo
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Abstract
The invention discloses a pythium splendens braun fluorescence quantitative PCR detection reagent, a detection kit and an application; the detection reagent comprises a pair of specific primers with sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and a specific fluorescence probe with a sequence as shown in SEQ ID NO: 3; an amplification target fragment has a length of 94 bp, and the nucleotide sequence of the amplification target fragment is shown in SEQ ID NO: 4; the detection kit comprises components of a CTAB extract, a PCR buffer containing the primers and the probe, TaqDNA polymerase, positive control liquid, and quantitative standard liquid; the detection method comprises the extraction of total RNA and the fluorescence PCR reaction; detection performed by using the pythium splendens braun fluorescence quantitative PCR detection reagent overcomes disadvantages of time consumption, easy pollution, and electrophoresis detection necessity after amplification of routine PCR, can realize rapid and accurate qualitative and quantitative detection of pythium splendens braun in a sample, and has the advantages of simplicity, easy operations, intuitive results, high sensitivity, good repeatability, and the like.
Description
Technical field
The present invention relates to the detection technique of pathogenic fungi, be specifically related to relate to oil palm da mping-off fungi fluorescence quantitative PCR detection reagent and detection kit and application.
Background technology
The oil palm da mping-off fungi (
Pythium splendens), claim that again magnificent corruption is mould, be a kind of important disease on the oil palm, this germ host range is very wide, remove infect oil palm (
Elaeisguineensis) in addition, can also infect corn (
Zeamays), Sunflower Receptacle (
Helianthusannuus), barley (
Hordeumvulgare), cucumber (
Cucumissativus), sweet potato (
Ipomoeabatatas), wheat (
Triticumaestivum), broad bean (
Viciafaba), cowpea (
Vignasinensis), Capsicum (
CapsicumSpp.), tobacco (
Nautilocalyxlynchei), Pelargonium (
PelargoniumSpp.), begonia (
BegoniaKind of plant surplus in the of 250 such as spp.) causes whole strain dehydration wilting of host seedling and root, basal part of stem, the otch browning rots and symptoms such as cortex comes off.At present; This germ mainly is distributed in Hawaii, Florida, the Pennsylvania of Belgium, France, Germany, Italy, Holland, Japan, Tanzania, the Congo and the U.S.; Once there was report in domestic Taiwan, Hainan; But other areas do not see and report, are China Plant Quarantine property harmful organism that enters the territory.
The detection method of oil palm da mping-off fungi comprises that mainly the traditional morphological characteristic combines biological assay and Auele Specific Primer PCR, ribosomal dna sequence analysis, the RFLP of Physiology and biochemistry proterties etc.During biological assay method consumption expenses of labour, and some morphological specificity is unstable, possibly degenerate or disappear, and the oil palm da mping-off fungi is the heterothally type, and unisexuality is cultivated can not produce sexual organ, has more increased its evaluation difficulty; The Auele Specific Primer PCR sensitivity of developments such as Wang is not very high, can't detect the DNA sample of lower aq; Ribosomal dna sequence is analyzed, though it is big to obtain the bioinformation amount, experimental implementation is loaded down with trivial details, and sense cycle is long; RFLP can save many loaded down with trivial details operations in the sequential analysis; But need carry out plurality of enzymes and cut also time-consuming taking a lot of work; And above molecular detecting method all will carry out PCR aftertreatments such as electrophoresis, and contact toxic reagent ethidium bromide also is prone to crossed contamination takes place and causes false positive results.
Quantitative fluorescent PCR (Real-time fluorescent PCR) is the method that the round pcr that grew up in recent years combines with fluoroscopic examination; Its principle is to use the fluorescent substance of ability specific mark nucleotide sequence; The accumulation that utilizes fluorescent signal is the whole PCR process of monitoring in real time, has that sense cycle is short, a specificity and highly sensitive and need not advantage such as PCR aftertreatment.With the TaqMan hydrolysis probes is example, probe one end mark fluorescent group, and the other end mark quenching group, under the common state, fluorophor and quenching group generation FRET phenomenon, the fluorescence of fluorophor is absorbed by quenching group.When PCR got into annealing stage, what fluorescent probe was special was combined between two primers, was cut by 5 of polysaccharase ' end 5 prime excision enzyme activity in the extension stage.Fluorophor separates with quenching group, and instrument detecting goes out fluorescence.The amplification that is accompanied by pcr template owing to fluorescent signal increases, and therefore can whether increase to confirm whether template increases according to fluorescent signal.The variation of fluorescent PCR appearance continuous detection fluorescent signal in reaction process, when fluorescent signal was strengthened to a certain threshold value, the cycle index (Ct) of this moment just went on record.The logarithmic value of starting template amount has strict linear relationship in this Ct value and the reaction system.Therefore, except can whether containing the target dna in qualitative definite reaction system, can also carry out relative quantification to the template DNA in the reaction system.Therefore, research set up special, responsive, can be to low levels
P.
SplendensThe method that directly detects has important application value at aspects such as quarantine, diagnosis, molecule epidemic disease-ology research.
Summary of the invention
Technical problem to be solved by this invention provide a kind of to the oil palm da mping-off fungi have the specificity height, susceptibility is strong, quantitatively and the fluorescence quantitative PCR detection reagent of rapid detection.
The present invention also provides detection kit and the application that contains this detection reagent.
The present invention is that selection oil palm da mping-off fungi ITS district conservative fragments is a target, uses primer Express 5.0 softwares, design synthetic primer and probe.The many of design are carried out the best pairing screening experiment to primer and probe, obtain only primer and probe.
Oil palm da mping-off fungi fluorescence quantitative PCR detection reagent of the present invention comprises a pair of Auele Specific Primer and a specificity fluorescent probe; Amplification target fragment length is 94bp; A pair of specific primer sequence pyspF:5 '-ttaaatggac agggtctttc tat-3 ' and pyspR:5 '-tgccgaagtc gccaaaag-3 ', specific probe sequence pyspT:5 '-FAM-cgagcaccac acttcacaca g-TAMRA-3 '.
The primer of oil palm da mping-off fungi fluorescence quantitative PCR detection reagent of the present invention and probe preparation:
One, selecting oil palm da mping-off fungi ITS district conservative fragments is target, and the amplification target nucleotide sequence of this conservative fragments is shown in SEQ ID NO:4;
Two, use primer Express 5.0 softwares, design synthetic primer and probe;
Three, the synthetic employing β-acetonitrile phosphamide chemical synthesis of primer and probe uses full-automatic dna synthesizer to carry out the synthetic of OligoDNA; It is synthetic that primer and probe are given birth to the worker by Shanghai;
Four, the synthetic two ends fluorescent mark that carries out simultaneously of probe, the fluorescence report group of probe 5 ' end mark is FAM, the fluorophor of 3 ' end mark is TAMRA; Fluorescence report group FAM also can use luminophores such as TET, VIC, JOE to replace;
Five, will design that synthetic is many primer and probe will be carried out the best pairing screening experiment after, tentatively confirm candidate's primer and probe;
Six,, simultaneous test preferred and proof test through a large amount of reaction conditions, and, obtain good described primer of amplification efficiency and specificity and described probe through the detection application evaluation of a large amount of actual samples.
The detection kit of forming by oil palm da mping-off fungi fluorescence quantitative PCR detection reagent of the present invention; Form by following component: CTAB extracting solution (solution I) 30 mL * 2 bottles; This CTAB extracting solution is to contain the NaC1 that final concentration is 0.7mol/L, the Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HC1) that final concentration is 100mmol/L, pH8.0, the YD 30 (EDTA) that final concentration is 20mmol/L, the Vinylpyrrolidone polymer 360 (PVP-360) that final concentration is 10g/L; Final concentration is that cetyl trimethylammonium bromide (CTAB) and the volume final concentration of 20g/L is the aqueous solution of the beta-mercaptoethanol of 2% (V:V); 2 * one one-step fluorescence quantitative PCR damping fluids (solution II), 1.25 mL that contain the said probe that said primer that concentration all is 1 μ mol/L and concentration is 0.4 μ mol/L; Concentration is Taq archaeal dna polymerase (solution III) the 50 μ L of 5U/ μ L; Sterilization deionized water (solution IV) 1.25 mL * 2, contain the oil palm da mping-off fungi (
P.
Splendens) positive control solution (solution V) the 50 μ L of positive DNA, do not contain
P.
SplendensThe negative controls of positive DNA (sterilization deionized water, solution VI) 50 μ L, containing concentration is 100ng/ μ L's
P.
SplendensQuantitative criterion liquid (solution VII) the 25 μ L of positive DNA.One one-step fluorescence quantitative PCR damping fluid, Taq archaeal dna polymerase are all purchased the company in TAKARA.Positive control solution and quantitative criterion liquid are to prepare according to the oil palm da mping-off fungi DNA that the CTAB method is extracted.
Method with oil palm da mping-off fungi fluorescence quantitative detection kit detection oil palm da mping-off fungi of the present invention the steps include:
1) total DNA extraction: the exsiccant mycelia 0.5g that learns from else's experience, put into 1.5mL and be immersed in the centrifuge tube in the liquid nitrogen, pulverize with the plastics pestle; Add 600 μ L CTAB extracting solutions again, 65 ℃ of water-bath 30min put upside down mixing frequently, the centrifugal 5min of 10000 r/min; Get supernatant, add isopyknic chloroform of this supernatant and primary isoamyl alcohol mixed solution, put upside down centrifuge tube to forming the milkiness shape, 4 ℃ of centrifugal 5min of 10000rpm, chloroform and primary isoamyl alcohol volume ratio are 24:1 in said chloroform and the primary isoamyl alcohol mixed solution; Get supernatant in new 1.5mL centrifuge tube, adding ice-cold ethanol and concentration is 3 mol/L, P
HBe 6.0 sodium-acetate (NaAc) solution, the volume of said sodium acetate soln is 1/10 of this supernatant volume, and said ice-cold alcoholic acid volume is 2.5 times of this supernatant volume; After putting upside down mixing, place-20 ℃ to preserve 30 min, 4 ℃ of 10000 centrifugal 10min of rpm; Abandon supernatant, the use mass concentration is 70% ethanol rinsing deposition secondary, after drying; Add 100 μ L sterilization deionized water dissolving deposition, obtain sample DNA, 4 ℃ of preservations are subsequent use;
2) PCR reaction: reaction system 25 μ L: 2 * one one-step fluorescence quantitative PCR damping fluids, the 12.5 μ L that contain the said probe that said primer that concentration all is 1 μ mol/L and concentration is 0.4 μ mol/L; Concentration is the Taq archaeal dna polymerase 0.5 μ L of 5U/ μ L; Sample DNA 0.5 μ L, the sterilization deionized water is supplied surplus; Reaction parameter: 50 ℃ of preheating 2 min; 95 ℃ of preparatory sex change 10 min; 95 ℃ of 15s, 60 ℃ of l min, 40 circulations; There is fluorescent signal then to be the oil palm da mping-off fungi as if sample DNA in the PCR reaction; Substitute sample DNA with positive control solution or negative controls simultaneously and carry out the PCR reaction, can further qualitative comparative analysis, substitute sample DNA with quantitative criterion liquid and carry out the PCR reaction, can carry out the regression curve of sample DNA and according to quantitative criterion liquid 10
-1～10
-7The sample DNA quantitative analysis is made in the doubly regression curve of dilution comparison.
Oil palm da mping-off fungi fluorescence quantitative PCR detection reagent of the present invention is that round pcr is combined with fluoroscopic examination; Make up and filter out above-mentioned amplification efficiency and good a pair of primer and the probe of specificity; Overcome conventional PCR time-consuming, be prone to pollute and amplification after need shortcoming such as electrophoresis detection; Can carry out fast and accurately qualitative and quantitative to the oil palm da mping-off fungi in the sample and detect, have advantages such as simple, easy to operate, visual result, susceptibility height, good reproducibility.Concrete advantage is following:
1, the present invention compares with the detection reagent of conventional PCR, has special, responsive advantage; Can be to low levels in the sample (can 8 times of dilutions)
P.
Splendens, latent disease or the host that carries disease germs accurately detect, and are a kind of very suitable
P.
SplendensThe method that detects;
2, the present invention compares with the detection reagent of conventional PCR, has advantage applied widely, safe in utilization; Can be in the plant
P.
SplendensCarry out rapid detection, applied widely; The present invention has overcome the product palpus electrophoresis detection of conventional PCR, the shortcoming of contact toxic reagent, has reduced Biosafety hidden danger, has improved safety in utilization;
3, the present invention compares with the detection reagent of conventional PCR, has the advantage of the big and rapid detection of detection limit; Can carry out a large amount of sample detection simultaneously, have high flux property, can reduce the risk of DNA or PCR product pollution; The detection time of conventional PCR is general more than 6 hours, the electrophoretic analysis after the present invention need not to increase, and the sample detection time is within 4 hours;
4, compare with conventional PCR, quantitative fluorescent PCR is made quantitative, an objective estimation, judges the positive, feminine gender and suspicious result, and the Ct value is 40.0 can confirm as positive with negative threshold value.It is few to the more important thing is that quantitative fluorescent PCR is specially adapted to content of molds, is difficult to the rapid detection of germ in the sample of separation and Culture;
5, the test kit of this reagent place composition can be made the qualitative and quantitative analysis result within 4 hours after receiving sample.Detect with this PCR kit for fluorescence quantitative
P.
SplendensBe a kind of special, sensitivity and stable detection method.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
1, design primer and probe
It is target that the present invention selects oil palm da mping-off fungi ITS district conservative fragments, through what report among the GenBank
P.
SplendensAnd allied species ITS sequence is carried out homology analysis relatively, selected oil palm da mping-off fungi ITS district's conservative fragments (94bp), application primer Express5.0 software, design primer and probe.The synthetic employing β of primer and probe-acetonitrile phosphorous acid amination synthesis method; Use full-automatic dna synthesizer to carry out the synthesising probing needle of OligoDNA; The synthetic two ends fluorescent mark that carries out simultaneously; The fluorescence report group of probe 5 ' end mark is FAM, 3 ' that hold mark is not fluorescent quenching group TAMRA.After will designing synthetic is many, primer and probe being carried out the best pairing screening experiment; Confirm a pair of primer and a probe that amplification efficiency and specificity are best; Primer sequence is respectively shown in SEQ ID NO:1 and SEQ ID NO:2; Probe sequence is shown in SEQ ID NO:3, and amplification target fragment length is 94bp, and its sequence is shown in SEQ ID NO:4.
2, stdn test kit component (25 μ L reaction * 100 times).
| Solution I | 30 mL * 2 bottle |
| Solution II | 1.25 mL |
| Solution III | 50μL |
| Solution IV | 1.25 mL * 2 |
| Solution V | 50μL |
| Solution VI | 50μL |
| Solution VII | 25μL |
3, CTAB method DNA extraction
Get oil palm da mping-off fungi mycelia 0.5g and put into 1.5mL and be immersed in the centrifuge tube in the liquid nitrogen, pulverize, add 600 μ L CTAB extracting solutions with the plastics pestle; 65 ℃ of water-bath 30 min, the centrifugal 5min of 10000 r/min gets supernatant; Add isopyknic chloroform/primary isoamyl alcohol mixed solution (v:v=24:1), put upside down mixing, 4 ℃ of 10000 centrifugal 5min of rpm; Get supernatant in new centrifuge tube; Add the NaAc solution (3 mol/L, pH 6.0) of this supernatant 1/10 volume and the ice-cold ethanol (saturated) of 2.5 times of volumes of this supernatant, place-20 ℃ to preserve 30min; 4 ℃ of 10000 centrifugal 10min of rpm abandons supernatant, and using mass concentration is 70 % ethanol rinsings deposition secondary; After drying; Add 100 μ L sterilization deionized water dissolving deposition, obtaining sample DNA or positive control and being made into concentration is 100ng/ μ L quantitative criterion liquid, and 4 ℃ of preservations are subsequent use; The final concentration of NaC1 is that the final concentration of 0.7mol/L, Tris-HC1 (pH8.0) is that the final concentration of 100mmol/L, EDTA is that the final concentration of 20mmol/L, PVP-360 is 10g/L in this CTAB extracting solution, and the final concentration of CTAB is that the volume final concentration of 20g/L and beta-mercaptoethanol is 2% (V:V).
4,
P.
SplendensFluorescent quantitative PCR
P.
SplendensQuantitative fluorescent PCR adopts 25 μ L volumetric reaction systems; In ABI7900 type quantitative real time PCR Instrument; Reaction system is: solution II 12.5 μ L, solution III 0.5 μ L, solution V or solution VI or solution VII or DNA sample 0.5 μ L; The sterilization deionized water is supplied surplus, carries out the PCR reaction by following reaction parameter: 50 ℃ of preheating 2 min; 95 ℃ of preparatory sex change 10min; 95 ℃ of 15s, 60 ℃ of l min, 40 circulations; With solution V and solution VI; Many to primer and probe best pairing screening experiment by above-mentioned reaction system and reaction parameter to what design; The primer of screening and the concentration of probe are accurately screened, to obtain minimum Ct value and higher fluorescence intensity increased value (Δ Rn).During each the detection, each sample detection is done 3 pipe parallel tests.Negative at negative control, when positive control is positive, whole test is effective, result's a pair of Auele Specific Primer of the present invention and a specificity fluorescent probe are that the Ct value is low, highly sensitive, are suitable primer and probe.Each sample must be done a plurality of backups, to carry out stability, replica test.
5,
P.
SplendensThe specificity of quantitative fluorescent PCR and sensitivity test: adopt the thorn corruption of allied species mould
P. spinosum, wide male rotten mould
P. dissotocum, different ancestor is rotten mould
P. heterothallium, the corruption of clock device is mould
P. vexans, abnormal female corruption is mould
P. irregulare, ultimate corruption is mould
P. ultimum, side is male rotten mould
P. paroecandram, the melon and fruit corruption is mould
P. aphanidermatum,
P. sylvaticum, P. takayamanum,
P. attrantheridiumAnd phytophthora Phytophthora nicotianae
Phytophthora nicotianae, palm mould
Phytophthora palmivoraWith some common root chain lattice spores
Alternaria radicina, Botrytis cinerea
Botrytis cinerea, subtilis
Bacillus subtilisThe DNA sample with
P.
SplendensThe DNA sample of bacterial strain carries out specificity and relatively detects.And will
P.
SplendensThe DNA sample of bacterial strain carries out 10 times of serial dilutions; The DNA sample of above-mentioned bacterial strains is carried out that conventional PCR detects (ordinary method) and by carrying out fluorescence quantitative PCR detection under above-mentioned steps 4 reaction systems and the same terms; The susceptibility that compares them, the primer that conventional PCR adopts is consistent with the primer of quantitative fluorescent PCR; Result's a pair of Auele Specific Primer of the present invention and a specificity fluorescent probe have only
P.
SplendensBacterial strain produces fluorescent signal, can be effective to 8 times of diluting reactions, so high specificity, highly sensitive.
6,
P.
SplendensThe stability of quantitative fluorescent PCR and replica test: when the circulation ratio of assessing fluorescence quantitative PCR detection oil palm da mping-off fungi method, stability; With positive and negative sample; Under by above-mentioned steps 4 reaction systems and the same terms; Carry out fluorescence quantitative PCR detection repeatedly, each sample of each test is done 6 parallel reaction tubess, repeats 12 times; Therefore a pair of Auele Specific Primer of the present invention and specificity fluorescent probe stability and good reproducibility.
7, the calculating of oil palm da mping-off fungi content in the foundation of the preparation of oil palm da mping-off fungi quantitative criterion liquid, typical curve and the sample: extract the DNA of oil palm da mping-off fungi with the CTAB method, with its OD of spectrophotometric determination
260It is quantitative that value is carried out dna profiling, OD
260Value is 1 to be equivalent to 50 μ g/mL double-stranded DNAs.Adopt the sterilization deionized water, the DNA that obtains extraction dilutes quantitatively to 100 ng/ μ L.These standard substance are carried out 10 times of serial dilutions, obtain 10
-1～10
-7Doubly the dna profiling of dilution is used for fluorescent quantitative PCR (by above-mentioned steps 4 reaction systems and reaction conditions), is the X axle with the logarithmic value of template concentrations, and the Ct value is made regression curve and drawn regression equation for the Y axle.Convert according to the result, can be worth it to contain the dna content of oil palm da mping-off fungi from the Ct of unknown sample.
8, the screening of primer and probe and concentration thereof: select the best concentration ratio of primer, probe, template, obtain the minimum Ct value and the highest Δ Rn of quantitative fluorescent PCR reaction, improve amplification efficiency and susceptibility.Primer concentration is increased progressively with 0.1 μ mol/L from 0.1 μ mol/L～1 μ mol/L, confirm the best final concentration of primer.Concentration and probe concentration increases progressively with 0.1 μ mol/L from 0.2 μ mol/L～1 μ mol/L, confirms the best final concentration of probe.The result is that the primer final concentration is 0.5 μ mol/L in the 25 μ L reaction systems, and the probe final concentration is that 0.2 μ mol/L is better.
9, to the detection of sample
To supplying test agent to carry out fluorescence quantitative PCR detection, the result shows that positive all has typical amplification curve, and the Ct value is less than 35.0, and negative control and negative sample all do not have typical amplification curve, do not have the Ct value yet.The stable and consistent as a result of three parallel tests.The proof fluorescence quantitative PCR detection
P.
SplendensHigh specificity, susceptibility height and good reproducibility.
9.1 total DNA extraction
(1) exsiccant mycelia 0.5 g that learns from else's experience puts into 1.5 mL and is immersed in the centrifuge tube in the liquid nitrogen, pulverizes with the plastics pestle;
(2) add 600 μ L solution I, 65 ℃ of water-bath 30 min put upside down mixing frequently, centrifugal 5 min of 10 000 r/min;
(3) get supernatant, add isopyknic chloroform/primary isoamyl alcohol (v:v=24:1), put upside down centrifuge tube to forming the not really fast layering of milkiness shape, 4 ℃ of 10 000 centrifugal 5 min of rpm;
(4) get supernatant in 1.5 new mL centrifuge tubes, add the NaAc (3 mol/L, pH 6.0) of this supernatant volume 1/10 and 2.5 times ice-cold ethanol, put upside down mixing after, place-20 ℃ to preserve 30 min.4 ℃ of 10000 centrifugal 10 min of rpm abandons supernatant;
(5) with 70 % ethanol rinsings deposition secondary, after drying, add 100 μ L sterilization deionized water dissolving deposition, 4 ℃ of preservations are subsequent use.
9.2PCR reaction
(1) 25 μ L reaction system: solution II 12.5 μ L, solution III 0.5 μ L, sample DNA 0.5 μ L, the sterilization deionized water is supplied surplus; Establish positive control and negative control 25 μ L reaction systems 2 pipes simultaneously;
(2) quantitative: as also to dilute laggard performing PCR reaction production standard curve, at least 7 extent of dilution of standard substance with standard substance.
9.3 amplification: in ABI7900 type quantitative real time PCR Instrument, undertaken: 50 ℃ of preheating 2 min by following reaction parameter; 95 ℃ of preparatory sex change 10 min; 95 ℃ of 15s, 60 ℃ of l min, 40 circulations.
9.4 interpretation of result and judgement
(1) interpretation of result condition enactment
The threshold line setting principle is adjusted according to the noise of instrument situation, is as the criterion with the vertex of threshold line just above normal negative sample amplification curve;
(2) quality control standard
Negative control does not have the Ct value, and does not have typical amplification curve.The Ct value of positive control should be less than 35.0, and typical amplification curve occurs, and 2 positive control amplification curves overlap basically, particularly near fluorescence threshold value (Ct value).Otherwise it is invalid that experiment this time is regarded as.The standard substance amplification curve of quantitative usefulness: typical amplification curve occurs, the exponential region is more obvious, 7 some tool favorable linearity scopes, and platform area is compiled in together, and relation conefficient is more than 0.99;
(3) result describes and judges
Negative: no Ct value or do not have amplification curve, represent no oil palm da mping-off fungi in the sample;
Positive: Ct value should be smaller or equal to 35.0, and typical amplification curve occurs, represent to have the oil palm da mping-off fungi in the sample;
Effective principle: the Ct value is reformed in 35.0 ~ 40.0 sample suggestion.The result that reforms does not have the Ct value or does not have typical amplification curve and is judged to feminine gender, otherwise is judged to the positive;
Quantitatively: the regression curve of sample DNA with according to the comparison of the regression curve of standard substance, it is quantitative to make sample DNA.
< 110>Ningbo Institute of Inspection and Quarantine Science Technology
< 120>oil palm da mping-off fungi fluorescence quantitative PCR detection reagent and detection kit and application
<160>?4
<170>?PatentIn?version?3.1
<210>?1
<211>?23
<212>?DNA
< 213>artificial sequence
<220>
< 223>be designed for the Auele Specific Primer that detects the oil palm da mping-off fungi according to ITS district conservative fragments
<400>?1
ttaaatggac?agggtctttc?tat 23
<210>2
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>be designed for the Auele Specific Primer that detects the oil palm da mping-off fungi according to ITS district conservative fragments
<400>2
tgccgaagtc?gccaaaag 18
<210>3
<211>?21
<212>?DNA
< 213>artificial sequence
<220>
< 223>be designed for the specific probe that detects the oil palm da mping-off fungi according to ITS district conservative fragments
<400>3
FAM-cgagcaccac?acttcacaca?g-TAMRA 21
<210>4
<211>?94
<212>?DNA
< 213>target fragment of amplification oil palm da mping-off fungi
<400>4
ttaaatggac agggtctttc tatggtctgt gtgaagtgtg gtgctcgaaa 50
ggcagtgatt ttcggatcgc tggcggcttt tggcgacttc ggca 94
Claims (3)
1. oil palm da mping-off fungi fluorescence quantitative PCR detection reagent; Comprise a pair of Auele Specific Primer and a specificity fluorescent probe; Amplification target fragment length is 94bp; The nucleotide sequence that it is characterized in that this a pair of Auele Specific Primer is shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequence of this specific probe is shown in SEQ ID NO:3, and 5 of this specific probe ' end is marked with fluorescence report group FAM; 3 ' end is marked with not fluorescent quenching group TAMRA, and the nucleotide sequence of amplification target fragment is shown in SEQ ID NO:4.
2. the test kit of forming by the described oil palm da mping-off fungi of claim 1 fluorescence quantitative PCR detection reagent; It is characterized in that forming: CTAB extracting solution 30mL * 2 bottles by following component; 2 * one one-step fluorescence quantitative PCR damping fluid 1.25mL that contain the said probe that said primer that concentration all is 1 μ mol/L and concentration is 0.4 μ mol/L; Concentration is the Taq archaeal dna polymerase 50 μ L of 5U/ μ L; Sterilization deionized water 1.25 mL * 2; The positive control solution 50 μ L that contain the positive DNA of oil palm da mping-off fungi; The negative controls 50 μ L that do not contain the positive DNA of oil palm da mping-off fungi; Contain concentration and be the quantitative criterion liquid 25 μ L of the positive DNA of oil palm da mping-off fungi of 100ng/ μ L, said CTAB extracting solution is to contain the NaC1 that final concentration is 0.7mol/L, the Tri(Hydroxymethyl) Amino Methane Hydrochloride that final concentration is 100mmol/L, the YD 30 that final concentration is 20mmol/L, the Vinylpyrrolidone polymer 360 that final concentration is 10g/L, and final concentration is that cetyl trimethylammonium bromide and the volume final concentration of 20g/L is the water-soluble of 2% beta-mercaptoethanol.
3. utilize the described oil palm da mping-off fungi of claim 2 fluorescence quantitative detection kit to detect the method for oil palm da mping-off fungi, it is characterized in that step is:
1) total DNA extraction: the exsiccant mycelia 0.5g that learns from else's experience, put into 1.5mL and be immersed in the centrifuge tube in the liquid nitrogen, pulverize with the plastics pestle; Add 600 μ L CTAB extracting solutions again, 65 ℃ of water-bath 30min put upside down mixing frequently, the centrifugal 5min of 10000 r/min; Get supernatant, add isopyknic chloroform of this supernatant and primary isoamyl alcohol mixed solution, put upside down centrifuge tube to forming the milkiness shape, 4 ℃, the centrifugal 5min of 10000rpm, chloroform and primary isoamyl alcohol volume ratio are 24:1 in said chloroform and the primary isoamyl alcohol mixed solution; Get supernatant in new 1.5mL centrifuge tube, adding ice-cold ethanol and concentration is 3 mol/L, P
HBe 6.0 sodium acetate soln, the volume of said sodium acetate soln is 1/10 of this supernatant volume, and said ice-cold alcoholic acid volume is 2.5 times of this supernatant volume; After putting upside down mixing, place-20 ℃ to preserve 30 min, 4 ℃, the centrifugal 10min of 10000rpm; Abandon supernatant, the use mass concentration is 70% ethanol rinsing deposition secondary, after drying; Add 100 μ L sterilization deionized water dissolving deposition, obtain sample DNA, 4 ℃ of preservations are subsequent use;
2) PCR reaction: reaction system 25 μ L: 2 * one one-step fluorescence quantitative PCR damping fluids, the 12.5 μ L that contain the said probe that said primer that concentration all is 1 μ mol/L and concentration is 0.4 μ mol/L; Concentration is the Taq archaeal dna polymerase 0.5 μ L of 5U/ μ L; Sample DNA 0.5 μ L, the sterilization deionized water is supplied surplus; Reaction parameter: 50 ℃ of preheating 2 min; 95 ℃ of preparatory sex change 10 min; 95 ℃ of 15s, 60 ℃ of l min, 40 circulations; There is fluorescent signal then to be the oil palm da mping-off fungi as if sample DNA in the PCR reaction.
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| CN102382890B CN102382890B (en) | 2013-06-19 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676659A (en) * | 2012-04-20 | 2012-09-19 | 宁波检验检疫科学技术研究院 | LAMP detection reagent and kit of oil palm damping-off fungi |
| CN109234432A (en) * | 2018-10-12 | 2019-01-18 | 南京农业大学 | A kind of primer, probe and kit based on recombinase polymeric enzymatic amplification method detection soybean samping off |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1851448A (en) * | 2005-12-08 | 2006-10-25 | 中国农业科学院作物科学研究所 | Green smut bug real-time fluorescent quantitative PCR test kit and its use |
| CN101381268A (en) * | 2008-10-24 | 2009-03-11 | 北京易众恒科技有限公司 | Broadspectrum pest control agent and preparation method thereof |
| CN101805794A (en) * | 2010-04-08 | 2010-08-18 | 中华人民共和国上海出入境检验检疫局 | Real-time fluorescent PCR detection method of L.maculans as well as primer and probe used for detection |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1851448A (en) * | 2005-12-08 | 2006-10-25 | 中国农业科学院作物科学研究所 | Green smut bug real-time fluorescent quantitative PCR test kit and its use |
| CN101381268A (en) * | 2008-10-24 | 2009-03-11 | 北京易众恒科技有限公司 | Broadspectrum pest control agent and preparation method thereof |
| CN101805794A (en) * | 2010-04-08 | 2010-08-18 | 中华人民共和国上海出入境检验检疫局 | Real-time fluorescent PCR detection method of L.maculans as well as primer and probe used for detection |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676659A (en) * | 2012-04-20 | 2012-09-19 | 宁波检验检疫科学技术研究院 | LAMP detection reagent and kit of oil palm damping-off fungi |
| CN109234432A (en) * | 2018-10-12 | 2019-01-18 | 南京农业大学 | A kind of primer, probe and kit based on recombinase polymeric enzymatic amplification method detection soybean samping off |
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