CN104195237B - A kind of PCR detects the method for nosema locustae disease - Google Patents
A kind of PCR detects the method for nosema locustae disease Download PDFInfo
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- CN104195237B CN104195237B CN201410397744.1A CN201410397744A CN104195237B CN 104195237 B CN104195237 B CN 104195237B CN 201410397744 A CN201410397744 A CN 201410397744A CN 104195237 B CN104195237 B CN 104195237B
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention relates to a kind of method that PCR detects nosema locustae disease, and a kind of PCR primer and kit thereof for detection of nosema locustae disease is provided. The present invention also provides a kind of PCR to detect the method for nosema locustae disease, and it is to utilize above-mentioned primer or kit to carry out PCR detection to locust. By content disclosed in this invention, Rapid identification locust infects nosema locustae situation in enormous quantities, detects required sample size and greatly reduces, and detect precision simultaneously and greatly improve, and credible result degree is high, and sensitivity can reach 7 × 103Spore/ml, compared with traditional Microscopical Method For Detection, reduces testing cost and workload greatly.
Description
Technical field
The present invention relates to technical field of biological, be specifically related to a kind of PCR of employing diagnostic techniquesDetect the PCR detection technique of nosema locustae disease.
Background technology
Polymerase chain reaction (PolymeraseChainReaction, PCR) is 20th century 80The isothermal DNA amplification that mid-nineties 90 grows up. It is good, quick that round pcr has specificityThe multiple advantages such as perception is high, productive rate is high, reproducible, quick, easy, easy automation; ShouldTechnology can in vitro expand genes of interest or a certain DNA fragmentation at one within the very short timeIncrease to 100,000 and even 1,000,000 times, result even can directly be observed and be judged by naked eyes; ByThis technology, technical staff can bleed from a hair,, even a cell, amplifyEnough DNA are for analysis and research and detect qualification. Even several weeks of days past just can accomplishThing, adopt pcr analysis technology just can complete at several hours. Round pcr is biological doctorDetection technique in revolutionary invention and milestone significance in field, it is raw from moleculeThing level is brought the development of advancing by leaps and bounds to detection field.
Microsporidian, is a kind of unicellular parasite by sporogenesis, and it belongs to mycota(Fungi), Microspora (Microsporidia). Have at present more than 1,000,000 kinds of micro-sporesWorm. Microsporidian can parasitic most of animal, and most infected insects, are the common cause of diseases of insectMicroorganism.
PCR molecular detection technology has been applied to the research of every field more and more at present,PCR molecular detection technology is in the method for the pathogenic microorganism such as microsporidian that detects silkwormTo application, but also do not adopt PCR diagnostic techniques to detect the relevant report of nosema locustae diseaseRoad. Nosema locustae is the pathogen with the effect that well continues to eliminate locusts. Current, for locustThe diagnosis of worm microsporidiosis, mainly adopts Microscopical Method For Detection. But because nosema locustae individuality is micro-Little, conventional microscopy exists observation morphological differences not obvious, and this method takes time and effort and sensitivityLower, also not relevant molecular biological method for quick. Therefore, how can be fastSpeed, accurate, Sensitive Detection nosema locustae disease, become problem demanding prompt solution.
Summary of the invention
The object of this invention is to provide a kind of according to nosema locustae polar tube protein (ptp) gene orderDesign primer, amplification nosema locustae specific gene fragment, sets up dividing of nosema locustaeSub-identification system, finally realize nosema locustae disease fast, accurately, Sensitive Detection.
To achieve these goals, the invention provides a kind of for detection of nosema locustae diseasePCR primer, described primer comprises:
Upstream primer Nlos-F5:5 '-GCTCCAAATACAGCGTAAATGC-3 '(SEQIDNO：1)；
Downstream primer Nlos-R5:5 '-GAGATTCGCATTCGCCACAGC-3 '(SEQIDNO：2)。
The present invention also provides the reagent for detection of nosema locustae disease that comprises above-mentioned primerBox.
Described kit also comprises dNTPs, TaqDNA polymerase, Mg2+, PCR reaction is slowRush one or more in liquid.
Described kit also comprises standard positive template.
A kind of method that the present invention also provides PCR to detect nosema locustae disease, it is on utilizingState primer or kit nosema locustae is carried out to PCR detection.
Said method comprising the steps of:
1) extract the total DNA in sample tissue;
2) taking step 1) in total DNA be template, Nlos-F5 and Nlos-R5 are primer, enterPerforming PCR amplified reaction;
3) analyze PCR product.
The PCR reaction system of described method is counted with 25 μ l:
2 × TaqMasterMix, 12.5 μ l; 10 μ M primer Nlos-F5,1 μ l; 10 μ M primersNlos-R5,1 μ l; DNA profiling, 1 μ l; ddH2O, 9.5 μ l; Overall reaction system is 25 μ l.
PCR reaction condition is: 95 DEG C, and 5min, 1 circulation; 94 DEG C, 30s, 66 DEG C,30s, 72 DEG C, 30s, 35 circulations; Last 72 DEG C are extended 10min, 1 circulation.
Beneficial effect of the present invention:
1. Rapid identification locust infects nosema locustae situation in enormous quantities.
2. detect required sample size and greatly reduce, detect precision simultaneously and greatly improve. And knotFruit is with a high credibility, and sensitivity can reach 7 × 103Spore/ml.
3. compared with traditional Microscopical Method For Detection, greatly reduce testing cost and workload.
Brief description of the drawings
Fig. 1 is the PCR testing result figure of nosema locustae, wherein: M:200bpDNALadderMarker (Tiangen); 1: the locust DNAPCR product of not catching an illness; 2: locust is micro-Sporozoite DNAPCR product.
Fig. 2 is the sensitivity result figure that the sick PCR of nosema locustae detects, wherein: M:200bpDNALadderMarker (Tiangen); 1: the locust DNAPCR product of not catching an illness; 2-12Be respectively nosema locustae DNA dilution 1.67 × 104Doubly, 2.5 × 104Doubly, 3.33 × 104Doubly,5×104Doubly, 1 × 105Doubly, 1.11 × 105Doubly, 1.25 × 105Doubly, 1.43 × 105Doubly, 1.67 × 105Doubly, 2.5 × 105Doubly, 5 × 105The product of PCR doubly.
Detailed description of the invention
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention. If notSpecialize, embodiment is all according to normal experiment condition, as the cloning experimentation of Sambrook equimolecularHandbook (SambrookJ&RussellDW, Molecularcloning:alaboratorymanual,2001), or according to the condition of manufacturer description suggestion.
The extraction of embodiment 1 nosema locustae sample DNA
Get nosema locustae, adopt CTAB method to extract DNA, be specially:
It is centrifugal that nosema locustae is got 1ml, removes supernatant. The electronic grinding rod ground sample of organizing20-30s; Every 1.5ml centrifuge tube adds 0.5mlCTAB buffer solution, mixes, and each pipe adds 20mg/mlProteinase K 20 μ l, on oscillator, shake 30s; 56 DEG C of water-bath 6h; Add equal-volume phenol/chlorineImitative/isoamyl alcohol (volume ratio 25:24:1) extracting is once; 12000rpm, 10min; Get supernatant,Add equal-volume chloroform extracting 1 time; 12000rpm, 10min; Get supernatant, add equal-volume isopropyl alcohol,Precipitate 20min on ice; 12000rpm, 10min, abandons supernatant; 75% ethanol 500 μ l washing 1 time,Slightly centrifugal, abandon supernatant; Absolute ethyl alcohol 500 μ l washing 1 time, slightly centrifugal, abandon supernatant; Super-clean benchBlow 10min, dry; Add 20 μ lddH2O dissolves, and obtains total DNA of nosema locustae,-20 DEG C of preservations.
The design of embodiment 2 primers
According to known array (gene order number: GQ397125), design primer:
Nlos-F5：5’-GCTCCAAATACAGCGTAAATGC-3’(SEQIDNO：1)；
Nlos-R5：5’-GAGATTCGCATTCGCCACAGC-3’(SEQIDNO：2)。
The pcr amplification program of embodiment 3 nosema locustaes
Taking sample DNA as template, taking Nlos-F5 and Nlos-R5 as forward and reverse primer, enterRow fragment amplification. PCR reaction system is: (a day root biochemical technology has 2 × TaqMasterMixLimit company), 12.5 μ l; 10 μ M primer Nlos-F5,1 μ l; 10 μ M primer Nlos-R5,1 μ l;DNA profiling, 1 μ l; ddH2O, 9.5 μ l; Overall reaction system is 25 μ l. Reaction condition is: 95 DEG C,5min, 1 circulation; 94 DEG C, 30s, 66 DEG C, 30s, 72 DEG C, 30s, 35 circulations;Last 72 DEG C are extended 10min, 1 circulation. Get PCR product 10 μ l, the agarose 1% is solidifyingElectrophoresis in glue [containing nucleic acid dye (10000 × GeneGreen)], voltage 100V, 40min,Under strong uviol lamp, observe PCR diagnostic result (the results are shown in Figure 1).
The sensitivity level that the sick PCR of embodiment 4 nosema locustaes detects
Nosema locustae sample DNA is carried out (diluting gradient and being respectively dilution after gradient dilution1.67×104Doubly, 2.5 × 104Doubly, 3.33 × 104Doubly, 5 × 104Doubly, 1 × 105Doubly, 1.11 × 105Doubly, 1.25 × 105Doubly, 1.43 × 105Doubly, 1.67 × 105Doubly, 2.5 × 105Doubly, 5 × 105Doubly) doFor template, taking Nlos-F5 and Nlos-R5 as forward and reverse primer, carry out fragment amplification. PCR is anti-The system of answering is: 2 × TaqMasterMix, 12.5 μ l; 10 μ M primer Nlos-F5,1 μ l; 10 μ MPrimer Nlos-R5,1 μ l; DNA profiling, 1 μ l; ddH2O, 9.5 μ l; Overall reaction system is 25 μ l.Reaction condition is: 95 DEG C, and 5min, 1 circulation; 94 DEG C, 30s, 66 DEG C, 30s, 72 DEG C,30s, 35 circulations; Last 72 DEG C are extended 10min, 1 circulation. Get PCR product 10 μ l,Electrophoresis in Ago-Gel [containing nucleic acid dye (10000 × GeneGreen)] 1%, electricityPress 100V, 40min observes PCR diagnostic result (the results are shown in Figure 2) under strong uviol lamp.
Although, above used general explanation, detailed description of the invention and test, to thisBrightly do detailed description, but on basis of the present invention, can make some modifications or improvements it,This will be apparent to those skilled in the art. Therefore, do not departing from spirit of the present inventionBasis on these modifications or improvements, all belong to the scope of protection of present invention.
Claims (8)
1. the Auele Specific Primer pair for detection of nosema locustae, is characterized in that,
The right sequence of described Auele Specific Primer is as follows:
Upstream primer Nlos-F5:5 '-GCTCCAAATACAGCGTAAATGC-3 '(SEQIDNO：1)；
Downstream primer Nlos-R5:5 '-GAGATTCGCATTCGCCACAGC-3 '(SEQIDNO：2)。
2. contain Auele Specific Primer claimed in claim 1 right for detection of the micro-spore of locustThe kit of parasitosis.
3. kit according to claim 2, is characterized in that, described kit alsoComprise dNTPs, TaqDNA polymerase, Mg2+, a kind of in PCR reaction buffer orMultiple.
4. according to the kit described in claim 2 or 3, it is characterized in that described reagentBox also comprises standard positive template.
5. the PCR of non-medical diagnosis on disease therapeutic purposes detects a method for nosema locustae,It is the Auele Specific Primer pair utilizing described in claim 1, or claim 2-4 any one instituteThe kit of stating carries out PCR detection to locust.
6. method according to claim 5, is characterized in that, comprises the following steps:
1) extract the total DNA in sample tissue;
2) taking step 1) in total DNA be template, Nlos-F5 and Nlos-R5 are primer, carry outPcr amplification reaction;
3) analyze PCR product.
7. according to the method described in claim 5 or 6, it is characterized in that, PCR reacts bodySystem counts with 25 μ l:
2 × TaqMasterMix, 12.5 μ l; 10 μ M primer Nlos-F5,1 μ l; 10 μ M drawThing Nlos-R5,1 μ l; DNA profiling, 1 μ l; ddH2O, 9.5 μ l; Overall reaction system is 25 μ l.
8. according to the method described in claim 5 or 6, it is characterized in that, PCR reacts barPart is: 95 DEG C, and 5min, 1 circulation; 94 DEG C, 30s, 66 DEG C, 30s, 72 DEG C, 30s,35 circulations; Last 72 DEG C are extended 10min, 1 circulation.
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| CN1128209C (en) * | 2001-05-28 | 2003-11-19 | 中国农业大学 | Locust, microsporidian and East-Asia migratory locust strain product and its preparing process |
| CN101492684A (en) * | 2008-12-11 | 2009-07-29 | 西南大学 | Cultivated silkworm microsporidian polar filament protein PTP2 gene and uses thereof |
| CN103131773B (en) * | 2013-01-24 | 2014-09-24 | 华南农业大学 | LAMP visual rapid detection kit of silkworm pathogenic micro spore worms and detection method thereof |
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