CN102363809A - Multiple PCR detection method of G. spinigerum, G. nipponicum and G. doloresi and primer for detection - Google Patents
Multiple PCR detection method of G. spinigerum, G. nipponicum and G. doloresi and primer for detection Download PDFInfo
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Abstract
The invention discloses a multiple PCR detection method of G. spinigerum, G. nipponicum and G. doloresi. The method comprises the following steps: 1, extracting DNA from Gnathostomas, and simultaneously designing specific primers of G. spinigerum, G. nipponicum and G. doloresi; 2, optimizing PCR reaction conditions of the designed primers to determine an optimal multiple PCR mode; and 3, experimenting according to the optimal multiple PCR mode, verifying the single PCR specificity, the multiple PCR specificity, the PCR sensitivity and the clinical sample operation feasibility of each primer. The invention also discloses a primer sequence for simultaneously detecting G. spinigerum, G. nipponicum and G. doloresi. Various verifications show that G. spinigerum, G. nipponicum and G. doloresi can be simultaneously identified through the method of the invention; and the method which has the characteristics of rapidness, sensitivity, specificity and synchronous identification of the three Gnathostomas can be applied to the identification and the quarantine of Gnathostomas of aquatic products.
Description
Technical field
The present invention relates to agricultural and Animal Quarantine technical field; Be specifically related to the detection method of a kind of gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode, be specifically related to the multi-PCR detection method of a kind of gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode.In addition, the invention still further relates to the primer that is used for detecting simultaneously gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode.
Background technology
Gnathostomiasis is to eat the infecting both domestic animals and human parasitosis that the living or underdone fresh-water fishes that contain Cheiracanthus (Gnathostoma) larva cause because of the people.The jaw mouth nematode belongs to has 13 effectively to plant; The pathogenic kind of having reported at present has gnathostoma siamense (G.spinigerum), just stings jaw mouth nematode (G.hispidum), Du Shi jaw mouth nematode (G.doloresi), Gnathostoma nipponicum (G.nipponicum), Malaysia jaw mouth nematode (G.malaysiae) and double-core jaw mouth nematode (G.binucleatum); Therefore, significant to the discriminating of the jaw mouth nematode larva worm kind that detects in the food to this sick diagnosis and treatment.The evaluation of jaw mouth nematode kind; Traditional method is to be identification beacon with the morphological specificity, but the life history of jaw mouth nematode is complicated, and the different developmental phases metamorphosis is bigger; The third phase larva plesiomorphism of some worm kind; The traditional form method be difficult to differentiate, thereby the accurate index that can not differentiate as the worm kind, and traditional method also wastes time and energy.
Easy, quick, sensitive advantage that round pcr has, at present, this technology is applied to the discriminating of parasite species gradually, and the goal gene of research is many to be main with rDNA (rDNA).The internal transcribed spacer district of eukaryote rDNA (internal transcribed spacerregion; ITS) comprise ITS-1 and ITS-2; It is very fast to evolve, between having kind specificity with plant in conservative property, be the excellent materials of classifying between plant; But the primer of designs specificity increases, and relatively the difference of ITS sequence is identified nondescript sibling species on the morphology.The ITS sequence is differentiated jaw mouth nematode worm kind with multiple PCR method, the present domestic relevant report of still not having.Gnathostoma siamense, Du Shi jaw mouth nematode, Gnathostoma nipponicum are pathogenic kind of the modal 3 kinds of jaw mouth nematodes in Asia; The present invention is according to the ITS-2 sequence; Design the special primer of these 3 kinds of jaw mouth nematodes respectively; Carry out multiplex PCR amplification, be intended to set up a kind of fast, sensitive, differentiate the method for jaw mouth nematode kind reliably.
Summary of the invention
The technical problem that the present invention will solve is to provide the multi-PCR detection method of a kind of gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode; It does not need numerous and diverse identification of morphology and order-checking step, just can differentiate gnathostoma siamense, Gnathostoma nipponicum, three kinds of jaw mouth nematodes of Du Shi jaw mouth nematode simultaneously.Problem such as consuming time when being intended to solve the jaw mouth nematode and identifying, that cost is high, workload is big is also for identifying simultaneously that further all 13 jaw mouth nematode worm kinds lay a good foundation.The foundation of this method can be satisfied and imports and exports fishery products quarantine, requirement fast and accurately, simultaneously, and in the research of China's infecting both domestic animals and human parasitosis and the food safety prevention and control a kind of new usability methods being provided.For this reason, the present invention also is provided for the detection primer of aforesaid method.
The present invention realizes through following technical scheme:
In one aspect of the invention, the multi-PCR detection method of a kind of gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode is provided, concrete steps comprise:
1 polypide DNA extraction
2 design of primers and screening
The 3PCR reaction condition optimization
4 single pcr amplification specificity checkings
The checking of 5 multiplex PCR specific amplifications
The checking of 6PCR susceptibility
The feasibility checking of 7 clinical worm kind authentication methods
In the step 1; Described gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode DNA extraction method are: preserve from 70% ethanol and take out single standard worm kind (3 strain standard worm kinds have been passed through morphology and molecular biology identification) the liquid; Place the 1.5mLEppendorf pipe; Extract jaw mouth nematode genomic dna according to QIAGEN company DNA extraction test kit (DNeasy Blood&Tissue kit) working instructions, it is subsequent use that the genomic dna that extracts is put-20 ℃ of preservations.
In the step 2; Described jaw mouth nematode design of primers and screening are specially: Application of DNA star7.1 and Primer5.0 equimolecular biological software compare to the ITS2 gene order between Cheiracanthus nematode kind; Select the bigger zone of base difference, at this scope design gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode Auele Specific Primer.The used primer of final experiment is confirmed in screening, guarantee that institute's primer that design can specific amplified goes out the title product of corresponding worm kind, and in implementing the multiplex PCR process, many does not promptly have homology, complementation to there not being interference between the primer, do not form hairpin structure etc.Final determined primer sequence is seen table 1.
Table 1 multiple PCR primer sequence
In the step 3, described PCR reaction condition optimization concrete grammar is: select the PCR reaction system of 25 μ L, system comprises 25mmol/L Mg
2+2.5 μ L, 10 * PCR buffer, 2.5 μ L, 2.5mmol/L dNTP 1 μ L, 5U/ μ LTaq polysaccharase 0.1 μ L, dna profiling 1 μ L, primer working concentration are 10 μ mol/L, according to becoming clear of multiplex PCR band, suitably adjust the ratio of 3 pairs of primer add-ons.Annealing temperature is carried out 51 ℃, 53 ℃, 55 ℃, 57 ℃, 59 ℃, 61 ℃ annealing temperature gradient tests according to primer Tm value scope.Confirm best multiplex PCR pattern at last.Final definite best multi-PRC reaction system is: 10 * buffer, 2.5 μ L in the 25 μ L reaction systems, Mg
2+2.5 μ L (25mmol), dNTP1 μ L (2.5mmol), each 0.3 μ L (10 μ mol) of G.doloresi and G.nipponicum upstream and downstream primer, each 0.4 μ L (10 μ mol) of G.spinigerum upstream and downstream primer.Taq polysaccharase 0.1 μ L (5U/ μ L), each 1 μ L of template DNA adds sterilization distilled water to 25 μ L.Best multi-PRC reaction condition is: 95 ℃ of 3min; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 5min.
In the step 4; Described single pcr amplification specificity checking concrete grammar is: with the single primer of G.spinigerum, G.doloresi or G.nipponicum; Respectively to the dna profiling of G.spinigerum, G.doloresi, G.nipponicum, palace fat nematode, Anisakid nematode, Echinostomatidae and Europe sparganum erinacei; Under equal conditions carry out the PCR reaction, to identify its specificity.
In the step 5; Described multiplex PCR specific amplification checking is specially: use 3 pairs of primers simultaneously; Respectively to the dna profiling of G.spinigerum, G.doloresi and G.nipponicum and various combination, palace fat nematode, Anisakid nematode, Echinostomatidae and Europe sparganum erinacei; Under equal conditions carry out the PCR reaction, to identify its specificity.
In the step 6; Described PCR susceptibility checking is specially: earlier 3 kinds of standard worm kind DNA are measured the template original liquid concentration with the nucleic acid-protein determinator; Respectively the dna profiling of G.spinigerum, G.doloresi, G.nipponicum3 kind jaw mouth nematode is diluted to 5 concentration gradients with 10 times of serial dilutions again; Carry out pcr amplification by above condition, measure the susceptibility of single PCR and multi-PRC reaction.
In the step 7, the evaluation verification method feasibility of described clinical worm kind is specially: the jaw mouth nematode polypide (all passing through morphology and molecular biology identification) that various places are detected, extract dna profiling, and increase by the multiple PCR method of above-mentioned foundation.
In another aspect of this invention, also be provided for detecting simultaneously the primer of gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode, its sequence is:
The Auele Specific Primer of gnathostoma siamense:
GS2-F：5’-GAGATGTCTAGCATCATCTCT-3’(SEQ?ID?NO.1)；
6S4-R：5’-ACTGTATCGGCGAAGCTG-3’(SEQ?ID?NO.2)；
The Auele Specific Primer of Du Shi jaw mouth nematode:
GD2-F：5’-AGATTTTGTTGCTGTTCGTT-3’(SEQ?ID?NO.3)；
GD3-R：5’-TATATACGGCCGCAGCTC-3’(SEQ?ID?NO.4)；
The Auele Specific Primer of Gnathostoma nipponicum:
6N1-F：5’-TCTACGACGACGAGAGGACT-3’(SEQ?ID?NO.5)；
GN5-R：5’-TGGAGTTGTCGATGTGTG-3’(SEQ?ID?NO.6)。
Above-mentioned 3 kinds of jaw mouth nematode multi-PCR detection methods have following advantage:
1. simple, directly perceived: 3 pairs of primers of the present invention's design, GC content is close, and the Tm value is close, and this has just guaranteed that under identical annealing temperature, G.spinigerum, G.doloresi and G.nipponicum can both well be increased.Its amplified fragments is respectively 282bp, 183bp, 358bp, and this makes and when detecting, can on same running gel, carry out, and just can judge the worm kind through the position of observing PCR product band.
2. high specificity: the detection method that the present invention sets up shows, no matter is single PCR or multiplex PCR, all can go out G.spinigerum, G.doloresi and G.nipponicum band by specific amplification; Reagent dosage is few simultaneously, and required cost is lower, and is cost-saved, reduces the manpower consumption.
3. react fast weak point consuming time: the inventive method is consuming time only to need 3 hours, reacts quick, abundant, takes weak point, has saved the time of order-checking process.
Compared with prior art; The present invention can identify 3 kinds of jaw mouth nematodes (gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode) simultaneously; And can carry out flexible combination as required, joint-detection, and simple to operate; Time spent short (entire reaction can be accomplished in 3 hours), the traditional morphological authentication method can't be accomplished this point with the order-checking authentication method.Utilize this invention can be fast, cheap, identify gnathostoma siamense, Du Shi jaw mouth nematode, Gnathostoma nipponicum exactly, it is quick, responsive, special and identify that synchronously the characteristics of 3 kinds of jaw mouth nematodes can be applied to the evaluation of jaw mouth nematode kind and the quarantine of fishery products jaw mouth nematode.
Description of drawings
Fig. 1 is the synoptic diagram as a result of single primer PCR specific amplification checking in the embodiment of the invention; Wherein, Figure 1A is the synoptic diagram as a result of G.spinigerum primer PCR, and wherein 1-7 is a template with G.spinigerum, G.doloresi, G.nipponicum, palace fat nematode, Anisakid nematode, Echinostomatidae, the palace tapeworm DNA that changes respectively, and 8 is blank; Figure 1B is the synoptic diagram as a result of G.doloresi primer PCR, and wherein 1-7 is a template with G.doloresi, G.spinigerum, G.nipponicum, palace fat nematode, Anisakid nematode, Echinostomatidae, the palace tapeworm DNA that changes respectively, and 8 is blank; Fig. 1 C is the synoptic diagram as a result of G.nipponicum primer PCR, and wherein 1-7 is a template with G.nipponicum, G.spinigerum, G.doloresi, palace fat nematode, Anisakid nematode, Echinostomatidae, the palace tapeworm DNA that changes respectively, and 8 is blank; M:DL500Marker.
Fig. 2 is the synoptic diagram as a result of multiplex PCR specific amplification checking in the embodiment of the invention; Wherein, M:DL500Marker; 1:G.spinigerum, G.doloresi, G.nipponicum hybrid template; 2:G.spinigerum, the G.doloresi hybrid template; 3:G.spinigerum, the G.nipponicum hybrid template; 4:G.doloresi, the G.nipponicum hybrid template; 5:G.spinigerum template; 6:G.doloresi template; 7:G.nipponicum template; 8-11 is respectively palace fat nematode, Anisakid nematode, Echinostomatidae, palace tapeworm dna profiling changes; 12 is blank.
Fig. 3 is the synoptic diagram as a result of PCR sensitivity test checking in the embodiment of the invention; Fig. 3 A is the synoptic diagram as a result of the single PCR sensitivity test of G.doloresi; Fig. 3 B is the synoptic diagram as a result of the single PCR sensitivity test of G.nipponicum; Fig. 3 C is the synoptic diagram as a result of the single PCR sensitivity test of G.spinigerum; Fig. 3 D is the synoptic diagram as a result of hybrid template multiplex PCR sensitivity test; Wherein, M:DL500Marker; 1-6 is respectively each dna profiling that 100-10-5 doubly dilutes.
Fig. 4 is that multiple PCR method is identified the sample result synoptic diagram in the embodiment of the invention; Wherein, M:DL500Marker; 1-8 is Indonesia's strain isolated, and 9-10 is Philippines's strain isolated, and 12-13 is the Heilungkiang strain isolated, and 11,14 is G.spinigerum, G.doloresi, the positive biased sample contrast of G.nipponicum, and 15 is blank.
Embodiment
Elaborate in the face of embodiments of the invention down: present embodiment is to implement under the prerequisite in technical scheme of the present invention; Provide detailed embodiment and concrete operating process, be not used in restriction scope of the present invention but present embodiment only is used to the present invention is described.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, for example J.Sambrook etc. is in the condition described in " molecular cloning experiment guide " (Science Press, 1992), or the condition of advising according to manufacturer.
Embodiment
1. at first extract gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode DNA
The DNA extraction method is described the DNA extraction method with the DNA extraction agent box (DNeasy Blood&Tissue kit) of QIAGEN company: (source of this three strains worm: G.spinigerum (Yizhou strain), G.doloresi (Fujian strain) are so kind as to give by eqpidemic disease prevention and control center, Guangxi Zhang Hongman chief physician three kinds of known polypides of picking wall scroll; G.nipponicum (Guangzhou strain) is separated from loach by the Shanghai inspection and quarantining for import/export and through morphology and molecular biology identification; Preserve in 70% ethanol) put into the sterilization centrifuge tube, add sterile purified water, repeated washing 3 times; After finishing, washing discards zero(ppm) water in the centrifuge tube; With the rod of milling polypide in the centrifuge tube is pulverized, add 180 μ l Buffer ATL, 20 μ L Proteinase Ks; Behind the mixing in 56 ℃ hatch 1h～3h to digestion fully, during concussion frequently rock.After the digestion fully, whirlpool concussion 15s adds 200 μ L Buffer AL, and the mixing of whirlpool concussion immediately adds 200 μ L ethanol (96%～100%) again, whirlpool concussion mixing.Centrifugal post places on the collection tube, with last one the step mixture suck in the centrifugal post, the centrifugal 1min of 8000r/min abandons collection tube.Centrifugal post is placed new collection tube, add 500 μ L Buffer AW1, the centrifugal 1min of 8000r/min abandons collection tube.Centrifugal post is placed new collection tube, add 500 μ L Buffer AW2, the centrifugal 3min of 14000r/min abandons collection tube.Centrifugal post is placed the 1.5mL centrifuge tube of a sterilization, add 200 μ L Buffer AE incubated at room 1min, the centrifugal 1min of 8000r/min, centrifugal gained DNA preserves subsequent use in-20 ℃ of refrigerators.(repeat this step and can enlarge the DNA recovery)
2. design of primers and screening
Application of DNA star7.1 and Primer5.0 equimolecular biological software compare to the ITS2 gene order between Cheiracanthus nematode kind; Select the bigger zone of base difference, at this scope design gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode Auele Specific Primer.The used primer of final experiment is confirmed in screening, guarantee that institute's primer that design can specific amplified goes out the title product of corresponding worm kind, and in implementing the multiplex PCR process, many does not promptly have homology, complementation to there not being interference between the primer, do not form hairpin structure etc.Final determined primer sequence is seen table 1.
Table 1 multiple PCR primer sequence
3.PCR reaction system and condition
PCR reagent used in the present invention is produced for TAKARA company.PCR reaction condition optimization concrete grammar is: select the PCR reaction system of 25 μ L, system comprises 25mmol/L Mg
2+2.5 μ L, 10 * PCR buffer, 2.5 μ L, 2.5mmol/L dNTP 1 μ L, 5U/ μ L Taq polysaccharase 0.1 μ L, dna profiling 1 μ L, primer working concentration are 10 μ mol/L, according to becoming clear of multiplex PCR band, suitably adjust the ratio of 3 pairs of primer add-ons.Annealing temperature is carried out 51 ℃, 53 ℃, 55 ℃, 57 ℃, 59 ℃, 61 ℃ annealing temperature gradient tests according to primer Tm value scope.Confirm best multiplex PCR pattern at last.Final definite best multi-PRC reaction system is: 10 * buffer, 2.5 μ L in the 25 μ L reaction systems, Mg
2+2.5 μ L (25mmol), dNTP 1 μ L (2.5mmol), each 0.3 μ L (10 μ mol) of G.doloresi and G.nipponicum upstream and downstream primer (seeing table 1), each 0.4 μ L (10 μ mol) of G.spinigerum upstream and downstream primer (seeing table 1).Taq polysaccharase 0.1 μ L (5U/ μ L), each 1 μ L of template DNA adds sterilization distilled water to 25 μ L.Best multi-PRC reaction condition is: 95 ℃ of 3min; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 5min.
4. single pcr amplification specificity checking
Single primer (seeing table 1) with G.spinigerum, G.doloresi or G.nipponicum; Respectively to the dna profiling of G.spinigerum, G.doloresi, G.nipponicum, palace fat nematode, Anisakid nematode, Echinostomatidae and Europe sparganum erinacei; According to above PCR reaction system and condition, under equal conditions carry out the PCR reaction.Behind the single pcr amplification, product detects through 3.0% agarose gel electrophoresis, and visible G.spinigerum, G.doloresi, G.nipponicum amplify the band of 1 282bp, 183bp, 358bp respectively, conforms to separately intended purposes band; 3 kinds of primers all do not amplify any band (see figure 1) to palace fat nematode, Anisakid nematode, Echinostomatidae and Europe sparganum erinacei.G.spinigerum, G.doloresi, the single PCR specific amplification products of G.nipponicum are behind sequencing analysis, and the result shows and the ITS2 target gene sequences of these 3 kinds of jaw mouth nematodes matches.It is thus clear that the specificity proof test of single PCR has confirmed that these 3 pairs of primers have very strong specificity, can detect gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode respectively.
5. multiplex PCR specific amplification checking
Use 3 pairs of primers (seeing table 1) simultaneously; Respectively to the dna profiling of G.spinigerum, G.doloresi and G.nipponicum and various combination, palace fat nematode, Anisakid nematode, Echinostomatidae and Europe sparganum erinacei; According to above PCR reaction system and condition, under equal conditions carry out the PCR reaction.Respectively the dna profiling of G.spinigerum, G.doloresi, G.nipponicum is carried out pcr amplification with 3 pairs of primers simultaneously, all amplify 1 intended purposes band; Dna profiling to G.spinigerum and G.doloresi, G.spinigerum and G.nipponicum and G.doloresi and 3 kinds of various combinations of G.nipponicum carries out pcr amplification; Amplify 2 intended purposes bands respectively, and palace fat nematode, Anisakid nematode, Echinostomatidae and Europe sparganum erinacei DNA are not all amplified respective strap; To the hybrid dna template of G.spinigerum, G.doloresi and 3 kinds of jaw mouth nematodes of G.nipponicum, amplify the purpose band (see figure 2) of 3 expection sizes.It is thus clear that the specificity proof test of multiplex PCR has confirmed that these 3 pairs of primers have very strong specificity, can detect gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode simultaneously.
6. sensitivity test checking
Be respectively 133ng/ μ L, 125ng/ μ L, 204ng/ μ L through nucleic acid-protein determinator mensuration G.doloresi, G.nipponicum, G.spinigerum dna profiling original content.Dna profiling is become 5 concentration gradients by 10-1～10-5 times of serial dilution; Carry out pcr amplification by above PCR reaction system and condition, single PCR is respectively 0.01ng/ μ L, 0.01ng/ μ L, 0.2ng/ μ L to the minimum detectable activity of G.doloresi, G.nipponicum and G.spinigerum.The identical (see figure 3) of hybrid template multiplex PCR minimum detectable activity with single PCR.It is thus clear that the PCR sensitivity test has verified that these 3 pairs of primers are higher to the susceptibility of template concentrations, considerably beyond the requirement of experiment of 1 jaw mouth nematode being put forward DNA concentration.
7. the feasibility of the evaluation verification method of clinical worm kind
To the jaw mouth nematode strain isolated of separation in 2010～2011, identify according to the multiple PCR method of setting up from Philippines, Indonesia import swamp eel and Heilungkiang loach.The dna profiling of 2 strain Philippines, 8 strain Indonesia jaw mouth nematode strain isolateds increases, and the band position consistency of electrophoretic band and G.spinigerum positive control is 282bp; Dna profiling to 2 strain Heilungkiang jaw mouth nematode strain isolateds increases, and electrophoretic band and G.nipponicum positive control band position consistency are the 358bp (see figure 4).It is thus clear that the multiplex PCR qualification result conforms to the Molecular Identification result with the raw sample identification of morphology fully, proves when this multiple PCR method carries out the evaluation of clinical sample, has verified the feasibility of this method.
In Fig. 4,8 strain Indonesia jaw mouth nematodes (1-8 among Fig. 4): Shanghai Entry-Exit Inspection and Quarantine bureau isolates in Indonesia import swamp eel, and is accredited as gnathostoma siamense through morphology and molecular biology order-checking.2 strain Philippines (9-10 among Fig. 4): Shanghai Entry-Exit Inspection and Quarantine bureau isolates from Philippines import swamp eel, and is accredited as gnathostoma siamense through morphology and molecular biology order-checking.2 strain Heilungkiang jaw mouth nematodes (12-13 among Fig. 4): Shanghai Entry-Exit Inspection and Quarantine bureau entrusts in the loach of Heilungkiang Entry-Exit Inspection and Quarantine Bureau available from the market of farm produce, Heilungkiang and isolates, and is accredited as Gnathostoma nipponicum through morphology and molecular biology order-checking.
Claims (10)
1. the multi-PCR detection method of a gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode is characterized in that concrete steps comprise as follows:
1. the polypide DNA extraction of gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode;
2. design of primers and screening;
3. to step 2. institute's designed primer carry out the PCR reaction condition optimization, obtain best multiplex PCR pattern;
4. carry out single pcr amplification specificity checking according to step best PCR reaction conditions 3.;
5. carry out the checking of multiplex PCR specific amplification according to step best PCR reaction conditions 3.;
6. carry out the checking of PCR susceptibility according to step best PCR reaction conditions 3.;
7. according to the multiple PCR method of setting up clinical worm kind authentication method is carried out the feasibility checking.
2. the multi-PCR detection method of gnathostoma siamense according to claim 1, Gnathostoma nipponicum, Du Shi jaw mouth nematode; It is characterized in that; Step 1. in; The polypide DNA extraction method of described gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode is: preserve from 70% ethanol and take out single standard worm kind the liquid; Adopt the DNA extraction test kit to extract gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode genomic dna, it is subsequent use that the genomic dna that extracts is put-20 ℃ of preservations.
3. the multi-PCR detection method of gnathostoma siamense according to claim 1, Gnathostoma nipponicum, Du Shi jaw mouth nematode; It is characterized in that; Design of primers and the screening of step described in 2. is specially: applied molecular biology software compares to the ITS2 gene order between Cheiracanthus nematode kind, selects the bigger zone of base difference, at this scope design gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode Auele Specific Primer; Primer is confirmed in screening; Guarantee that the primer that designs is necessary for Auele Specific Primer, a pair of primer can only amplify the sequence fragment of corresponding worm kind, and and other primer between do not disturb; Promptly do not have homology, complementation, do not form hairpin structure; Said designed primer sequence is:
The Auele Specific Primer of gnathostoma siamense:
GS2-F：5’-GAGATGTCTAGCATCATCTCT-3’(SEQ?ID?NO.1)；
GS4-R：5’-ACTGTATCGGCGAAGCTG-3’(SEQ?ID?NO.2)；
The Auele Specific Primer of Du Shi jaw mouth nematode:
GD2-F：5’-AGATTTTGTTGCTGTTCGTT-3’(SEQ?ID?NO.3)；
GD3-R：5’-TATATACGGCCGCAGCTC-3’(SEQ?ID?NO.4)；
The Auele Specific Primer of Gnathostoma nipponicum:
GN1-F：5’-TCTACGACGACGAGAGGACT-3’(SEQ?ID?NO.5)；
GN5-R：5’-TGGAGTTGTCGATGTGTG-3’(SEQ?ID?NO.6)。
4. the multi-PCR detection method of gnathostoma siamense according to claim 1, Gnathostoma nipponicum, Du Shi jaw mouth nematode; It is characterized in that; Step 3. in, described PCR reaction condition optimization concrete grammar is: select the PCR reaction system of 25 μ L, system comprises 25mmol/L Mg
2+2.5 μ L, 10 * PCR buffer, 2.5 μ L, 2.5mmol/L dNTP 1 μ L; 5U/ μ L Taq polysaccharase 0.1 μ L, dna profiling 1 μ L, primer working concentration are 10 μ mol/L; According to becoming clear of multiplex PCR band, the suitably ratio of set-up procedure primer add-on 2.; Annealing temperature is carried out 51 ℃, 53 ℃, 55 ℃, 57 ℃, 59 ℃, 61 ℃ annealing temperature gradient tests according to primer Tm value scope, confirms best multiplex PCR pattern at last.
5. according to the multi-PCR detection method of claim 1 or 4 described gnathostoma siamenses, Gnathostoma nipponicum, Du Shi jaw mouth nematode; It is characterized in that; Step best multiplex PCR pattern 3. is specially: best multi-PRC reaction system: 10 * buffer, 2.5 μ L in the 25 μ L reaction systems, Mg
2+2.5 μ L, dNTP 1 μ L, each 0.3 μ L of Du Shi jaw mouth nematode and Gnathostoma nipponicum upstream and downstream primer, each 0.4 μ L of gnathostoma siamense upstream and downstream primer; Taq polysaccharase 0.1 μ L, each 1 μ L of template DNA adds sterilization distilled water to 25 μ L; Best multi-PRC reaction condition is: 95 ℃ of 3min; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 5min.
6. the multi-PCR detection method of gnathostoma siamense according to claim 1, Gnathostoma nipponicum, Du Shi jaw mouth nematode; It is characterized in that; Step 4. in; Described single pcr amplification specificity checking concrete grammar is: with the single primer of 2. gnathostoma siamense of step, Gnathostoma nipponicum, Du Shi jaw mouth nematode, respectively to the dna profiling of gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode, palace fat nematode, Anisakid nematode, Echinostomatidae and Europe sparganum erinacei, according to step best multiplex PCR pattern 3.; Under equal conditions carry out the PCR reaction, to identify its specificity.
7. the multi-PCR detection method of gnathostoma siamense according to claim 1, Gnathostoma nipponicum, Du Shi jaw mouth nematode; It is characterized in that; Step 5. in; The checking of described multiplex PCR specific amplification is specially: simultaneously with step gnathostoma siamense, Gnathostoma nipponicum, 3 pairs of primers of Du Shi jaw mouth nematode 2., respectively to the dna profiling of gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode and various combination thereof, palace fat nematode, Anisakid nematode, Echinostomatidae and Europe sparganum erinacei, according to step best multiplex PCR pattern 3.; Under equal conditions carry out the PCR reaction, to identify its specificity.
8. the multi-PCR detection method of gnathostoma siamense according to claim 1, Gnathostoma nipponicum, Du Shi jaw mouth nematode; It is characterized in that; Step 6. in; Described PCR susceptibility checking is specially: earlier gnathostoma siamense, Gnathostoma nipponicum, 3 kinds of standard worms of Du Shi jaw mouth nematode kind DNA are measured the template original liquid concentration with the nucleic acid-protein determinator; Respectively the dna profiling of gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode is diluted to 5 concentration gradients with 10 times of serial dilutions again, 3. best multiplex PCR pattern is carried out pcr amplification set by step, measures the susceptibility of single PCR and multi-PRC reaction.
9. the multi-PCR detection method of gnathostoma siamense according to claim 1, Gnathostoma nipponicum, Du Shi jaw mouth nematode; It is characterized in that; Step 7. in; Described clinical worm kind authentication method carries out feasibility checking and is specially: with the jaw mouth nematode polypide that various places detect, extract dna profiling, increase by the multiple PCR method of above-mentioned foundation.
10. be used for detecting simultaneously the primer of gnathostoma siamense, Gnathostoma nipponicum, Du Shi jaw mouth nematode, it is characterized in that its sequence is:
The Auele Specific Primer of gnathostoma siamense:
GS2-F：5’-GAGATGTCTAGCATCATCTCT-3’(SEQ?ID?NO.1)；
GS4-R：5’-ACTGTATCGGCGAAGCTG-3’(SEQ?ID?NO.2)；
The Auele Specific Primer of Du Shi jaw mouth nematode:
GD2-F：5’-AGATTTTGTTGCTGTTCGTT-3’(SEQ?ID?NO.3)；
GD3-R：5’-TATATACGGCCGCAGCTC-3’(SEQ?ID?NO.4)；
The Auele Specific Primer of Gnathostoma nipponicum:
GN1-F：5’-TCTACGACGACGAGAGGACT-3’(SEQ?ID?NO.5)；
GN5-R：5’-TGGAGTTGTCGATGTGTG-3’(SEQ?ID?NO.6)。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102879563A (en) * | 2012-10-19 | 2013-01-16 | 中国疾病预防控制中心寄生虫病预防控制所 | Immunochromatography test strip for detecting anisakis disease and preparation method thereof |
| CN103361401A (en) * | 2012-03-30 | 2013-10-23 | 舟山市疾病预防控制中心 | TagMan PCR detection method for anisakis simplexes in marine products |
| CN104561265A (en) * | 2014-11-21 | 2015-04-29 | 深圳市疾病预防控制中心 | Real-time fluorescence PCR detection kit and detection method of anisakis |
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| CHARINTHON NGARMAMONPIRAT, ET AL.: "Analysis of sequence variation in Gnathostoma spinigerum mitochondrial DNA by single-strand conformation polymorphism analysis and DNA sequence", 《PARASITOLOGY INTERNATIONAL》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103361401A (en) * | 2012-03-30 | 2013-10-23 | 舟山市疾病预防控制中心 | TagMan PCR detection method for anisakis simplexes in marine products |
| CN102879563A (en) * | 2012-10-19 | 2013-01-16 | 中国疾病预防控制中心寄生虫病预防控制所 | Immunochromatography test strip for detecting anisakis disease and preparation method thereof |
| CN104561265A (en) * | 2014-11-21 | 2015-04-29 | 深圳市疾病预防控制中心 | Real-time fluorescence PCR detection kit and detection method of anisakis |
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