CN103952467A - Special primers and identification method for rapidly identifying anoplocephalidae tapeworms parasitizing on Equine - Google Patents
Special primers and identification method for rapidly identifying anoplocephalidae tapeworms parasitizing on Equine Download PDFInfo
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Abstract
The present invention provides special primers for rapidly identifying anoplocephalidae tapeworms parasitizing on Equine, wherein the upstream primer is 5'-GCAGAGTTTTGCTGATTTTATGAAG-3', and the downstream primer is 5'-CCACTATACTCAGTATTAAATCCACTAACAAG-3'. The present invention further provides a method for rapidly identifying anoplocephalidae tapeworms parasitizing on Equine, wherein the method comprises: adopting the primers to carry out PCR amplification on NAD1 of anoplocephalidae tapeworm, sequencing, comparing the sequencing result with sequences in SEQIDNo.1 and SEQIDNo.2, and judging. With the method, the defects that the general morphology observation can not distinguish gravid proglottids and the microscopic observation can not distinguish eggs are solved. The PCR method of the present invention can be used for molecule identification of two anoplocephalidae tapeworms, and can further be used for epidemiological investigation of the Equine anoplocephalidae tapeworm.
Description
Technical field
The present invention relates to a kind of quick differential host in primer special and the discrimination method thereof of naked tapeworm of equus.
Background technology
Two kinds of naked tapeworms of equus: anoplocephala perfoliata (Anoplocephala perfoliata) and anoplocephala magna (A. magna) parasitize in the intestine and small intestine of equus, worldwide distribution, all there is report in China various places, in northwest and Pastoral Areas in Inner Mongolia, be often region popular especially.Entomophytic position can cause mucous membrane inflammation and oedema, mucosa injury, and the proliferative annular hemorrhagic ulcer of formative tissue, once perforated ulcer just causes acute peritonitis, causes death.A large amount of while infecting anoplocephala perfoliata, return, blind, colon all spreads all over ulcer.Go back to blind narrow portion and block, mucous enteritis and mucous membrane occur and come off, often cause death.When severe infection anoplocephala magna, can cause Catarrhal or hemorrhagic enteritis.Clinical visible maldigestion, intermittent colic and diarrhoea, and cause that improving is become thin and anaemia.Carry out stool examination, can find a large amount of worm's ovums or gravid proglottid.Worm's ovum or gravid proglottid feature are almost in full accord, rely on morphological specificity to be difficult to be differentiated.Teniasis has had a strong impact on the sound development of animal husbandry economy.Therefore, strengthen Species identification and the epidemiology survey of naked teniasis Endemic Area tapeworm of equus, specify the advantage worm kind of naked the tapeworm in each department, instruct relevant department to formulate correct prevention, control and control techniques and that clinical patients is carried out to scientific and effective treatment is all significant.
Current diagnostic method can only, in conjunction with clinical symptom, by stool examination, be observed and whether have worm's ovum or gravid proglottid.It is that this method can not be distinguished for which kind of cestode infection, can not make a definite diagnosis infected insect species.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of quick differential host in the method for naked tapeworm of equus, determines naked the tapeworm of advantage infecting, for epidemiology survey and the clinical diagnosis of naked teniasis provide technical guarantee.For this reason, the present invention also provides quick differential host in the primer special of naked tapeworm of equus.
The invention provides quick differential host in the primer special of naked tapeworm of equus, described primer is: upstream primer is 5 '-GCAGAGTTTTGCTGATTTTATGAAG-3 ', and downstream primer is 5 '-CCACTATACTCAGTATTAAATCCACTAACAAG-3 '.
The present invention also provides the primer special of naked teniasis of rapid differential diagnosis equus, described primer is: upstream primer is 5 '-GCAGAGTTTTGCTGATTTTATGAAG-3 ', and downstream primer is 5 '-CCACTATACTCAGTATTAAATCCACTAACAAG-3 '.
The 3rd object of the present invention is to provide the NAD1 nucleotide sequence of naked the tapeworm that parasitizes equus, described naked tapeworm is anoplocephala perfoliata and anoplocephala magna, the NAD1 nucleotide sequence of described anoplocephala perfoliata is referring to SEQ ID No.1, and the NAD1 nucleotide sequence of described anoplocephala magna is referring to SEQ ID No.2.
The 4th object of the present invention is to provide quick differential host in the method for naked tapeworm of equus, application rights requires the NAD1 of naked tapeworm of primer pair described in 2 to carry out pcr amplification, order-checking, sequence alignment in sequencing result and SEQ ID No.1 and SEQ ID No.2, if the nucleotide sequencing result of testing sample and anoplocephala perfoliata nucleotide sequence similarity are high, it is anoplocephala perfoliata; If nucleotide sequencing result and anoplocephala magna nucleotide sequence similarity are high, it is anoplocephala magna.
Preferably, the condition of described pcr amplification is: 95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 53 DEG C of annealing 30s, 72 DEG C are extended 30s, totally 30 circulations; After last loop ends, 72 DEG C are extended 10min.
Preferably, the NAD1 of described naked tapeworm extracts from gravid proglottid or worm's ovum.
The method of the described NAD1 that extracts naked tapeworm from worm's ovum is first to collect and collect by floating method by the precipitator method.
Preferably, step is as follows:
1) application rights requires the NAD1 of naked tapeworm of primer pair described in 2 to carry out pcr amplification, obtains PCR product;
2) through agarose gel electrophoresis, reclaim PCR object product, PCR object product is connected and is spent the night at 16 DEG C with T-easy carrier, transfection JM109 competent cell;
3) selecting single bacterium colony cultivates, PCR is accredited as positive recombinant plasmid and checks order, sequencing result and two kinds of tapeworms separately distinctive nucleotide sequence are compared, if the nucleotide sequencing result of testing sample and anoplocephala perfoliata nucleotide sequence similarity are high, it is anoplocephala perfoliata; If nucleotide sequencing result and anoplocephala magna nucleotide sequence similarity are high, it is anoplocephala magna.
The applicant, by anoplocephala perfoliata or anoplocephala magna extracting genome DNA, mitochondrion sequencing, has obtained the NAD1 nucleotide sequence of anoplocephala perfoliata and anoplocephala magna, referring to SEQ ID No.1 in sequence table and SEQ ID No.2.Choose conservative sequence area design primer, the object product size of amplification should be 507 bp.
First the applicant carries out extracting genome DNA, pcr amplification object fragment and order-checking to unknown sample.Then with DNAStar software, sequencing result and anoplocephala perfoliata that we provide and the NAD1 nucleotide sequence of anoplocephala magna are compared respectively, determine the kind of naked tapeworm according to the height of sequence similarity.
The applicant has carried out pcr amplification and order-checking to two kinds naked tapeworm NAD1 gene, and its nucleotide sequence is analyzed, and finds that the homology of the two is 87.2%, referring to Fig. 2, can be used as the target gene of the two differential diagnosis.At present the NAD1 sequence of anoplocephala magna, for we check order first, is not found the report of correlated series.Differentiate two kinds of naked tapeworms, only need pair of primers to increase to the template DNA of tapeworm, Zai Song biotech firm order-checking, compares sequence and the reference sequences that we obtain, according to just distinguishing two kinds of naked tapeworms with reference sequences homology.Aforesaid method has solved common morphological observation can not distinguish for which kind of gravid proglottid, and microscopic examination cannot be distinguished the shortcoming of which kind of worm's ovum.PCR method involved in the present invention, has reached susceptibility and the specificity of molecular diagnosis, has advantages of fast, responsive, special.Meanwhile, method of the present invention is simple, accurate, easy to operate, not only can be used for the molecule discriminating of two kinds of naked tapeworms, but also can be for the epidemiology survey of naked tapeworm of equus.
Brief description of the drawings
Fig. 1 is tapeworm agarose gel electrophoresis figure; Wherein, 1 is anoplocephala perfoliata electrophorogram; 2 is anoplocephala magna electrophorogram; M is DL2000 Marker;
Fig. 2 is anoplocephala perfoliata and anoplocephala magna nucleotide sequence comparison diagram.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
embodiment 1
Quick differential host of the present invention is as follows in the method steps of naked tapeworm of equus:
1. sample collection: polypide gravid segment (abbreviation gravid proglottid) is collected: often excrete with gravid segment full wafer the entozoic tapeworm of digestive tube, the nodal plate the greater in ight soil, is very easy to find.To less, first by collecting dung in basin, add 5-10 clear water doubly, stir, leave standstill and treat natural sedimentation, then supernatant liquid is gone, rejoin clear water, stir, repeatable operation, until supernatant liquid is limpid.Finally upper strata liquid is inclined, find polypide with naked eyes.
2. the genomic extraction of DNA: get prepared by 20mg left and right step (1) polypide gravid segment, move in mortar, firmly grind to form homogenate.Add gentle grinding 30s after the Buffer PBS (purchased from AXYGEN company) of 350 μ L and 0.9 μ LRNase A, collect and in ground tissue homogenate 350 μ L, add the Buffer C-L of 150 μ L and the Proteinase K of 20 μ L, vortex oscillation 1min mixes, and puts 56 DEG C of water-bath 30min-60min.Then the Buffer P-D that adds 350 μ L, vortex oscillation 30s mixes, the centrifugal 10min of 12000 × g.Collection supernatant enters DNA to be prepared in pipe, and the centrifugal 1min of 12000 × g, abandons filtrate; Add the Buffer W1 of 500 μ L, the centrifugal 1min of 12000 × g, abandons filtrate; The Buffer W2 washing that adds 700 μ L, the centrifugal 1min of 12000 × g, abandons filtrate, and repeated washing is once; DNA is prepared to pipe in the centrifugal 1min of 12000 × g, add the elutriant of 40 μ L, the centrifugal 1min of 12000 × g, is DNA extraction liquid.
3. pcr amplification: upstream primer is 5 '-GCAGAGTTTTGCTGATTTTATGAAG-3 ', downstream primer is 5 '-CCACTATACTCAGTATTAAATCCACTAACAAG-3 ' (TaKaRa company is synthetic), pcr amplification is 50 μ L reaction systems, comprises 2 × Premix rTaq solution damping fluid, 25 μ L (purchased from TaKaRa company), concentration is the upstream primer 2 μ L of 40 μ mol/L, downstream primer 2 μ L, above-mentioned DNA extraction liquid 2 μ L, the ddH that concentration is 40 μ mol/L
2o 19 μ L; Amplification reaction condition is: 95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 53 DEG C of annealing 30s, 72 DEG C are extended 30s, totally 30 circulations; After last loop ends, 72 DEG C are extended 10min, obtain PCR product; Agarose gel electrophoresis reclaims object band, and size is approximately 507bp, as shown in Figure 1.
4. ligation: after getting object fragment 3 μ L that pGEM-T easy carrier 1 μ L (50 μ g/ μ L) and glue reclaims and mixing, add 2 × Rapid ligase buffer, 5 μ L, T4 DNA ligase 1 μ L (3U/ μ L), mixes rearmounted 16 DEG C of connections and spends the night.
5. connect the conversion of product: (1) is got 10 μ L and connected product, adds in JM109 competent cell under aseptic condition, gentle shake mixes, and ice bath is placed 30min.(2) 42 DEG C of water-baths, heat shock 90s, ice bath makes it cooling immediately afterwards.(3) add the LB nutrient solution of 800 μ L, 37 DEG C, the gentle vibration of 150r/min 1h.(4), by centrifugal culture 5000r/min 5min, abandon 600 μ L supernatants.(5) mix remaining 200 μ L culture and cell precipitations, then add the chloro-3-indoles-D-of the bromo-4-of 16 μ L X-gal(5-galactoside, 20mg/mL), 4 μ L IPTG(isopropylthio-β-D-galactosides, 200mg/mL) mix, evenly coat on the LB agar plate containing penbritin (100 μ g/mL).
6. extract recombinant plasmid: (1), with aseptic toothpick single bacterium colony of picking white from LB agar plate, is inoculated in 3mL containing in the LB nutrient solution of penbritin (100 μ g/mL), 37 DEG C, 230r/min, violent jolting 12 ~ 16h.(2) the aseptic 1.5mL of pipetting bacterium liquid is put in eppendorf pipe, and the centrifugal 3min of 12000r/min, abandons supernatant, is inverted and eliminates residual liquid.(3) extract plasmid (purchased from AXYGEN company) with plasmid extraction kit (AxyPrep Plasmid Miniprep Kit).
7. the qualification of recombinant plasmid: it is template that recombinant plasmid is got 1 μ L, add 5 μ L 10 × PCR damping fluids, 2 μ L dNTP (2.5mmol/L) mixtures, 0.5 μ L Taq archaeal dna polymerase (5U/ μ L), 0.5 μ L upstream and downstream primers (50pmol/L), mend distilled water to 50 μ L volume and carry out PCR reaction, the same step of PCR reaction conditions (3), uses DL2000 Marker as reference.Be that template is as negative control with blank plasmid vector simultaneously.Electrophoresis observation result.
8. the order-checking of recombinant plasmid: pcr amplification is accredited as to positive clone, send TaKaRa company by plasmid, check order with dideoxy chain termination.
9. nucleotide sequence comparison: with DNAStar software to check order row with our specification sheets in sequence carry out respectively homology comparison.The NAD1 nucleotide sequence of anoplocephala perfoliata is referring to SEQ ID No.1, and the NAD1 nucleotide sequence of described anoplocephala magna is referring to SEQ ID No.2.If the nucleotide sequencing result of testing sample and anoplocephala perfoliata nucleotide sequence similarity are high, it is anoplocephala perfoliata; If nucleotide sequencing result and anoplocephala magna nucleotide sequence similarity are high, it is anoplocephala magna; So just clearly surveyed area equus in district office's is that anoplocephala perfoliata or anoplocephala magna or two kinds of tapeworms all infect.
embodiment 2
The difference of the present embodiment and embodiment 1 is:
1. sample collection: worm's ovum is collected: get ight soil 10g, more than adding clear water 100mL, stir into liquid manure, filter by 250 μ m copper sieves, filtrate collection is in Erlenmeyer flask or beaker, staticly settle 20-40min, the upper strata liquid that inclines, retains sediment, add water and mix again, redeposition, so repeatable operation until supernatant liquid transparent after, upper strata liquid inclines.Add saturated brine 100mL, mix, leave standstill half an hour, worm's ovum floating.Draw the liquid that contains polypide in upper strata in the centrifuge tube of another 50mL, add PBS(pH7.2) to cumulative volume 45mL, the centrifugal 10min of 5000 × g.Collecting precipitation is worm's ovum.In prior art, collect the worm's ovum precipitator method or floating method, the worm's ovum of collection can be for microscopic examination.The present invention, for the worm's ovum of collecting is carried out to differential diagnosis, need to reduce the pollution of impurity, and therefore we combine two kinds of methods, makes the worm's ovum of acquisition cleaner.
The genomic extraction of 2.DNA: get prepared by 20mg left and right step (1) worm's ovum, move in mortar, firmly grind to form homogenate.
All the other steps are all identical with embodiment 1.
Finally it should be noted that: the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement.Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
sequence table
<110> Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
The quick differential host of <120> is in primer special and the discrimination method thereof of naked tapeworm of equus
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 507
<212> DNA
<213> anoplocephala perfoliata
<400> 1
gcagagtttt gctgatttta tgaagttggt ttttaagttt aagatttatt tttttcagag 60
tcggagatat atagggttac taggtgtctt tttattagtt tgtttggttg ttttttattg 120
cttaatatat ggaggttatt ataggttgag ttatagtggt ttttcatttc tttgattttt 180
ggtggttact agttttacta gttattcttt actctgtgct ggttgaggta gctatagaga 240
gtattcgttt ttaggtgcta tgcgttcagc atttgggtct gttagatttg aagcatgttt 300
tatgagagtg attatgtttt gtgctttatg ttatagtggt tattgtttgt gtgattattt 360
ttattgggga agatgcgttt ttatggtgtt tccagttttg tatattttgt gtttaatctg 420
tatactgtgt gagactaatc gtactccttt tgattatggg gaatcggaga gagaacttgt 480
tagtggattt aatactgagt atagtgg 507
<210> 2
<211> 507
<212> DNA
<213> anoplocephala magna
<400> 2
gcagagattt gctgatttta tgaagttggt ttttaagtat aagatttatt tttttcagag 60
tcggaggtat gttggattgt ttggtgtttt tttattaatt tgtctggttg ttttttactg 120
tttaatatat gggggctatt atagactgag atacagtgaa ttttcttttc tttgattttt 180
ggtagtgact agttttacta gttactcttt actttgtatt ggttggggta ggtatagaaa 240
gtattcattt ttgggtgcta tgcgttcggc atttggttct gttaggtttg aagcgtgttt 300
tatgagggtt atagtgtttt gtggtttgtg ttatggtggg tattgtttaa gtgattattt 360
ttattgggga gggtgtattt ttatgatatt tcctgcttta tatattttgt gcttaatttg 420
tatattgtgt gagacaaatc gtactccttt tgattatgga gagtcggaga gagagcttgt 480
tagtggattt aatactgagt atagagg 507
sequence table
<110> Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
The quick differential host of <120> is in primer special and the discrimination method thereof of naked tapeworm of equus
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 507
<212> DNA
<213> anoplocephala perfoliata
<400> 1
gcagagtttt gctgatttta tgaagttggt ttttaagttt aagatttatt tttttcagag 60
tcggagatat atagggttac taggtgtctt tttattagtt tgtttggttg ttttttattg 120
cttaatatat ggaggttatt ataggttgag ttatagtggt ttttcatttc tttgattttt 180
ggtggttact agttttacta gttattcttt actctgtgct ggttgaggta gctatagaga 240
gtattcgttt ttaggtgcta tgcgttcagc atttgggtct gttagatttg aagcatgttt 300
tatgagagtg attatgtttt gtgctttatg ttatagtggt tattgtttgt gtgattattt 360
ttattgggga agatgcgttt ttatggtgtt tccagttttg tatattttgt gtttaatctg 420
tatactgtgt gagactaatc gtactccttt tgattatggg gaatcggaga gagaacttgt 480
tagtggattt aatactgagt atagtgg 507
<210> 2
<211> 507
<212> DNA
<213> anoplocephala magna
<400> 2
gcagagattt gctgatttta tgaagttggt ttttaagtat aagatttatt tttttcagag 60
tcggaggtat gttggattgt ttggtgtttt tttattaatt tgtctggttg ttttttactg 120
tttaatatat gggggctatt atagactgag atacagtgaa ttttcttttc tttgattttt 180
ggtagtgact agttttacta gttactcttt actttgtatt ggttggggta ggtatagaaa 240
gtattcattt ttgggtgcta tgcgttcggc atttggttct gttaggtttg aagcgtgttt 300
tatgagggtt atagtgtttt gtggtttgtg ttatggtggg tattgtttaa gtgattattt 360
ttattgggga gggtgtattt ttatgatatt tcctgcttta tatattttgt gcttaatttg 420
tatattgtgt gagacaaatc gtactccttt tgattatgga gagtcggaga gagagcttgt 480
tagtggattt aatactgagt atagagg 507
Claims (8)
1. quick differential host is in the primer special of naked tapeworm of equus, it is characterized in that: described primer is: upstream primer is 5 '-GCAGAGTTTTGCTGATTTTATGAAG-3 ', downstream primer is 5 '-CCACTATACTCAGTATTAAATCCACTAACAAG-3 '.
2. the primer special of naked teniasis of rapid differential diagnosis equus, it is characterized in that: described primer is: upstream primer is 5 '-GCAGAGTTTTGCTGATTTTATGAAG-3 ', downstream primer is 5 '-CCACTATACTCAGTATTAAATCCACTAACAAG-3 '.
3. parasitize the NAD1 nucleotide sequence of naked tapeworm of equus, described naked tapeworm is anoplocephala perfoliata and anoplocephala magna, it is characterized in that: the NAD1 nucleotide sequence of described anoplocephala perfoliata is referring to SEQ ID No.1, and the NAD1 nucleotide sequence of described anoplocephala magna is referring to SEQ ID No.2.
4. quick differential host is in the method for naked tapeworm of equus, it is characterized in that: application rights requires the NAD1 of naked tapeworm of primer pair described in 2 to carry out pcr amplification, order-checking, sequence alignment in sequencing result and SEQ ID No.1 and SEQ ID No.2, if the nucleotide sequencing result of testing sample and anoplocephala perfoliata nucleotide sequence similarity are high, it is anoplocephala perfoliata; If nucleotide sequencing result and anoplocephala magna nucleotide sequence similarity are high, it is anoplocephala magna.
5. method according to claim 4, is characterized in that: the condition of described pcr amplification is: 95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 53 DEG C of annealing 30s, 72 DEG C are extended 30s, totally 30 circulations; After last loop ends, 72 DEG C are extended 10min.
6. method according to claim 4, is characterized in that: the NAD1 of described naked tapeworm extracts from gravid proglottid or worm's ovum.
7. method according to claim 7, is characterized in that: the method for the described NAD1 that extracts naked tapeworm from worm's ovum is first to collect and collect by floating method by the precipitator method.
8. method according to claim 4, is characterized in that: step is as follows:
(1) application rights requires the NAD1 of naked tapeworm of primer pair described in 2 to carry out pcr amplification, obtains PCR product;
(2) through agarose gel electrophoresis, reclaim PCR object product, PCR object product is connected and is spent the night at 16 DEG C with T-easy carrier, transfection JM109 competent cell;
(3) selecting single bacterium colony cultivates, PCR is accredited as positive recombinant plasmid and checks order, sequencing result and two kinds of tapeworms separately distinctive nucleotide sequence are compared, if the nucleotide sequencing result of testing sample and anoplocephala perfoliata nucleotide sequence similarity are high, are anoplocephala perfoliata; If nucleotide sequencing result and anoplocephala magna nucleotide sequence similarity are high, it is anoplocephala magna.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107326082A (en) * | 2017-08-01 | 2017-11-07 | 中国农业科学院兰州兽医研究所 | A kind of universal primer, nucleotide sequence and its discrimination method of three-type-person source tapeworm ep45 genes |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107326082A (en) * | 2017-08-01 | 2017-11-07 | 中国农业科学院兰州兽医研究所 | A kind of universal primer, nucleotide sequence and its discrimination method of three-type-person source tapeworm ep45 genes |
| CN107326082B (en) * | 2017-08-01 | 2020-12-15 | 中国农业科学院兰州兽医研究所 | Universal primer of human cestode ep45 gene and identification method thereof |
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