CN107326082A - A kind of universal primer, nucleotide sequence and its discrimination method of three-type-person source tapeworm ep45 genes - Google Patents
A kind of universal primer, nucleotide sequence and its discrimination method of three-type-person source tapeworm ep45 genes Download PDFInfo
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- 101100389553 Xenopus laevis serpina6 gene Proteins 0.000 title claims abstract description 54
- 241000242722 Cestoda Species 0.000 title claims abstract description 50
- 239000002773 nucleotide Substances 0.000 title claims abstract description 15
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 15
- 238000012850 discrimination method Methods 0.000 title abstract 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims abstract description 21
- 238000012408 PCR amplification Methods 0.000 claims abstract description 15
- 238000001976 enzyme digestion Methods 0.000 claims description 28
- 241000244155 Taenia Species 0.000 claims description 20
- 241000244157 Taenia solium Species 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 15
- 239000000499 gel Substances 0.000 claims description 13
- 238000011084 recovery Methods 0.000 claims description 13
- 241000866681 Taenia asiatica Species 0.000 claims description 11
- 239000002299 complementary DNA Substances 0.000 claims description 9
- 108020004414 DNA Proteins 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 6
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- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
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- 238000012163 sequencing technique Methods 0.000 abstract description 4
- 238000001962 electrophoresis Methods 0.000 abstract description 3
- 241000894007 species Species 0.000 abstract description 2
- 230000000692 anti-sense effect Effects 0.000 abstract 1
- 230000002068 genetic effect Effects 0.000 abstract 1
- 238000013507 mapping Methods 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 4
- 208000004441 taeniasis Diseases 0.000 description 4
- 108050000761 Serpin Proteins 0.000 description 3
- 102000008847 Serpin Human genes 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000003001 serine protease inhibitor Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 201000000077 Cysticercosis Diseases 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 229910001868 water Inorganic materials 0.000 description 2
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 241000244159 Taenia saginata Species 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
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- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
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- 238000003199 nucleic acid amplification method Methods 0.000 description 1
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- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
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- 210000000813 small intestine Anatomy 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 244000054366 zoonotic parasite Species 0.000 description 1
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Abstract
The present invention relates to field of molecular biotechnology, a kind of universal primer, nucleotide sequence and its discrimination method of three-type-person source tapeworm ep45 genes are disclosed.The primer includes such as SEQ ID NO:Sense primer and SEQ ID NO shown in 1:Anti-sense primer shown in 2；Wherein, SEQ ID NO:1：5’‑AGAGACAAAGGAAAGGCAGCTG‑3’；SEQ ID NO:2：5’‑GGGGCTCAGGCACCTTGAT‑3’；Embodiments of the present invention are by designing the universal primers of three-type-person source tapeworm ep45 genes, RT PCR amplifications obtain the ep45 genetic fragments of tapeworm, then according to the difference of restriction endonuclease sites between three ep45, digestion processing is carried out with EcoR I and/or Aar I, the difference (1/2/3 band) of collection of illustrative plates is occurred according to digestion products electrophoresis species identification is carried out to three-type-person source tapeworm, the time of PCR primer sequencing identification is substantially reduced, and it is more accurate, reliable according to the result of determination of restriction enzyme mapping.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to universal primers and nucleotide sequences of three human cestode ep45 genes and an identification method thereof.
Background
Taenia solium, Taenia asialica and Taenia bovis seu Bubali are important zoonotic parasites, which all parasitize in human small intestine and cause taeniasis and even cysticercosis in human. Therefore, the identification of the species has important significance in the related research and the comprehensive prevention and treatment of the taeniasis/cysticercosis. The three tapeworms parasitizing in human intestinal tracts are very similar in shape, and the most important difference between the three is whether the knob is provided with a tip protrusion or a small hook. Thus, the conventional methods are distinguished by microscopic observation of the head segment after tabletting. Unfortunately, the head segment of the worm body is usually lost, and the worm body is difficult to identify by simply relying on the segment or the worm egg, and usually needs to be identified by a molecular biological method. The most common identification method is to amplify 18s or mitochondrial gene sequences of three polypide by PCR respectively, then carry out sequencing analysis and identify according to the difference of target sequences. However, this method has the disadvantage that sequencing is time consuming and that accurate interspecific identification is not possible when the sequence similarity is close (hybrids).
Disclosure of Invention
The embodiment of the invention aims to provide three methods for identifying human tapeworms, which not only overcome the defects of the traditional microscopic examination, but also carry out single-gene PCR amplification and enzyme digestion identification through a universal primer, can realize the molecular identification of the tapeworms without gene sequencing, greatly accelerate the speed of the tapeworm identification, and simultaneously enable the identification result of the tapeworms to be more accurate and reliable through a method for comparing enzyme digestion maps.
In order to solve the technical problems, the embodiment of the invention provides three universal primers of human tapeworm ep45 gene, which comprise an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2; wherein, SEQ ID NO: 1: 5'-AGAGACAAAGGAAAGGCAGCTG-3', respectively; 2, SEQ ID NO: 5'-GGGGCTCAGGCACCTTGAT-3' are provided.
The embodiment of the invention also provides nucleotide sequences of three human tapeworms ep45, wherein the three human tapeworms are taenia solium, taenia bovis and taenia asiana; the nucleotide sequence of ep45 of taenia solium is shown in SEQ ID NO 3; the nucleotide sequence of ep45 of Taenia bovis is set forth in SEQ ID NO 4; the nucleotide sequence of ep45 of Taenia Asiatica is shown in SEQ ID NO. 5.
The embodiment of the invention also provides three identification methods of the human tapeworm, which comprise the following steps:
(1) extracting three kinds of total RNA of the human tapeworm to be identified, and carrying out reverse transcription on the total RNA by the universal primer of claim 1 to synthesize cDNA of the human tapeworm to be identified;
(2) carrying out PCR amplification by using the cDNA of the human tapeworm to be identified as a template ep45 gene;
(3) carrying out agarose gel electrophoresis on the PCR amplification product, and then carrying out gel recovery; obtaining an ep45 glue recovery product;
(4) carrying out enzyme digestion treatment on the recovered product of the ep45 gel by using EcoR I and/or Aar I restriction endonuclease;
(5) and (4) carrying out agarose gel electrophoresis on the enzyme digestion product, and identifying the human tapeworm according to the enzyme digestion map.
Compared with the prior art, the embodiment of the invention can obtain the ep45 gene fragment of three human tapeworms by RT-PCR amplification by designing the universal primer of the ep45 gene, then carry out enzyme digestion treatment by using EcoR I and/or Aar I according to the difference of restriction enzyme sites among the three, and carry out interspecies identification on the human tapeworms according to the difference (1 strip/2 strips/3 strips) of the appearance map of enzyme digestion product electrophoresis, thereby greatly shortening the identification time of the human tapeworms, and the judgment result of the enzyme digestion map is more reliable than that of sequence comparison.
Preferably, the conditions for PCR amplification of the ep45 gene are: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 60 ℃ for 1min, and extension at 72 ℃ for 90s for 30 cycles; after the end of the last cycle, the extension was carried out for 10min at 72 ℃.
Preferably, the reaction system for PCR amplification of the ep45 gene is as follows: rTaq texture: 25.0 μ L; 1, SEQ ID NO: 1.0 μ L; 2, SEQ ID NO: 1.0 μ L; and (3) identifying human tapeworm cDNA: 1.0 μ L; DEPC H2O: 22.0. mu.L.
Preferably, the gel recovery is performed using a DNA gel in step 3.
Preferably, the total RNA of the human tapeworm to be identified is extracted from a tapeworm segment or an egg.
Preferably, the enzyme cutting system of the ep45 gene is as follows: the ep45 glue recovers 5 μ L of the product; EcoR I or/Aar I1. mu.L; buffer 2. mu.L; 11.5 μ L deionized water.
Drawings
FIG. 1 is a cleavage map of a single cleavage of a recovered product of a gel according to a second embodiment of the present invention.
1. TS control 12; 2. 11, TSA control; 3. TAS control 10; 4, TSep45 EcoR I enzyme cutting result; 5: TSAep45 EcoR I enzyme cutting result; 6, enzyme cutting result of TASep45 EcoR I; 7, carrying out enzyme digestion on TSep45 Aar I; 8, performing enzyme digestion on the TSAep45 Aar I; 9, TASep45 Aar I digestion result.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The first embodiment of the invention relates to universal primers of three human cestode ep45 genes, which comprise an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2; wherein,
SEQ ID NO:1：5’-AGAGACAAAGGAAAGGCAGCTG-3’
SEQ ID NO:2：5’-GGGGCTCAGGCACCTTGAT-3’。
the second embodiment of the invention relates to a method for identifying three human tapeworms, which comprises the following steps:
(1) sequence analysis of three human cestodes ep45
Downloading the nucleotide sequences of ep45 genes of three Tapeworms (TS), Taenia bovis (TSA) and Taenia Asiatica (TAS) from a Taenia genomic database of Lanzhou veterinary institute, and comparing the nucleotide sequences of ep45 of the three tapeworms by Blast analysis, wherein the nucleotide sequence of ep45 of the Taenia suis is shown in SEQ ID NO. 3; the nucleotide sequence of ep45 of Taenia bovis is set forth in SEQ ID NO 4; the nucleotide sequence of ep45 from Taenia Asiatica is shown in SEQ ID NO 5;
(2) design of universal primers for three human cestodes ep45
Primers are designed by Primer Premier 5.0 software, the upstream Primer (ep45UPM-F) is at 18-40 sites of ORF, the downstream Primer (ep45UPM-R) is at 1305-1329 sites, the length of amplified fragments is 1306bp,
ep45UPM-F (i.e., SEQ ID NO:1):
5’-AGAGACAAAGGAAAGGCAGCTG-3’；
ep45UPM-R (i.e., SEQ ID NO:2):
5’-GGGGCTCAGGCACCTTGAT-3’；
(3) PCR amplification of ep45 Gene
Collecting about 30mg of human tapeworm section or worm egg to be detected, extracting total RNA by Trizol, then carrying out reverse transcription by using ep45UPM-R (SEQ ID NO:2) as a primer, synthesizing a cDNA first chain, and further carrying out PCR amplification by using cDNA as a template, wherein the conditions of the PCR amplification are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 60 ℃ for 1min, and extension at 72 ℃ for 90s for 30 cycles; after the end of the last cycle, the extension was carried out for 10min at 72 ℃.
The reaction system for the PCR amplification of the ep45 gene is as follows:
rTaq texture (available from TaKaRa Co.): 25.0 μ L;
SEQ ID NO:1：1.0μL；
SEQ ID NO:2：1.0μL；
and (3) identifying human tapeworm cDNA: 1.0 μ L;
DEPC H2O 22.0μL。
(4) recovery and purification of PCR product of ep45 gene
The PCR product was observed by 1% agarose gel electrophoresis, and the gel strip of the target fragment was cut off and the target fragment was recovered by a DNA gel recovery kit.
(5) Digestion reaction of PCR product
Carrying out enzyme digestion treatment on the glue recovery product in the step 4 by using EcoR I and/or Aar I restriction endonuclease; wherein,
an enzyme digestion reaction system:
the reason why the EcoR I and/or Aar I restriction enzymes are used in the present embodiment is as follows: restriction enzyme sites of the three ep45 sequences were analyzed using DNAstar-seqBuilder restriction site software. Because the nucleotide sequence of the ep45 of the taenia solium has EcoR I and Aar I2 enzyme cutting sites, the nucleotide sequence of the ep45 of the taenia solium has only one EcoR I enzyme cutting site, and the nucleotide sequence of the ep45 of the taenia solium does not have the 2 enzyme cutting sites. Therefore, single-enzyme digestion or double-enzyme digestion reaction can be carried out on the ep45 PCR product of the sample to be identified by using EcoR I and/or Aar I restriction enzymes, then agarose gel electrophoresis is carried out on the enzyme digestion product, and three human tapeworms are identified according to the number or size (enzyme digestion pattern) of the bands.
(6) Band identification
If the gel recovery product in the step 4 is subjected to EcoR I single enzyme digestion treatment and then 2 bands (766bp and 540bp) appear, the sample to be identified is Taenia Solium (TS), and if 1 band (1306bp) appears, the sample is Taenia Solium (TSA) or Taenia Asiana (TAS);
when the recovered product of the gel in step 4 is digested with Aar I, it is TSA (697bp, 609bp) if 2 bands appear, and TAS if 1 band (1306bp) appears, as shown in FIG. 1.
If 3 bands (609bp, 540bp and 157bp) appear after the gel recovery product in the step 4 is subjected to two enzyme digestion treatment by EcoR I and Aar I (shown in the table 1), the sample to be detected is Taenia Solium (TS);
if 2 bands (697bp, 609bp) appear after the gel recovery product in the step 4 is subjected to two enzyme digestion treatment by EcoR I and Aar I, the sample to be detected is taenia bovis (TSA);
if 1 band (1306bp) appears after the gel recovery product in the step 4 is subjected to two enzyme digestion treatment by EcoR I and Aar I, the sample to be detected is Taenia Asiatica (TAS) (not shown in the figure). Therefore, the glue recovery product in the step 4 can be treated by the EcoR I or Aar I alone or in combination, and the three human tapeworms can be subjected to differential diagnosis.
| Human tapeworm | EcoR I | Aar I | EcoR I and Aar I |
| TS | 766bp,540bp | 609bp,697bp | 609bp,540bp,157bp |
| TSA | 1306bp | 609bp,697bp | 609bp,697bp |
| TAS | 1306bp | 1306bp | 1306bp |
TABLE 1 enzyme digestion maps of three human cestode ep45 gene fragments
Compared with the prior art, in the research of serpin inhibitor (serpins) gene family, it is found that the (ep45) gene which is one of serpins is very well conserved in Taenia suis (Taenia solium), Taenia asians (Taenia asitica) and Taenia bovis (Taenia saginata), and the Identity (Identity) of the three genes on the nucleic acid level is up to 96.4%, and the similarity (similarity) is up to 99.6%. Therefore, in the embodiment, on the basis of cloning and sequence analysis of the ep45 genes of the three human tapeworms, the ep45 gene segments (1306bp) of the three human tapeworms can be obtained by selecting a very conservative sequence to design a universal primer of the ep45 gene and performing RT-PCR. And then carrying out enzyme digestion treatment by using EcoR I or Aar I according to the difference of restriction enzyme sites among the three, and carrying out interspecies identification on three human tapeworms according to the difference (1/2/3 bands) of the appearance maps of the enzyme digestion product electrophoresis, thereby greatly shortening the time of traditional molecular identification, and the judgment result of the enzyme digestion maps is more reliable than that of sequence comparison.
It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples for carrying out the invention, and that various changes in form and details may be made therein without departing from the spirit and scope of the invention in practice.
SEQUENCE LISTING
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> universal primers and nucleotide sequences of three human cestode ep45 genes and identification method thereof
<130>2017
<160>5
<170>PatentIn version 3.5
<210>1
<211>22
<212>DNA
<213> Artificial sequence
<400>1
agagacaaag gaaaggcagc tg 22
<210>2
<211>19
<212>DNA
<213> Artificial sequence
<400>2
ggggctcagg caccttgat 19
<210>3
<211>1350
<212>DNA
<213> taenia solium
<400>3
atgatttctc tacgtatgag agacaaagga aaggcagctg catctgagcc tgagagaccc 60
aagtctactt tttcgaaaag cgtcaaagtc aatccggtcg actatctgaa gcacagggac 120
ttcaacaaca aatattgctt cggtgttcat gccattaatg acatcaccaa gaagtcgggt 180
cactcgccaa caacgatacg cttcctcctg acagtgttgg tgggctcgaa ggcggcacgg 240
ggcactagtg cggaccagat cacacaggca ctcagcacca cgaactcacg tgacgtgggc 300
accgaatgca ccagcctcgt ggacagcgcg cttgacgagt gggctctgct ctctactgcg 360
ggtctaagcg atataccact cgatcgcatt gaggaagggc gcctctgtcg ctttcagtct 420
gctatcttcg tccccatgga tgatctcacc ttcaaggccg agttccagtg gttgatcacc 480
aaccgcatgg gtgtcgtctg gactcagact tcgagaaagg atttcgccca tgctcagaag 540
tggttatcca aagtgtcaaa aggtttattc actcaccact ttccaaaaca atgtgccaac 600
tctattgtac ttgccacagc attacaattt aaaggcaggt ggactcaacc tttggaactc 660
tacggtagtt ccaagggatc attcgaagtg tcaccccact cgaagatcga tgttcctatg 720
ctaaaaattt caacaaaggt tatttactat aaggatacca aaaaaggctt ccatttggtt 780
ggaattccac ttaaggatgc gcggttcgca atcgcatttc tattaccctt ggtgccgcac 840
aagttcgtag aggtggagca tcgctttaca gagggtttga actacggtct cttcaacagc 900
tcgcgactgc atttctgcag catgcacgtg gtcattccta tgttccgagt gcaaacggag 960
gtggacctct gtaaggcctt gcccttcctc ggcatgagca atccattcga ttctgatcgg 1020
gcagacttct ccggtatcag cgatgtagag aatctgcata tcaacagcgg caaggagtcg 1080
gcctttctgc aggtctccag aagcgggatc cgactgctct cagtgggtac tctcaatctg 1140
gagacgaacc agcaccagtt taggcatcct gacctctttg aggtgccaac cctcgaggca 1200
cccggggacg tggacccaaa ggggttgcac tctttcttag tgaatcagcc ctttgctgtc 1260
atcctcattg atcgcgagtc cggctgcgtc ctataccaag ccagaatcaa ggtgcctgag 1320
ccccccccca actccacact ctgccactaa 1350
<210>4
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<213> Taenia bovis
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atgatttctc tacatatgag agacaaagga aaggcagctg catctgagcc tgagagaccc 60
aaatctactg tttcgaaaag cgtcaaagtc aatccggtcg actatctgaa gcacagggac 120
ttcaacaaca aatattgctt cggtgttcat gccattaatg acatcaccaa gaagtcgggt 180
cactcgccaa caacgatacg cttcctcctg acagtgttgg tgggctcgaa ggcggcacgg 240
ggcaccagtg cggaccagat cacacaggca ctcagcacca cgaactcacg tgatgtgggc 300
accgaatgca ccagcctcgt ggacagcgcg cttgatgagt gggctctgct ctctactgcg 360
ggtctaagcg atataccact cgatcgcatt gaggaagggc gcctctgtcg tttccagtct 420
gctatcttcg tccccatgga tgatctcacc ttcaaagccg agttccagtg gctgatcacc 480
aaccgcatgg gtgtcgtctg gactcagact tcgagaaatg atttcgccca tgctcagaag 540
tggttatcca aagtgtcaaa aggtttattc actcaccact ttccaaaaca atgtaccaac 600
tctcttgtac ttgccacagc attacaattt aaaggcaggt ggactcaacc tttggagctc 660
tacggtagtt ccaatggatc attcgaagtg tcaccccact cgaggataga tattcctatg 720
ctaaaaattt caacaaaggt tatttactac aaggatacca aaaaaggctt ccatttggtt 780
ggaatcccac ttaaggatac acggttcgca atcgcatttc tattaccctt ggtgccgcac 840
aagttcgtag aggtggagca tcgctttaca gagggtttga actacggtct cttcaacagc 900
tcgcgactgc atttctgcag catgcacgtg gtcattccta tgtttcgagt gcaaacggag 960
gtggacctct gtaaggcctt gcccttcctc ggcatgagca atccattcga ctctgatcgg 1020
gccgacttct ccggtatcag cgatgtagag aatctgcata tcaacagcgg caaggagtcg 1080
gcctttctgc aggtctccag aagcgggatc cgactgctct cagtgggtac tctcaatctg 1140
gagacgaacc agcaccagtt taggcatcct gacctctttg aggtgccaac tctcgaggca 1200
cccggggacg tggacccaaa ggaggtgcac tttttcttag tgaatcagcc ctttgctgtc 1260
atcctcattg atcgcgagtc cgactgcgtc ctataccaag ccagaatcaa ggtgcctgag 1320
cccctctcca actccacact gcctgccact aacttacaat aa 1362
<210>5
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<213> Asian taenia
<400>5
atgatttctc tacatataag agacaaagga aaggcagctg catctgagcc tgagagaccc 60
aaatctactg tttcgaaaag cgtcaaagtc aatccggtcg actctctgaa gcacagggac 120
ttcaacaaca aatactgctt cggtgttcat gccattaatg acatcaccaa gaagtcgggt 180
cactcgccaa caacgatacg cttcctcctg acagtgttgg tgggctcgaa ggcggcacgg 240
ggcaccagtg cggaccagat cacacaggca ctcagcacca cgaactcacg tgatgtgggc 300
accgaatgca ccagcctcgt ggacagcgcg cttgatgagt gggctctgct ctctactgcg 360
ggtctaagcg atataccact cgatcgcatt gaggaagggc gcctctgtcg tttccagtct 420
gctatcttcg tccccatgga tgatctcacc ttcaaagccg agttccagtg gctgatcacc 480
aaccgcatgg gtgtcgtctg gactcagact tcgagaaatg atttcgccca tgctcagaag 540
tggttatcca aagtgtcaaa aggtttattc actcaccact ttccaaaaca atgtaccaac 600
tctcttgtac ttgccacagc attacaattt aaaggtaggt ggactcaacc tttggagctc 660
tacggtagtt ccaagggatc attcgaagtg tcaccccact cgaggataga tattcctatg 720
ctaaaaattt caacaaaggt tatttactac aaggatacca aaaaaggctt ccatttggtt 780
ggaatcccac ttaaggatac acggttcgca atcgcatttc tattaccctt ggtgccgcac 840
aagttcgtag aggtggagca tcgctttaca gagggtttga actacggtct cttcaacagc 900
tcgcgactgc atttctgcag catgcacgtg gtcattccta tgtttcgagt gcaaacggag 960
gtggacctct gtaaggcctt gcccttcctc ggcatgagca atccattcga ctctgatcgg 1020
gccgacttct ccggtatcag cgatgtagag aatctgcata tcaacagcgg caaggagtcg 1080
gcctttctgc aggtctccag aagcgggatc cgactgctct cagtgggtac tctcagtctg 1140
gagacgaacc agcaccagtt taggcatcct gacctctttg aggtgccaac cctcgaggca 1200
cccggggacg tggacccaaa ggaggtgcac tttttcttag tgaatcagcc ctttgctgtc 1260
atcctcattg atcgcgagtc cgactgcgtc ctataccaag ccagaatcaa ggtgcctgag 1320
cccctctcca actccacact gcctgccact aacttacaat aa 1362
Claims (8)
1. A universal primer of three human cestode ep45 genes is characterized by comprising an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2; wherein,
SEQ ID NO:1：5’-AGAGACAAAGGAAAGGCAGCTG-3’；
SEQ ID NO:2：5’-GGGGCTCAGGCACCTTGAT-3’。
2. nucleotide sequences of three human tapeworm ep45 genes, wherein the tapeworms are taenia solium, taenia bovis and taenia asialis; wherein the nucleotide sequence of ep45 of taenia solium is shown in SEQ ID NO 3; the nucleotide sequence of ep45 of Taenia bovis is shown in SEQ ID NO 4; the nucleotide sequence of ep45 of Taenia Asiatica is shown in SEQ ID NO. 5.
3. The identification method of three human tapeworms is characterized by comprising the following steps:
(1) extracting three kinds of total RNA of the human tapeworm to be identified, and carrying out reverse transcription on the total RNA by the universal primer of claim 1 to synthesize cDNA of the human tapeworm to be identified;
(2) carrying out PCR amplification by using the cDNA of the human tapeworm to be identified as a template ep45 gene; obtaining a PCR amplification product;
(3) carrying out agarose gel electrophoresis on the PCR amplification product, and then carrying out gel recovery; obtaining an ep45 glue recovery product;
(4) carrying out enzyme digestion treatment on the recovered product of the ep45 glue by using EcoR I and/or Aar I;
(5) and (4) carrying out agarose gel electrophoresis on the enzyme digestion product, and identifying the human tapeworm according to the enzyme digestion map.
4. The method for identifying three human tapeworms of claim 3, wherein the PCR amplification conditions for the ep45 gene are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 60 ℃ for 1min, and extension at 72 ℃ for 90s for 30 cycles; after the end of the last cycle, the extension was carried out for 10min at 72 ℃.
5. The method for identifying three human tapeworms according to claim 4, wherein the reaction system for PCR amplification of the ep45 gene is as follows:
rTaq mixture：25.0μL；
SEQ ID NO:1：1.0μL；
SEQ ID NO:2：1.0μL；
and (3) identifying human tapeworm cDNA: 1.0 μ L;
DEPC H2O：22.0μL。
6. the method for identifying three human tapeworms of claim 3, wherein the DNA is recovered and purified by gel electrophoresis in step 3.
7. The method for identifying three human tapeworms according to claim 3, wherein the total RNA of the human tapeworms to be identified is extracted from tapeworm segments or worm eggs.
8. The method for identifying three human tapeworms according to claim 3, wherein the enzyme digestion system of the ep45 gene is as follows:
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