CN108950020A - The application of Echinococcus granulosus mitochondria ND6 gene - Google Patents
The application of Echinococcus granulosus mitochondria ND6 gene Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, disclose a series of new opplications of Echinococcus granulosus mitochondria ND6 gene.The present invention is using Echinococcus granulosus mitochondria ND6 gene as molecular diagnostic markers, detect whether dog infects Echinococcus granulosus by PCR, with very high reliability, specificity and sensitivity, and the Echinococcus granulosus in dog excrement can be detected in time in infection early stage, it can be used for the epidemiological survey of dog infection Echinococcus granulosus.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to the application of Echinococcus granulosus mitochondria ND6 gene.
Background technique
The history of life of Echinococcus granulosus (Echinococcus granulosus, Eg) is complex, to pass through intermediate place
Owner or certain domestic animals (such as ox, sheep herbivore) and final host canid are completed jointly.Echinococcus granulosus
Middle silk ribbon phase larva-Echinococcus Granulosus Cysts main parasitic is in the liver and lungs of intermediate host, so as to cause echinococcosis granulosa
(cystic echinococcosis) (Echinococcosis).The disease is a kind of infecting both domestic animals and human parasitic disease, is in worldwide distribution, wherein China
Belong to one of hotspot, Major Epidemic is in Qinghai, Xinjiang, Tibet, Northwest Sichuan pastoral area and Ningxia, and China is at least at present
368 popular counties.Now the disease has been cited as China's " long-term animal epidemic control program in country " and preferentially prevents in (2012-2020) year
Control the animal epidemic with guard key.
Dog is the most important final host of Echinococcus granulosus and the infection sources.Dog eats after echinococcus, and protoscolex is small at its
Enteral can be developed for adult for 40~50 days, and adult is 5~6 months in the dog intracorporal service life.Adult gravid proglottid and worm's ovum are with dog
Excrement is discharged and pollutes foodstuff, water source and environment, and intermediate host orally eats worm's ovum and is infected, so as to cause bladder type Echinococcus hydatid cyst
Disease.As can timely and accurately detecting the case where dog infects Echinococcus granulosus, there is weight to the prevention and control of humans and animals echinococcosis
Want meaning.
The conventional method of detection dog infection Echinococcus granulosus mainly has: dissect method, arecaline purgative therapy and worm's ovum floating
Inspection technique.Dissect method testing result is intuitive, but cumbersome, in fact it could happen that missing inspection situation；Arecaline purgative therapy has preferable
Application value, but it is led the rate of letting out and there was only 70% or so, thereby increases and it is possible to it will cause environmental pollution.Though it is simple and convenient that worm's ovum floats inspection technique
It is easy, but still will appear missing inspection situation.In addition, due to Echinococcus tapeworm worm's ovum and other Taeniidaes worm's ovum morphologically
It is difficult to distinguish, therefore causes the method specificity not high.
The principle of excrement antigen ELISA detection method is that polypide parasitizes in host intestine, metabolin, secretion, nodal plate, worm
The body tissues such as ovum (claiming excrement antigen) can be discharged in vitro with host's excrement, can detect excrement antigen by preparing specific antibody.The method
It is simple and easy, it is not stringent to detection sample requirement, it is suitble to promote the use of in base；And rising sun etc. was once reported, when dog infects 5
The positive can be detected after it infects 16d when polypide, there is early diagnosis value.Though the specificity of the method up to 97%,
Sensibility is but infected the influence of degree, wherein infection polypide number at 100 or more sensibility up to 92.0%, but
At 100 or less, sensibility only has 29.0%.In addition, the method may go out between the polypide similar in Taeniidae kind
Existing serious cross reaction, therefore, excrement antigen ELISA method testing result is likely to occur higher false positive.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amp lification, LAMP) is directed to
4 pairs of primers are designed in 6 regions of target gene, using the Bst archaeal dna polymerase with strand-displacement activity at constant temperature (65 DEG C)
Lower about 1h can amplify target fragment.Equipment needed for the method is simple, and rapid reaction, product detection is convenient, high specificity.Chen Lu
Deng once report when dog infection Echinococcus granulosus be greater than 10000 when, 18d can be detected after its infection.In addition, to dog
When excrement is detected, Bst archaeal dna polymerase activity hardly influenced by excrement mortifier, this but also LAMP method spirit
Sensitivity is higher than Standard PCR.But also because of its excessively high sensitivity, a small amount of gene contamination will lead to the method experimental result in false sun
Property, need other detection method to this verifying；And the electrophoretogram of LAMP method is multi-ribbon, even if there is the production of non-specific amplification
It is raw, it is also not easy to recognize.Meanwhile also because of non-single band, LAMP method can not carry out Genotyping by gene sequencing.Separately
Outside, which requires the control of target sequence length hereinafter, more demanding to primer, to filter out suitable primer in 300bp and need to consume
Take a large amount of work.
Animal wastes are easy to the substantial amounts of the source of infection in Collection and conservation and excrement, and pathogen inhereditary material can be with source
In the worm's ovum of helminth or the cell of helminth polypide and fragment of tissue etc., purpose base only can be extracted with a small amount of excrement
Cause, therefore excrement PCR detection method has been increasingly becoming the important channel of detection Animal diseases.Compared to pathogeny detection and it is immunized
Learn detection method, PCR detection method have many advantages, such as quickly, it is accurate, sensitivity, special, can also realize and form is difficult to differentiate between
The identification of Taeniidae.Echinococcus multilocularis sense is detected from fox excrement DNA from Bretagne etc. (1993) report round pcr
After dye, which is also widely used in the detection of dog excrement echinococcus ovum.Dinkel etc. (1998) applies nest-type PRC
Detection method expands Echinococcus multilocularis 12S rRNA segment (373bp), as a result lowest detection to 1 worm's ovum, and thin
For 11 kinds of other tapeworms such as grain echinococcus without non-specific amplification, specificity is 100%.Abbasi etc. (2003) is to target weight
Complex sequences (EgG1 Hae III) (133bp) has carried out PCR amplification, and susceptibility is very high, minimum also to can detect that a worm's ovum.
But its specificity is poor, while also 133bpDNA having the same is heavy for the Taenia of amplification, bull band tapeworm, sheep band tapeworm
Complex sequences.Stefania etc. (2004) carries out PCR amplification to G1 plants of Echinococcus granulosus of 12SrDNA segment (255bp), sensitive
Property reach detection to a worm's ovum, while other 5 kinds of genotype of Echinococcus granulosus and other 14 kinds of tapeworm genes are carried out
Amplification, it is as a result only specific to amplify Echinococcus granulosus G1 type, testing result non-false positive.In addition, Dinkel etc.
(2004) also according to G1 plants of mitochondria 12S rRNA gene order (254bp) design primers of Echinococcus granulosus, it authenticated tapeworm
Do not belong to Echinococcus granulosus different genotype (totally 16 kinds), specificity is up to 100%, while its susceptibility also reaches
The DNA content of 0.25pg.
Although round pcr due to sensibility with higher, specificity the features such as be widely used in clinical detection,
It is that properly reliable molecular diagnostic markers are highly important to the detection of Animal diseases for selection.
Summary of the invention
In view of this, the purpose of the present invention is to provide the new opplication of Echinococcus granulosus mitochondria ND6 gene, so that institute
When stating molecular diagnostic markers of the ND6 gene as detection Echinococcus granulosus, have higher accuracy, sensitivity and special
Property, and Echinococcus granulosus can be detected earlier.
To achieve the goals above, the invention provides the following technical scheme:
Application of the Echinococcus granulosus mitochondria ND6 gene as the molecular diagnostic markers of detection Echinococcus granulosus；
Echinococcus granulosus mitochondria ND6 gene (ND6) complete sequence length is short (456bp), the simple intronless of structure, into
It is moderate to change rate, sequence variations are abundant, and mutation rate is significantly higher than core DNA, has been widely used in yak, fish and insect at present
Genetic Constitution of Population analysis, but have not been reported for dog infection Echinococcus granulosus detection in.The present invention utilizes PCR skill
Art using the ND6 complete genome sequence filtered out from mitochondrial genomes as molecular diagnostic markers and expands the gene,
Establish a kind of PCR method for detecting Echinococcus granulosus DNA in dog excrement.
In China, the tapeworm of dog small intestine endoparasitism is in addition to Echinococcus granulosus, and most common there are also Echinococcus multilocularis, more
Headband tapeworm, blister band tapeworm, Taenia Pisiformis, diphlidium caninum, Meng Shi change other tapeworms such as palace tapeworm.In addition, dog bow is first
Roundworm is also very common helminth.The present invention respectively carries out above eight kinds of helminths using ND6 gene as molecular diagnostic markers
PCR detection, the results showed that do not amplify purpose band in addition to Echinococcus granulosus, illustrate using ND6 gene as molecular diagnosis
The species specificity with height is marked, cross reaction will not occur.This species specificity in order to further increase, the present invention with
For main molecular diagnostic markers simultaneously, the part of part tRNA-GLU sequence (38bp) and downstream of its upstream is added in ND6 gene
TRNA-TYR sequence (64bp) composition more preferably molecular diagnostic markers.
When measurement is using ND6 gene as the sensitivity of molecular diagnostic markers, target gene is diluted into (273ng- in proportion
4pg), the results showed that with gradually decreasing for DNA concentration, purpose band brightness also be increasingly diminished until be diluted to 4pg when sun
Property band just completely disappears.In existing report, Abbasi (2003) once detected the gene of single Echinococcus granulosus worm's ovum
(EgG1 Hae III) DNA content is 8pg, using ND6 gene as molecular diagnostic markers sensitivity with higher, i.e., this is indicated that
Be detected only 1 Echinococcus granulosus worm's ovum can also.
In addition, be verifying using ND6 gene as the reliability of molecular diagnostic markers, dog of the present invention to artificial challenge protoscolex
Excrement has carried out PCR detection, the results showed that when about 50000 protoscolexs of dog artificial challenge, PCR the 13rd day can be from dog in infection
Target gene is amplified in excrement, shows that dog Echinococcus granulosus can be being early diagnosed out by molecular diagnostic markers of ND6 gene
Infection conditions.
It is a variety of excellent effects of molecular diagnostic markers based on ND6 gene, the invention also provides following various new
Using:
Echinococcus granulosus mitochondria ND6 gene answering in the molecular diagnostic markers of preparation detection Echinococcus granulosus
With；
Echinococcus granulosus mitochondria ND6 gene detects the reagent of Echinococcus granulosus as molecular diagnostic markers in preparation
Application in box；
Expand examination of the upstream and downstream primer in preparation detection Echinococcus granulosus of Echinococcus granulosus mitochondria ND6 gene
Application in agent box；
Wherein, the Echinococcus granulosus mitochondria ND6 gene is ND6 wild type gene；Or on ND6 wild type gene
Swim the sequence of 1-100bp, ND6 wild type gene and ND6 wild type gene downstream 1-100bp composition；Or in ND6 wild type gene
The sequence of one or more bases is lacked, adds or replaced in sequence；Or to the sequence that ND6 wild type gene is modified；Institute
It states missing, addition or one or more bases and modification is replaced to will not influence and draw using the amplification that ND6 gene is designed as target gene
The PCR amplification of object.On ND6 wild type gene downstream 1-100bp sequence be respectively part tRNA-GLU sequence and part tRNA-
TYR sequence.
According to above-mentioned application, the present invention also provides a kind of kits for detecting Echinococcus granulosus, including amplification particulate
The upstream and downstream primer of echinococcus mitochondria ND6 gene；The upstream and downstream of the amplification Echinococcus granulosus mitochondria ND6 gene
Primer can be designed according to existing software such as Primer Premier 5.0, in the specific embodiment of the invention, the present invention
It provides by ND6 wild type gene upstream 1-100bp, ND6 wild type gene and ND6 wild type gene downstream 1-100bp group
At the upstream and downstream primer that is designed for target gene of sequence, the upstream and downstream primer is as shown in SEQ ID NO:1-2；It also mentions simultaneously
The upstream and downstream primer only designed using ND6 wild type gene as target gene, the upstream and downstream primer such as SEQ ID NO:3-4 are supplied
It is shown；
In addition, the kit may also include the enzyme for PCR amplification, dNTP, ddH2O, one of stabilizer or two
Kind or more component.In the specific embodiment of the invention, the kit includes SEQ ID NO:1-2 or SEQ ID NO:3-4
Shown upstream and downstream primer, Taq PCR MasterMix, ddH2O and stabilizer BSA.
From the above technical scheme, the present invention is led to using Echinococcus granulosus mitochondria ND6 gene as molecular diagnostic markers
It crosses whether PCR detection dog infects Echinococcus granulosus, there is very high reliability, specificity and sensitivity, and can infect
Early stage detects the Echinococcus granulosus in dog excrement in time, can be used for the epidemiological survey of dog infection Echinococcus granulosus.
Detailed description of the invention
Fig. 1 show the PCR amplification gel figure of 18 age in days child worm DNA；Wherein, M is DL2000 DNA molecular amount standard；1
For 18 age in days child worm DNA；2 be positive control；3 be negative control；
Fig. 2 show the PCR amplification gel figure of different tapeworm DNA samples；Wherein, M is DL2000 DNA molecular amount standard；
1 is Echinococcus granulosus DNA；2 be Echinococcus multilocularis DNA；3 be bull band tapeworm DNA；4 change palace tapeworm DNA for Meng Shi；5 are
Taenia Pisiformis DNA；6 be blister band tapeworm DNA；7 be diphlidium caninum DNA；8 be Toxocara canis；9 be negative control；
Fig. 3 show bull band tapeworm dog excrement and 2 parts of the dog excrement of 5 parts of natural infection Taenia Pisiformis, 5 parts of natural infections
PCR amplification gel figure of the blister of natural infection with tapeworm dog excrement；Wherein, M is DL2000 DNA molecular amount standard；1 is the positive
Control；2-6 is Taenia Pisiformis dog excrement DNA；7-11 is bull band tapeworm dog excrement DNA；12-13 is blister band tapeworm dog excrement DNA；
Fig. 4 show the PCR amplification gel figure of 24 parts of clinical dog excrement；Wherein, M is DL2000 DNA molecular amount standard；1-
24 be clinical dog excrement DNA；
Fig. 5 show the PCR amplification gel figure of the Echinococcus granulosus DNA of different dilutions；Wherein, M DL2000DNA
Molecular weight standard；1 is initial DNA；2-11 is followed successively by 1:10,1:100,1:1000,1:2000,1:4000,1:8000,1:
16000, the DNA of 1:32000,1:64000,1:128000 doubling dilution；
Fig. 6 show the PCR amplification gel figure of 2 artificial challenge's dog 3d-18d excrement；Wherein, (a) and (b) generation respectively
2 artificial challenge's dog 3d-18d excrement of table as a result, M be DL2000 DNA molecular amount standard；1-16 is followed successively by artificial challenge
3rd day to the 18th day dog excrement DNA.
Specific embodiment
The invention discloses the application of Echinococcus granulosus mitochondria ND6 gene, those skilled in the art can use for reference this
Literary content, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to art technology
It is it will be apparent that they are considered as being included in the present invention for personnel.Application of the present invention, which has passed through, preferably to be implemented
Example is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to it is as described herein apply into
Row change or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The application of Echinococcus granulosus mitochondria ND6 gene provided by the present invention is described further below.
Embodiment 1: design of primers
According to the ND6 gene sequence of Echinococcus granulosus mitochondria full-length genome (sequence number: KJ559023.1) in GenBank
Column, using 5.0 software design 1 of Primer Premier to primer.Upstream primer A1: 5 '-TTTCGTGCTGTAGATGGT-3 '
(shown in SEQ ID NO:1), downstream primer A2: 5 '-CACAGATTTCAAAGGGTT-3 ' (shown in SEQ ID NO:2) expand piece
Section is the (part of complete ND6 wild type gene 456bp and its part tRNA-GLU sequence 38bp of upstream, downstream 558bp
TRNA-TYR sequence 64bp composition, sequence is as shown in SEQ ID NO:5).Primer sequence is by raw work engineering (Shanghai) share of biology
Co., Ltd's synthesis.
Embodiment 2:PCR amplification
Amplification system is 25 μ L:2 × Taq PCR MasterMix 12.5 μ L, primer A1、A2Each 1.0 μ L, template DNA
1.0 μ L, ddH28.7 0.8 μ L (BSA is stabilizer) of μ L, 0.4%BSA of O, mixes gently rear brief centrifugation 15s, meanwhile, with
ddH2O replaces template DNA to make blank control.Amplification condition: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45s, 58 DEG C of annealing 45s, 72
DEG C extend 30s, totally 30 circulation；Last 72 DEG C of extensions 6min.10 μ L amplified productions, 1% Ago-Gel is taken after reaction
Electroresis appraisal.Parallel test is repeated 3 times.
Embodiment 3: Echinococcus granulosus DNA PCR amplification
With the primer A of synthesis1、A2, using the DNA extracted from 18 age in days child worms as template, expanded according to 2 method of embodiment, knot
Fruit sees Fig. 1, and stripe size is consistent with expected results conjunction, and band is clear, and negative control is without any band；Furthermore public by reagent
Gene sequencing is taken charge of, expection is as a result met.
Embodiment 4: specific assay
1, the specific test of different tapeworm DNA samples
Echinococcus granulosus, Meng Shi change palace tapeworm (Spirometra mansoni), Taenia Pisiformis (Taenia
Pisiformis), diphlidium caninum (Dipylidium caninum), bull band tapeworm (Taeniamulticeps) and blister
Band tapeworm (Taenia hydatigena) sample DNA and Toxocara canis sample DNA (Toxocara canis) are by sichuan agriculture
University animal parasitic disease research center provides；Echinococcus multilocularis (Echinococcus multilocularis) sample DNA
By the Chinese Shanghai CDC, institute of parasitic doctor Dang Zhisheng is given.
With the primer A of synthesis1、A2, according to 2 method of embodiment to above-mentioned Echinococcus granulosus, Echinococcus multilocularis, Meng Shi
Repeatedly palace tapeworm, Taenia Pisiformis, diphlidium caninum, bull are expanded with tapeworm, blister with the DNA of tapeworm and Toxocara canis
Increase, as a result sees Fig. 2.
Echinococcus granulosus DNA sample pcr amplification reaction generates single purpose band, and Echinococcus multilocularis, Meng Shi
The repeatedly DNA sample and yin of palace tapeworm, Taenia Pisiformis, diphlidium caninum, bull with tapeworm, blister with tapeworm and Toxocara canis
Property control without this amplified band.
2, the detection of different fecal specimens
The dog excrement of Taenia Pisiformis (5 parts), bull with tapeworm (5 parts) and blister with tapeworm (2 parts) is by Sichuan Agricultural University
Animal parasitosis research center provides；
24 parts of clinical dog excrement pick up from Sichuan Province Ganzi echinococcosis Endemic Area, by Ganzi animal epidemic prevention and control center
It provides, result is the positive after the detection of dog excrement echinococcous antigen detection kit.
Excrement is extracted according to Qiagen DNeasy PowerSoil kit (Qiagen, Carlsbad, CA) specification
DNA.The DNA extracted carry out immediately PCR use or be stored in -20 DEG C it is spare.
New upstream and downstream primer (the SEQ ID of ND6 wild type gene is directed to using 5.0 software design of Primer Premier
Shown in NO:3-4), after carrying out PCR detection to 24 parts of clinical dog excrement according to 2 method of embodiment, having 3 parts of dog excrement samples is PCR positive
(Fig. 3).This 24 parts clinical dog excrement are detected again using loop-mediated isothermal amplification technique, as a result with result one of the invention
It causes, this has also confirmed existing excrement antigen ELISA detection method and has haveed the defects that false positive is high.
And to the bull of the dog excrement of 5 parts of natural infection Taenia Pisiformis, 5 parts of natural infections band tapeworm dog excrement and 2 parts of natures
When the blister band tapeworm dog excrement of infection carries out PCR detection, amplification property band (Fig. 4) is presented in no sample.
Embodiment 5: sensitivity determination
Particulate is measured using Thermo Scientific NanoDrop spectrophotometer (Thermo, New York, USA)
The initial concentration of echinococcus template DNA, later by the template DNA extracted by 1:10,1:100,1:1000,1:2000,1:
4000,1:8000,1:16000,1:32000,1:64000,1:128000 doubling dilution is soft using Primer Premier 5.0
Part design is directed to the new upstream and downstream primer (shown in SEQ ID NO:3-4) of ND6 wild type gene, and examines according to 2 method of embodiment
It surveys, as a result sees Fig. 5.
After measured, the initial DNA content of Echinococcus granulosus template is 273ng, with the increase of dilution, worm's ovum in template
Content it is lower and lower, the brightness of DNA band is gradually weakening, dilution be 1/64000 i.e. (4pg) when can expand to one
Very light band, but amplified when dilution is 1/128000 (i.e. 2pg) without band.
Embodiment 6: the dog excrement PCR measurement of artificial challenge protoscolex
After carrying out deworm to 2 domesticated dogs, the protoscolex of every feeding about 50000.After infecting 3d, receive respectively daily
The excrement for collecting 2 dogs places it in -80 DEG C of progress harmless treatments in 2 weeks or more.Dog is euthanized and is dissected in 18d
Small intestine collects virgin worm.DNA extraction is successively carried out to the dog excrement of different infection number of days and detects (primer A according to 2 method of embodiment1、
A2), as a result see Fig. 6.
After measured, when infecting protoscolex 3d~11d, the PCR amplification result of 2 dog excrement is feminine gender, in 12d
When, the PCR result of only one dog is the positive, and when infection 13d~18d, there is positive purpose item in two dog PCR results
Band (Fig. 6).
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Sichuan Agricultural University
<120>application of Echinococcus granulosus mitochondria ND6 gene
<130> MP1815798
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
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tttcgtgctg tagatggt 18
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cacagatttc aaagggtt 18
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<213>artificial sequence (Artificial Sequence)
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ttggcttttc gtgctgtaga tggt 24
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aacttcacag atttcaaagg gtt 23
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tttcgtgctg tagatggttg tttaattaga cagattttat gttgttggaa gtttttatag 60
ttatgtattt ttgtgtattg gtgttttttt gttttactag ccattgtatt tattattgtg 120
ttatgttggt ggtgaatgcg ttattggcta gttgtatttg ttatttggtt tatggattta 180
gttggtattc gttactttta tgtttggtgt atgtgggggg tgtttatgtt ttgtttattt 240
ttgtgtcggt gtttagtcct aataggaatt ttgttttgta ttatagtgtt tgggaggttg 300
gtatatgttt gtggtttggt tttgggttgt ttatttgtgt gttgatttat tatttgttgg 360
ttggtagtga gtttagtggc atgttgtgta aagttagtga gggttggttg tatttatgtt 420
tatgtttgtc tttggttttt ggatttttgg ttttgagggt tgttgtgaga agtaaggtga 480
aattttatcg ttagggttga ttttagctgg tttagcatat atttaatgtg gtgggttgta 540
aaccctttga aatctgtg 558
Claims (10)
1. application of the Echinococcus granulosus mitochondria ND6 gene as the molecular diagnostic markers of detection Echinococcus granulosus.
2. applying according to claim 1, which is characterized in that the Echinococcus granulosus mitochondria ND6 gene is that ND6 is wild
Type gene；It or is ND6 wild type gene upstream 1-100bp, ND6 wild type gene and ND6 wild type gene downstream 1-100bp group
At sequence；Or the sequence of one or more bases is lacked, adds or replaced on ND6 wildtype gene sequence；Or to the open country ND6
The sequence that raw type gene is modified；The missing, addition replace one or more bases and modification to will not influence with ND6
The PCR amplification for the amplimer that gene designs for target gene.
3. application of the Echinococcus granulosus mitochondria ND6 gene in the molecular diagnostic markers of preparation detection Echinococcus granulosus.
4. applying according to claim 3, which is characterized in that the Echinococcus granulosus mitochondria ND6 gene is that ND6 is wild
Type gene；It or is ND6 wild type gene upstream 1-100bp, ND6 wild type gene and ND6 wild type gene downstream 1-100bp group
At sequence；Or the sequence of one or more bases is lacked, adds or replaced on ND6 wildtype gene sequence；Or to the open country ND6
The sequence that raw type gene is modified；The missing, addition replace one or more bases and modification to will not influence with ND6
The PCR amplification for the amplimer that gene designs for target gene.
5. the kit that Echinococcus granulosus mitochondria ND6 gene detects Echinococcus granulosus as molecular diagnostic markers in preparation
In application.
6. applying according to claim 5, which is characterized in that the Echinococcus granulosus mitochondria ND6 gene is that ND6 is wild
Type gene；It or is ND6 wild type gene upstream 1-100bp, ND6 wild type gene and ND6 wild type gene downstream 1-100bp group
At sequence；Or the sequence of one or more bases is lacked, adds or replaced on ND6 wildtype gene sequence；Or to the open country ND6
The sequence that raw type gene is modified；The missing, addition replace one or more bases and modification to will not influence with ND6
The PCR amplification for the amplimer that gene designs for target gene.
7. a kind of kit for detecting Echinococcus granulosus, which is characterized in that including expanding Echinococcus granulosus mitochondria ND6 base
The upstream and downstream primer of cause.
8. kit according to claim 7, which is characterized in that further include enzyme, dNTP, ddH for PCR amplification2O, stabilization
One or more of agent component.
9. expanding reagent of the upstream and downstream primer in preparation detection Echinococcus granulosus of Echinococcus granulosus mitochondria ND6 gene
Application in box.
10. applying according to claim 9, which is characterized in that the upstream and downstream primer as shown in SEQ ID NO:1-2 or
As shown in SEQ ID NO:3-4.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811009391.8A CN108950020B (en) | 2018-08-31 | 2018-08-31 | Application of Echinococcus granulosus mitochondrion ND6 gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811009391.8A CN108950020B (en) | 2018-08-31 | 2018-08-31 | Application of Echinococcus granulosus mitochondrion ND6 gene |
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