CN109762910A - It is a kind of for detecting the primer and kit of amphitypy echinococcosis simultaneously - Google Patents
It is a kind of for detecting the primer and kit of amphitypy echinococcosis simultaneously Download PDFInfo
- Publication number
- CN109762910A CN109762910A CN201910022916.XA CN201910022916A CN109762910A CN 109762910 A CN109762910 A CN 109762910A CN 201910022916 A CN201910022916 A CN 201910022916A CN 109762910 A CN109762910 A CN 109762910A
- Authority
- CN
- China
- Prior art keywords
- primer
- echinococcus
- kit
- echinococcosis
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a kind of for detecting the primer and kit of amphitypy echinococcosis simultaneously, and primer is with freeze-dried powder or the form being dissolved in buffer solution loaded in kit.Primer includes three pairs of specific primers pair, be respectively as follows: Echinococcus granulosus specific primer to, Canadian echinococcus specific primer to and Echinococcus multilocularis specific primer pair.Using three pairs of primers in kit of the present invention, the independent or mixed infection of detectable three kinds of cause of diseases is expanded by a wheel PRC, can determine whether tested sample has infection and infected insect species according to the particular bands occurred in amplified production electrophoresis.It using the kit in the present invention, not only operates quickly, conveniently, but also being capable of Accurate Diagnosis and the identification echinococcosis as caused by three kinds of Echinococcus infections.
Description
Technical field
The present invention relates to a kind of primer and kits, and in particular to it is a kind of for simultaneously detect amphitypy echinococcosis primer and
Kit.
Background technique
Echinococcosis is to seriously endanger human health as caused by worm kinds multiple in Echinococcus infection, cause huge society's warp
The great Arbo infectious disease for burden of helping.Echinococcosis is broadly divided into bladder type and two kinds of alveolitoid, the former distribution is extremely wide, the whole world
Patient disease burden reaches 1,009,662 ten thousand DALYs (Disability adjusted life years), and China's direct economic loss is caused to exceed every year
3000000000 yuan；The latter can lead to liver cancer sample lesion also referred to as " worm cancer ", and lethality is high, does not treat the death rate within 10 years and reaches
94%.China is whole world echinococcosis prevalence one of the countries with the most serious ..., and existing illness calculates that number reaches 11.5 ten thousand, annual to suffer from
Patient and his family raise more than 50,000,000, caused by people and animals' economic loss account for about global 40%, rank the first in the world.Echinococcosis is divided extensively
It is distributed in northwest China portion minority area, is related to the popular county, 350,9 province in addition to Tibet, compromised population is more than 50,000,000 people,
It is the major reason that the Endemic Area people drive into poverty by medical crises, back into poverty by medical crises.
The prevention and treatment of the emphasis parasitic disease such as echinococcosis is to ensure body health of people, push forward Health China construction comprehensively
One of important link.Echinococcus is the pathogen of people and animals' echinococcosis, is made of 9 worm kinds, wherein 4 worm kinds are deposited in China
In different degrees of prevalence, but only wherein 3 kinds --- cause the narrow sense Echinococcus granulosus of cystic echinococcosis
(Echinococcus granulosus sensu strict) and Canadian echinococcus (Echinococcus
Canadensis), and cause the Echinococcus multilocularis (Echinococcus multilocularis) of alveolar echinococcosis to people
Body has pathogenic, is the main object of current China's echinococcosis prevention and treatment and research.Due to amphitypy echinococcosis principle of reatment, drug
Difference significant in efficacy, three worm kind history of life, propagation characteristic have notable difference, therefore, the Accurate Diagnosis of amphitypy echinococcosis and
Identification effectively treated as echinococcosis, the basis of science bridle, it appears it is particularly important.
Currently, the diagnosis of echinococcosis is relied primarily in the image technologies such as B ultrasound, CT and pathologic finding, because method itself limits,
Easily cause small lesion, atypia lesion mistaken diagnosis, fail to pinpoint a disease in diagnosis with parting difficulty, especially for echinococcosis Major Epidemic nationality partially
Remote area is easier to the problems such as mistaken diagnosis occur due to horizontal universal relatively low of the above diagnostic techniques；Endemic Area low for echinococcosis or
Non- Endemic Area, due to contact case it is less, lack correlation experience, even if in some Grade A hospitals, equally exist error diagnosis or
The case where parting.Serologic detection is that another uses more hydatidosis diagnosis method, but existing detection kit at present
False positive is high, cross contamination is serious, and testing result is only capable of being provided with referring to as auxiliary foundation.Currently, examining for echinococcosis
Disconnected method, in addition to accuracy deficiency, it is difficult to other than the timeliness and effect that guarantee treatment, it is often more important that, it is currently used for Echinococcus hydatid cyst
In other words the identification that the detection methods of disease can not achieve worm kind level can not judge ring according to the variation of patient's infected insect species
The growth and decline situation that worm kind is propagated in border, to adjust prevention and control strategy in time.For these reasons, there is an urgent need to study to establish one kind
Accurately, result standardization judgement, the detection method that inter-species identification can be carried out can be achieved, to meet the work need of echinococcosis prevention and treatment
It asks.
The features such as round pcr is quick, accurate, sensitive because of its, is employed for all kinds of parasitic diseases more and more at present
Diagnosis, in detection, and achieve preferable application effect, such as PCR detection method has had been established in malaria, toxoplasma, and
The wide popularization and application in preventing and controlling, show round pcr played in the diagnostic field of parasitic disease it is increasingly heavier
The effect wanted.As the currently the only method that can be carried out Hydatid Disease Infection species identified, round pcr can be realized to human body and various
Intermediate, terminal animal reservoir infection detection, is current hydatidosis diagnosis, the developing direction of identification and research hotspot.However, existing
Some is directed to the PCR detection method of echinococcus based on Single tube amplification, and pair of primers can only detect correspondence by single reaction
A kind of echinococcus, or identified using sequencing, for the area popular there are the mixing of multiple worm kinds, method process is cumbersome, consumption
When consumptive material, be not able to satisfy the demand of quick diagnosis and large-scale inquiry.According to data according to open report sum number, it there is no at present
It can be used for detecting the multiple PCR primer pair of particulate, more rooms and Canadian echinococcus simultaneously, and only look into and see have for other worm kinds
Multiplexed PCR amplification method, the preventing and controlling not being suitable under the current echinococcus prevalence situation in China.
Summary of the invention
For the above-mentioned prior art, the present invention provide it is a kind of for and meanwhile detect the primer of amphitypy echinococcosis and comprising this
The kit of primer, to achieve the purpose that quick, Accurate Diagnosis and identify echinococcosis.
In order to achieve the above object, the technical scheme adopted by the invention is that: provide and a kind of be used for while detecting amphitypy packet
The primer of parasitosis, primer include three pairs of specific primers pair, in which:
Primer pair 1: sequence is shown in SEQ ID NO.1 and SEQ ID NO.2；
Primer pair 2: sequence is shown in SEQ ID NO.3 and SEQ ID NO.4；
Primer pair 3: sequence is shown in SEQ ID NO.5 and SEQ ID NO.6.
The present invention is also based on the primer and constructs a kind of kit, and obtained primer is with freeze-dried powder or is dissolved in buffer solution
Form be present in kit.
In kit of the invention other than primer, in kit further include containing Taq archaeal dna polymerase, dNTPs,
MgCl2, reaction buffer, 2 × Taq PCR amplification reagent of gel loading fuel and nuclease-free water.
The beneficial effects of the present invention are:
1. by using primer provided by the invention and kit, can popular all three existing to China have to human body
The echinococcus kind of infectious carries out detection and identification, while will not expand to other nontarget organisms or region, method
Specific good, high sensitivity, and result accuracy of judgement, be easy to standardize, it is accurate effectively to overcome existing hydatidosis diagnosis method
Property it is insufficient, cannot achieve kind of the horizontal defect identified, avoid current hydatidosis diagnosis method due to personnel specialty level difference
Parting is failed to pinpoint a disease in diagnosis and is difficult in caused mistaken diagnosis.
2. can be reacted by Single-tube multiplex-PCR using primer provided by the invention and kit, detection China has been sent out at present
Existing popular all 3 kinds have pathogenic particulate, more rooms and Canadian echinococcus to human body, to substance and mixed infection into
Row identifies simultaneously, effectively shortens experimental period, method operation efficiently, it is easy, economical, quick, suitable for China echinococcus
The quick diagnosis of echinococcosis and accurate prevention and control under prevalence situation.
Detailed description of the invention
Fig. 1 is that 3 pairs of specific detection primers carry out spy to independent to three kinds of purpose echinococcus respectively or hybrid dna template
The PCR result electrophoretogram of specific amplification；
Fig. 2 is 3 pairs of specific detection primers to respectively to three kinds of purpose echinococcus and the closer non-mesh of seven kinds of affinities
Worm kind DNA profiling carry out specific detection PCR result electrophoretogram；
Fig. 3 is that the PCR that 3 pairs of primers carry out sensitivity technique to three kinds of echinococcus DNA profilings of various concentration respectively is tied
Fruit electrophoretogram；
Fig. 4 is 3 pairs of primer pairs using human body, livestock, small-sized mammalian lesion sample as DNA profiling respectively when different
Between carry out three repeated experiments PCR result electrophoretogram.
Specific embodiment
The present invention is that realize can be two caused by or mixed infection independent to three echinococcus kinds by one tube PCR amplification
Type echinococcosis carries out the purpose of diagnosis different, develops a kind of kit, includes three species-specific primers in kit, can pass through
Single tube, which once expands, simultaneously detects narrow sense Echinococcus granulosus, Canadian echinococcus and Echinococcus multilocularis, can show
Write the diagnostic accuracy and efficiency for promoting echinococcosis.
Below with reference to embodiment, specific embodiments of the present invention will be described in detail.
The design of one primer of embodiment
The narrow sense Echinococcus granulosus (NC_008075.1) announced on GenBank, Canadian echinococcus are downloaded in retrieval
The reference of (NC_011121.1, AB235848.1, MG597240.1) and Echinococcus multilocularis (NC_000928.2) chondriogen
Sequence, using Primer Premier 6.0,7 software of Oligo by the way of manually combining, design is directed to above 3 worm kinds
The specific primer of gene, and primer quality is assessed.It is final to determine that 3 pairs of primers be quick, quasi- by multi-turns screen
Really, detection and identification, primer sequence are carried out to 3 kinds of echinococcus simultaneously are as follows:
(1) narrow sense Echinococcus granulosus
Upstream primer F1:5 '-gtctgtgtttcttaccattg-3 ' (SEQ ID NO.1)
Downstream primer R1:5 '-gacccgtacaaacatatatcaac-3 ' (SEQ ID NO.2)
(2) Canadian echinococcus
Upstream primer F2:5 '-gtaagtctaagttggttattattcac-3 ' (SEQ ID NO.3)
Downstream primer R2:5 '-cttattaaacaacacaaaaatactaaatg-3 ' (SEQ ID NO.4)
(3) Echinococcus multilocularis
Upstream primer F3:5 '-ttgttctttgtgttactgtagga-3 ' (SEQ ID NO.5)
Downstream primer R3:5 '-ctatacagacattgattaccata-3 ' (SEQ ID NO.6)
The foundation of the multiple PRC amplification method of embodiment two
1. the extraction of sample gene to be tested group DNA
15~25mg body tissue or human body, zoogenetic infection lesion are taken, is extracted according to tissue DNA extracts kit specification
Sample complete genome DNA.
2. amplification system
Using 25 μ l reaction systems, 2 × Taq PCR amplification reagent, 12.5 μ l, 10uM F1/R1 and F2/R2 primer pair is added
Each 0.5 μ l, 10uM F3/R3 primer pair upstream and downstream primer of upstream and downstream primer each 1.5 μ l, 6.5 μ l of nuclease-free water, sample form
DNA 1μl.Using nuclease-free water as template, system preparation is carried out according to the method described above, as negative control.
3. reaction condition
Above-mentioned prepared PCR reaction tube is put into PCR instrument, response procedures: 94 DEG C of initial denaturations are set by the following conditions
2min；94 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 1min, 40 recycle；72 DEG C of lengthenings extend 5min.
The identification of three PCR reaction product of embodiment
Take 10 μ l pcr amplification products in the Ago-Gel of 2% (w/v) with 3~5V/cm constant pressure electrophoresis 30~
60min, in Labworks image acquisition and analysis software observation as a result, using DNA molecular amount standard as reference, according to the presence or absence of purpose band and
Size determines whether there is the echinococcus type of infection and infection.
If amplified product band length is 811bp, it is determined as that narrow sense echinococcus granulosus infection is positive, if amplified production
Band length is 315bp, then is determined as that Canadian Echinococcus infections are positive, if amplified product band length is 457bp, sentences
It is set to the echinococcus multilocularis infection positive；If both the above or three kinds of length bands occurs simultaneously in amplified production, it is determined as phase
Answer two or three of echinococcus mixed infection positive；If amplified production is determined as feminine gender without band.If negative control occurs
Amplified band, then experiment is invalid.
The determination of example IV specificity
Using three pairs of primer pairs provided in the present invention and multiplexed PCR amplification method, using following three mode and according to
Operation in embodiment two~tri- carries out multiplexed PCR amplification, detection method specificity:
(1) PCR amplification is carried out as template using the DNA of particulate, Canada, Echinococcus multilocularis respectively；
(2) particulate, Canada, at least two in Echinococcus multilocularis are selected, are template progress PCR using their DNA
Amplification；
(3) select seven kinds of China common popular, with particulate, Canada, Echinococcus multilocularis affiliation it is closer its
Its helminth, including Shiqu echinococcus, taeniarhynchus saginatus, taeniasis suis, Lilium asiatic hybrids, taenia serrata, taenia crassicollis and
Hymenolepis diminuta carries out PCR amplification by template of their DNA.
Amplification is as depicted in figs. 1 and 2.Fig. 1 is 3 pairs of specific detection primers provided by the invention respectively to three kinds of mesh
Echinococcus individually or hybrid dna template carries out the PCR result electrophoretogram of specific amplification, in which: 1~3 swimming lane is respectively
The PCR amplification of particulate, more rooms and Canadian echinococcus DNA profiling is as a result, 4~6 swimming lanes are respectively particulate and more room spine ball silk ribbons
The PCR amplification of worm, particulate and Canadian echinococcus, more rooms and Canadian echinococcus hybrid dna as a result, 7 swimming lanes be particulate,
For the PCR amplification of Canada and Echinococcus multilocularis hybrid dna as a result, 8 swimming lanes are negative control, M is DNA molecular amount standard
Marker II.Fig. 2 is 3 pairs of specific detection primers provided by the invention to respectively to three kinds of purpose echinococcus and seven kinds of parents
The closer non-purpose worm kind DNA profiling of edge carries out the PCR result electrophoretogram of specific detection, wherein 1~3 swimming lane is respectively
The PCR amplification of particulate, more rooms and Canadian echinococcus DNA profiling is as a result, 4~10 swimming lanes are respectively Shiqu echinococcus, pig
With tapeworm, Lilium asiatic hybrids, taeniarhynchus saginatus, taenia serrata, taenia crassicollis, Hymenolepis diminuta DNA profiling PCR as a result, 11
Swimming lane is negative control, and M is DNA molecular amount standard Marker II.As can be seen from the figure: three pairs in kit of the present invention
Specific primer is to specifically capable of detecting particulate, Canada and Echinococcus multilocularis simultaneously, particulate and Canadian spine ball silk ribbon
Worm, particulate and Echinococcus multilocularis, Canada and Echinococcus multilocularis and particulate, Canada and Echinococcus multilocularis it is mixed
Infection is closed, and only amplifies the purpose band of corresponding worm kind, in addition, three pairs of primer pairs are not sent out with other 7 kinds of non-targeted helminths
Raw reaction, no band generate；Three pairs of primers for showing that invention provides are capable of detecting when corresponding three kinds of target echinococcus, mutually
Between no cross reaction, and nonspecific reaction does not occur between other closer helminths of common affinity, have compared with
Good specificity.
The confirmation of five accuracy of embodiment
Particulate, Canada and the PCR product of Echinococcus multilocularis in example four are sent to biotech firm and carry out bidirectional sequencing.
Sequence results are uploaded to NCBI and carry out BLAST comparison, are confirmed as corresponding worm kind.Detailed sequence information is shown in sequence table SEQ ID
NO.7~9, wherein SEQ ID NO.7 is narrow sense Echinococcus granulosus sequencing result, and SEQ ID NO.8 is Echinococcus multilocularis survey
Sequence is as a result, SEQ ID NO.9 is Canadian echinococcus sequencing result.
The determination of six PCR amplification sensitivity of embodiment
It is dense with micro ultraviolet specrophotometer measurement primary template DNA respectively using three kinds of purpose echinococcus DNA as template
Degree, is diluted, making template final concentration in PCR system is respectively 0.4ng/ μ l, 0.2ng/ μ with DNA extraction kit eluent
l、0.04ng/μl、0.036ng/μl、0.032ng/μl、0.028ng/μl、0.024ng/μl、0.02ng/μl、0.016ng/μl、
0.012ng/ μ l, 0.008ng/ μ l, 0.004ng/ μ l carry out multiplexed PCR amplification, inspection according to the operation in embodiment two~tri-
Survey method sensitivity.
Electrophoresis result is as shown in figure 3, Fig. 3 is 3 pairs of primers provided by the invention respectively to three kinds of spine ball silk ribbons of various concentration
The PCR result electrophoretogram of worm DNA profiling progress sensitivity technique, wherein A is the PCR amplification knot of narrow sense Echinococcus granulosus DNA
Fruit, B is the PCR amplification of Echinococcus multilocularis DNA as a result, C is the PCR amplification of Canadian echinococcus DNA as a result, three kinds of spine balls
The final concentration of tapeworm DNA profiling from swimming lane 1~12 be respectively 0.4ng/ μ l, 0.2ng/ μ l, 0.04ng/ μ l, 0.036ng/ μ l,
0.032ng/μl、0.028ng/μl、0.024ng/μl、0.02ng/μl、0.016ng/μl、0.012ng/μl、0.008ng/μl、
0.004ng/ μ l, 13 swimming lanes are negative control, and M is DNA molecular amount standard Marker II.As can be seen that provided by the present invention
Three kinds of purpose worm kind DNA detection limits can reach 0.004ng/ μ l, illustrate sensitivity with higher.
Seven primer of embodiment is applied and repeatability comparison
4 patients, 3 portions of livestocks and 3 parts of small-sized mammalian lesions are collected, DNA is extracted, infection worm is identified by sequencing
Kind.Using this 10 parts of DNA clearly identified as template, according to the operation in embodiment two~tri-, multiplexed PCR amplification is carried out, and
Divide the different time in triplicate.
Electrophoresis result is as shown in Figure 4, wherein Fig. 4 A-C is respectively different time using identical patient, livestock and small-sized
Mammal amounts to PCR result electrophoretogram of 10 parts of lesion samples as DNA profiling；1,2,5,7,8 swimming lanes are narrow in electrophoretogram
The PCR amplification of adopted Echinococcus granulosus DNA profiling is as a result, 3,6,9,10 swimming lanes are the PCR amplification of Echinococcus multilocularis DNA profiling
As a result, the inspection of 4 swimming lanes is the PCR amplification of Canadian echinococcus DNA profiling as a result, 11 swimming lanes are negative control, M is DNA molecular
Amount standard Marker II.It can be seen from the figure that the amplification of primer pair provided by the present invention and sequencing result complete one
It causes, illustrates that the primer pair can be effectively applied to clinical and field samples detections.What different time carried out repeats detection knot three times
Fruit is consistent, shows there is good repeatability.
Embodiment eight constructs kit
Kit body in the present invention is formed by multiple partitions, to accommodate fixed multiple such as containers of pipe or bottle, is held
Primer is loaded in device respectively, contains Taq archaeal dna polymerase, dNTPs, MgCl2, reaction buffer, gel loading fuel 2 ×
Taq PCR amplification reagent and nuclease-free water etc..Primer is contained in container in the form of freeze-dried powder, or primer pair is molten
In TE buffer, then it is contained in container.Test sample need to be only added using this kit, pcr amplification reaction can be carried out, instead
Answer product directly loading can carry out gel electrophoresis analysis.
Although in conjunction with the embodiments and attached drawing is described in detail a specific embodiment of the invention, should not be understood
For the restriction of the protection scope to this patent.In range described by claims, those skilled in the art are without creation
Property the various modifications that can make of labour and deformation still belong to the protection scope of this patent.
<110>disease prevention and control center, Sichuan Province
<120>a kind of for detecting the primer and kit of amphitypy echinococcosis simultaneously
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (narrow sense Echinococcus granulosus upstream primer F1)
<400> 1
gtctgtgttt cttaccattg 20
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (narrow sense Echinococcus granulosus downstream primer R1)
<400> 2
gacccgtaca aacatatatc aac 23
<210> 3
<211> 26
<212> DNA
<213>artificial sequence (Canadian echinococcus upstream primer F2)
<400> 3
gtaagtctaa gttggttatt attcac 26
<210> 4
<211> 29
<212> DNA
<213>artificial sequence (Canadian echinococcus downstream primer R2)
<400> 4
cttattaaac aacacaaaaa tactaaatg 29
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Echinococcus multilocularis upstream primer F3)
<400> 5
ttgttctttg tgttactgta gga 23
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Echinococcus multilocularis downstream primer R3)
<400> 6
ctatacagac attgattacc ata 23
<210> 7
<211> 778
<212> DNA
<213>Echinococcus granulosus (Echinococcus granulosus)
<400> 7
taatcaacct aatgatcgac tgaaaaatct aaaatcctag acacaatgta ccaatggatt 60
aatacaacaa aaacttcata aaaaaacaat ccaactaaaa ccaaccctca tcaacaatta 120
acaaaacaca aattacccaa tgccactgca ccaaaacacc ccaacctaat attaataaaa 180
ggacgcaaaa ccaatacaac aggacgtata acataactaa tagactcggc caagcacact 240
aaaaaacata tccataaagg agttcctagc gggacaaaac aagcaaaaaa tccattaact 300
ttattaaaaa tacgacacat aaataacgac acaaacatca caaaaaccac acaaaacaaa 360
aaaacggaaa acaaataaac cctataacaa tatggcaaac gataaaccat aaaccacatt 420
aataccaacg ccaataaaac aaaatagtaa tatgacacac gaccaaacac caacatataa 480
accaaaccaa ttaaagaaca aaaatcatta acaacaacca tcaaactcaa ccagacacaa 540
gaatgtgtat aatagggaca tgaaccctac agtttcatta acttaccagt ctctctaaca 600
aaagttatct ccaacgcttc aaattgacgc tttaatttaa gctaaaagaa atttaacgga 660
taactaatct ccttcatgac caaaacacaa aatgcaaaaa cacctcatta aaccactata 720
gcacaactat cgcgaaataa caccaaaccc ctaaataaca caaaaaaaaa taataaca 778
<210> 8
<211> 418
<212> DNA
<213>Echinococcus multilocularis (Echinococcus multilocularis)
<400> 8
tgattaccat aaaatctaat taacaataac aaaaaatccc aaaatcagta aacataaaca 60
aacctctcag aaaaatttca caaataaatc ataacgaaca cgaggtaacg ttgcccgagc 120
tcacataaaa aataataaat taaacaacaa cataaacaaa ccaacaaaac caccaccaac 180
catcattaca acaatcaacc atgaaaacac atatacaact atatactcac aagcaaacaa 240
acacgtaaag tatataccgc tatactcaac attaaaccca ctaactaact ccctttcaga 300
ctccccataa tcaaatggta tacgattagt ctcacatagc acacacacca aaaacaatcc 360
ataaatcaat ggaaacaaca acaagcttca tcaacaacta taataaaaat caatcaaa 418
<210> 10
<211> 291
<212> DNA
<213>Canadian echinococcus (Echinococcosis canadensis)
<400> 10
gttggttatt attcactgac ctggtttttt agctgtgata ttggtttttt tggtttattt 60
gccttttgca tcatgcttgg cggagttttg taaagtattt ttttataccg aaaggctctg 120
atcttgagtt taattgtttg gcattgtttt aaacttgggt aaagtttttt aaattaaatt 180
catggagtga taagtaagtc gtagatcttt ataactgttt agttgttggt tatttatagt 240
aatgtatgtg gctagaaggt catttatatt tttatacatt tagtattttt g 291
Claims (4)
1. a kind of for detecting the primer of amphitypy echinococcosis simultaneously, which is characterized in that including three pairs of specific primers pair, in which:
Primer pair 1: sequence is shown in SEQ ID NO.1 and SEQ ID NO.2；
Primer pair 2: sequence is shown in SEQ ID NO.3 and SEQ ID NO.4；
Primer pair 3: sequence is shown in SEQ ID NO.5 and SEQ ID NO.6.
2. a kind of amphitypy echinococcosis is quickly detected with kit, it is characterised in that: including primer as described in claim 1.
3. kit according to claim 2, it is characterised in that: the primer is with freeze-dried powder or is dissolved in buffer solution
Form is loaded in the kit.
4. kit according to claim 3, it is characterised in that: further include polymerizeing in the kit containing Taq DNA
Enzyme, dNTPs, MgCl2, reaction buffer, 2 × Taq PCR amplification reagent of gel loading fuel and nuclease-free water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910022916.XA CN109762910B (en) | 2019-01-10 | 2019-01-10 | Primer and kit for simultaneously detecting two types of echinococcosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910022916.XA CN109762910B (en) | 2019-01-10 | 2019-01-10 | Primer and kit for simultaneously detecting two types of echinococcosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109762910A true CN109762910A (en) | 2019-05-17 |
| CN109762910B CN109762910B (en) | 2023-06-09 |
Family
ID=66453809
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910022916.XA Active CN109762910B (en) | 2019-01-10 | 2019-01-10 | Primer and kit for simultaneously detecting two types of echinococcosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109762910B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235285A (en) * | 2020-03-04 | 2020-06-05 | 南京亿科人群健康研究院有限公司 | Kit for detecting echinococcosis diagnosis |
| CN111607656A (en) * | 2020-06-28 | 2020-09-01 | 哈密市动物疫病预防控制中心 | Triple PCR detection primer, method and kit for tapeworm and sporozoon in canine feces |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001075448A2 (en) * | 2000-04-04 | 2001-10-11 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention | Methods and compositions for detecting larval taenia solium with a cloned diagnostic antigen |
| JP2008187900A (en) * | 2007-01-31 | 2008-08-21 | Hokkaido Univ | New protein derived from echinococcus multilocularis |
| CN102168089A (en) * | 2011-02-14 | 2011-08-31 | 中国疾病预防控制中心寄生虫病预防控制所 | Echinococcus granulosus calreticulin gene and application thereof |
| CN103173554A (en) * | 2013-03-27 | 2013-06-26 | 中国农业科学院兰州兽医研究所 | Detection kit for detecting and distinguishing multiple kinds of echinococcus |
| CN103374615A (en) * | 2012-04-20 | 2013-10-30 | 广州世优生物科技有限公司 | PCR (Polymerase Chain Reaction) detection kit for cystic echinococcosis of dog |
| CN105018613A (en) * | 2015-07-17 | 2015-11-04 | 上海师范大学 | Method for detecting Echinococcus multilocularis from fox excrement |
| CN106399549A (en) * | 2016-10-28 | 2017-02-15 | 青海大学 | Method for PCR (polymerase chain reaction) detection of and detection kit |
| CN106591456A (en) * | 2016-12-22 | 2017-04-26 | 青海大学 | Real-time quantitative PCR (Polymerase Chain Reaction) method for detecting echinococcus multilocularis and echinococcus granulosus by types based on MGB (Minor Groove Binder) probe and detection kit |
| CN107164479A (en) * | 2017-05-27 | 2017-09-15 | 四川省疾病预防控制中心 | The primer pair of echinococcosis cause of disease is organized to combine and kit with surveyor for detecting |
| CN107365849A (en) * | 2017-08-10 | 2017-11-21 | 西南民族大学 | The kit of dog particulate, Echinococcus multilocularis based on POCKIT Micro fluorescent PCR platforms and application |
-
2019
- 2019-01-10 CN CN201910022916.XA patent/CN109762910B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001075448A2 (en) * | 2000-04-04 | 2001-10-11 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention | Methods and compositions for detecting larval taenia solium with a cloned diagnostic antigen |
| JP2008187900A (en) * | 2007-01-31 | 2008-08-21 | Hokkaido Univ | New protein derived from echinococcus multilocularis |
| CN102168089A (en) * | 2011-02-14 | 2011-08-31 | 中国疾病预防控制中心寄生虫病预防控制所 | Echinococcus granulosus calreticulin gene and application thereof |
| CN103374615A (en) * | 2012-04-20 | 2013-10-30 | 广州世优生物科技有限公司 | PCR (Polymerase Chain Reaction) detection kit for cystic echinococcosis of dog |
| CN103173554A (en) * | 2013-03-27 | 2013-06-26 | 中国农业科学院兰州兽医研究所 | Detection kit for detecting and distinguishing multiple kinds of echinococcus |
| CN105018613A (en) * | 2015-07-17 | 2015-11-04 | 上海师范大学 | Method for detecting Echinococcus multilocularis from fox excrement |
| CN106399549A (en) * | 2016-10-28 | 2017-02-15 | 青海大学 | Method for PCR (polymerase chain reaction) detection of and detection kit |
| CN106591456A (en) * | 2016-12-22 | 2017-04-26 | 青海大学 | Real-time quantitative PCR (Polymerase Chain Reaction) method for detecting echinococcus multilocularis and echinococcus granulosus by types based on MGB (Minor Groove Binder) probe and detection kit |
| CN107164479A (en) * | 2017-05-27 | 2017-09-15 | 四川省疾病预防控制中心 | The primer pair of echinococcosis cause of disease is organized to combine and kit with surveyor for detecting |
| CN107365849A (en) * | 2017-08-10 | 2017-11-21 | 西南民族大学 | The kit of dog particulate, Echinococcus multilocularis based on POCKIT Micro fluorescent PCR platforms and application |
Non-Patent Citations (13)
| Title |
|---|
| CONG-NUAN LIU等: "Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay", 《PLOS NEGL TROP DIS》 * |
| CONG-NUAN LIU等: "Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay", 《PLOS NEGL TROP DIS》, vol. 09, no. 09, 22 September 2015 (2015-09-22), pages 6 - 7 * |
| GHALIA BOUBAKER等: "A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa", 《MOLECULAR AND CELLULAR PROBES》 * |
| GHALIA BOUBAKER等: "A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa", 《MOLECULAR AND CELLULAR PROBES》, vol. 30, no. 04, 31 August 2016 (2016-08-31), pages 1 - 2 * |
| M. ROSTAMI NEJAD等: "Molecular identification of animal isolates of Echinococcus granulosus from Iran using four mitochondrial genes", 《JOURNAL OF HELMINTHOLOGY》 * |
| M. ROSTAMI NEJAD等: "Molecular identification of animal isolates of Echinococcus granulosus from Iran using four mitochondrial genes", 《JOURNAL OF HELMINTHOLOGY》, vol. 86, no. 04, 31 December 2012 (2012-12-31), pages 487 * |
| NAKAO,M.等: "Accession NO: AB018440.2 Echinococcus multilocularis mitochondrial DNA, complete genome", GENBANK DATABASE * |
| 于浩等: "《生物信息学实验指导》", 30 September 2009, 吉林大学出版社, pages: 124 - 127 * |
| 徐振波: "《葡糖球菌生物被摸的分子机制研究》", 30 June 2018, 华南理工大学出版社, pages: 22 * |
| 李双男 等: "加拿大棘球绦虫的基因分型与分子流行病学研究进展", 《中国人兽共患病学报》, vol. 32, no. 04, 15 April 2016 (2016-04-15), pages 392 - 399 * |
| 李润乐 等: "我国包虫病基础研究现状及存在的问题", 《中国高原医学与生物学杂志》, vol. 38, no. 04, 31 December 2017 (2017-12-31), pages 217 - 218 * |
| 胡丹丹 等: "青藏高原细粒棘球绦虫的分子鉴定与遗传变异分析", 《畜牧兽医学报》, vol. 44, no. 09, 15 September 2013 (2013-09-15), pages 1438 - 1444 * |
| 谭贵良，赖心田主编: "《现代分子生物学及组学技术在食品安全检测中的应用》", 30 June 2014, 中山大学出版社, pages: 25 - 26 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235285A (en) * | 2020-03-04 | 2020-06-05 | 南京亿科人群健康研究院有限公司 | Kit for detecting echinococcosis diagnosis |
| CN111607656A (en) * | 2020-06-28 | 2020-09-01 | 哈密市动物疫病预防控制中心 | Triple PCR detection primer, method and kit for tapeworm and sporozoon in canine feces |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109762910B (en) | 2023-06-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hum et al. | Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies | |
| Berri et al. | The detection of Coxiella burnetii from ovine genital swabs, milk and fecal samples by the use of a single touchdown polymerase chain reaction | |
| Lebech et al. | Diagnostic value of PCR for detection of Borrelia burgdorferi DNA in clinical specimens from patients with erythema migrans and Lyme neuroborreliosis | |
| Kaynak-Onurdag et al. | Screening Brucella spp. in bovine raw milk by real-time quantitative PCR and conventional methods in a pilot region of vaccination, Edirne, Turkey | |
| CN104946749B (en) | A kind of universal primer for field quick detection brucella and probe and kit | |
| CN105002173A (en) | Kit for identifying Brucella S2 vaccine strain and wild strain | |
| CN104152584B (en) | Capripoxvirus virus Taqman-MGB probe multiple real-time fluorescence quantitative PCR detection primer, method and test kit | |
| WO2020034317A1 (en) | Dual real-time fluorescent quantitative pcr detection reagent and reagent kit for seneca virus a and foot-and-mouth disease virus | |
| CN107365849A (en) | The kit of dog particulate, Echinococcus multilocularis based on POCKIT Micro fluorescent PCR platforms and application | |
| CN108048588A (en) | The detection primer and detection kit of a kind of Arcanobacterium pyogenes | |
| Singh et al. | 16S rRNA and omp31 gene based molecular characterization of field strains of B. melitensis from aborted foetus of goats in India | |
| CN111518877B (en) | One-tube method nest type real-time quantitative PCR detection kit for detecting echinococcus multilocularis and echinococcus granulosus by parting trace samples | |
| CN109762910A (en) | It is a kind of for detecting the primer and kit of amphitypy echinococcosis simultaneously | |
| US20110287965A1 (en) | Methods and compositions to detect clostridium difficile | |
| JP2011522533A (en) | Molecular diagnostic kit for detection of virulent strains of Helicobacter pylori | |
| CN114790490A (en) | Molecular marker capable of distinguishing Brucella melitensis and detection method | |
| CN104911269B (en) | Differentiate primer, probe and the kit of brucella A19 vaccine strains in aerosol | |
| Taylor et al. | Use of non-radioactive DNA probes for detection of Campylobacter jejuni and Campylobacter coli in stool specimens | |
| CN113846181A (en) | Kit and method for rapidly and visually detecting PRRSV (porcine reproductive and respiratory syndrome Virus) based on Cas12a protein | |
| AU611630B2 (en) | Detection and differentiation of coxiella burnetii in biological fluids | |
| Xie et al. | An analysis of the Malassezia species distribution in the skin of patients with pityriasis versicolor in Chengdu, China | |
| CN111500774B (en) | Epidemic hemorrhagic disease virus and serotype identification RT-PCR kit | |
| CN113637778A (en) | Kit and method for detecting brucella | |
| CN102168137A (en) | Primer set for detecting oocyst deoxyribonucleic acid (DNA) of Cryptosporidium by using loop-mediated isothermal amplification technology, kit, detection method and application | |
| CN108913790B (en) | Recombinase polymerase isothermal amplification method for detecting coxiella burnetii, special primer and probe and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |