CN107164479A - The primer pair of echinococcosis cause of disease is organized to combine and kit with surveyor for detecting - Google Patents
The primer pair of echinococcosis cause of disease is organized to combine and kit with surveyor for detecting Download PDFInfo
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- 208000009366 Echinococcosis Diseases 0.000 title claims abstract description 41
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 39
- 201000010099 disease Diseases 0.000 title claims abstract description 35
- 206010014096 Echinococciasis Diseases 0.000 title claims abstract description 34
- 241000244170 Echinococcus granulosus Species 0.000 claims abstract description 65
- 201000003808 Cystic echinococcosis Diseases 0.000 claims abstract description 63
- 238000000034 method Methods 0.000 claims abstract description 42
- 241000244163 Echinococcus multilocularis Species 0.000 claims abstract description 38
- 238000001962 electrophoresis Methods 0.000 claims description 13
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- 238000012408 PCR amplification Methods 0.000 claims description 11
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- 206010019659 Hepatic echinococciasis Diseases 0.000 description 2
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Abstract
It can detect and identify simultaneously that Echinococcus granulosus G1 G3 types, Echinococcus multilocularis and Echinococcus granulosus G6 G10 type three-type-persons organize the multiple PCR method of echinococcosis cause of disease the invention discloses a kind of.The present invention designs 3 pairs of specific primers for the chondriogen of 3 kinds of cause of diseases, add in same PCR reaction systems, specific primer is combined with the target gene complementation of corresponding worm kind, passes through amplified reaction, different worm kinds produce purpose band of different sizes, are separated and are detected through gel electrophoresis.It is not interfere with each other three primers of the present invention, and cross reaction does not occur with 8 kinds of nearer tapeworms of affiliation, or does not disturb result interpretation.This method high specificity, sensitivity are high, can efficiently and accurately realize the synchronous detection to 3 kinds of cause of diseases, greatly save detection time and cost, effectively improve operating efficiency, Classification Identification and echinococcosis molecule epidemic disease-ology research available for Human Hydatidosis cause of disease.
Description
Technical field
The invention belongs to medical diagnosis on disease field, and in particular to one kind is used to detecting and identifying that a variety of people organize echinococcosis cause of disease
Primer pair combination and its kit.
Background technology
Echinococcosis (Hydatidosis) is being commonly called as hydatidosis (echinococcosis), is the children by echinococcus
Worm parasitizes parasitic zoonoses caused by human body.The Echinococcus that the whole world was found in the past is divided into 6 kinds, i.e.,
Echinococcus granulosus (Echinococcus granulosus), Echinococcus multilocularis (Echinococcus
Multilocularis), Fu Shi echinococcus (Echinococcus vogeli), less section echinococcus (Echinococcus
Oligarthus), Echinococcus felids and Shiqu echinococcus (Echinococcus shiquicus), wherein carefully
Grain echinococcus (E.granulosus) is divided into 10 genotype, i.e. Echinococcus granulosus G1-G10 types by genotype.With point
Application and development of the sub- biology techniques in echinococcus is studied, the result that people obtain according to Phylogenetic analysis, again
10 genotype of original Echinococcus granulosus (E.granulosus) are divided into 4 new worm kinds, they are respectively
E.granulosus sensu stricto (G1-G3 types), E.equinus (G4), E.ortleppi (G5) and
E.canadensis(G6–G10).Echinococcosis is widely current in all over the world, falls ill to have over nearly 20 years and increases trend.China is complete
The popular the most serious country of ball echinococcosis, district occurred frequently accounts for the 44% of national total area, and compromised population exceedes 5,000,000.
According to existing literature, what China had now been found that causes the echinococcosis cause of disease of human infection to have three kinds, wherein, carefully
In the highest flight, Echinococcus granulosus G6-G10 types are in various regions for grain echinococcus G1-G3 types and the prevalence of Echinococcus multilocularis
Show distributed state, but its infection risk also can not be ignored.The adult and its larva of echinococcus not of the same race are in form, biology
Feature, pathogenic aspect have differences.Wherein, the children of Echinococcus granulosus G1-G3 types and Echinococcus granulosus G6-G10 types
Worm can cause the cystic echinococcosis of people, and the larva of Echinococcus multilocularis can cause the alveolar echinococcosis of people.Cystic echinococcosis can be sent out
Life is in the multiple internal organs of whole body, and the most common with liver, lung, alveolar echinococcosis primary lesion is nearly all located at liver.Detection and identification
The species pop disease of echinococcosis cause of disease is learned research, the prevention and control of disease and treatment and all had great importance.
At present, existing PCR detection method by specific amplification it is minimally invasive/operation patients lesion tissue in target DNA sequence,
To be confirmed whether to infect echinococcosis, and confirm the echinococcosis cause of disease species of infection.Existing PCR detection method mainly has substance
PCR method and multiple PCR method.(the A PCR system for identification of such as Dinkel A
Echinococcus species and genotypes,with reference to the epidemiological
situation in eastern Africa[J].2004:48-48.) establish the PCR detection sides of detection Echinococcus granulosus
Method；(the Development of Three PCR Assays for the Differentiation such as Boufana B
between Echinococcus shiquicus,E.granulosus(G1genotype),and E.multilocularis
DNA in the Co-Endemic Region of Qinghai-Tibet plateau,China[J].American
Journal of Tropical Medicine&Hygiene,2013,88(4):795-802) establish detection particulate spine ball silk ribbon
Worm G1 types, Echinococcus multilocularis and 3 kinds of substance PCR methods of Shiqu echinococcus；(the Detection of such as Can H
Echinococcus granulosus and Echinococcus multilocularis in cyst samples using
a novel single tube multiplex real-time polymerase chain reaction.[J]
.Mikrobiyoloji Bulteni,2016,50(2):266.) establish while detecting Echinococcus multilocularis and particulate spine ball silk ribbon
The real-time fluorescence multiple PCR method of worm；Cong nuan Liu et al. (Discrimination between E.granulosus
sensu stricto,E.multilocularis and E.shiquicus Using a Multiplex PCR Assay.
[J].Plos Neglected Tropical Diseases,2015,9(9):) and Lou Zhongzi et al. (patent e0004084.
CN201310102181.4) establish while detecting Echinococcus granulosus G1-G3 types, Shiqu echinococcus and Echinococcus multilocularis
Multiple PCR method, wherein Shiqu echinococcus do not find human infection also at present, and its infection host is mainly Xiaoxinganling mountains
Animal and canid.
In the above method, one reaction system of substance PCR method can only detect a kind of cause of disease, and detection program is cumbersome, time-consuming
Arduously, it is unfavorable for the quick detection of extensive sample.Multiple PCR method can be to two under same system, same reaction condition
Or more than two different target genes synchronize the reaction system of detection, i.e., one can detect a variety of cause of diseases simultaneously, greatly improve
The efficiency of method.But, three-type-person's echinococcosis disease that on the one hand existing multiple PCR method not finds directed entirely to China
Original (Echinococcus granulosus G1-G3 type E.granulosus sensu stricto, Echinococcus multilocularis E.Multilocularis
With Echinococcus granulosus G6-G10 type E.canadensis) and design, it is impossible to meet detection and identify Human Hydatidosis cause of disease
Demand；On the other hand, the design of primers difficulty of multiplex PCR is far longer than substance PCR, because adding multipair draw in one system
Thing, easily occurs cross reaction with the nearer cause of disease of affiliation, occurs that complementation also easily occurs between false positive results, and primer pair
With reference to, be also easy to produce disturbing reaction, therefore, existing echinococcosis multiple PCR method has that specific poor, method is unreliable etc. to ask
Topic.
Therefore, the present invention is directed to Echinococcus granulosus G1-G3 types (the E.granulosus sensu of China's human infection
Stricto), Echinococcus multilocularis (E.Multilocularis) and Echinococcus granulosus G6-G10 types (E.canadensis) three
The chondriogen conservative region design specific primer of kind of cause of disease, constructing a kind of can synchronously detect a variety of echinococcosis cause of diseases
Multiple PCR method.This method high specificity, sensitivity are high, and with high flux, simplicity, quick advantage, can significantly improve
Operating efficiency, reduces time, financial cost, in the further identification parting and echinococcosis disease of minimally invasive or operation echinococcosis patient
People's Molecule Epidemiology Investigation field is with a wide range of applications.
The content of the invention
In view of the deficienciess of the prior art, the present invention first purpose be provide one kind can and meanwhile be used for detect and
Identify that a variety of people organize the primer pair of echinococcosis cause of disease to combine, primer pair combination is carried out in same system, under identical conditions
Non-interference during multiplexed PCR amplification, high specificity, sensitivity are high, for three kinds of cause of diseases (particulate spine ball of China's human infection
Tapeworm G1-G3 types, Echinococcus multilocularis and Echinococcus granulosus G6-G10 types, hereinafter also referred to as three kinds purpose echinococcus)
And design.
Second object of the present invention is to provide a kind of kit, and the kit contains can be while being used to detect and reflecting
Fixed a variety of people organize the primer pair of echinococcosis cause of disease to combine.
Third object of the present invention is the application method for providing mentioned reagent box.
To achieve these goals, technical scheme is as follows：
A kind of to organize the primer pair of echinococcosis cause of disease to combine with surveyor for detecting, the primer pair combination includes：
Echinococcus granulosus G1-G3 type-special primers pair：Gtp1F is as shown in sequence table SEQ ID NO.1, and gtp1R is such as
Shown in sequence table SEQ ID NO.2；
Echinococcus multilocularis specific primer pair：Mtb42F is as shown in sequence table SEQ ID NO.3, mtb42R such as sequence tables
Shown in SEQ ID NO.4；
Echinococcus granulosus G6-G10 type-special primers pair：Cox1F is as shown in sequence table SEQ ID NO.5；Cox1R is such as
Shown in sequence table SEQ ID NO.6.
Further, it is above-mentioned to be used to detect the purpose piece for organizing the primer pair combination of echinococcosis cause of disease to amplify with surveyor
Segment length is followed successively by 240bp, 152bp and 441bp.
A kind of kit, containing it is above-mentioned be used for detect and surveyor organize the primer pair of echinococcosis cause of disease to combine.
Further, the mentioned reagent box also PCR reagent containing DNA cloning.
Further, PCR reagent described in mentioned reagent box includes archaeal dna polymerase mixture and aseptic double-distilled water.
The application method of kit described above, including：People's biopsy DNA to be checked is extracted, and is added as template
Reagent in the kit enters performing PCR amplification, and the PCR primer point sample of amplification carries out electrophoresis into 2% Ago-Gel, sees
Examine electrophoresis result.
Further, the application method of mentioned reagent box, the condition of the PCR amplifications is 94 DEG C of pre-degeneration 3min；94℃
It is denatured 30s；56 DEG C of annealing 30s；72 DEG C of extension 40s；Totally 35 circulations；72 DEG C of extension 5min.
The design and screening of primer are the keys of multiple PCR method, and the specificity of primer and sensitivity directly determine whole
The specificity of individual method and sensitivity.The design of primers difficulty of multiplex PCR is far longer than substance PCR, because adding in one system
Multipair primer is entered, has easily occurred cross reaction with the nearer cause of disease of affiliation, occur between false positive results, and primer pair
Easily occur complementary combination, be also easy to produce disturbing reaction.In addition, to require that amplified production length there must be certain poor for multiple PCR method
It is different, band can be made a distinction by electrophoresis, this restrictive condition also increases the difficulty of design of primers.Three couple of the present invention
It is not interfere with each other between primer, high specificity, cross reaction, or cross reaction production does not occur with 8 kinds of nearer tapeworms of affiliation
Carded sliver band is of different sizes in purpose band, and result interpretation is not disturbed.Sensitivity is high, and three kinds of cause of disease DNA detection limit is above
0.005ng/μl.Multiplex PCR detection is carried out to sample according to the method for the present invention using the specific primer, China is may be implemented in
The synchronization of the Echinococcus granulosus G1-G3 types of human infection, Echinococcus multilocularis and Echinococcus granulosus G6-G10 3 kinds of cause of diseases of type
Detection, greatlys save testing cost and time compared with conventional detection method, improves detection efficiency and accuracy, in echinococcosis disease
The Classification Identification of people's particularly suspected case and echinococcosis molecule epidemic disease-ology research field are with a wide range of applications.
Brief description of the drawings
Fig. 1 is the pcr amplification product electrophoretogram of the specificity experiments 1 of embodiment 1；
Fig. 2 is the pcr amplification product electrophoretogram of the specificity experiments 2 of embodiment 1；
Fig. 3 is the pcr amplification product electrophoretogram of the specificity experiments 3 of embodiment 1；
Fig. 4 is the sensitivity experiment result of embodiment 2；
Fig. 5 be in embodiment 3 10 patient's surgery excision tissue DNA to be checked respectively with the multiple of 3 pairs of mix primers reaction
PCR testing results；
Fig. 6 is comparative example experimental result picture.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.For those of ordinary skills, embodiment can be replaced according to present invention/
Combination, and these should all belong to invention which is intended to be protected.
In following embodiments, all unreceipted specific experiment conditions, be according to routine well known to those skilled in the art
Condition, the molecular cloning of the work such as Sambrook：Laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989), experimental animal routine handbook (National Laboratory Animal specification center, in November, 2004)：Substantially
Condition and experimental procedure described in technical manual the 5th edition (John Wiley＆Sons, Inc, 2005), or according to manufacturer
Proposed condition and experimental procedure.Material and reagent in embodiment, non-specified otherwise, can be by conventional method through business
Approach is obtained.
Experimental method
Primer pair of the present invention：
Echinococcus granulosus G1-G3 type-special primers,
Sense primer gtp1F：TGGTTTGGTTTATATGTTGGTGTTT；
Anti-sense primer gtp1R：ATCCATAAAGGAGTTCCTAGCG
Echinococcus multilocularis specific primer,
Sense primer mtb42F：ACCAACTGAGGGTGGCACTA；
Anti-sense primer mtb42R：CCATTACTACCCTAAACAACTGACA
Echinococcus granulosus G6-G10 type-special primers,
Sense primer cox1F：TTTTATTTACGTTTGGGGGCG；
Anti-sense primer cox1R：CCACCAAACCAAAAGACCTG
Standard DNA template：
The DNA of three kinds of purpose echinococcus and other nearer tapeworms of affiliation is extracted respectively
Echinococcus granulosus G1-G3 types (E.granulosus sensu stricto), Echinococcus multilocularis
(E.Multilocularis), Echinococcus granulosus G6-G10 types (E.canadensis), Shiqu echinococcus
(E.shiquicus), blister band tapeworm (T.hydatigena), taenia crassicollis (T.taeniaformis), Lilium asiatic hybrids
(T.asiatica), taeniarhynchus saginatus (T.saginata), taeniasis suis (T.solium), Hymenolepis diminuta (H.diminuta) and
Taenia serrata (T.serialis), carries out complete genome DNA extraction, to extraction according to DNA extraction kit operating instruction
DNA enters performing PCR and detects and be sequenced, and sequence is compared with the corresponding sequence in NCBI gene pools, determines the DNA samples after species
This is DNA profiling reference material, stand-by.
Patient dna to be measured：
Patient's tumour hair tonic layer tissue to be measured or 15~25mg of cyst fluid are chosen, tissue DNA extracts kit is used after shredding
Complete genome DNA is extracted to specifications.
PCR amplification system and the observation of condition and result：
PCR reaction solutions are prepared in PCR reaction tubes, using 50uL reaction systems.Including：25uL archaeal dna polymerases are mixed
Thing, concentration is more than 50ng/uL DNA profiling 5uL (the specificity experiments concentration, sample to be tested detection is not limited to this), special
Each 1.5uL of specific primer (10uM), distilled water supplies 50uL.
PCR reaction tubes are put into PCR instrument and reacted, thermocycling program is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation
30s；56 DEG C of annealing 30s；72 DEG C of extension 40s；Totally 35 circulations；72 DEG C of extension 5min, 4 DEG C of preservations after the completion of reaction.PCR reacts
Product (contains 0.5 μ g/ml golden view) in 2% Ago-Gel carries out electrophoresis, voltage 80-110v, through ultraviolet solidifying
Glue imager observes result.
According to whetheing there is electrophoretic band and electrophoretic band size determines whether to infect echinococcosis, and infection worm species
Type.
The specificity experiments of embodiment 1
3 groups of experiments are designed altogether investigates the technology for detection of the present invention and the specificity of identification echinococcosis cause of disease, 3 groups of experiment difference
For：
1st, the specificity that 3 pairs of mix primers of the invention are expanded to the DNA profiling of three kinds of purpose echinococcus respectively
Experiment；
2nd, the specificity that 3 pairs of mix primers of the invention are expanded to the DNA hybrid templates of three kinds of purpose echinococcus
Experiment；
3rd, 3 pairs of mix primers of the invention are respectively to three kinds of purpose echinococcus and the nearer silk ribbon of other 8 kinds of affiliations
The specificity experiments that worm DNA profiling is expanded.
Concrete operations are as described in previous experiments method, and electrophoresis result is referring to Fig. 1~3.
In Fig. 1：1,2,3 swimming lane is that three kinds of mix primers expand Echinococcus granulosus G1-G3 types, Echinococcus multilocularis respectively
With the PCR results of Echinococcus granulosus G6-G10 type DNA profilings；4 swimming lanes are negative control；M is DNA molecular amount standard
markerII。
In Fig. 2：1 swimming lane is three kinds of mix primer amplification Echinococcus granulosus G1-G3 types, Echinococcus multilocularis and particulate spine
The PCR results of ball tapeworm G6-G10 type hybrid dna templates；2 swimming lanes are negative control；M is DNA molecular amount standard markerII.
In Fig. 3：1-3 swimming lanes are that mix primer expands purpose worm kind Echinococcus granulosus G1-G3 types, many room spine ball silk ribbons respectively
Worm and the PCR results of Echinococcus granulosus G6-G10 types；4-11 swimming lanes are amplification Shiqu echinococcus, blister with tapeworm, huge neck
Tapeworm, Lilium asiatic hybrids, taeniarhynchus saginatus, taeniasis suis, the PCR results of Hymenolepis diminuta and taenia serrata；M is DNA molecular
Amount standard markerII.
According to experimental result, 3 pairs of primers of the invention amplify the specific single of 240bp, 152bp and 441bp respectively
Purpose band, is corresponding in turn to Echinococcus granulosus G1-G3 types, Echinococcus multilocularis, Echinococcus granulosus G6-G10 types, expands piece
Duan great little unanimously, illustrates that this method can specifically detect three kinds of purpose echinococcus with being expected；After three pairs of primer mixing
It will not interfere, and can only amplify the purpose band of correspondence worm kind；And the present invention can only detect corresponding three kinds
The stripe size that purpose worm kind, the 8 kind worm kind no cross reactions nearer with other affiliations, or cross reaction are formed is non-
Purpose band does not influence the judgement of testing result, therefore with very strong specificity.
The sensitivity experiment of embodiment 2
Respectively using three kinds of purpose echinococcus DNA as template, template final concentration is respectively：0.7ng/ μ l, 0.35ng/ μ l,
0.18ng/ μ l, 0.09ng/ μ l, 0.04ng/ μ l, 0.02ng/ μ l, 0.01ng/ μ l, 0.005ng/ μ l, according to previous experiments method
Expanded.
Electrophoresis result is as shown in figure 4, the template of 1-8 swimming lanes is Echinococcus granulosus G1-G3 types, and the template of 9-16 swimming lanes is
Echinococcus multilocularis, the template of 17-24 swimming lanes is Echinococcus granulosus G6-G10 types.Template final concentration is respectively in reaction system
0.7ng/ μ l, 0.35ng/ μ l, 0.18ng/ μ l, 0.09ng/ μ l, 0.04ng/ μ l, 0.02ng/ μ l, 0.01ng/ μ l, 0.005ng/
μl.The present invention is above 0.005ng/ μ l to three kinds of purpose worm kind DNA detection limit, illustrate the present invention with very high sensitive
Degree.
The application that embodiment 3 is detected to body foci's tissue samples
Choose 10 operation patients censorship liver lesion tissues and carry out Echinococcus infections detection.Patient dna to be measured is extracted
Carried out with multiplexed PCR amplification according to previous experiments method.
Echinococcus granulosus G1-G3 types, Echinococcus multilocularis, the amplified band size point of Echinococcus granulosus G6-G10 types
Not Wei 240bp, 152bp and 441bp, electrophoresis result as shown in figure 5, wherein：1-10 swimming lanes are sample P CR results；11 swimming lanes are
The PCR amplifications of kind of the purpose worm kind hybrid dna template of positive control, i.e., 3 and 3 pairs of mix primers；12 swimming lanes are negative control；
M is DNA molecular amount standard markerII.
1st, 3,7,10 swimming lane bands are 240bp, therefore correspondence patient is that the infection of Echinococcus granulosus G1-G3 types is positive；2、4、
5th, 9 swimming lane bands are 152bp, therefore correspondence patient is that echinococcus multilocularis infection is positive；8 swimming lane bands are 441bp, therefore the patient
Infected for Echinococcus granulosus G6-G10 types positive；6 swimming lanes do not have band to be feminine gender, therefore the non-Hydatid Disease Infection of the patient.
Comparative example
By the multi-PCR detection method of the present invention respectively with contrasting 1：Cong-Nuan Liu et al. (Liu C N, Lou Z
Z,Li L,et al.Discrimination between E.granulosus sensu stricto,
E.multilocularis and E.shiquicus Using a Multiplex PCR Assay[J].Plos
Neglected Tropical Diseases,2015,9(9):E0004084.) and contrast 2：Patent CN201310102181.4
That delivers is compared for Echinococcus granulosus G1-G3 types, Echinococcus multilocularis and Shiqu echinococcus multi-PCR detection method
Compared with.
Echinococcus granulosus G1-G3 types, Echinococcus multilocularis, Echinococcus granulosus is respectively adopted in the DNA profiling of the present invention
G6-G10 types and three kinds of purpose worm kind DNA mixtures；Operation is according to previous experiments method.
Contrast 1 and contrast 2 respectively using Echinococcus granulosus G1-G3 types, Echinococcus multilocularis, Shiqu echinococcus and
Three kinds of worm kind DNA mixtures；Operation is according to respective literature method.
Electrophoresis result is as shown in fig. 6, wherein, 1-4 swimming lanes expand purpose worm kind particulate spine ball respectively for the method for the present invention
The PCR results of tapeworm G1-G3 types, Echinococcus multilocularis and Echinococcus granulosus G6-G10 types and three kinds of purpose worm kind hybrid dnas,
5-8 swimming lanes expand Echinococcus granulosus G1-G3 types, Echinococcus multilocularis, Shiqu echinococcus and three kinds respectively for 1 method of contrast
The PCR results of worm kind hybrid dna, 9-12 swimming lanes expand Echinococcus granulosus G1-G3 types, many room spine balls respectively for 2 methods of contrast
The PCR results of tapeworm, Shiqu echinococcus and three kinds of worm kind hybrid dnas.
It can be seen that, the present invention can detect each purpose worm kind respectively, and three worm kind mixed infections can be also detected simultaneously.Contrast
1 method can not detect Echinococcus multilocularis, and 8 swimming lanes expected three purpose bands do not occur, two band only occur,
Stripe size is respectively 219bp and 471bp, is Echinococcus granulosus G1-G3 types and Shiqu echinococcus, illustrates that this method can not
Three kinds of worm kind mixed infections are detected simultaneously.In the method for contrast 2, specific amplification stripe size is respectively 196bp, 584bp and
There are 3 band in 471bp, 10 swimming lanes, wherein one is more than 500bp, two other and Echinococcus granulosus G1-G3 types and Shiqu
The purpose band of echinococcus is approached, but does not occur 584bp single goal band equally as expected, illustrates that this method is detected
The poor specificity of Echinococcus multilocularis, and the detection of Echinococcus granulosus G1-G3 types and Shiqu echinococcus can be disturbed.12 swimming
Only there are two purpose bands in road, illustrates that this method can not realize the mixed infection detection of three kinds of worm kinds.
In summary, the present invention only passes through primary first-order equation, you can, can also to detect the independent infection of three kinds of purpose worm kinds
The mixed infection of three kinds of worm kinds of detection, purpose worm inter-species no cross reaction, band good separating effect, Detection results are better than existing
Echinococcosis multiple PCR method, overcoming prior art can not be while three kinds of Echinococcus hydatid cyst disease pest kinds to China's human infection be examined
The defect surveyed and identified.
SEQUENCE LISTING
<110>Disease prevention and control center of Sichuan Province
<120>The primer pair of echinococcosis cause of disease is organized to combine and kit with surveyor for detecting
<130> 201705
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence（gtp1F）
<220>
<221>Echinococcus granulosus G1-G3 type sense primers
<222> (1)..(25)
<400> 1
tggtttggtt tatatgttgg tgttt 25
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence（gtp1R）
<220>
<221>Echinococcus granulosus G1-G3 type anti-sense primers
<222> (1)..(22)
<400> 2
atccataaag gagttcctag cg 22
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence（mtb42F）
<220>
<221>Echinococcus multilocularis sense primer
<222> (1)..(20)
<400> 3
accaactgag ggtggcacta 20
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence（mtb42R）
<220>
<221>Echinococcus multilocularis anti-sense primer
<222> (1)..(25)
<400> 4
ccattactac cctaaacaac tgaca 25
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence（cox1F）
<220>
<221>Echinococcus granulosus G6-G10 type sense primers
<222> (1)..(21)
<400> 5
ttttatttac gtttgggggc g 21
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence（cox1R）
<220>
<221>Echinococcus granulosus G6-G10 type anti-sense primers
<222> (1)..(20)
<400> 6
ccaccaaacc aaaagacctg 20
Claims (7)
1. a kind of organize the primer pair of echinococcosis cause of disease to combine for detecting with surveyor, the primer pair combination includes：
Echinococcus granulosus G1-G3 type-special primers pair：Gtp1F is as shown in sequence table SEQ ID NO.1, gtp1R such as sequences
Shown in table SEQ ID NO.2；
Echinococcus multilocularis specific primer pair：Mtb42F is as shown in sequence table SEQ ID NO.3, mtb42R such as sequence table SEQs
Shown in ID NO.4；
Echinococcus granulosus G6-G10 type-special primers pair：Cox1F is as shown in sequence table SEQ ID NO.5；Cox1R such as sequences
Shown in table SEQ ID NO.6.
2. according to claim 1 organize the primer pair of echinococcosis cause of disease to combine for detecting with surveyor, its feature exists
In, Echinococcus granulosus G1-G3 type-special primers to, Echinococcus multilocularis specific primer pair and Echinococcus granulosus G6-
G10 type-special primers are followed successively by 240bp, 152bp and 441bp to the purpose fragment length amplified.
3. a kind of kit, it is characterised in that detected containing being used for described in claim 1 or 2 and surveyor organizes echinococcosis
The primer pair combination of cause of disease.
4. kit according to claim 3, it is characterised in that the kit also PCR reagent containing DNA cloning.
5. kit according to claim 4, it is characterised in that the PCR reagent includes archaeal dna polymerase mixture and nothing
Bacterium distilled water.
6. the application method of the kit as described in claim 3~5 any one, it is characterised in that including：Extract people to be checked
Biopsy DNA, and the reagent added as template in the kit enters performing PCR amplification, the PCR primer point sample of amplification is extremely
Electrophoresis is carried out in 2% Ago-Gel, electrophoresis result is observed.
7. application method according to claim 6, it is characterised in that the condition of the PCR amplifications is 94 DEG C of pre-degenerations
3min；94 DEG C of denaturation 30s；56 DEG C of annealing 30s；72 DEG C of extension 40s；Totally 35 circulations；72 DEG C of extension 5min.
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| CN201710392941.8A CN107164479B (en) | 2017-05-27 | 2017-05-27 | Primer pair combination and kit for detecting and identifying human tissue hydatid pathogen |
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| CN106868153A (en) * | 2017-03-14 | 2017-06-20 | 西南民族大学 | The detection kit of the cattle and sheep echinococcosis granulosa based on POCKIT Micro fluorescent PCR platforms and application |
| CN108950020A (en) * | 2018-08-31 | 2018-12-07 | 四川农业大学 | The application of Echinococcus granulosus mitochondria ND6 gene |
| CN109097482A (en) * | 2018-08-31 | 2018-12-28 | 四川农业大学 | The application of Echinococcus granulosus mitochondria ND6 gene |
| CN109762910A (en) * | 2019-01-10 | 2019-05-17 | 四川省疾病预防控制中心 | It is a kind of for detecting the primer and kit of amphitypy echinococcosis simultaneously |
| CN110656187A (en) * | 2019-10-28 | 2020-01-07 | 中国疾病预防控制中心寄生虫病预防控制所 | Kit and method for detecting echinococcus in lesion tissues or canine feces by using multiple RAA and multiple PCR (polymerase chain reaction) |
| CN110846309A (en) * | 2018-08-20 | 2020-02-28 | 中国农业科学院兰州兽医研究所 | Primer pair and primer probe set for identifying echinococcus multilocularis and application of primer pair and primer probe set |
| 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 |
| CN111733254A (en) * | 2019-03-25 | 2020-10-02 | 中国疾病预防控制中心寄生虫病预防控制所 | Free DNA sequence from Echinococcus granulosus and application thereof |
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| CN106868153A (en) * | 2017-03-14 | 2017-06-20 | 西南民族大学 | The detection kit of the cattle and sheep echinococcosis granulosa based on POCKIT Micro fluorescent PCR platforms and application |
| CN110846309B (en) * | 2018-08-20 | 2023-03-24 | 中国农业科学院兰州兽医研究所 | Primer pair and primer probe set for identifying echinococcus multilocularis and application of primer pair and primer probe set |
| CN110846309A (en) * | 2018-08-20 | 2020-02-28 | 中国农业科学院兰州兽医研究所 | Primer pair and primer probe set for identifying echinococcus multilocularis and application of primer pair and primer probe set |
| CN109097482B (en) * | 2018-08-31 | 2021-09-14 | 四川农业大学 | Application of Echinococcus granulosus mitochondrion ND6 gene |
| CN108950020A (en) * | 2018-08-31 | 2018-12-07 | 四川农业大学 | The application of Echinococcus granulosus mitochondria ND6 gene |
| CN109097482A (en) * | 2018-08-31 | 2018-12-28 | 四川农业大学 | The application of Echinococcus granulosus mitochondria ND6 gene |
| CN108950020B (en) * | 2018-08-31 | 2021-09-14 | 四川农业大学 | Application of Echinococcus granulosus mitochondrion ND6 gene |
| CN109762910A (en) * | 2019-01-10 | 2019-05-17 | 四川省疾病预防控制中心 | It is a kind of for detecting the primer and kit of amphitypy echinococcosis simultaneously |
| CN111733254A (en) * | 2019-03-25 | 2020-10-02 | 中国疾病预防控制中心寄生虫病预防控制所 | Free DNA sequence from Echinococcus granulosus and application thereof |
| CN111733254B (en) * | 2019-03-25 | 2023-08-18 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Biomarker DNA molecular fragment for detecting echinococcus granulosus and application thereof |
| CN110656187A (en) * | 2019-10-28 | 2020-01-07 | 中国疾病预防控制中心寄生虫病预防控制所 | Kit and method for detecting echinococcus in lesion tissues or canine feces by using multiple RAA and multiple PCR (polymerase chain reaction) |
| CN110656187B (en) * | 2019-10-28 | 2023-06-02 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Kit for detecting pathological tissues or echinococcus in canine feces by multiple RAA and multiple PCR and detection method |
| 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 |
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