CN109234410A - A method of the detection echinococcosis based on loop-mediated isothermal amplification technique - Google Patents
A method of the detection echinococcosis based on loop-mediated isothermal amplification technique Download PDFInfo
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Abstract
The invention belongs to the measurement comprising enzyme or microorganism or method of inspection technical field, a kind of method of detection echinococcosis based on loop-mediated isothermal amplification technique is disclosed, aseptic aspiration bubble cyst fluid is put into and is transferred to laboratory in hot bottle and is detected；Or it is sterile take lens capsule tissue, rinsed 5 times with physiological saline, -4 DEG C of alcohol of 95% preservations be added；The sterile packing tissue 25mg for taking echinococcosis patient or infection animal is put into culture dish, with nucleic acid free water cleansing tissue sample to remove the ethyl alcohol for remaining in surface, is then placed in 1.5mL centrifuge tube；Nucleic acid extraction is carried out to pretreated sample；To treated, sample carries out LAMP detection.The present invention is easy, quick, accurate, low price gene amplification method；It is a kind of completely new nucleic acid amplification method, has the characteristics that simple, quick, high specificity.
Description
Technical field
The invention belongs to the measurement comprising enzyme or microorganism or method of inspection technical fields, more particularly to a kind of ring that is based on to be situated between
The method for leading the detection echinococcosis of isothermal amplification technique.
Background technique
Echinococcosis is being commonly called as hydatidosis (echinococcosis), is to be drawn by the parasitized larvae of echinococcus in human body
The parasitic zoonoses risen.Mainly there is the larva of Echinococcus granulosus (echinococcus granulosus) in China
Cause capsule echinococcosis (cystic echinococcosis, CE), Echinococcus multilocularis (echinococcus
Multilocularis larva) causes alveolar echinococcosis (alveolar echinococcosis, AE).
Currently, the detection method of existing Human Hydatidosis mainly has amynologic diagnostic method and helminth and pathological tissue
It checks.Amynologic diagnostic method has indirect hemagglutination test (HA test) (IHA), enzyme-linked immunosorbent assay (ELISA), PVC film
Rapid ELISA, wherein ELISA is the most commonly used and sensitive.Existing echinococcosis immunological testing method is in sensibility and specificity
It is upper to there is very big difference.Test result is influenced by many factors: the property and quality of antigen；The experimental system of detection,
Quantity, position and the vigor of hydatidocystis；The difference of different geography worm strains, the difference of individual immunity responsing reaction.About 10%-
The patients with hydatidosis that 40% operation is made a definite diagnosis can't detect specific antibody with the antigen being currently known.Helminth and pathological tissue
Inspection needs experienced technical staff, is not suitable for basic medical unit and carries out detection.
In existing detection method, although not using PCR detection method, needed according to traditional PCR technique anti-
The cyclic amplification of heating and cooling and the gel electrophoresis analysis after PCR are answered as a result, taking a long time, carrying pollution is easy to produce, uses
Dyestuff (EB etc.) has carcinogenicity, and its detection sensitivity is still to be improved.Real-time fluorescent PCR technology is not necessarily to electrophoretic analysis, but
It is to need using specific probe and instrument and equipment costly, higher cost, operator needs by professional training, because
This is unfavorable for large-scale use.
In conclusion problem of the existing technology is:
(1) there is on sensibility and specificity very big difference in existing echinococcosis immunological testing method.
(2) patients with hydatidosis that the operation of existing about 10%-40% is made a definite diagnosis can't detect specifically with the antigen being currently known
Property antibody.
(3) helminth and pathologic diagnosis need experienced technical staff in the prior art, are not suitable for base doctor
Treat institutions conduct detection.
(4) it in existing detection method, takes a long time, is easy to produce carrying pollution, the dyestuff (EB etc.) used, which has, to be caused
It is carcinous, and its detection sensitivity is still to be improved.
(5) existing real-time fluorescent PCR technology is needed using specific probe and instrument and equipment costly, cost
Higher, operator needs by professional training, therefore is unfavorable for large-scale use.
Solve the difficulty and meaning of above-mentioned technical problem:
LAMP technology uses a plurality of primer, identifies different regions, and any region will not be expanded with primer mismatch, because
This is with high specificity；Do not need preparatory double-stranded DNA thermal denaturation, avoid temperature cycles and caused by the loss of time,
Detection time is shorter；And expensive fluorescence quantitative PCR instrument is not needed, it is only necessary to which a water-bath can be tested, cost
It is lower, easily carried out extensively in basic medical unit.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of detection packet based on loop-mediated isothermal amplification technique
The method of parasitosis.
The invention is realized in this way a method of the detection echinococcosis based on loop-mediated isothermal amplification technique, it is described
The method of detection echinococcosis based on loop-mediated isothermal amplification technique the following steps are included:
Step 1, aseptic aspiration bubble cyst fluid are put into hot bottle and are transferred to laboratory and are detected；Or it is sterile
Lens capsule tissue is taken, is rinsed 5 times with physiological saline, 95% -4 DEG C of alcohol preservations are added；
Step 2, the sterile packing tissue 25mg for taking echinococcosis patient or infection animal are put into culture dish, use nucleic
Acid free water cleansing tissue sample is then placed in 1.5mL centrifuge tube with removing the ethyl alcohol for remaining in surface；
Step 3 carries out nucleic acid extraction to pretreated sample；
Step 4, to treated, sample carries out LAMP detection.
Further, the primer sequence of the step 1 is as follows:
—CE-F3:5'-ATAGAGTTAGGTATAGTGGTCTT-3'；
—CE-B3:5'-CCTACAACAACACAAAGCA-3'；
—CE-FIP:5'-ACCTGCGCACAACAAAGAATTCTGTATTATGATTTTTAGCTGCTG-3'；
—CE-BIP:5'-TCTTTTTTAAGGTCGGTTCGATGTCACACATAAAACAAGCCTCAA-3'；
—CE-LF:5'-TGCTAGAAATTCTAG--3'；
—CE-LB:5'-GCTTTTGGATCTGTTAGG--3'；
—AE-F3:5'-ATTTTCTTTTGTATTCTCTGTGTT-3'；
—AE-B3:5'-GCACAGACGATAATCCACAC-3'；
—AE-FIP:5'-GCACAACTCCAAAAAAACCCAAAA-GTTTTGTTTTTTGTGTTGTGTAGT-3'；
—AE-BIP:5'-TATTCCTTCATTTTTTGTTGGGGC-TTACATAGCTCAACAAAGACCTA-3'；
Further, the positive control, negative control of the step 2 and blank control difference are as follows:
Positive control is using the echinococcosis positive sample containing detection sequence echinococcosis genomic DNA or plasmid or retention
The template that nucleic acid is reacted as LAMP；
The template that negative control uses the genomic DNA without containing testing gene sequence to react as LAMP；
Set up two blank controls: that sets up when extracting DNA extracts the blank control of blank control and LAMP reaction.
Further, the LAMP reaction system of the step 3 are as follows: tested using the commercial kit obtained, each
Pattern detection can do two parallel pipes, 25 μ L, 2 × LAMP mixed liquor of overall reaction system 12.5 μ L, 1 μ L of fluorescent dye, detection dye
Expect 1 μ L, Bst archaeal dna polymerase, 1 μ L, 2 μ L of primer mixed liquor；FIP and BIP primer concentration 40pmol, LF and LB primer concentration
20pmol, F3 and B3 primer concentration 5pmol, DNA profiling 5 μ L, ddH2O complements to 25 μ L.
Further, bubble tumour liquid sample nucleic acid is extracted in the nucleic acid extraction of the step 3 can be used the examination of commercialization
Agent box QIAamp DNA mini kit；Extracting lens capsule tissue sample nucleic acid can be used the kit QIAamp tissue of commercialization
Kit is prepared according to kit operation.
Further, the step 4 LAMP detection the following steps are included:
Step 1 carries out design of primers to aim sequence；
Step 2, setting positive control, negative control and blank control；
Step 3 constructs LAMP reaction system；
Step 4, set LAMP reaction condition, 63 DEG C of incubation 60min, can according to condition optimizing appropriate adjustment temperature and when
Between.
Further, need to carry out quality control in the LAMP detection process of the step 4, any control if there is
Non- following normal outcomes, should reform experiment；That is blank control: liquid is purple in reaction tube；Negative control: liquid in reflection pipe
It is purple；Positive control: liquid is blue in reflection pipe.
Further, the corresponding sample of the positive findings occurred after the step 4 detection should send qualified laboratory to carry out
Reinspection or identification, determining should be immediately to inspection or sample presentation unit reports after positive findings.
Further, the waste in the step 4 checkout procedure should be collected, and collect laggard horizontal high voltage steam sterilizing
Processing impregnates 30min or more using 0.5% liquor natrii hypochloritis.
In conclusion advantages of the present invention and good effect are as follows:
(1) method of the detection echinococcosis provided by the invention based on loop-mediated isothermal amplification technique has ring mediation etc.
Warm amplification technique, can be under the conditions of isothermal (60-65 DEG C), (usual 1 hour) progress nucleic acid amplification in the short time, are a kind of " letters
Just, gene amplification method quickly, accurately, at a low price ".Relative to traditional round pcr or real-time fluorescent PCR technology, ring is situated between
Leading isothermal duplication method is a kind of completely new nucleic acid amplification method, has the characteristics that simple, quick, high specificity.Ring mediated isothermal
Nucleic acid amplification technologies are using two pairs of special primers specifically corresponding to 6 constant gene segment Cs in target sequence, in Bst archaeal dna polymerase
Isothermal nucleic acid amplification reaction is carried out to target sequence under effect, macroscopic magnesium pyrophosphate white precipitate is formed, can sentence accordingly
Determine the positive reaction in pipe；Can also directly be detected by an unaided eye result by the method that dyestuff is added in final product simultaneously.With
Real-time fluorescence quantitative PCR is compared, and detection is limited up to 10.0fg.Detection is greater than 10pg or more DNA, and detection sensitivity 100% is special
Different degree is relatively good, has not seen amplified production from other samples (cysticercus bovis, schistosoma bovis) without containing echinococcosis.
(2) method of the detection echinococcosis provided by the invention based on loop-mediated isothermal amplification technique has detection method
Sensitivity, see the table below:
(3) method of the detection echinococcosis provided by the invention based on loop-mediated isothermal amplification technique, the spy with primer
Different degree testing result, see the table below；
Detailed description of the invention
Fig. 1 is the method flow of the detection echinococcosis provided in an embodiment of the present invention based on loop-mediated isothermal amplification technique
Figure.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
As shown in Figure 1, the method for the detection echinococcosis provided in an embodiment of the present invention based on loop-mediated isothermal amplification technique
The following steps are included:
S101: sample acquisition: aseptic aspiration bubble cyst fluid is put into hot bottle and is transferred to laboratory and is detected；
Or it is sterile take lens capsule tissue, rinsed 5 times with physiological saline, -4 DEG C of alcohol of 95% preservations be added；
S102: pre-process to sample: the sterile packing tissue 25mg for taking echinococcosis patient or infection animal is put into training
It supports in ware, with nucleic acid free water cleansing tissue sample to remove the ethyl alcohol for remaining in surface, is then placed in
In 1.5mL centrifuge tube；
S103: nucleic acid extraction is carried out to pretreated sample；
S104: to treated, sample carries out LAMP detection.
As the preferred embodiment of the present invention, extracting bubble tumour liquid sample nucleic acid in the nucleic acid extraction of step S103 can
Using the kit QIAamp DNA mini kit of commercialization；Extracting lens capsule tissue sample nucleic acid can be used the kit of commercialization
QIAamp tissue kit (QIAGEN, Germany) is prepared according to kit operational manual.
As the preferred embodiment of the present invention, the LAMP detection of step S104 the following steps are included:
Step 1 carries out design of primers to aim sequence；
Step 2, setting positive control, negative control and blank control；
Step 3 constructs LAMP reaction system；
Step 4, set LAMP reaction condition, 63 DEG C of incubation 60min, can according to condition optimizing appropriate adjustment temperature and when
Between.
As the preferred embodiment of the present invention, need to carry out quality control in the LAMP detection process of step S104, it is any
Kind control should reform experiment if there is non-following normal outcomes；That is blank control: liquid is purple in reaction tube；It is negative right
According to: liquid is purple in reflection pipe；Positive control: liquid is blue in reflection pipe.
As the preferred embodiment of the present invention, the corresponding sample of the positive findings occurred after step S104 detection should send money
The laboratory of matter rechecked or identified, determining should be immediately to inspection or sample presentation unit reports after positive findings.
As the preferred embodiment of the present invention, the waste in step S104 checkout procedure should be collected, and be collected laggard
The processing of horizontal high voltage steam sterilizing uses 0.5% liquor natrii hypochloritis to impregnate 30min with other equivalent processing methods such as upper.
As the preferred embodiment of the present invention, the primer sequence of step S101 is as follows:
—CE-F3:5'-ATAGAGTTAGGTATAGTGGTCTT-3'；
—CE-B3:5'-CCTACAACAACACAAAGCA-3'；
—CE-FIP:5'-ACCTGCGCACAACAAAGAATTCTGTATTATGATTTTTAGCTGCTG-3'；
—CE-BIP:5'-TCTTTTTTAAGGTCGGTTCGATGTCACACATAAAACAAGCCTCAA-3'；
—CE-LF:5'-TGCTAGAAATTCTAG--3'；
—CE-LB:5’-GCTTTTGGATCTGTTAGG--3’。
—AE-F3:5'-ATTTTCTTTTGTATTCTCTGTGTT-3'；
—AE-B3:5'-GCACAGACGATAATCCACAC-3'；
—AE-FIP:5'-GCACAACTCCAAAAAAACCCAAAA-GTTTTGTTTTTTGTGTTGTG TAGT-3'；
—AE-BIP:5'-TATTCCTTCATTTTTTGTTGGGGC-TTACATAGCTCAACAAAGACCTA-3'；
As the preferred embodiment of the present invention, positive control, negative control and the blank control of step S102 is distinguished as follows:
Positive control is used containing detection sequence echinococcosis (echinococcus) genomic DNA (or plasmid) or the echinococcosis retained
The template that the nucleic acid of positive sample is reacted as LAMP；
The template that negative control uses the genomic DNA without containing testing gene sequence to react as LAMP；
Set up two blank controls: the extraction blank control (sample is replaced with water) and LAMP reaction set up when extracting DNA
Blank control (with DNA solution replace DNA profiling).
As the preferred embodiment of the present invention, the LAMP reaction system of step S103 are as follows: using the commercial reagents obtained
Box (LAMP kit, Mast Company, South Africa) is tested, and each pattern detection can make two parallel pipes, always
25 μ L, 2 × LAMP mixed liquor of reaction system 12.5 μ L, 1 μ L of fluorescent dye (real-time monitoring use) detect dyestuff 1 μ L, Bst
1 μ L of archaeal dna polymerase, 2 μ L of primer mixed liquor (FIP and BIP primer concentration 40pmol, LF and LB primer concentration 20pmol, F3 and B3
Primer concentration 5pmol), DNA profiling 5 μ L, ddH2O complement to 25 μ L.
Application principle of the invention is further described combined with specific embodiments below.
Embodiment 1；
1, bio-security
(1) personal protection in work is detected referring to GB19489
(2) laboratory should meet GB 19489 and WS 233 to the bio-safety in the laboratory bio-safety second level (BSL-2)
It is required that.
(3) used experimental article should carry out harmless treatment in accordance with processing requirement of the GB 19489 to waste.
(4) anti-pollution measure is executed by the regulation of SN/T 1193.
2, instrument and equipment
Key instrument is as follows:
II grades of Biohazard Safety Equipments；Vortex oscillator；Superpure water machine or distilled water device；Constant-temperature incubation instrument；Micro centrifuge；It is high
Fast refrigerated centrifuge；Electronic balance (d=1mg；Refrigerator: 4 DEG C, -20 DEG C, -70 DEG C；High-pressure sterilizing pot；Adjustable micro sample adding appliance
(10 μ L, 100 μ L, 1000 μ L) and supporting belt filter core pipette tips.
3, main agents
Main agents are as follows:
Nucleic acid reagent preparation: QIAGEN QIAamp DNA mini kit or QIAamp tissue kit；
Primer should be the purifying of PAGE or more rank, primer sequence；
LAMP reagent: LAMP kit, Mast Company, South Africa.
4, program is detected
The acquisition of 4.1 samples and processing
Aseptic aspiration bubble cyst fluid is put into hot bottle and is transferred to laboratory and is detected.Or sterile take capsule group
It knits, is rinsed 5 times with physiological saline, 95% -4 DEG C of alcohol preservations are added.
4.2 laboratory inspection
4.2.1 sample preprocessing
The sterile packing tissue 25mg for taking echinococcosis patient or infection animal is put into culture dish, with nucleic acid
Free water cleansing tissue sample is then placed in 1.5mL centrifuge tube with removing the ethyl alcohol for remaining in surface.
4.2.2 nucleic acid extraction
Extracting bubble tumour liquid sample nucleic acid can be used the kit QIAamp DNA mini kit of commercialization；It extracts
The kit QIAamp tissue kit (QIAGEN, Germany) of commercialization can be used in lens capsule tissue sample nucleic acid
It is prepared according to kit operational manual.
4.2.3 LAMP is detected
4.2.3.1 primer
Primer sequence is as follows:
Capsule echinococcosis primer
—CE-F3:5'-ATAGAGTTAGGTATAGTGGTCTT-3'；
—CE-B3:5'-CCTACAACAACACAAAGCA-3'；
—CE-FIP:5'-ACCTGCGCACAACAAAGAATTCTGTATTATGATTTTTAGCTGCTG-3'；
—CE-BIP:5'-TCTTTTTTAAGGTCGGTTCGATGTCACACATAAAACAAGCCTCAA-3'；
—CE-LF:5'-TGCTAGAAATTCTAG--3'；
—CE-LB:5’-GCTTTTGGATCTGTTAGG--3’。
Alveolar echinococcosis primer
—AE-F3:5'-ATTTTCTTTTGTATTCTCTGTGTT-3'；
—AE-B3:5'-GCACAGACGATAATCCACAC-3'；
—AE-FIP:5'-GCACAACTCCAAAAAAACCCAAAA-GTTTTGTTTTTTGTGTTGTGTAGT-3'；
—AE-BIP:5'-TATTCCTTCATTTTTTGTTGGGGC-TTACATAGCTCAACAAAGACCTA-3'；
4.2.3.2 positive control, negative control and the setting of blank control
(1) positive control
Positive control is used containing detection sequence echinococcosis (echinococcus) genomic DNA (or plasmid) or the echinococcosis retained
The template that the nucleic acid of positive sample is reacted as LAMP.
(2) negative control
The template that negative control uses the genomic DNA without containing testing gene sequence to react as LAMP.
(3) two blank controls are set up
The extraction blank control set up when-extraction DNA (sample is replaced with water)
The blank control of-LAMP reaction (DNA profiling is replaced with DNA solution)
4.2.3.3 the composition of LAMP reaction system
The commercial kit (LAMP kit, Mast Company, South Africa) that acquisition can be used is tested,
Each pattern detection can be two parallel pipes, 25 μ L of overall reaction system.2 × LAMP mixed liquor, 12.5 μ L, 1 μ L of fluorescent dye are (real
When monitoring use), detect 1 μ L, Bst archaeal dna polymerase of dyestuff, 1 μ L, 2 μ L (FIP and BIP primer concentration of primer mixed liquor
40pmol, LF and LB primer concentration 20pmol, F3 and B3 primer concentration 5pmol), DNA profiling 5 μ L, ddH2O complement to 25 μ L.
4.2.3.4 reaction condition
LAMP reaction condition: 63 DEG C of incubation 60min can suitably be adjusted because of the performance difference of different instruments according to condition optimizing
Whole temperature and time.
4.2.4 quality controls
Basic principle: the various control LAMP testing results of lab setup should meet following situations, otherwise, any right
According to if there is non-following normal outcomes, experiment should be reformed.
Blank control: liquid is purple in reaction tube.
Negative control: liquid is purple in reflection pipe.
Positive control: liquid is blue in reflection pipe.
4.2.5 result judgement and report
Visual results, blue indicate positive, and purple indicates negative.Sample to be examined does 2 Duplicate Samples.
Inspection result of the present invention judges and is reported as follows:
-- the pipe of liquid at least one is blue in 2 parallel samples reaction tubes, while various experiment contrast results are normal, can sentence
The sample system primary dcreening operation that breaks is positive；
-- liquid is purple in 2 parallel samples reaction tubes, while various experiment contrast results are normal, can determine whether the sample
This is primary dcreening operation feminine gender.
5, positive findings are disposed
It is detected using LAMP method, the corresponding sample for positive findings occur should send qualified laboratory to be rechecked
Or identification, determining should be immediately to inspection or sample presentation unit reports after positive findings.
6, the disposition of waste
Waste in checkout procedure collects laggard horizontal high voltage steam sterilizing processing or molten using 0.5% sodium hypochlorite
Liquid impregnates 30min and waits other equivalent processing methods with upper.
Below with reference to gene order table, the present invention will be described in further detail；
(1) capsule echinococcosis primer
S1:SEQ ID NO:1
S2:SEQ ID NO:2
S3:SEQ ID NO:3
S4:SEQ ID NO:4
S5:SEQ ID NO:5
S6:SEQ ID NO:6
S1—CE-F3:5'-ATAGAGTTAGGTATAGTGGTCTT-3'；
S2—CE-B3:5'-CCTACAACAACACAAAGCA-3'；
S3
—CE-FIP:5'-ACCTGCGCACAACAAAGAATTCTGTATTATGATTTTTAGCTGCTG-3'；
S4
—CE-BIP:5'-TCTTTTTTAAGGTCGGTTCGATGTCACACATAAAACAAGCCTCAA-3'；
S5—CE-LF:5'-TGCTAGAAATTCTAG--3'；
S6—CE-LB:5’-GCTTTTGGATCTGTTAGG--3’。
(2) alveolar echinococcosis primer
S1:SEQ ID NO:7
S2:SEQ ID NO:8
S3:SEQ ID NO:9
S4:SEQ ID NO:10
S1—AE-F3:5'-ATTTTCTTTTGTATTCTCTGTGTT-3'；
S2—AE-B3:5'-GCACAGACGATAATCCACAC-3'；
S3
—AE-FIP:5'-GCACAACTCCAAAAAAACCCAAAA-GTTTTGTTTTTTGTGTTGTGTAGT-3'；
S4
—AE-BIP:5'-TATTCCTTCATTTTTTGTTGGGGC-TTACATAGCTCAACAAAGACCTA-3'；
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
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Claims (9)
1. a kind of method of the detection echinococcosis based on loop-mediated isothermal amplification technique, which is characterized in that described to be mediated based on ring
The method of the detection echinococcosis of isothermal amplification technique the following steps are included:
Step 1, aseptic aspiration bubble cyst fluid are put into hot bottle and are transferred to laboratory and are detected；Or sterile take capsule
Tissue is rinsed 5 times with physiological saline, and 95% -4 DEG C of alcohol preservations are added；
Step 2, the sterile packing tissue 25mg for taking echinococcosis patient or infection animal are put into culture dish, use nucleic
Acid free water cleansing tissue sample is then placed in 1.5mL centrifuge tube with removing the ethyl alcohol for remaining in surface；
Step 3 carries out nucleic acid extraction to pretreated sample；
Step 4, to treated, sample carries out LAMP detection.
2. the method for the detection echinococcosis based on loop-mediated isothermal amplification technique as described in claim 1, which is characterized in that institute
The primer sequence for stating step 1 is as follows:
—CE-F3:5'-ATAGAGTTAGGTATAGTGGTCTT-3'；
—CE-B3:5'-CCTACAACAACACAAAGCA-3'；
—CE-FIP:5'-ACCTGCGCACAACAAAGAATTCTGTATTATGATTTTTAGCTGCTG-3'；
—CE-BIP:5'-TCTTTTTTAAGGTCGGTTCGATGTCACACATAAAACAAGCCTCAA-3'；
—CE-LF:5'-TGCTAGAAATTCTAG--3'；
—CE-LB:5'-GCTTTTGGATCTGTTAGG--3'；
—AE-F3:5'-ATTTTCTTTTGTATTCTCTGTGTT-3'；
—AE-B3:5'-GCACAGACGATAATCCACAC-3'；
—AE-FIP:5'-GCACAACTCCAAAAAAACCCAAAA-GTTTTGTTTTTTGTGTTGTGTAGT-3'；
—AE-BIP:5’-TATTCCTTCATTTTTTGTTGGGGC-TTACATAGCTCAACAAAGACCTA-3’。
3. the method for the detection echinococcosis based on loop-mediated isothermal amplification technique as described in claim 1, which is characterized in that institute
Positive control, negative control and the blank control difference for stating step 2 are as follows:
Positive control uses the nucleic acid of the echinococcosis positive sample containing detection sequence echinococcosis genomic DNA or plasmid or retention
Template as LAMP reaction；
The template that negative control uses the genomic DNA without containing testing gene sequence to react as LAMP；
Set up two blank controls: that sets up when extracting DNA extracts the blank control of blank control and LAMP reaction.
4. the method for the detection echinococcosis based on loop-mediated isothermal amplification technique as described in claim 1, which is characterized in that institute
State the LAMP reaction system of step 3 are as follows: tested using the commercial kit obtained, each pattern detection can do two
Parallel pipe, 25 μ L, 2 × LAMP mixed liquor of overall reaction system 12.5 μ L, 1 μ L of fluorescent dye, detection 1 μ L, Bst DNA of dyestuff polymerization
1 μ L of enzyme, 2 μ L of primer mixed liquor；FIP and BIP primer concentration 40pmol, LF and LB primer concentration 20pmol, F3 and B3 primer is dense
Spend 5pmol, DNA profiling 5 μ L, ddH2O complements to 25 μ L.
5. the method for the detection echinococcosis based on loop-mediated isothermal amplification technique as described in claim 1, which is characterized in that institute
Stating extraction bubble tumour liquid sample nucleic acid in the nucleic acid extraction of step 3 can be used the kit QIAamp DNA of commercialization
mini kit；The kit QIAamp tissue kit that commercialization can be used in extraction lens capsule tissue sample nucleic acid is grasped according to kit
It is prepared.
6. the method for the detection echinococcosis based on loop-mediated isothermal amplification technique as described in claim 1, which is characterized in that institute
State step 4 LAMP detection the following steps are included:
Step 1 carries out design of primers to aim sequence segment；
Step 2, setting positive control, negative control and blank control；
Step 3 constructs LAMP reaction system；
Step 4 sets LAMP reaction condition, and 63 DEG C of incubation 60min can be according to condition optimizing appropriate adjustment temperature and time.
7. the method for the detection echinococcosis based on loop-mediated isothermal amplification technique as described in claim 1, which is characterized in that institute
It states and needs to carry out quality control in the LAMP detection process of step 4, any control is answered if there is non-following normal outcomes
Reform experiment；That is blank control: liquid is purple in reaction tube；Negative control: liquid is purple in reflection pipe；Positive control:
Reflect that liquid is blue in pipe.
8. the method for the detection echinococcosis based on loop-mediated isothermal amplification technique as described in claim 1, which is characterized in that institute
The corresponding sample for stating the positive findings occurred after step 4 detection should send qualified laboratory to be rechecked or identified, determine sun
It should be immediately to inspection or sample presentation unit reports after property result.
9. the method for the detection echinococcosis based on loop-mediated isothermal amplification technique as described in claim 1, which is characterized in that institute
Stating the waste in step 4 checkout procedure should be collected, and collect laggard horizontal high voltage steam sterilizing processing or using 0.5%
Liquor natrii hypochloritis impregnates 30min or more.
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