CN109735642A - A kind of primer, probe and the detection method of RAA Fluorometric assay Giardia lamblia - Google Patents
A kind of primer, probe and the detection method of RAA Fluorometric assay Giardia lamblia Download PDFInfo
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Abstract
It is high specificity, high sensitivity, reproducible the invention discloses primer, probe and the detection method of a kind of RAA Fluorometric assay Giardia lamblia；Energy fast and easy accurately identifies Giardia lamblia, and easy to operate, detection time is short, and detection is completed in 15min.Detection method of the invention is quick, high throughput easy to accomplish, and testing cost substantially reduces, and high sensitivity, and every reaction detection sensitivity reaches 100Copies/ reaction.
Description
Technical field
The present invention relates to technical field of molecular biological detection, more particularly to a kind of RAA Fluorometric assay is blue
Primer, probe and the detection method of family name's giardia lamblia stiles.
Background technique
Giardia lamblia is that a kind of main parasitic in the top of people and the small intestine of certain mammals answers door parasitic protozoa,
Any age groups can infect, and children, old age, weak and immunodeficiency person are particularly susceptible infection.Lan Shi merchant's flagellum
Worm variance in form and excretes after breeding with excrement, the drinking water or food that human or animal's intake is polluted by packing living and
It is infected, cause using diarrhea and indigestion as the lambliasis of cardinal symptom.Giardia lamblia is with packing
Form be present in water, about 8~10 μm of size.Being encapsulated in 25 DEG C of water can keep 2 weeks infectious, and can in 4 DEG C of water
It keeps infectious to be up to 11 weeks as long as, Giardia lamblia is 10~25 packings living to the pathogenic dosage of human body.Lan Shi merchant
Flagellosis prevalence is found everywhere through the world, most with Perenniporia martius, and developing country's number of the infected is about 2.5 hundred million；In
Also very extensively, for various regions infection rate between 0.48%~10%, summer and autumn disease incidence is higher for state's distribution.Giardia lamblia
Popular outburst and local economy and social development levels, the degree received an education, life style and health consciousness etc. have closely
Relationship.Estimate according to the World Health Organization, human population worldwide's Giardia lamblia infection rate is 1%~30%, wherein children
Infection rate highest, thus the parasitic protozoa be classified as by the World Health Organization endanger human health important parasite it
One.
Giardia lamblia is resided in extensively in the Different Waters of the Nature, and packing can be with long-term existence in water body
In, people once drinks the water polluted by Giardia lamblia, just very likely falls ill.Giardia lamblia mostlys come from
Farm, farm and slaughterhouse, by the polluted water of discharge, to be polluted to other water environments.By optimizing water process
Technique can play certain removal effect, but since it has stronger resistance to traditional chlorhexidine-containing disinfectant, deposit in water environment
Live time is long, therefore can not be completely eliminated.Only reinforce real-time monitoring, is taken according to monitoring result and targetedly arranged
It applies, the harm of Giardia lamblia can just be effectively reduced.
It is mainly at present three kinds to the detection method of Giardia lamblia, respectively pathogeny detection, immunology detection
And molecular Biological Detection.Wherein, " goldstandard " of diagnosis is pathogeny detection method, by detecting in excrement under microscope
Packing or trophozoite, to make a definite diagnosis Giardia lamblia, but this method have it is time-consuming and laborious, sensibility, specificity
It is not high, and the shortcomings that cannot distinguish worm kind and genotype.And molecular Biological Detection technology is mainly real-time fluorescence quantitative PCR skill
Art, principle, by fluorescence signal, are measured in real time to PCR process during PCR amplification.This method is sensitive, special
It is different, recall rate is high, but high to personnel requirement and equipment requirement, detection time is 1.5~2 hours.In addition, the common Lan Shi in water source
Giardia lamblia stiles detection method is US EPA1623 method.This method mainly comprises the following steps filtering, elution, is concentrated, isolates and purifies, is glimmering
Light colour developing and microscopy, it is larger that the major defect of this method is needed volume of water sample, and operates extremely complex, and step is more, behaviour
It is required very high, error is also larger when microscopy, is easy missing inspection and generates false negative, fixed kind of effect of tracing to the source is undesirable, completes entire inspection
Survey needs 2-3 days time.In addition, if adopting this method, the testing cost of each sample is very high.
The disadvantages of long, inconvenient for operation and false negative is high there are the time in view of existing detection technique, provides a kind of RAA fluorescence
Method detects primer, probe and the detection method of Giardia lamblia, makes that its is quick, sensitive, easy to operate, is suitable for laboratory
And field quick detection, the problem of being those skilled in the art's urgent need to resolve.
Summary of the invention
In view of this, the present invention provides primer, probe and the detections of a kind of RAA Fluorometric assay Giardia lamblia
Method can complete the detection of Giardia lamblia for 5-15 minutes under the conditions of 39 DEG C, have quick, sensitive, easy to operate, suitable
The characteristics of for laboratory and field quick detection.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of primer of RAA Fluorometric assay Giardia lamblia, the primer sequence are as follows:
Upstream primer: 5 '-TCAGGAAGGAGGCCCTCAAGAGCCTGAACG-3 '；SEQ ID NO.3；
Downstream primer: 5 '-AGCTTCGTGTTTGTGAGCGCTTCTGTCGTG-3 '；SEQ ID NO.5.
Further, the use concentration of the upstream primer and downstream primer is 5~20 μM.
Preferably, the use concentration of the upstream primer and downstream primer is 10 μM.
Further, a kind of probe of RAA Fluorometric assay Giardia lamblia, the probe sequence are as follows:
5’-TYGCCACGGAGAACGCMGARAGGAAGAAGATGTAYGACCAGC
TCAACG-3'；SEQ ID NO.8；
The probe is modified using fluorescent reporter group and fluorescent quenching group, and fluorescent reporter group is modified in probe
On position of the sequence from 5 ' end base number 31bp；Fluorescent quenching group modification is in position of the probe sequence from 5 ' end base number 33bp
On, 1 bases G is spaced between fluorescent reporter group and quenching group, wherein the G on the position from 5 ' end base number 32bp is with four
The replacement of hydrogen furans residue.
Probe after modification are as follows:
TYGCCACGGAGAACGCMGARAGGAAGAAGA(FAM-dT)(THF)(BHQ1-dT)AYGACCAGCTCAACG。
Further, the fluorescent reporter group is FAM, HEX, TET, JOE or VIC；The fluorescent quenching group be BHQ1,
BHQ2 or BHQ3.
Preferably, the fluorescent reporter group is FAM；The fluorescent quenching group is BHQ1.
Further, the concentration of the probe is 5~40 μM.
Preferably, the concentration of the probe is 10 μM.
Further, a kind of method of RAA Fluorometric assay Giardia lamblia, the specific steps are as follows:
(1) sample to be examined DNA is extracted, DNA extracting solution is obtained；
(2) constant temperature fluorogene detector is powered on and is preheated, response parameter is configured；
(3) the 2 μ L primers and 0.5 μ L probe that concentration is 10 μM are added in 42.5 μ L reaction buffers, after being sufficiently mixed
It is added in the reaction reagent of RAA fluorescence basis and mixes, obtains reaction premixed liquid；
(4) DNA extracting solution obtained in step (1) described in 5 μ L and reaction premixed liquid obtained in the step (3) are filled
Divide mixing, obtained reaction system is put into constant temperature fluorogene detector test fluorescence signal；
(5) positive is determined as when by slope value K >=20 according to the positive determination method in detecting instrument；Slope value K <
It is determined as feminine gender when 20.
Further, the response parameter is set as 39 DEG C, the reaction time: 15min.
It can be seen via above technical scheme that compared with prior art, the present disclosure provides a kind of inspections of RAA fluorescence method
Survey primer, probe and the detection method of Giardia lamblia, it is high specificity, high sensitivity, reproducible；It is suitble to scene high
Flux contamination-freely quickly detects Giardia lamblia.Method energy fast and easy of the invention accurately identifies Lan Shi merchant the
Flagellate, easy to operate, detection time is short, and detection is completed in 15min；Make DNA without passing through 95 DEG C of denaturation of high temperature as PCR
It untwists, then anneals at 50~60 DEG C, finally extend in 72 DEG C of completions, only need to carry out isothermal duplication at 39 DEG C can be completed inspection
It surveys；Without being expanded using 4-6 primer at 65 DEG C as LAMP technology and be easy to produce false positive.This hair
Bright detection method is quick, high throughput easy to accomplish, and testing cost substantially reduces, and high sensitivity, every reaction detection sensitivity
Reach 100Copies/ reaction.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 attached drawing is various concentration Giardia lamblia Plasmid DNA sensitivity technique result of the present invention；
Fig. 2 attached drawing is the specific detection result of Giardia lamblia of the present invention；
Fig. 3 attached drawing is Giardia lamblia cyst sample DNA testing result of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1 selects Giardia lamblia β-giardin gene conserved sequence to carry out primer, probe design
According to Gene Name Giardia lamblia β-giardin gene, corresponding full genome is found in genebank
Sequence (www.ncbi.nlm.nih.gov) carries out homology analysis with DNASTAR software and b1ast sequence is analyzed, sifts out
Giardia lamblia β-giardin gene highly conserved sequence is as follows:
GCAAACTTCCGCAAGTCCCTTGCGGAGATGGGCGACACACTCAACAACGTTGAGACAAATCTCCAGAA
CCAGATCGCCATCCATAACGACGCCATCGCGGCTCTCAGGAAGGAGGCCCTCAAGAGCCTGAACGATCTCGAGACG
GGCATTGCCACGGAGAACGCAGAAAGGAAGAAGATGTACGACCAGCTCAACGAGAAGGTCGCAGAGGGCTTCGCCC
GCATCTCCGCCGCGATCGAGAAGGAGACGATCGCCCGCGAGAGGGCCGTTAGCGCTGCCACGACAGAAGCGCTCAC
AAACACGAAGCTCGTCGAG；SEQ ID NO.1；
Using highly conserved sequence as testing goal gene, synthesizes positive plasmid and carry out primer, probe design；
According to above Giardia lamblia β-giardin gene conserved sequence student on commission's work bioengineering (Shanghai) share
Co., Ltd carries out synthetic DNA plasmid, plasmid size 315bp.
(1) design of primers
It is designed using RAA technology design of primers principle, upstream primer and downstream primer length 30-35bp, according to indigo plant
Family name's giardia lamblia stiles β-giardin gene conserved sequence, design of primers include upstream primer and downstream primer, and upstream primer is under
Trip primer respectively designs 3, and primer sequence is as follows:
RAA-F1:5 '-TCCAGAACCAGATCGCCATCCAYAACGACGCCAT-3 '；SEQ ID NO.2；
RAA-F2:5 '-TCAGGAAGGAGGCCCTCAAGAGCCTGAACG-3 '；SEQ ID NO.3；
RAA-F3:5 '-CTGAACGAYCTCGAGACRGGCATYGCCACGGA-3 '；SEQ ID NO.4；
RAA-R1:5 '-AGCTTCGTGTTTGTGAGCGCTTCTGTCGTG-3 '；SEQ ID NO.5；
RAA-R2:5 '-CTGTCGTGGCRGCGCTRACGGCCCTCTCGC-3 '；SEQ ID NO.6；
RAA-R3:5 '-TSGCRGCGGAGATGCGGGCGAAGCCCTCTG-3 '；SEQ ID NO.7；
Above-mentioned primer is combined into 3 × 3 totally 9 primer combinations, primer combination is shown in Table 1；
1 Giardia lamblia specific primer of table
Note: where " Y " representative " CT ", " R " representative " AG ", " S " representative " GC ".
By screening and evaluating, determines that two pairs of primer sets are best primer sets, be shown in Table 2.
The combination of the best primer of table 2
| RAA-F2 | TCAGGAAGGAGGCCCTCAAGAGCCTGAACG |
| RAA-R1 | AGCTTCGTGTTTGTGAGCGCTTCTGTCGTG |
| RAA-F1 | TCCAGAACCAGATCGCCATCCAYAACGACGCCAT |
| RAA-R2 | CTGTCGTGGCRGCGCTRACGGCCCTCTCGC |
Through further repeatability, stability, sensitivity, the assessment of specificity, more preferably RAA-F2/RAA-R1, tool
Body are as follows:
RAA-F2:5 '-TCAGGAAGGAGGCCCTCAAGAGCCTGAACG-3 '；SEQ ID NO.3；
RAA-R1:5 '-AGCTTCGTGTTTGTGAGCGCTTCTGTCGTG-3 '；SEQ ID NO.5.
(2) probe designs
1) it is designed using RAA technology probe design principle, it is conservative according to Giardia lamblia β-giardin gene
Sequence, the probe sequence of design are as follows:
5’-TYGCCACGGAGAACGCMGARAGGAAGAAGATGTAYGACCAGC
TCAACG-3'；SEQ ID NO.8.
2) fluorescent decoration group and fluorescent quenching group are selected
The RAA-F1620 fluorescent base produced according to laboratory apparatus using Wuxi surprise day biology scientific instrument Co., Ltd
Because of detector, fluorescence detected is FAM fluorescence, therefore fluorescent decoration group is selected as FAM, and fluorescent quenching group is selected as BHQ1；
The performance that fluorescence can also be detected according to instrument, is selected as HEX, TET, JOE or VIC for fluorescent decoration group；It is glimmering
Optical quenching group selects BHQ2 or BHQ3；
But it is preferred that fluorescent decoration group is FAM, preferably fluorescent quenching group is BHQ1.
3) probe is modified using fluorescent reporter group and fluorescent quenching group, and fluorescent reporter group modification is being visited
On position of the needle sequence from 5 ' end base number 31bp；Fluorescent quenching group modification is in position of the probe sequence from 5 ' end base number 33bp
It sets, 1 bases G is spaced between fluorescent reporter group and quenching group, wherein the G on the position from 5 ' end base number 32bp is used
The replacement of tetrahydrofuran residue.
Probe after modification are as follows:
TYGCCACGGAGAACGCMGARAGGAAGAAGA(FAM-dT)(THF)(BHQ1-dT)AYGACCAGCTCAACG。
(3) primer, probe and plasmid entrust Sangon Biotech (Shanghai) Co., Ltd.) limited liability company synthesized.
(4) detection reagent of Giardia lamblia, including RAA fluorescence basic reaction are quickly detected based on RAA fluorescence method
Reagent, reaction buffer, positive quality control product, negative quality-control product, primer and probe；
RAA fluorescence basis reaction reagent is the freeze-dried powder by frozen drying, is purchased from Jiangsu Qi Tian gene biological section
Skill Co., Ltd, article No. F00001, reaction specification are 50 μ L, carry out molten, the reaction buffer of weight with reaction buffer using preceding
For RAA fluorescence basis reaction reagent matched reagent.
Positive quality control product is the plasmid containing Giardia lamblia β-giardin gene, and concentration is 1 × 104Copies/
μL；Plasmid is cultivated and is extracted by Escherichia coli of transferring, and obtains carrying out concentration survey with ultramicron ultraviolet specrophotometer after plasmid
It is fixed, and copy number calculating is carried out, 1.0 × 10 are prepared by concentration gradient dilution1Copies/μL-1.0×1010Copies/ μ L mark
Quasi- product are spare.
Negative quality-control product is ddH2O or purified water.
The concentration of upstream primer and downstream primer is 10 μM；The concentration of probe is 10 μM.
Embodiment 2
A kind of method of RAA Fluorometric assay Giardia lamblia, includes the following steps:
(1) sample to be examined is Giardia lamblia cyst or trophozoite, using cracking, enrichment with magnetic bead, washing, elution etc.
Step extracts nucleic acid；- 20 DEG C save backup；
(2) constant temperature fluorogene detector RAA-F1620 is powered on and is preheated, response parameter is configured,
Response parameter is set as 39 DEG C, the reaction time: 15min.
(3) the 2 μ L primers and 0.5 μ L probe that concentration is 10 μM are added in 42.5 μ L reaction buffers, after being sufficiently mixed
It is added in the reaction reagent of RAA fluorescence basis and mixes, obtains reaction premixed liquid；
(4) by reaction premixed liquid obtained in nucleic acid extraction liquid obtained in step (1) described in 5 μ L and the step (3)
It is sufficiently mixed, obtained reaction system is put into constant temperature fluorogene detector RAA-F1620 detection fluorescence signal；
(5) positive is determined as when by slope value K >=20 according to the positive determination method in RAA-F1620 detecting instrument,
It is determined as feminine gender when slope value K < 20；It can also determine have the judgement obviously expanded for the positive, no amplification by amplification curve
Judgement be feminine gender.
3 sensitivity experiment of embodiment
(1) primer, probe and negative quality-control product are same as Example 1.
(2) working standard is prepared:
By 5 μ L plasmids switching Escherichia coli, cultivates and the concentration extracted is 1010Copies/ μ L, plasmid is made not
With the working standard of gradient, it is respectively as follows:
Working standard 1 contains 1.0 × 106The target gene DNA fragmentation of Copies/ μ L Giardia lamblia.
Working standard 2 contains 1.0 × 105The target gene DNA fragmentation of Copies/ μ L Giardia lamblia.
Working standard 3 contains 1.0 × 104The target gene DNA fragmentation of Copies/ μ L Giardia lamblia.
Working standard 4 contains 1.0 × 103The target gene DNA fragmentation of Copies/ μ L Giardia lamblia.
Working standard 5 contains 1.0 × 102The target gene DNA fragmentation of Copies/ μ L Giardia lamblia.
(3) detection sensitivity specific implementation step:
Step 1 prepares reaction solution:
The RAA basic agent box purchased from Jiangsu Qi Tian gene Biotechnology Co., Ltd, article No. are as follows: anti-in F00001
It answers and draws the preprepared 1.5mL EP pipe of 255 μ L addition in buffer, be separately added into 12 μ L probes, 3 μ L primers be (probe
Concentration is 10 μM, and the concentration of primer is 10 μM), it mixes well, the reaction solution after must mixing.
Step 2, RAA fluorescence basis reaction reagent weight are molten
Prepare 6 RAA fluorescence basis reaction reagents, draws the reaction buffer mixed in 45 μ L steps 1 every time and add respectively
Enter into ready 6 RAA fluorescence basis reaction reagent pipe, so that freeze-dried powder is sufficiently dissolved and is mixed, become RAA reactant
System, and mark.
Step 3, sample-adding reaction
5 μ L feminine gender quality-control products of addition in the reaction reagent test tube of above 6 prepared RAA fluorescence basis, its
5 μ L working standards 5, working standard 4, working standard 3, working standard 2, work are separately added into his 5 reaction tubes
Standard items 1；Often plus what a sample just covers pipe lid, has added each reaction tube after sample to be mixed well, each reaction tube
Total volume is 50 μ L.
Step 4, detection and result
6 reaction tubes of mixing are put into constant temperature fluorogene detector RAA-F1620, set reaction temperature as 39
DEG C, the reaction time 15 minutes.
It is determined as the positive, slope when by slope value K >=20 according to the positive determination method in RAA-F1620 detecting instrument
It is determined as feminine gender when value K < 20；It can also determine have the judgement obviously expanded for the positive by amplification curve, no amplification is sentenced
It is set to feminine gender.
Testing result can detect work as shown in Figure 1, all standards work product have amplification in 10 minutes as the result is shown
Standard items 5 contain 1.0 × 102The fluorescence signal of Copies/ μ L, sensitivity reach 102Copies/ reaction.
4 specificity experiments of embodiment
(1) the primer, probe are same as Example 1.
(2) samples sources and DNA are extracted
All samples are provided by Jiangsu Prov. Bilharziasis Prevention and Control Inst., and Giardia lamblia sample type is packing,
Other samples for being used for specificity experiments are respectively as follows: campylobacter jejuni, salmonella, Shigella, enteropathogenic E. Coli
O157:H7, the above bacterial strain are all made of the nucleic acid that the reference culture of buying extracts, and Cryptosporidium sample is egg capsule；
DNA extract using commercialization tissue samples DNA extraction kit, extraction operation step by kit specification into
Row, DNA-80 DEG C extracted save backup.
(3) specificity experiments implementation method:
Step 1 prepares reaction solution:
The RAA basic agent box purchased from Jiangsu Qi Tian gene Biotechnology Co., Ltd, article No. are as follows: anti-in F00001
It answers and draws the preprepared 1.5ml EP pipe of 297.5 μ L addition in buffer, be separately added into 14 μ L probes, 3.5 μ L primers (are visited
The concentration of needle is 10 μM, and the concentration of primer is 10 μM), it mixes well, the reaction solution after must mixing.
Step 2, RAA fluorescence basis reaction reagent weight are molten
Prepare 7 RAA fluorescence basis reaction reagents, draw the reaction buffer that mixes in 45 μ L steps 1 every time, respectively plus
Enter into ready 7 RAA fluorescence basis reaction reagent pipe, so that freeze-dried powder is sufficiently dissolved and is mixed, become RAA reactant
System, and mark.
Step 3, sample-adding reaction
5 μ L feminine gender quality-control products of addition in the reaction reagent test tube of above 7 prepared RAA fluorescence basis, its
Campylobacter jejuni, salmonella, Shigella, enteropathogenic E. Coli that 5 μ L have been extracted are separately added into his 5 reaction tubes
The Lan Shi of extracting is added in the last one reaction tube for the nucleic acid that the nucleic acid and Cryptosporidium parvum oocysts suspended of O157:H7 reference culture extract
Giardia lamblia stiles nucleic acid, often plus what a sample just covers pipe lid, has added each reaction tube after sample to be mixed well, each
Reaction tube total volume is 50 μ L.
Step 4, detection and result
7 reaction tubes of mixing are put into constant temperature fluorogene detector RAA-F1620, set reaction temperature as 39
DEG C, the reaction time 15 minutes.
Testing result as shown in Fig. 2, obviously have amplification in 10 minutes in addition to Giardia lamblia nucleic acid as the result is shown,
Belong to the positive, other are as equal such as campylobacter jejuni, salmonella, Shigella, enteropathogenic E. Coli O157:H7, Cryptosporidium
It does not expand, for feminine gender, illustrates that the primer of the present invention, probe specificity are good.
The detection of 5 actual sample of embodiment
(1) the primer, probe are same as Example 1.
(2) samples sources and DNA are extracted
Sample is provided by Jiangsu Prov. Bilharziasis Prevention and Control Inst., and Giardia lamblia sample type is packing, and DNA is mentioned
It takes using the tissue samples DNA extraction kit being commercialized, extraction operation step is carried out by kit specification, extracted
DNA-80 DEG C saves backup.
(3) implementation method
Step 1 prepares reaction solution:
The RAA basic agent box purchased from Jiangsu Qi Tian gene Biotechnology Co., Ltd, article No. are as follows: anti-in F00001
It answers and draws the preprepared 1.5mL EP pipe of 127.5 μ L addition in buffer, be separately added into 6 μ L probes, 1.5 μ L primers (are visited
The concentration of needle is 10 μM, and the concentration of primer is 10 μM), it mixes well, the reaction solution after must mixing.
Step 2, RAA fluorescence basis reaction reagent weight are molten
Prepare 3 RAA fluorescence basis reaction reagents, draw the reaction buffer that mixes in 45 μ L steps 1 every time, respectively plus
Enter into ready 3 RAA fluorescence basis reaction reagent pipe, so that freeze-dried powder is sufficiently dissolved and is mixed, become RAA reactant
System, and mark.
Step 3, sample-adding reaction
5 μ L feminine gender quality-control products of addition in the reaction reagent test tube of above 3 prepared RAA fluorescence basis, its
5 μ L samples 1 are separately added into his 2 reaction tubes, the nucleic acid that sample 2 extracts；Often plus what a sample just covers pipe lid, adds
Each reaction tube is mixed well after sample, and each reaction tube total volume is 50 μ L.
4, detection and result
3 reaction tubes of mixing are put into constant temperature fluorogene detector RAA-F1620, set reaction temperature as 39
DEG C, the reaction time 15 minutes.
Testing result obviously has amplification in 10 minutes, can be used for indigo plant as shown in figure 3, sample 1, sample 2 as the result is shown
The detection of family name's giardia lamblia stiles sample packing.
Primer provided by the invention, probe compositions can detect Giardia lamblia fastly, easy to operate, in 15min
It is interior to obtain testing result.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Sequence table
<110>Jiangsu Prov. Bilharziasis Prevention and Control Inst. Jiangsu Qi Tian gene Biotechnology Co., Ltd
<120>a kind of primer, probe and the detection method of RAA Fluorometric assay Giardia lamblia
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 315
<212> DNA
<213> Artificial Sequence
<400> 1
gcaaacttcc gcaagtccct tgcggagatg ggcgacacac tcaacaacgt tgagacaaat 60
ctccagaacc agatcgccat ccataacgac gccatcgcgg ctctcaggaa ggaggccctc 120
aagagcctga acgatctcga gacgggcatt gccacggaga acgcagaaag gaagaagatg 180
tacgaccagc tcaacgagaa ggtcgcagag ggcttcgccc gcatctccgc cgcgatcgag 240
aaggagacga tcgcccgcga gagggccgtt agcgctgcca cgacagaagc gctcacaaac 300
acgaagctcg tcgag 315
<210> 2
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 2
tccagaacca gatcgccatc cayaacgacg ccat 34
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 3
tcaggaagga ggccctcaag agcctgaacg 30
<210> 4
<211> 32
<212> DNA
<213> Artificial Sequence
<400> 4
ctgaacgayc tcgagacrgg catygccacg ga 32
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 5
agcttcgtgt ttgtgagcgc ttctgtcgtg 30
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 6
ctgtcgtggc rgcgctracg gccctctcgc 30
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 7
tsgcrgcgga gatgcgggcg aagccctctg 30
<210> 8
<211> 48
<212> DNA
<213> Artificial Sequence
<400> 8
tygccacgga gaacgcmgar aggaagaaga tgtaygacca gctcaacg 48
Claims (10)
1. a kind of primer of RAA Fluorometric assay Giardia lamblia, which is characterized in that the primer sequence is as follows:
Upstream primer: 5 '-TCAGGAAGGAGGCCCTCAAGAGCCTGAACG-3 '；SEQ ID NO.3；
Downstream primer: 5 '-AGCTTCGTGTTTGTGAGCGCTTCTGTCGTG-3 '；SEQ ID NO.5.
2. a kind of primer of RAA Fluorometric assay Giardia lamblia according to claim 1, which is characterized in that institute
The use concentration for stating upstream primer and downstream primer is 5~20 μM.
3. a kind of primer of RAA Fluorometric assay Giardia lamblia according to claim 2, which is characterized in that institute
The use concentration for stating upstream primer and downstream primer is 10 μM.
4. a kind of probe of RAA Fluorometric assay Giardia lamblia, which is characterized in that the probe sequence is as follows:
5'-TYGCCACGGAGAACGCMGARAGGAAGAAGATGTAYGACCAGCTCAACG-3'；SEQ ID NO.8；
The probe is modified using fluorescent reporter group and fluorescent quenching group, and fluorescent reporter group is modified in probe sequence
On position from 5 ' end base number 31bp；Fluorescent quenching group is modified on position of the probe sequence from 5 ' end base number 33bp,
1 bases G is spaced between fluorescent reporter group and quenching group, wherein the G tetrahydro on the position from 5 ' end base number 32bp
The replacement of furans residue.
5. a kind of probe of RAA Fluorometric assay Giardia lamblia according to claim 4, which is characterized in that institute
Stating fluorescent reporter group is FAM, HEX, TET, JOE or VIC；The fluorescent quenching group is BHQ1, BHQ2 or BHQ3.
6. a kind of probe of RAA Fluorometric assay Giardia lamblia according to claim 5, which is characterized in that institute
Stating fluorescent reporter group is FAM；The fluorescent quenching group is BHQ1.
7. according to a kind of described in any item probes of RAA Fluorometric assay Giardia lamblia of claim 4-6, feature
It is, the concentration of the probe is 5~40 μM.
8. a kind of probe of RAA Fluorometric assay Giardia lamblia according to claim 7, which is characterized in that institute
The concentration for stating probe is 10 μM.
9. a kind of method of RAA Fluorometric assay Giardia lamblia, which is characterized in that specific step is as follows:
(1) sample to be examined DNA is extracted, DNA extracting solution is obtained；
(2) constant temperature fluorogene detector is powered on and is preheated, response parameter is configured；
(3) the 2 μ L primers and 0.5 μ L probe that concentration is 10 μM are added in 42.5 μ L reaction buffers, are added after being sufficiently mixed
It is mixed into RAA fluorescence basis reaction reagent, obtains reaction premixed liquid；
(4) reaction premixed liquid obtained in DNA extracting solution obtained in step (1) described in 5 μ L and the step (3) is sufficiently mixed
It closes, obtained reaction system is put into constant temperature fluorogene detector test fluorescence signal；
(5) positive is determined as when by slope value K >=20 according to the positive determination method in detecting instrument；When slope value K < 20
It is determined as feminine gender.
10. a kind of method of RAA Fluorometric assay Giardia lamblia according to claim 9, which is characterized in that institute
It states response parameter and is set as 39 DEG C, the reaction time: 15min.
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Cited By (2)
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| CN110643693A (en) * | 2019-10-10 | 2020-01-03 | 首都儿科研究所 | Primer, probe, kit and detection method for detecting Mcr by RAA fluorescence method |
| CN111321233A (en) * | 2020-04-10 | 2020-06-23 | 四川国际旅行卫生保健中心(成都海关口岸门诊部) | Fluorescent RAA primer, probe and detection method for detecting schistosoma japonicum |
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| CN110643693A (en) * | 2019-10-10 | 2020-01-03 | 首都儿科研究所 | Primer, probe, kit and detection method for detecting Mcr by RAA fluorescence method |
| CN111321233A (en) * | 2020-04-10 | 2020-06-23 | 四川国际旅行卫生保健中心(成都海关口岸门诊部) | Fluorescent RAA primer, probe and detection method for detecting schistosoma japonicum |
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Application publication date: 20190510 |