CN109880924A - It is a kind of for quickly detecting primer sets, kit and the detection method of dirofilariaimmitis and burnt worm - Google Patents
It is a kind of for quickly detecting primer sets, kit and the detection method of dirofilariaimmitis and burnt worm Download PDFInfo
- Publication number
- CN109880924A CN109880924A CN201910155028.5A CN201910155028A CN109880924A CN 109880924 A CN109880924 A CN 109880924A CN 201910155028 A CN201910155028 A CN 201910155028A CN 109880924 A CN109880924 A CN 109880924A
- Authority
- CN
- China
- Prior art keywords
- dirofilariaimmitis
- primer
- worm
- kit
- burnt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of for quickly detecting primer sets, kit and the detection method of dirofilariaimmitis (Dirofilaria immitis) and burnt worm (Babesia spp.), belongs to molecular biology field；The present invention combines recombinase-polymerase nucleic acid amplification technologies with detection technique of fluorescence, specificity detects the hereditary material DNA of dirofilariaimmitis and burnt worm, cross reaction will not be generated with other dog helminths, the present invention provide one it is simple, quickly, be not necessary to expensive device can scene carry out the screening mode of dirofilariaimmitis and burnt insect infection, has the characteristics that highly sensitive, specificity, suitable for instant quick diagnosis, primary first-order equation can identify dirofilariaimmitis and burnt worm respectively, and the quick detection of First Line can be provided for pet.
Description
Technical field
It is the present invention relates to molecular biology field, in particular to a kind of for quickly detecting the primer of dirofilariaimmitis and burnt worm
Group, kit and detection method.
Background technique
The cause of disease of dirofilariaimmitis disease is heartworm (Dirofilaria immitis), during the vector of dirofilariaimmitis is
A variety of mosquitos such as magnificent anopheles, aedes albopictus, Culex pipiens pallens, canine by the mosquito bite containing infective larvae due to being infected.The heart
The adult of filaria parasitizes at the right H&L artery of dog cat and the mankind, and adult can give birth to larva, (female adult directly generates larva,
As microfilaria) recycled in blood, mosquito bite infect heartworm disease dog cat after i.e. carry heartworm larva, larva in
Mosquito salivary gland, which is changed in quality, becomes infectious young filaria, then infects via other dog cats of mosquito bite and the mankind.Dog cat can
This disease of superinfection.Since adult parasitizes in heart, heart is caused to destroy, gradually influences dog cat and human health, fall ill it
First dog cat is without significant discomfort, but after infecting a period of time, gradually appear movement intolerant to, cough, be short of breath, the past is often by mistaken diagnosis
For tracheitis or general heart disease.There is cardiopulmonary failure, anaemia, wet lung, ascites, jaundice, liver renal failure death in stage.Cause
This, the early screening of heartworm disease is particularly important.
Burnt worm (Babesia spp.) can infect via tick (Ticks) or blood transfusion, under immunosuppressive situation, meeting
Increase the risk of infection.Babesia canis infection mainly leads to the destruction and anaemia of red blood cell, including being commonly called as big burnt worm
Babesia canis, and it is commonly called as the Babesia gibsoni of small burnt worm.The length of big coke worm is at 4~7 μm, in Western pyriform
Shape generally all only shows slight or unobvious at dog unless just will cause serious clinical symptoms under immunosuppressive situation
Clinical symptoms, but for puppy then it is serious mostly, it is relatively conventional in Southern Taiwan, china at present.Small coke worm (2.5 μm) is outside
Seeing is in roundlet round, and major part infection can all cause serious clinical symptoms at dog: lethargic sleep torpescence, anorexia, mucous membrane be pale,
Fever vomits, is amber to brown urine, spleen enlargement, jaundice, weight loss, accelerated breathing, palpitating speed.Therefore, Babesia canis
Early screening the protective action of First Line is played to the healthy development of pet.
Currently, examine dirofilariaimmitis with coke the common method of insect infection be to be observed under the microscope after blood smear, but
Its sensitivity is bad, and omission factor is high.Heartworm Adult Antigens detection kit is also commonly used to the heart silk in screening blood
Worm antigen, but its sensitivity and specificity are bad, and false negative rate is higher.The molecule of dirofilariaimmitis and burnt worm examine mainly with
Real time-PCR method detects the helminth nucleic acid in serum, although this method specificity and high sensitivity, detection time
Need at least 60-90min, and need complex instrument, be not particularly suited for point-of-care detection (point-of-care test,
POCT it) uses.Therefore, need to research and develop one kind can quick and precisely, simply, without expensive instrument, can be used for field quick detection
The kit and detection method of dirofilariaimmitis and burnt worm.
Summary of the invention
Being designed to provide for invention is a kind of for quickly detecting primer sets, kit and the detection of dirofilariaimmitis and burnt worm
Method solves the problems in background technique.
The present invention uses recombinase-polymerase nucleic acid amplification technologies and detection technique of fluorescence, can provide first for pet
The quick detection of line, and dirofilariaimmitis, big burnt worm and small burnt worm can be segmented, it is made a definite diagnosis with providing medical treatment, and specificity is high, no
Cross reaction can be generated with other dog helminths, have the characteristics that highly sensitive and specificity.
According to one aspect of the invention, it provides a kind of for quickly detecting the primer sets of dirofilariaimmitis and burnt worm, described
Primer sets under the conditions of suitable primer concentration, can be independent of each other in primary amplification and meanwhile detect dirofilariaimmitis, big coke
Worm and small burnt worm.
The invention is realized in this way a kind of for quickly detecting the primer sets of dirofilariaimmitis and burnt worm, which is
A set of dirofilariaimmitis and burnt worm characteristic primer sets, a set of dirofilariaimmitis and burnt worm characteristic primer sets are by three pairs of primer sets
At three pairs of primers are respectively primer pair one, primer pair two and primer pair three, and each pair of primer includes a forward primer and one
A reverse primer,
The primer pair one is to detect the primer pair of heartworm, and the primer pair two is the primer pair of the big burnt worm of detection, institute
The primer pair that primer pair three is the small burnt worm of detection is stated,
The sequence of three pairs of primers is respectively as follows:
Primer pair one (heartworm):
Forward primer: AAC TTT TTA TCC TCC TTT GAG TGT AGA GG；
Reverse primer: TTC AAA CAA ACA TAC TAA TCT GAT CTA AAG；
Primer pair two (big coke worm):
Forward primer: CTT GGC TTT CGG TGA TTC ATA ATA AAC；
Reverse primer: CCT TCC TTA GAT GTG GTA GCC GTC TCT C；
Primer pair two (small coke worm):
Forward primer: CTA CTT GCC TTG TCT GGT TTC GCT TTT G；
Reverse primer: ATG CCC CCA ACC GTT CCT ATT AAC CAT TAC.
The primer is the pathogenic genes sequence and TwistDx that inventor refers to NCBI gene database
Instruction manual and Primer-BLAST design of primers principle, a plurality of primer of design and synthesis.In design process
In, consider not only the formation that avoid primer dimer, hairpin structure, it is also contemplated that different target gene is anti-at one
The problem of answering coamplification in system.Primer screening experiment shows that the primer specificity is strong, amplification efficiency is high, can effectively, quickly
Detect dirofilariaimmitis, big burnt worm and small burnt worm in ground.
According to a further aspect of the present invention, provide it is a kind of containing above-mentioned primer sets for quickly detect dirofilariaimmitis with
The kit of burnt worm.
Preferably, the kit is for dirofilariaimmitis, the detection of big burnt worm and small burnt worm.
Further, the kit further includes recombinase, polymerase, DNA binding protein, buffer solution and fluorescence dye
Material.
Preferably, the recombinase is T4uvsX/uvsY, and the polymerase is Bsu archaeal dna polymerase, and the DNA is combined
Albumen is T4gp32；
The buffer solution includes Tris-HCl buffer, potassium acetate, magnesium acetate, dithiothreitol (DTT), dNTPs, ATP, phosphorus
Creatine acid, creatine kinase and Carbowax20M；
The fluorescent dye is SYBR-Green.
Preferably, the Tris-HCl buffer concentration is 50mM, the Tris-HCl pH of buffer 7.9, the acetic acid
Potassium concn is 100mM, and the acetic acid magnesium density is 14mM, and the dithiothreitol (DTT) concentration is 2mM, and the dNTPs concentration is
200uM, the ATP concentration are 3mM, and the phosphocreatine concentration is 50mM, and the creatine kinase concentration is 100ng/ μ L, described
Carbowax20M concentration is 5%.
Further, the kit further includes ddH2O。
Another aspect according to the present invention provides a kind of detection using mentioned reagent box detection dirofilariaimmitis and burnt worm
Method, this method are recombination enzymatic polymerization enzyme amplification method, comprising the following steps:
(1) DNA of sample to be tested is extracted as detection template；
(2) constant-temperature amplification is carried out to the DNA and obtains DNA amplification sequence；
(3) fluorescence detection is carried out to the DNA amplification sequence and obtains solubility curve Tm value.
(4) testing result is judged according to solubility curve Tm value.
Preferably, sample to be tested described in the step (1) is dog serum sample.
The invention has the following beneficial effects:
1, the present invention combines recombinase-polymerase nucleic acid amplification technologies with detection technique of fluorescence, can carry out simultaneously
The detection of dirofilariaimmitis, big burnt worm and small burnt worm, and specificity is high, will not generate cross reaction with other dog helminths；
2, the present invention provide one it is simple, quickly, be not necessary to expensive device can scene carry out the sieve of dirofilariaimmitis and burnt worm
Procuratorial organ's formula has the characteristics that highly sensitive, specificity, is suitable for immediately quickly detection, it is possible to provide the quick detection of dog First Line,
And dirofilariaimmitis, big burnt worm and small burnt worm can be identified, to provide medical treatment detection.
Detailed description of the invention
It in order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology and advantage, below will be to implementation
Example or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, the accompanying drawings in the following description is only
It is only some embodiments of the present invention, for those of ordinary skill in the art, without creative efforts,
It can also be obtained according to these attached drawings other attached drawings.
Fig. 1 is to carry out recombinase polymeric enzymatic amplification combination fluorescence detection dirofilariaimmitis and coke using kit of the present invention
The result figure of worm.In figure: a is negative control, and b is heartworm, and c is big burnt worm, and d is small burnt worm.
Fig. 2 is the DNA that recombinase polymeric enzymatic amplification detection dirofilariaimmitis and burnt worm are carried out using kit of the present invention
Glue figure.In figure: 1 is negative control, and 2 be heartworm, and 3 be big burnt worm, and 4 be small burnt worm.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but the following example is merely to illustrate
The present invention, and should not be taken as limiting the scope of the invention.Reagent is commercial products in following embodiment, and the primer is in Shanghai
The synthesis of Sheng Gong Biotechnology Co., Ltd.
Embodiment 1: for detecting the preparation of the primer sets of dirofilariaimmitis and burnt worm
1, design of primers
According to the respiratory tract infection pathogenic genes sequence of NCBI gene database, with reference to TwistDx instruction
Manual and Primer-BLAST design of primers principle, design primer.Primer length is about 30-35nt, due to currently without needle
To the primer-design software of RPA, is designed during previous work of the present invention and synthesized a large amount of primers, be screened out from it a set of sensitive
The primer sets that degree is high and specificity is good are for the present invention, primer sets base sequence group as shown in SEQ ID NO:1-8 in table 1
At.
Table 1
2, primer synthesizes
According to base sequence shown in SEQ ID NO:1-8 in table 1, primer sequence synthesis is carried out.
Embodiment 2: for detecting the establishment of the kit of dirofilariaimmitis and burnt worm
The kit for detecting dirofilariaimmitis and burnt worm is set up, makes that it includes following components:
(1) upstream primer described in table 1 and downstream primer；
(2) recombinase, polymerase and DNA binding protein, wherein the recombinase is T4 uvsX/uvsY, the polymerization
Enzyme is Bsu archaeal dna polymerase, and the DNA binding protein is T4gp32；
(3) buffer solution and fluorescent dye, wherein the buffer solution contains following components: 50mM Tris-HCl buffering
Liquid, 100mM potassium acetate, 14mM magnesium acetate, 2mM dithiothreitol (DTT), 200uM dNTPs, 3mM ATP, 50mM phosphocreatine,
100ng/ μ L creatine kinase, 5%Carbowax20M, wherein the pH value of the Tris-HCl buffer is 7.9, the fluorescence dye
Material is SYBR-Green, for carrying out fluorescence detection to amplification of nucleic acid.
Embodiment 3: dirofilariaimmitis and the coke worm of clinical sample are detected
Using the detection dirofilariaimmitis of kit described in embodiment 2 and burnt worm, detection method is recombination enzymatic polymerization enzyme constant temperature base
Gene-amplification method, the specific steps are as follows:
1, clinical sample prepares
Collect dog serum sample, collection condition are as follows: check after (cultivation or Bio-molecular analysis method) really through laboratory
Think (55), positive clinical sample with the dirofilariaimmitis positive clinical samples (32) with burnt worm, and after inspection really
Think the negative clinical sample (30) of no dirofilariaimmitis and burnt insect infection.Collected residue sample will be first with 95 DEG C of heating 10
Minute deactivation, has infection doubt to avoid transport process.Clinical sample is placed in -80 DEG C of preservations.
2, the nucleic acid extraction of clinical sample
The present embodiment carries out clinical sample using blood DNA pillar extracts kit or automation sample nucleic acid extraction apparatus
Nucleic acid extraction.
3, reaction system configures
According to following every proportional arrangement reaction system:
| Sample nucleic acid | 2ug |
| Positive and reverse primer | Each 1 μ L |
| Buffer solution | 25μL |
| T4uvsX/uvsY, 120ng/ μ L | 1μL |
| Bsu archaeal dna polymerase, 30ng/ μ L | 1μL |
| T4gp32,900ng/ μ L | 1μL |
| ddH2O | Complement to 50 μ L |
4, after concussion mixes reaction system made from step 3, sample carries out RPA amplification in 39 DEG C of isothermal reaction 20min.
5, fluorescent dye SYBR-Green is added, solubility curve Tm value is obtained to the DNA sequence dna progress fluorescence detection of amplification.
6, testing result is judged according to solubility curve Tm value.
7, result interpretation
Dirofilariaimmitis, burnt worm and negative control fluorescence detection the result is shown in Figure 1 are carried out using primer sets designed by the present invention
With table 2.Dirofilariaimmitis, burnt worm and negative control DNA glue testing result, which are carried out, using primer sets designed by the present invention sees Fig. 2.
Table 2
Note: Tm is the temperature of DNA double chain unwinding 50%, and IC is interior Quality Control (Internal Control)
Table 3
As shown in Table 3, high specificity of the present invention, high sensitivity can effectively and quickly examine dirofilariaimmitis and burnt worm
It surveys.
The above embodiments are merely illustrative of the technical solutions of the present invention rather than limiting the scope of the invention, although ginseng
The present invention is explained in detail according to preferred embodiment, those skilled in the art should understand that, it can be to of the invention
Technical solution is modified or replaced equivalently, without departing from the spirit and scope of technical solution of the present invention.
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.
Claims (8)
1. a kind of for quickly detecting the primer sets of dirofilariaimmitis and burnt worm, which is characterized in that the primer sets are a set of dog heart silk
Worm and burnt worm characteristic primer sets, a set of dirofilariaimmitis are made of with burnt worm characteristic primer sets three pairs of primers, and described three
It is respectively primer pair one, primer pair two and primer pair three to primer, each pair of primer includes that a forward primer and one reversely draw
Object,
The primer pair one is to detect the primer pair of heartworm, and the primer pair two is the primer pair of the big burnt worm of detection, described to draw
Object is the primer pair for detecting small burnt worm to three,
The sequence of three pairs of primers is respectively as follows:
Primer pair one:
Forward primer: AAC TTT TTA TCC TCC TTT GAG TGT AGA GG；
Reverse primer: TTC AAA CAA ACA TAC TAA TCT GAT CTA AAG；
Primer pair two:
Forward primer: CTT GGC TTT CGG TGA TTC ATA ATA AAC；
Reverse primer: CCT TCC TTA GAT GTG GTA GCC GTC TCT C；
Primer pair three:
Forward primer: CTA CTT GCC TTG TCT GGT TTC GCT TTT G；
Reverse primer: ATG CCC CCA ACC GTT CCT ATT AAC CAT TAC.
2. a kind of for quickly detecting the kit of dirofilariaimmitis and burnt worm, which is characterized in that the kit includes claim 1
Described is a kind of for quickly detecting the primer sets of dirofilariaimmitis and burnt worm.
3. according to claim 2 a kind of for quickly detecting the kit of dirofilariaimmitis and burnt worm, which is characterized in that should
Kit further includes recombinase, polymerase, DNA binding protein, buffer solution and fluorescent dye.
4. according to claim 3 a kind of for quickly detecting the kit of dirofilariaimmitis and burnt worm, which is characterized in that institute
Stating recombinase is T4 uvsX/uvsY, and the polymerase is Bsu archaeal dna polymerase, and the DNA binding protein is T4 gp32；Institute
Stating buffer solution includes Tris-HCl buffer, potassium acetate, magnesium acetate, dithiothreitol (DTT), dNTPs, ATP, phosphocreatine, creatine
Kinases and Carbowax20M；The fluorescent dye is SYBR-Green.
5. according to claim 4 a kind of for quickly detecting the kit of dirofilariaimmitis and burnt worm, which is characterized in that institute
Stating Tris-HCl buffer concentration is 50mM, and the Tris-HCl pH of buffer 7.9, the acetic acid potassium concn is 100mM, described
Acetic acid magnesium density is 14mM, and the dithiothreitol (DTT) concentration is 2mM, and the dNTPs concentration is 200uM, and the ATP concentration is
3mM, the phosphocreatine concentration are 50mM, and the creatine kinase concentration is 100ng/ μ L, and the Carbowax20M concentration is
5%.
6. it is a kind of for quickly detecting the kit of dirofilariaimmitis and burnt worm according to claim 3-5 any one,
It is characterized in that, the kit further includes ddH2O。
7. a kind of detection method using the detection dirofilariaimmitis of kit described in claim 6 and burnt worm, which is characterized in that the party
Method is recombination enzymatic polymerization enzyme amplification method, comprising the following steps:
(1) DNA of sample to be tested is extracted as detection template；
(2) constant-temperature amplification is carried out to the DNA and obtains DNA amplification sequence；
(3) fluorescence detection is carried out to the DNA amplification sequence and obtains solubility curve Tm value.
(4) testing result is judged according to solubility curve Tm value.
8. a kind of detection method using the detection dirofilariaimmitis of kit described in claim 7 and burnt worm, which is characterized in that described
Sample to be tested described in step (1) is dog serum sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910155028.5A CN109880924A (en) | 2019-03-01 | 2019-03-01 | It is a kind of for quickly detecting primer sets, kit and the detection method of dirofilariaimmitis and burnt worm |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910155028.5A CN109880924A (en) | 2019-03-01 | 2019-03-01 | It is a kind of for quickly detecting primer sets, kit and the detection method of dirofilariaimmitis and burnt worm |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109880924A true CN109880924A (en) | 2019-06-14 |
Family
ID=66930220
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910155028.5A Withdrawn CN109880924A (en) | 2019-03-01 | 2019-03-01 | It is a kind of for quickly detecting primer sets, kit and the detection method of dirofilariaimmitis and burnt worm |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109880924A (en) |
-
2019
- 2019-03-01 CN CN201910155028.5A patent/CN109880924A/en not_active Withdrawn
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Junqueira et al. | Comparison of the polymerase chain reaction with two classical parasitological methods for the diagnosis of Chagas disease in an endemic region of north-eastern Brazil | |
| CN110551846A (en) | cpf1 kit for quickly detecting African swine fever virus nucleic acid and detection method thereof | |
| Khalil et al. | High-throughput isolation of giant viruses in liquid medium using automated flow cytometry and fluorescence staining | |
| Prevedel et al. | Identification, localization, and quantification of HIV reservoirs using microscopy | |
| CN107828914A (en) | Infectious subcutaneous and haematopoietic necrosis virus（IHHNV）RAA constant temperature fluorescence detection method and reagent | |
| Rizzo et al. | A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis | |
| CN115478120A (en) | Method for simultaneously detecting nodavirus and decapod iridovirus 1 of macrobrachium rosenbergii | |
| Halecker et al. | Virological and parasitological characterization of Mini-LEWE minipigs using improved screening methods and an overview of data on various minipig breeds | |
| Dimitrov et al. | Molecular characterization of haemosporidian parasites (Haemosporida) in yellow wagtail (Motacilla flava), with description of in vitro ookinetes of Haemoproteus motacillae | |
| CN102108414B (en) | Real-time fluorescence transcription-polymerase chain reaction (RT-PCR) detection method and kit for pike fry rhabdovirus (PFRV) | |
| CN108148890A (en) | The multiple PCR detection primer of duck newcastle disease, duck plague and duck tembusu virus disease | |
| CN109880922A (en) | A kind of primer, kit and detection method detecting mycobacterium tuberculosis TB | |
| CN107974514A (en) | A kind of reagent, detection method and application for pig A type Senecan viral diagnosis | |
| CN109880924A (en) | It is a kind of for quickly detecting primer sets, kit and the detection method of dirofilariaimmitis and burnt worm | |
| US20230250497A1 (en) | One-step nested pcr primers set and kit modified with locked nucleic acid for detecting african swine fever virus | |
| CN110257561A (en) | Reagent, detection method and application for epizootic hemorrhagic disease virus of deer (EHDV) detection | |
| CN104878112A (en) | Fluorescence detection primer of culex wolbachia, detection method thereof and detection kit | |
| Waliullah et al. | Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry | |
| Olsen et al. | Laboratory reporting accuracy of polymerase chain reaction testing for psittacine beak and feather disease virus | |
| Fernandez i Marti et al. | A novel, precise and high-throughput technology for viroid detection in cannabis (MFDetectTM) | |
| Ogbuigwe et al. | A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum | |
| CN110257560B (en) | Reagent for bluetongue virus type 8 detection, detection method and application | |
| Meyers et al. | A perspective: molecular detections of new agents in finfish—interpreting biological significance for fish health management | |
| CN106521038A (en) | High-sensitivity BHV-2 (bovine herpes virus 2) quantitative real-time PCR (polymerase chain reaction) detection method and kit | |
| Zhang et al. | Detection and characterization of naturally acquired West Nile virus infection in a female wild turkey |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20190614 |