CN112226517B - Primer group for detecting and distinguishing echinococcosis granulosa and echinococcosis multifilialis through loop-mediated isothermal amplification - Google Patents
Primer group for detecting and distinguishing echinococcosis granulosa and echinococcosis multifilialis through loop-mediated isothermal amplification Download PDFInfo
- Publication number
- CN112226517B CN112226517B CN202011002756.1A CN202011002756A CN112226517B CN 112226517 B CN112226517 B CN 112226517B CN 202011002756 A CN202011002756 A CN 202011002756A CN 112226517 B CN112226517 B CN 112226517B
- Authority
- CN
- China
- Prior art keywords
- echinococci
- reaction
- primer
- granulosa
- echinococcosis
- 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.)
- Active
Links
- 238000007397 LAMP assay Methods 0.000 title claims abstract description 19
- 206010014096 Echinococciasis Diseases 0.000 title claims description 40
- 208000009366 Echinococcosis Diseases 0.000 title claims description 40
- 238000006243 chemical reaction Methods 0.000 claims abstract description 56
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 20
- 230000003321 amplification Effects 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 201000010099 disease Diseases 0.000 claims description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 8
- 101000686153 Homo sapiens Ras-related GTP-binding protein A Proteins 0.000 claims description 7
- 102000009609 Pyrophosphatases Human genes 0.000 claims description 7
- 108010009413 Pyrophosphatases Proteins 0.000 claims description 7
- 102100025001 Ras-related GTP-binding protein A Human genes 0.000 claims description 7
- 238000003745 diagnosis Methods 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- 102100031822 Optineurin Human genes 0.000 claims description 6
- 101710131459 Optineurin Proteins 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000007795 chemical reaction product Substances 0.000 claims description 4
- 239000012154 double-distilled water Substances 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- AUJJPYKPIQVRDH-UHFFFAOYSA-N antimony potassium Chemical compound [K].[Sb] AUJJPYKPIQVRDH-UHFFFAOYSA-N 0.000 claims description 3
- 229960005070 ascorbic acid Drugs 0.000 claims description 3
- 235000010323 ascorbic acid Nutrition 0.000 claims description 3
- 239000011668 ascorbic acid Substances 0.000 claims description 3
- VLAPMBHFAWRUQP-UHFFFAOYSA-L molybdic acid Chemical compound O[Mo](O)(=O)=O VLAPMBHFAWRUQP-UHFFFAOYSA-L 0.000 claims description 3
- IIQJBVZYLIIMND-UHFFFAOYSA-J potassium;antimony(3+);2,3-dihydroxybutanedioate Chemical compound [K+].[Sb+3].[O-]C(=O)C(O)C(O)C([O-])=O.[O-]C(=O)C(O)C(O)C([O-])=O IIQJBVZYLIIMND-UHFFFAOYSA-J 0.000 claims description 3
- 229940095064 tartrate Drugs 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- 229960003237 betaine Drugs 0.000 claims description 2
- 235000013601 eggs Nutrition 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000000434 Melocanna baccifera Nutrition 0.000 claims 1
- 241001497770 Melocanna baccifera Species 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 108020004414 DNA Proteins 0.000 abstract description 33
- 201000003808 Cystic echinococcosis Diseases 0.000 abstract description 15
- 241000244170 Echinococcus granulosus Species 0.000 abstract description 15
- 241000244160 Echinococcus Species 0.000 abstract description 14
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 108091092878 Microsatellite Proteins 0.000 abstract description 2
- 108091081062 Repeated sequence (DNA) Proteins 0.000 abstract 1
- 238000003753 real-time PCR Methods 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 241000242677 Schistosoma japonicum Species 0.000 description 6
- 241000244155 Taenia Species 0.000 description 5
- 239000007850 fluorescent dye Substances 0.000 description 5
- 206010011732 Cyst Diseases 0.000 description 4
- 208000031513 cyst Diseases 0.000 description 4
- 238000001506 fluorescence spectroscopy Methods 0.000 description 4
- 238000011895 specific detection Methods 0.000 description 4
- 241000534000 Berula erecta Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 208000030852 Parasitic disease Diseases 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 241000754688 Cercaria Species 0.000 description 2
- 241000254173 Coleoptera Species 0.000 description 2
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 2
- 244000208060 Lawsonia inermis Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000002591 computed tomography Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011901 isothermal amplification Methods 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- -1 BIP-1 Proteins 0.000 description 1
- 241001222599 Clania variegata Species 0.000 description 1
- 241000244163 Echinococcus multilocularis Species 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 241000239368 Euphausia Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000864398 Hydrangea sect. Schizophragma Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000244174 Strongyloides Species 0.000 description 1
- 206010000269 abscess Diseases 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011841 epidemiological investigation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 208000037971 neglected tropical disease Diseases 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000009589 serological test Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer group for detecting and distinguishing echinococci granulosa and echinococci multiflori through loop-mediated isothermal amplification. The primer group comprises two pairs of outer primers and two pairs of inner primers, and the primers are designed according to the repeated sequence of echinococcus granulosus and the EmTorrple 83 microsatellite sequence of echinococcus multinomus. The primer group for detecting and distinguishing the echinococcus granulosus and the echinococcus multinomus through loop-mediated isothermal amplification can distinguish that the positive sample contains the genome DNA of the echinococcus granulosus or the echinococcus multinomus through two reactions, and the template DNA with the minimum detectable rate of 1.6fg has high detection sensitivity and strong specificity.
Description
Technical Field
The invention relates to the technical field of molecular detection of parasites, in particular to a primer group for detecting and distinguishing echinococcosis granulosa and echinococcosis in a loop-mediated isothermal amplification manner.
Background
Echinococcosis is a parasitic disease of both human and veterinary species seriously affecting the health of people and the development of animal husbandry, and is caused by infection of larvae of echinococcosis, belonging to a neglected tropical disease. The world health organization indicates that the worldwide population is about 1.93 ten thousand deaths each year due to echinococcosis, and the disease burden is about 87.1 ten thousand disabilities regulate life years (disability adjusted life year, DALY). In addition, echinococcosis severely affects the development of animal husbandry, with a global economic loss of up to $20 billion per year due to the disease. The population threatened by echinococcosis in China is about five millions, the serological positive rate is about 12.04%, and the Xinjiang and inner Mongolia are as high as more than 20%. The disease rate of the echinococcosis is 1.08% when the B ultrasonic examination is carried out, and the patients are about 38 ten thousands of people. The average infection rate of livestock is about 50%, and economic losses of over 8 hundred million yuan are caused to agricultural and livestock products in China each year. Echinococcosis has become one of the important reasons for affecting the health of farmers and pasture in western areas of China and restricting the economic development of pasture areas. Humans are infected with this disease by ingestion of echinococcosis eggs contained in contaminated food, water sources or soil or direct contact with animal hosts. Echinococcosis is mainly characterized by four forms of cyst, bubble, single cyst and multiple cyst, wherein the cyst and bubble are two most prominent forms of human infection, and the echinococcosis pathogen is echinococcosis granulosa (Echinococcus granulosus) and the bubble echinococcosis pathogen is echinococcosis multiforme (Echinococcus multilocularis). Diagnosis of echinococcosis is based mainly on imaging examinations such as ultrasound, and Computer Tomography (CT) and/or Magnetic Resonance (MRI) scanning aid diagnosis or diagnosis. Serological tests are of great importance for supporting diagnosis of echinococcosis and epidemiological investigation. Biopsy and ultrasound guided puncture can be used for differential diagnosis of a bag worm and abscess or tumor. The above examination method has the defects of low sensitivity and omission, especially in asymptomatic incubation period of echinococcosis.
In order to effectively control echinococcosis, highly sensitive, specific and simple detection techniques need to be established. The Polymerase Chain Reaction (PCR) has extremely high sensitivity and specificity and is applied to diagnosis of various parasitic diseases, but the PCR needs to be matched with a precise instrument, and cannot meet the requirements of basic level and field detection; in addition, the existing method for detecting the echinococci multiflori and the echinococci granulosa by using the MGB probe through real-time quantitative PCR typing requires professional experiment technical operators and special detection instruments (fluorescent quantitative PCR instruments) and cannot meet the detection requirements of the basic layer and the site.
Notomi Tsukuno et al developed a novel isothermal nucleic acid amplification method in 2000, namely Loop-mediated isothermal amplification (LAMP-Mediated Isothermal Amplification), which has the advantages of high sensitivity and specificity, simple operation, low requirements on personnel and instruments, and capability of realizing reaction by one water bath or incubator, and meeting the detection requirements of a base layer and a site. The LAMP principle utilizes the strand displacement activity of large fragment DNA polymerase of bacillus stearothermophilus (Bacillus stearothermophilus, bst) and two pairs of special inner primers (FIP is composed of F1C and F2; BIP is composed of B1C and B2) and outer primers (F3 and B3) designed according to different target sequences, specifically recognizes 6 independent regions on the target sequences, and carries out efficient and rapid amplification of the target sequences under isothermal conditions.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a novel rapid detection primer for the echinococcosis granulosa and the echinococcosis multinomi nucleic acid, which ensures that the nucleic acid detection of the echinococcosis granulosa and the echinococcosis multinomi is rapid, has high sensitivity and strong specificity, and can distinguish the echinococcosis granulosa and the echinococcosis multinomi; simple operation, low cost and easy interpretation of the result.
The invention firstly provides a primer group for detecting and distinguishing echinococcus granulosus and echinococcus multinomis through loop-mediated isothermal amplification, which comprises two pairs of outer primers and two pairs of inner primers, and the sequences are as follows:
a first pair of outer primers:
outer primer F3-1:5'-gcatgtgtgtgaatgcaagc-3' the number of the individual pieces of the plastic,
outer primer B3-1:5'-gggcaatcgcagtgaagt-3';
first pair of inner primers:
inner primer FIP-1:5'-aactacctccacagcacggcagcagatgcctacccatcc-3' the number of the individual pieces of the plastic,
inner primer BIP-1:5'-taagacatcggtgcgagcactctgccttcgttaggtggagat-3';
second pair of outer primers:
outer primer F3-2:5'-aaccaccaacctttcggtta-3' the number of the individual pieces of the plastic,
outer primer B3-2:5'-ggaatgggaaggtgatggc-3';
second pair of inner primers:
inner primer FIP-2:5'-gcagtgtagcgcgtggcacagccgaacgcgctaac-3' the number of the individual pieces of the plastic,
inner primer BIP-2:5'-gcaagccgccgcctcttctgatggtgaggtagtgttgca-3'.
The invention also provides application of the primer group in identifying echinococci granulosa and echinococci multiflori. Wherein, the detection sample is the worm body or ovum of the echinococci granulosa and the echinococci multiforme, and the detection can be carried out as long as genome DNA of the echinococci granulosa and the echinococci multiforme exists.
The invention also provides a detection kit for identifying echinococcosis granulosa and echinococcosis multifilialis, which comprises the primer group.
The invention also provides a method for identifying echinococcosis granulosa and echinococcosis multifilialis, which comprises the following steps:
(1) Extracting genome DNA of a sample to be detected;
(2) Taking the genomic DNA extracted in the step (1) as an amplification template, carrying out loop-mediated isothermal amplification by using the primer group, dividing the amplification reaction into two parts, wherein the first reaction uses a first pair of outer primers and a first pair of inner primers, and the second reaction uses a second pair of outer primers and a second pair of inner primers;
(3) Judging according to the amplification results of the two reactions in the step (2), and if the amplification results of the first reaction and the second reaction are positive, determining that the sample to be detected contains echinococcosis granulosa; if the first reaction amplification result is negative and the second reaction amplification result is positive, the sample to be detected contains echinococcosis; if the amplification results of the first reaction and the second reaction are negative, the sample to be detected does not contain echinococci granulosa and echinococci multiflori.
The loop-mediated isothermal amplification reaction procedure was 40min at 64 ℃.
The reaction system of loop-mediated isothermal amplification is as follows: 2. Mu.L of genomic DNA, 5pmol of outer primer and 40pmol of inner primer, LAMP buffer, 8U of Bst DNA polymerase, 8U of pyrophosphatase, 1.5mM of dNTP,7.5mM of MgSO 4 、ddH 2 O，
Wherein the LAMP buffer comprises a solution of Tris-HCl at a concentration of 20mM, KCl at a concentration of 10mM, and (NH) 4 ) 2 SO 4 Tween-20 and betaine of 1M with mass fraction of 0.1%.
The detection of the loop-mediated isothermal amplification reaction product in the step (2) can be detected by using various methods, for example, by adding a fluorescent dye into the reaction solution and performing the reaction on a fluorescent quantitative PCR instrument, and judging the reaction result by collecting fluorescent signals. Preferably, after the loop-mediated isothermal amplification reaction in the step (2) is finished, mixing a reaction product with a detection solution, wherein the amplification result is positive if the solution turns blue, and the amplification result is negative if the solution is colorless, and the detection solution is: a mixed solution composed of 4. Mu.L of a solution of potassium antimony molybdate-tartrate containing 21mM molybdic acid, 2mM potassium antimony tartrate and 5M sulfuric acid, 106. Mu.L of distilled water, 2. Mu.L of 10% ascorbic acid and 106. Mu.L of double distilled water. The pyrophosphatase is used for detection, the detection result can be judged through color change, the visualization of the detection result is realized, a fluorescent quantitative PCR instrument is not needed, the operation is simple and convenient, and the application range is wide.
The primer group for detecting and distinguishing the echinococcus granulosus and the echinococcus multinomus through loop-mediated isothermal amplification can distinguish that the positive sample contains the genome DNA of the echinococcus granulosus or the echinococcus multinomus through two reactions, and the template DNA with the minimum detectable rate of 1.6fg has high detection sensitivity and strong specificity.
Drawings
FIG. 1 shows the results of specific detection of the first outer primer pair and the first inner primer pair. 1-7 are respectively echinococci granulosa, echinococci multiflori, taenia muricata, echinococci guangzhou isolate, echinococci henna isolate, schistosoma japonicum, guangdou beetle genome DNA and 8 is distilled water.
FIG. 2 shows the results of specific detection of the second outer primer pair and the second inner primer pair. 1-7 are respectively echinococci granulosa, echinococci multiflori, taenia muricata, echinococci guangzhou isolate, echinococci henna isolate, schistosoma japonicum, guangdou beetle genome DNA and 8 is distilled water.
FIG. 3 shows the results of the sensitivity test of the first outer primer pair and the first inner primer pair. 1-7 are 1.6pg,160fg,16fg,1.6fg,0.16fg,0.016fg,0.0016fg echinococci granulosa genomic DNA, respectively, 8 is distilled water.
FIG. 4 shows the results of the sensitivity test of the second outer primer pair and the second inner primer pair. 1-7 are 140pg,14pg,1.4pg,140fg,14fg,1.4fg and 0.14fg, respectively, of the genomic DNA of echinococci many rooms.
FIG. 5 shows the detection results of pyrophosphatase detection sensitivity. 1-4 are 160fg,16fg,1.6fg,0.16fg echinococcus granulosus genomic DNA, respectively, 5 is distilled water.
Detailed Description
Example 1: primer design
Primer design was performed based on the echinococcus granulosus repeat sequence Repeat Region Sequence (RRS) (Gene bank: KR 347168.1) and the echinococcus multinomi EmTorrple 83 microsatellite sequence (NCBI: AF 492849.1), providing a total of 2 sets of 8 primer sets required for the isothermal amplification reaction of echinococcus, including outer primers F3-1, B3-1, F3-2, B3-2, inner primers FIP-1, BIP-1, FIP-2, BIP-2.
A first pair of outer primers:
outer primer F3-1:5'-gcatgtgtgtgaatgcaagc-3' the number of the individual pieces of the plastic,
outer primer B3-1:5'-gggcaatcgcagtgaagt-3';
first pair of inner primers:
inner primer FIP-1:5'-aactacctccacagcacggcagcagatgcctacccatcc-3' the number of the individual pieces of the plastic,
inner primer BIP-1:5'-taagacatcggtgcgagcactctgccttcgttaggtggagat-3';
second pair of outer primers:
outer primer F3-2:5'-aaccaccaacctttcggtta-3' the number of the individual pieces of the plastic,
outer primer B3-2:5'-ggaatgggaaggtgatggc-3';
second pair of inner primers:
inner primer FIP-2:5'-gcagtgtagcgcgtggcacagccgaacgcgctaac-3' the number of the individual pieces of the plastic,
inner primer BIP-2:5'-gcaagccgccgcctcttctgatggtgaggtagtgttgca-3'.
Example 2: specific detection of first outer primer pair and first inner primer pair
Using Echinococcus multinomial, echinococcus granulosus, echinococcus mucrotalarius, echinococcus schizophragma, schistosoma japonicum, and Euphausia guanylate genomic DNA as templates, adding each template into a reaction tube, adding primer sets F3-1, B3-1, FIP-1, BIP-1 (5 pmol of outer primer and 40pmol of inner primer), 8U of Bst 2.0DNA polymerase, 1.5mM dNTP,7.5mM MgSO 4 And ddH 2 O, 1. Mu.l of SYTO13 fluorescent dye at 25. Mu.M was added thereto, and the reaction was carried out on a Berle fluorescent quantitative PCR apparatus by the following procedure: the reaction was carried out at 65℃for 1.5 hours and fluorescence data were collected, and the reaction was carried out at 95℃for 5 minutes.
As shown in FIG. 1, the primer set can detect genome DNA of echinococcus granulosus, and can not detect genome DNA of echinococcus multinomial, taenia, cercaria guangzhou isolate, cercaria Henan isolate, schistosoma japonicum and Guangzhou circular worm, thus indicating that the primer has good specificity.
Example 3: specific detection of second outer primer pair and second inner primer pair
Taking echinococci, echinococci granulosa, taenia, echinococci guangzhou isolate, echinococci Henan isolate, schistosoma japonicum and Guangzhou circular worm genome DNA as templates, adding each templateThe reaction tube was then filled with the primer sets F3-2, B3-2, FIP-2, BIP-2 (5 pmol of outer primer and 40pmol of inner primer), 8U Bst 2.0DNA polymerase, 1.5mM dNTP,7.5mM MgSO 4 And ddH 2 O, 1. Mu.l of SYTO13 fluorescent dye at 25. Mu.M was added thereto, and the reaction was carried out on a Berle fluorescent quantitative PCR apparatus by the following procedure: the reaction was carried out at 65℃for 1.5 hours and fluorescence data were collected, and the reaction was carried out at 95℃for 5 minutes.
As shown in FIG. 2, the primer set can detect echinococcus granulosus and echinococcus multinomus genome DNA simultaneously, and can not detect taenia muricata, echinococcus merogenes, schistosoma japonicum, and strongyloides guangdou genome DNA, which indicates that the primer has good specificity.
Example 4: sensitivity detection of first outer primer pair and first inner primer pair
The genome DNA of echinococcus granulosus was diluted to 1.6pg,160fg,16fg,1.6fg,0.16fg,0.016fg,0.0016fg and added to the reaction tubes, respectively, to which were added primers F3-1, B3-1, FIP-1, BIP-1 (5 pmol of outer primer and 40pmol of inner primer), 8U of Bst 2.0DNA polymerase, 1.5mM dNTP,7.5mM MgSO 4 And ddH 2 O, 1. Mu.l of SYTO13 fluorescent dye at 25. Mu.M was added thereto, and the reaction was carried out on a Berle fluorescent quantitative PCR apparatus by the following procedure: the reaction was carried out at 65℃for 1.5 hours and fluorescence data were collected, and the reaction was carried out at 95℃for 5 minutes.
As shown in FIG. 3, the primer and the reaction system can detect 1.6fg of echinococci granulosa genome DNA at the lowest, and have good detection sensitivity.
Example 5: sensitivity detection of second outer primer pair and second inner primer pair
The genome DNA of echinococci multiplexing was diluted 140pg,14pg,1.4pg,140fg,14fg,1.4fg and 0.14fg respectively into a reaction tube to which primers F3-2, B3-2, FIP-2, BIP-2 (5 pmol of outer primer and 40pmol of inner primer) were added, 8U Bst 2.0DNA polymerase, 1.5mM dNTP,7.5mM MgSO4 and ddH2O, and 1. Mu.l of SYTO13 fluorescent dye at 25. Mu.M was further added, and the reaction was performed on a Bole fluorescent quantitative PCR apparatus according to the following specific reaction procedure: the reaction was carried out at 65℃for 1.5 hours and fluorescence data were collected, and the reaction was carried out at 95℃for 5 minutes.
As shown in FIG. 4, the primer and the reaction system can detect 1.4fg of echinococcosis multiplex genome DNA at the lowest, and have good detection sensitivity.
Example 6: detection of pyrophosphatase detection sensitivity
The genome DNA of echinococcus granulosus was diluted to 160fg,16fg,1.6fg,0.16fg and added to the reaction tubes, respectively, and primers F3-1, B3-1, FIP-1, BIP-1 (5 pmol outer primer and 40pmol inner primer), 8U Bst 2.0DNA polymerase, 1.5mM dNTP,7.5mM MgSO were also added to the tubes 4 And ddH 2 O, 8U of pyrophosphatase was additionally added thereto, and the reaction was allowed to react in a water bath at 63℃for 40 minutes, and then the reaction product was thoroughly mixed with 4. Mu.L of a solution of potassium antimony molybdate-tartrate (21 mM molybdic acid, 2mM potassium antimony tartrate, 5M sulfuric acid) and 106. Mu.L of a distilled water solution in the reaction tube 2, and 2. Mu.L of a solution of 10% ascorbic acid and 106. Mu.L of double distilled water in the reaction tube 3, and was positive if the solution turned blue and negative if colorless was maintained. As a result, as shown in FIG. 5, the pyrophosphatase detection system was able to detect 1.6fg of echinococcus granulosus genomic DNA at the lowest.
Sequence listing
<110> Hangzhou medical college
Tibet Autonomous Region Center for Disease Control and Prevention
Institute for parasitic disease control and prevention, China Center for Disease Control and Prevention
<120> primer set for loop-mediated isothermal amplification detection and differentiation of echinococci granulosa and echinococci multiflori
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gcatgtgtgt gaatgcaagc 20
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gggcaatcgc agtgaagt 18
<210> 3
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
aactacctcc acagcacggc agcagatgcc tacccatcc 39
<210> 4
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
taagacatcg gtgcgagcac tctgccttcg ttaggtggag at 42
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
aaccaccaac ctttcggtta 20
<210> 6
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
ggaatgggaa ggtgatggc 19
<210> 7
<211> 35
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gcagtgtagc gcgtggcaca gccgaacgcg ctaac 35
<210> 8
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gcaagccgcc gcctcttctg atggtgaggt agtgttgca 39
Claims (8)
1. The primer group for detecting and distinguishing echinococcosis granulosa and echinococcosis in a loop-mediated isothermal amplification manner is characterized by comprising two pairs of outer primers and two pairs of inner primers, and the sequences are as follows:
a first pair of outer primers:
outer primer F3-1:5'-gcatgtgtgtgaatgcaagc-3' the number of the individual pieces of the plastic,
outer primer B3-1:5'-gggcaatcgcagtgaagt-3';
first pair of inner primers:
inner primer FIP-1:5'-aactacctccacagcacggcagcagatgcctacccatcc-3' the number of the individual pieces of the plastic,
inner primer BIP-1:5'-taagacatcggtgcgagcactctgccttcgttaggtggagat-3';
second pair of outer primers:
outer primer F3-2:5'-aaccaccaacctttcggtta-3' the number of the individual pieces of the plastic,
outer primer B3-2:5'-ggaatgggaaggtgatggc-3';
second pair of inner primers:
inner primer FIP-2:5'-gcagtgtagcgcgtggcacagccgaacgcgctaac-3' the number of the individual pieces of the plastic,
inner primer BIP-2:5'-gcaagccgccgcctcttctgatggtgaggtagtgttgca-3'.
2. Use of the primer set according to claim 1 for the identification of echinococci granulosa and echinococci multiflori, said use being for the purpose of non-disease diagnosis.
3. The use according to claim 2, wherein the test sample is echinococci granulosa and echinococci multiflori bodies or eggs.
4. A test kit for the identification of echinococci granulosa and echinococci mulis comprising the primer set of claim 1.
5. A method for identifying echinococci granulosa and echinococci multiflori, said method for identifying echinococci granulosa and echinococci multiflori being for the purpose of non-disease diagnosis, comprising the steps of:
(1) Extracting genome DNA of a sample to be detected;
(2) Using the genome DNA extracted in the step (1) as an amplification template, performing loop-mediated isothermal amplification by using the primer set in the claim 1, wherein the amplification reaction is divided into two, a first reaction uses a first pair of outer primers and a first pair of inner primers, and a second reaction uses a second pair of outer primers and a second pair of inner primers;
(3) Judging according to the amplification results of the two reactions in the step (2), and if the amplification results of the first reaction and the second reaction are positive, determining that the sample to be detected contains echinococcosis granulosa; if the first reaction amplification result is negative and the second reaction amplification result is positive, the sample to be detected contains echinococcosis; if the amplification results of the first reaction and the second reaction are negative, the sample to be detected does not contain echinococci granulosa and echinococci multiflori.
6. The method of claim 5, wherein the loop-mediated isothermal amplification reaction procedure is 40 minutes at 64 ℃.
7. The method of claim 5, wherein the loop-mediated isothermal amplification reaction system is: 2. Mu.L of genomic DNA, 5pmol of outer primer and 40pmol of inner primer, LAMP buffer, 8U of Bst DNA polymerase, 8U of pyrophosphatase, 1.5mM of dNTP,7.5mM of MgSO 4 、ddH 2 O，
Wherein the LAMP buffer comprises a solution of Tris-HCl at a concentration of 20mM, KCl at a concentration of 10mM, and (NH) 4 ) 2 SO 4 Tween-20 and betaine of 1M with mass fraction of 0.1%.
8. The method of claim 7, wherein after the loop-mediated isothermal amplification reaction of step (2) is completed, the reaction product is mixed with a detection solution, the amplification result is positive if the solution turns blue, the amplification result is negative if the solution remains colorless,
the detection liquid comprises the following components: a mixed solution composed of 4. Mu.L of a solution of potassium antimony molybdate-tartrate containing 21mM molybdic acid, 2mM potassium antimony tartrate and 5M sulfuric acid, 106. Mu.L of distilled water, 2. Mu.L of 10% ascorbic acid and 106. Mu.L of double distilled water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011002756.1A CN112226517B (en) | 2020-09-22 | 2020-09-22 | Primer group for detecting and distinguishing echinococcosis granulosa and echinococcosis multifilialis through loop-mediated isothermal amplification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011002756.1A CN112226517B (en) | 2020-09-22 | 2020-09-22 | Primer group for detecting and distinguishing echinococcosis granulosa and echinococcosis multifilialis through loop-mediated isothermal amplification |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112226517A CN112226517A (en) | 2021-01-15 |
| CN112226517B true CN112226517B (en) | 2023-10-20 |
Family
ID=74107441
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011002756.1A Active CN112226517B (en) | 2020-09-22 | 2020-09-22 | Primer group for detecting and distinguishing echinococcosis granulosa and echinococcosis multifilialis through loop-mediated isothermal amplification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112226517B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173554A (en) * | 2013-03-27 | 2013-06-26 | 中国农业科学院兰州兽医研究所 | Detection kit for detecting and distinguishing multiple kinds of echinococcus |
| CN108796091A (en) * | 2018-06-04 | 2018-11-13 | 青海大学 | Loop-mediated isothermal amplification is applied to the genotyping detection method and detection kit of more room/Echinococcus Granulosus Cysts |
| CN110656185A (en) * | 2019-10-30 | 2020-01-07 | 浙江省医学科学院 | Primer group for detecting trichina gene through loop-mediated isothermal amplification, application and method |
| CN111518877A (en) * | 2020-05-12 | 2020-08-11 | 青海大学 | One-tube method nest type real-time quantitative PCR detection kit for detecting echinococcus multilocularis and echinococcus granulosus by parting trace samples |
-
2020
- 2020-09-22 CN CN202011002756.1A patent/CN112226517B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173554A (en) * | 2013-03-27 | 2013-06-26 | 中国农业科学院兰州兽医研究所 | Detection kit for detecting and distinguishing multiple kinds of echinococcus |
| CN108796091A (en) * | 2018-06-04 | 2018-11-13 | 青海大学 | Loop-mediated isothermal amplification is applied to the genotyping detection method and detection kit of more room/Echinococcus Granulosus Cysts |
| CN110656185A (en) * | 2019-10-30 | 2020-01-07 | 浙江省医学科学院 | Primer group for detecting trichina gene through loop-mediated isothermal amplification, application and method |
| CN111518877A (en) * | 2020-05-12 | 2020-08-11 | 青海大学 | One-tube method nest type real-time quantitative PCR detection kit for detecting echinococcus multilocularis and echinococcus granulosus by parting trace samples |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112226517A (en) | 2021-01-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Akhavan et al. | Leishmania species: detection and identification by nested PCR assay from skin samples of rodent reservoirs | |
| Sandoval et al. | A new PCR-based approach for the specific amplification of DNA from different Schistosoma species applicable to human urine samples | |
| Crowder et al. | Genotypic variation and mixtures of Lyme Borrelia in Ixodes ticks from North America and Europe | |
| CN108796131B (en) | Double-fluorescence RT-LAMP detection group for visually identifying foot-and-mouth disease viruses and bluetongue viruses, kit and application thereof | |
| Schatzberg et al. | Use of a multiplex polymerase chain reaction assay in the antemortem diagnosis of toxoplasmosis and neosporosis in the central nervous system of cats and dogs | |
| Ayan et al. | PREVALANCE AND MOLECULAR CHARACTERIZATION OF GIARDIA DUODENALIS IN LIVESTOCK IN VAN, TURKEY.. | |
| WO2012117431A1 (en) | Method and reagent kit for the identification of biological fluids in a sample | |
| Nie et al. | Recombinase polymerase amplification with lateral flow strip for detecting Babesia microti infections | |
| CN111088380A (en) | Brucella LF-RPA detection primer, probe and detection kit | |
| CN114457197A (en) | Primer probe group and kit for detecting FPV by RAA fluorescence method | |
| CN112226517B (en) | Primer group for detecting and distinguishing echinococcosis granulosa and echinococcosis multifilialis through loop-mediated isothermal amplification | |
| CN109762910B (en) | Primer and kit for simultaneously detecting two types of echinococcosis | |
| Hidalgo et al. | A PCR-RFLP assay for discrimination of Echinococcus granulosus sensu stricto and Taenia spp. in dogs stool | |
| Sellon | Update on molecular techniques for diagnostic testing of infectious disease | |
| Che-Engku-Chik et al. | Detection of tuberculosis (TB) using gold standard method, direct sputum smears microscopy, PCR, qPCR and electrochemical DNA sensor: A mini review | |
| Mahmood et al. | Application of serum based PCR and fluorescence polarization assay for diagnosis of brucellosis among people occupationally at risk to disease | |
| CN112708685A (en) | Eva Green fluorescent quantitative PCR kit for detecting ehrlichia and detection method | |
| CN116670301A (en) | Composition for diagnosing tsutsugamushi disease and kit comprising the same | |
| KR20130142220A (en) | Improved method for diagnosing mycobacterium tuberculosis using one-tube nested real-time pcr | |
| RU2636458C1 (en) | Method for increase of real time polymerase chain reaction sensitivity in detection of pathogenic bacteria dna | |
| KR102494777B1 (en) | A Primer set for Diagnosis of Dengue Virus Infectious Disease based on Multiple Isothermal Amplification, A Composition for Diagnosing Denque Virus Infectious Disease including the same, and A Visual Diagnosis method for Dengue Virus Infectious Disease using the same | |
| RU2795987C1 (en) | Set of oligonucleotide primers and fluorescently labeled probes and a method for detecting brucellosis pathogen dna | |
| RU2744665C1 (en) | Method for detecting lujo virus rna by reverse transcription and polymerase chain reaction, taking into account real-time results | |
| RU2700477C1 (en) | Test system for detecting the genome of the causative agent of bordetella bronchiseptica dna in farm livestock | |
| RU2809735C1 (en) | Method for identifying mycobacterium tuberculosis bacteria using loop isothermal amplification (lamp) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |