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
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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.
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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 |
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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 |
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