CN111850153B - Primer group for detecting prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus and kit containing primer group - Google Patents
Primer group for detecting prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus and kit containing primer group Download PDFInfo
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- 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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The invention discloses a primer group for detecting acute hepatopancreatic necrosis disease-vibrio parahaemolyticus of prawns and a kit containing the primer group, and belongs to the technical field of aquatic product pathogenic organism detection reagents. The primer group for detecting the acute hepatopancreatic necrosis disease-vibrio parahaemolyticus of the prawn is designed according to a nucleic acid sequence shown in SEQ ID No. 1. The primer group is used for detecting the prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus, so that the problems of dependence on large expensive instruments and complex detection operation in the prior art are solved; in addition, the primer group has high detection sensitivity and good specificity, and is suitable for rapid determination of simple and crude conditions of aquaculture farms and the like.
Description
Technical Field
The invention belongs to the technical field of aquatic pathogenic organism detection reagents, and particularly relates to a primer group for detecting prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus and a kit containing the primer group.
Background
Acute hepatopancreatic necrosis disease (AHPND), also known as early death syndrome (EMS), is a new epidemic disease that seriously jeopardizes prawn culture in recent years. The disease firstly appears in the culture of the penaeus vannamei and Chinese penaeus before and after 2010, then is spread to southeast Asia, central America and the like, the disease has hepatopancreas atrophy and yellowing after the seedling release within 7 to 31 days, the death rate reaches 90 percent, and the worldwide penaeus vannamei aquaculture industry loses nearly 10 hundred million dollars every year; seriously affecting the development of prawn industry. For AHPND, toAs yet unequivocal, the currently more recognized pathogen is a unique Vibrio parahaemolyticus infecting AHPND (VpAHPND) which carries a gene containing a binary Pir-like toxinpirA vp AndpirB vp the 69 kbp plasmid (pAP 1); it was found that the Vibrio parahaemolyticus is lethal to shrimp because it contains plasmids encoding PirA and PirB toxins.
The rapid diagnosis of the aquatic pathogen has great significance in the aspects of fine variety breeding of aquaculture, disease prevention, import and export quarantine, aquatic product food safety and the like. Currently, common pathogen detection methods in aquatic pathogen control include histopathological section observation, virus isolation culture, antigen-serum reaction, enzyme-linked immunosorbent assay (ELISA), nucleic acid hybridization technology, general Polymerase Chain Reaction (PCR) technology, and the like. The existing detection methods have the defects of dependence on precise instruments, high detection cost, poor sensitivity and specificity, complicated operation procedures, difficult popularization and the like; and most of the conventional pathogen detection technologies are not suitable for the on-site rapid detection of the culture base.
LAMP technology is a novel DNA amplification method, which relies on an automatic cycle strand displacement reaction to amplify 10 DNA fragments in less than 1 hour under isothermal conditions by using primers recognizing 6 specific regions on a target sequence and DNA polymerase having strand displacement 9 The target sequence copy has the characteristics of specificity, sensitivity, rapidness, simplicity and the like, and compared with the common PCR and fluorescent quantitative PCR technology, the LAMP has the advantages that: (1) Expensive precise instruments are not needed, and the constant temperature state is achieved; (2) the amplification efficiency is high, and a large amount of amplification products can be obtained; (3) has high specificity; (4) By adding the Loop primer, the reaction can be accelerated, and the time is shortened by half; (5) The result can be observed by naked eyes due to the generation of a large amount of by-product magnesium pyrophosphate white precipitate by amplification. Therefore, the method can detect pathogenic organisms such as pathogenic bacteria, viruses, parasites, fungi and the like in aquatic economic animals, and is suitable for on-site rapid detection of culture substrates.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a primer group for detecting the pathogeny of the prawn acute hepatopancreatic necrosis, namely vibrio parahaemolyticus and a kit containing the primer group, wherein the primer group is used for detecting the pathogeny of the prawn acute hepatopancreatic necrosis, namely vibrio parahaemolyticus, so that the problems of dependence on large expensive instruments and complex detection operation in the prior art are solved; in addition, the primer group has high detection sensitivity and good specificity, and is suitable for rapid determination of simple and crude conditions of aquaculture farms and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
the primer group for detecting the acute hepatopancreatic necrosis disease-vibrio parahaemolyticus of the prawn has the sequence as follows:
F3：5’- GTCGTGGTTTCTGTACAATC -3’；
B3：5’- GCCAAAAGATATTCCATAACGT -3’；
FIP：5’- AGGGTTAAATTCCGTCAAAGATGACTAAGAAGGTGCTCACATGAC -3’；
BIP：5’- CTATGCTTTCAAAGCAATGGTTTCAATAAAAAACCACCCGCGTAA -3’；
or
F3：5’- TGATAATGCATTCTATCATCAGC -3’；
B3：5’- ATTTGAAAGACCAAATGAAACC -3’；
FIP：5’- GTGAGCACCTTCTTAGTGGTAATAGTTGTAATTAACAATGGCGCTAG -3’；
BIP：5’-TGACGGAATTTAACCCTAACAATGCGCTTTGAAAGCATAGTTAGGATC -3’；
Or
F3：5’- CAATACCAATGGGGTGCG -3’；
B3：5’- TCGTTAGTCATGTGAGCAC -3’；
FIP：5’- CAGGGCGTTGTAAATGGTAAGTCTGGAAAGTGGCTAAATCAC -3’；
BIP：5’-GCATTCTATCATCAGCGTATTGTTGAGTGGTAATAGATTGTACAGAAAC-3’；
LF：5’- TTCATCACGTTGTACCACAT -3’
LB：5’- ATTAACAATGGCGCTAGTCG -3’。
The primer set sequence is designed according to the nucleotide sequences 305 to 538, 255 to 489 or 160 to 359 of the nucleotide sequences shown in SEQ ID No. 1, or the complementary base sequences or the transcribed RNA sequences thereof.
The primer group is applied to the preparation of a reagent for detecting prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus.
A prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus detection kit comprises at least one group of primer groups.
On the basis of the scheme, the detection kit for the prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus further comprises reaction buffer solution, bst DNA polymerase, calcein, dNTPs and ddH 2 O (RNase-free) and a positive control.
On the basis of the scheme, the positive control is a T cloning vector containing a nucleic acid sequence shown as SEQ ID No. 1.
On the basis of the scheme, when the kit is used for detecting the acute hepatopancreatic necrosis disease-vibrio parahaemolyticus of the prawn, the reaction temperature is 63 ℃, and the reaction time is 60min.
The invention has the beneficial effects that:
the primer group is based on a segment of sequence SEQ ID No. 1 in a gene of vibrio parahaemolyticus which is a pathogen of prawn acute hepatopancreatic necrosis disease; 3 sets of LAMP specific primer combinations designed by PrimeExploer V4 software are respectively AHPND-1, AHPND-2 and AHPND-3. Each set of primers comprises at least two inner primers (FIP and BIP), two outer primers (F3 and B3), and one or two loop primers (LF and/or LB).
Based on the primer group, the invention adopts loop-mediated isothermal amplification (LAMP) technology to detect the pathogeny of the prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus, through preliminary screening, AHPND-2 and AHPND-3 are determined to be used for detecting the prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus, and the specificity and the sensitivity of the AHPND-2 and the AHPND-3 are verified respectively, the specificity detection result shows that the primer group AHPND-2 and the AHPND-3 have no non-specificity amplification to water, grouper iridovirus, grouper nervous necrosis virus, white spot syndrome virus, prawn infectious hypodermal and hematopoietic necrosis virus, vibrio harveyi, prawn iridovirus, prawn enterocytozoonosis and fusarium, and the sensitive detection result shows that the minimum detection limit of the AHPND-2 is 1 ag/muL-10 muL/muL, and the minimum detection limit of the AHagD-3 is 1 agP/muL-10 muL/muL.
Drawings
FIG. 1 shows the results of preliminary amplification of each primer set;
FIG. 2 shows the preliminary amplification results of each set of primers under natural light (from left to right, the primers and corresponding templates in each tube are AHPND-1+ positive plasmid, AHPND-1+ AP1, AHPND-1+ water (parallel 1), AHPND-1+ water (parallel 2), AHPND-2+ positive plasmid, AHPND-2+ AP1, AHPND-2+ water (parallel 1), AHPND-2+ water (parallel 2), AHPND-3+ positive plasmid, AHPND-3+ AP1, AHPND-3+ water (parallel 1), and AHPND-3+ water (parallel 2));
FIG. 3 shows the preliminary amplification results of each primer set under UV light (the primers and corresponding templates in each tube from left to right are AHPND-1+ positive plasmid, AHPND-1+ AP1, AHPND-1+ water (parallel 1), AHPND-1+ water (parallel 2), AHPND-2+ positive plasmid, AHPND-2+ AP1, AHPND-2+ water (parallel 1), AHPND-2+ water (parallel 2), AHPND-3+ positive plasmid, AHPND-3+ APD 1, AHPND-3+ water (parallel 1), AHPND-3+ water (parallel 2));
FIG. 4 shows the result of sensitivity detection of AHPND-2 primer set;
FIG. 5 shows the result of the sensitivity detection of the AHPND-2 primer set under natural light (the template in each tube from left to right is 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L, 1 fg/. Mu.L, 100 ag/. Mu.L, 10 ag/. Mu.L, 1 ag/. Mu.L, 0.1 ag/. Mu.L, and water, in that order);
FIG. 6 shows the result of the sensitivity detection of AHPND-2 primer set under UV light (the template in each tube from left to right is 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L, 1 fg/. Mu.L, 100 ag/. Mu.L, 10 ag/. Mu.L, 1 ag/. Mu.L, 0.1 ag/. Mu.L, water, in that order);
FIG. 7 shows the result of the sensitivity detection of AHPND-3 primer set;
FIG. 8 shows the results of sensitivity detection of AHPND-3 primer set under natural light (the templates in the tubes from left to right are, in order, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L, 1 fg/. Mu.L, 100 ag/. Mu.L, 10 ag/. Mu.L, 1 ag/. Mu.L, and water);
FIG. 9 shows the result of the sensitivity detection of the AHPND-3 primer set under UV light (the templates in each tube from left to right are, in order: 10pg/μ L, 1 pg/μ L, 100 fg/μ L, 10 fg/μ L, 1 fg/μ L, 100 ag/μ L, 10ag/μ L, 1ag/μ L, and water);
FIG. 10 result of detection of the specificity of the AHPND-2 primer set;
FIG. 11 shows the result of specific detection of AHPND-2 primer set under natural light (the templates in the tubes from left to right are positive plasmid, water, epinephelus coioides Rainbow virus, epinephelus neuronecrosis virus, white spot syndrome virus, prawn infectious hypodermal and hematopoietic necrosis virus, vibrio harveyi, prawn iridovirus, prawn enterobacter hepaticus, and Fusarium);
FIG. 12 shows the result of specific detection of AHPND-2 primer set under UV light (the templates in each tube from left to right are positive plasmid, water, epinephelus coioides Rainbow virus, epinephelus neuronecrosis virus, white spot syndrome virus, prawn infectious hypodermal and hematopoietic necrosis virus, vibrio harveyi, prawn iridovirus, prawn enterocytozoon, and Fusarium);
FIG. 13 detection results of the specificity of AHPND-3 primer set;
FIG. 14 shows the result of specific detection of AHPND-3 primer set under natural light (the templates in each tube from left to right are positive plasmid, water, epinephelus coioides iridovirus, epinephelus neuronecrosis virus, white spot syndrome virus, prawn infectious hypodermal and hematopoietic necrosis virus, vibrio harveyi, prawn iridovirus, prawn enterocytozoon, and Fusarium);
FIG. 15 shows the result of specific detection of AHPND-3 primer set under ultraviolet light (from left to right, the templates in the tubes are positive plasmid, water, epinephelus coioides iridovirus, epinephelus neuronecrosis virus, white spot syndrome virus, prawn infectious hypodermal and hematopoietic necrosis virus, vibrio harveyi, prawn iridovirus, prawn enterocytozoon, and Fusarium).
Detailed Description
The sample in the present invention may be a sample containing Vibrio parahaemolyticus of prawn, such as cells isolated from infection experiments or the like and culture solutions thereof, or a sample derived from living body and cultured cells, or a living body sample suspected of infecting Vibrio parahaemolyticus organism of prawn, and these samples may be subjected to pretreatment such as isolation, extraction, concentration, purification, or the like.
Various reagents required for detection of nucleic acid amplification using the primer of the present invention may be combined in advance to form a kit, and specifically, various kinds of oligonucleic acids required as the primer or loop primer of the present invention, 4 kinds of dNTPs as substrates for nucleic acid synthesis, DNA polymerase for nucleic acid synthesis, buffers and salts providing conditions suitable for the enzyme reaction, positive control nucleic acid, protective reagents for stabilizing the enzyme and template, and reagents required for detection of a reaction product to be used as needed are provided in the form of a kit.
Example 1
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention is described in further detail below with reference to specific examples and data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
1. Design and Synthesis of primers
According to a sequence SEQ ID No. 1, the LAMP primer is designed by adopting PrimeExplorer V4 software, and the specific design principle is as follows: selecting a primer as a primary screening primer, wherein the Tm value of an inner primer FIP/BIP is about 65 ℃, the temperature of an outer primer F3/B3 is about 60 ℃, the 5 'end dataG value of the FIP/BIP is less than or equal to-4 kcal/mol, the 3' end dataG value of the F3/B3 is less than or equal to-4 kcal/mol, the GC content is 40-60%, and the amplification fragment of the primer is about 200bp, and on the basis of the design principle, the skilled person can perform selection according to experience; the invention designs three groups of primers which are respectively marked as AHPND-1, AHPND-2 and AHPND-3, wherein each group of primers respectively comprises two inner primers (FIP and BIP) and two outer primers (F3 and B3); some also contain one or two loop primers (LF and/or LB).
SEQ ID No:1
1 atgagtaacaatataaaacatgaaactgactattctcacgattggactgtcgaaccaaac 60
61 ggaggcgtcacagaagtagacagcaaacatacacctatcatcccggaagtcggtcgtagt 120
121 gtagacattgagaatacgggacgtggggagcttaccattcaataccaatggggtgcgcca 180
181 tttatggctggcggctggaaagtggctaaatcacatgtggtacaacgtgatgaaacttac 240
241 catttacaacgccctgataatgcattctatcatcagcgtattgttgtaattaacaatggc 300
301 gctagtcgtggtttctgtacaatctattaccactaagaaggtgctcacatgactaacgaa 360
361 tacgttgtaacaatgtcatctttgacggaatttaaccctaacaatgctcgtaaaagttat 420
421 ttatttgataactatgaagttgatcctaactatgctttcaaagcaatggtttcatttggt 480
481 ctttcaaatattccttacgcgggtggttttttatcaacgttatggaatatcttttggcca 540
541 aatacgccaaatgagccagatattgaaaacatttgggaacaattacgtgacagaatccaa 600
601 gatttagtagatgaatcgattatagatgccatcaatggaatattggatagcaaaatcaaa 660
661 gagacacgcgataaaattcaagacattaatgagactatcgaaaacttcggttatgctgcg 720
721 gcaaaagatgattacattggtttagttactcattacttgattggacttgaagagaacttt 780
781 aagcgcgagctagacggtgatgaatggcttggttatgcgatattgcctctattagcaaca 840
841 actgtaagtcttcaaattacttacatggcttgtggtctggattataaggatgaattcggt 900
901 ttcaccgattctgatgtgcataagctaacacgtaatattgataagctttatgatgatgta 960
961 tcgtcttacattacagaactcgctgcgtgggctgataacgactcttacaataatgcaaac 1020
1021 caagataacgtgtatgatgaagtgatgggtgctcgtagttggtgtacggttcacggcttt 1080
1081 gaacatatgcttatttggcaaaaaatcaaagagttgaaaaaagttgatgtgtttgttcac 1140
1141 agtaatttaatttcatattcacctgctgttggttttcctagtggtaatttcaactatatt 1200
1201 gctacaggtacggaagatgaaatacctcaaccattaaaaccaaatatgtttggggaacgt 1260
1261 cgaaatcgtattgtaaaaattgaatcatggaacagtattgaaatacattattacaatcgc 1320
1321 gtaggtcgacttaaactaacttatgaaaatggggaagtggtagaactaggcaaggctcat 1380
1381 aaatatgacgagcattaccaatctattgagttaaacggcgcttacattaaatatgttgat 1440
1441 gttattgccaatggacctgaagcaattgatcgaatcgtatttcatttttcagatgatcga 1500
1501 acatttgttgttggtgaaaactcaggcaagccaagtgtgcgtttgcaactggaaggtcat 1560
1561 tttatttgtggcatgcttgcggatcaagaaggttctgacaaagttgccgcgtttagcgtg 1620
1621 gcttatgaattgtttcatcccgatgaatttggtacagaaaagtag 1665
The nucleotide sequences of the Lamp primer group are respectively as follows:
AHPND-1
F3：5’- GTCGTGGTTTCTGTACAATC -3’（SEQ ID No:2）；
B3：5’- GCCAAAAGATATTCCATAACGT -3’ （SEQ ID No:3）；
FIP：5’- AGGGTTAAATTCCGTCAAAGATGACTAAGAAGGTGCTCACATGAC -3’ （SEQ ID No:4）；
BIP：5’- CTATGCTTTCAAAGCAATGGTTTCAATAAAAAACCACCCGCGTAA -3’ （SEQ ID No:5）；
AHPND-2
F3：5’- TGATAATGCATTCTATCATCAGC -3’ （SEQ ID No:6）；
B3：5’- ATTTGAAAGACCAAATGAAACC -3’ （SEQ ID No:7）；
FIP：5’- GTGAGCACCTTCTTAGTGGTAATAGTTGTAATTAACAATGGCGCTAG -3’ （SEQ ID No:8）；
BIP：5’-TGACGGAATTTAACCCTAACAATGCGCTTTGAAAGCATAGTTAGGATC -3’ （SEQ ID No:9）；
AHPND-3
F3：5’- CAATACCAATGGGGTGCG -3’ （SEQ ID No:10）；
B3：5’- TCGTTAGTCATGTGAGCAC -3’ （SEQ ID No:11）；
FIP：5’- CAGGGCGTTGTAAATGGTAAGTCTGGAAAGTGGCTAAATCAC -3’ （SEQ ID No:12）；
BIP：5’-GCATTCTATCATCAGCGTATTGTTGAGTGGTAATAGATTGTACAGAAAC-3’ （SEQ ID No:13）；
LF：5’- TTCATCACGTTGTACCACAT -3’ （SEQ ID No:14）；
LB：5’- ATTAACAATGGCGCTAGTCG -3’ （SEQ ID No:15）。
the primer set sequence of the invention is synthesized by Shanghai biological engineering Co., ltd.
2. Construction of prawn acute hepatopancreatic necrosis pathogen-vibrio parahaemolyticus positive plasmid
The positive plasmid is obtained by constructing a nucleic acid sequence shown in SEQ ID No. 1 into a cloning vector pMD18-T, transforming and screening.
3. Preliminary screening of primers
Performing LAMP reaction at 63 ℃ for 60 minutes by respectively combining AHPND-1, AHPND-2 and AHPND-3 primers with the prepared positive plasmid of prawn acute hepatopancreatic necrosis pathogen-vibrio parahaemolyticus, prawn acute hepatopancreatic necrosis pathogen-vibrio parahaemolyticus (AP 1) and water as templates, wherein the dosage of each primer is FIP, BIP 40pmol, F3 and B3 pmol respectively, and the dosage of the loop primer is 20pmol (the loop primer is not added in the primer group without the loop primer); after the reaction was completed, the amplification result was analyzed by observing the amplification curve.
The LAMP reaction system (25. Mu.L) was:
wherein the 2 × reaction buffer is: 20 mM Tris-HCl pH 8.8, 10 mM (NH) 4 ) 2 SO 4 ，10 mM KCl，2 mM MgSO 4 0.1% Triton X-100 and 0.8M betaine.
The results are shown in FIGS. 1-3: three sets of primers AHPND-1, AHPND-2 and AHPND-3 of FD are added, positive plasmids can be detected, and the water contrast is normal; the AHPND-2 and AHPND-3 primers were able to detect AP1, whereas the AHPND-1 primer was unable to detect AP1.
4. Sensitivity detection
4.1 Sensitive detection of AHPND-2
The Vibrio parahaemolyticus positive plasmid synthesized as described above was diluted in the order of concentration gradient such as 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L, 1 fg/. Mu.L, 100 ag/. Mu.L, 10 ag/. Mu.L, 1 ag/. Mu.L, 0.1 ag/. Mu.L, etc., and used as a sensitivity test.
Adopting an AHPND-2 primer group, respectively taking vibrio parahaemolyticus positive plasmids with the concentrations of 1 ng/mu L, 100 pg/mu L, 10 pg/mu L, 1 pg/mu L, 100 fg/mu L, 10 fg/mu L, 1 fg/mu L, 100 ag/mu L, 10 ag/mu L, 1 ag/mu L and 0.1 ag/mu L as templates, and taking water as a negative control; mu.L of the template was added to the 25. Mu.L system and reacted for 60 minutes at 63 ℃ on a LAMP real-time turbidimeter. After the reaction was completed, the amplification result was analyzed by observing the amplification curve.
The test results are shown in FIGS. 4-6: amplification can be detected when the content of the vibrio parahaemolyticus positive plasmid is 1 ng/muL-10 ag/muL, amplification is not seen in a negative control, and the lowest detection limit of the AHPND-2 primer group is 1 ag/muL-10 ag/muL, namely the lowest detection limit is 0.21 copies/muL-2.1 copies/muL.
4.2 Sensitive detection of AHPND-3
The Vibrio parahaemolyticus positive plasmid synthesized as described above was diluted in the order of concentration gradient of 100 pg/μ L, 10pg/μ L, 1 pg/μ L, 100 fg/μ L, 10 fg/μ L, 1 fg/μ L, 100 ag/μ L, 10ag/μ L, 1ag/μ L, etc., and used as a sensitivity test, and the concentrations of 1ng/μ L and 100 pg/μ L were high, so these two concentration gradients were omitted.
Adopting an AHPND-3 primer group, respectively taking vibrio parahaemolyticus positive plasmids with the concentrations of 10 pg/mu L, 1 pg/mu L, 100 fg/mu L, 10 fg/mu L, 1 fg/mu L, 100 ag/mu L, 10 ag/mu L and 1 ag/mu L as templates, and taking water as a negative control; mu.L of the template was added to the 25. Mu.L system and reacted for 60 minutes at 63 ℃ on a LAMP real-time turbidimeter. After the reaction was completed, the amplification result was analyzed by observing the amplification curve.
The test results are shown in FIGS. 7-9: amplification can be detected when the content of the vibrio parahaemolyticus positive plasmid is 10 pg/muL-10 ag/muL, amplification is not seen in a negative control, and the lowest detection limit of the AHPND-3 primer group is 1 ag/muL-10 ag/muL, namely the lowest detection limit is 0.21 copies/muL-2.1 copies/muL.
5. Specificity test
5.1 AHPND-2 specificity assay
Adopting an AHPND-2 primer group, respectively taking positive plasmids, water, grouper iridovirus, grouper nervous necrosis virus, white spot syndrome virus, prawn infectious hypodermal and hematopoietic necrosis virus, vibrio harveyi, prawn iridovirus, prawn enterocytozoon and fusarium as templates, and carrying out LAMP reaction for 60 minutes at 63 ℃ under the condition of adding FD. After the reaction was completed, the amplification result was analyzed by observing the amplification curve.
The results of the tests are shown in FIGS. 10-12: the AHPND-2 primer group can effectively detect positive plasmids, but can not detect water, grouper iridovirus, grouper nervous necrosis virus, white spot syndrome virus, prawn infectious hypodermal and hematopoietic necrosis virus, vibrio harveyi, prawn iridovirus, prawn enterocytozoon, fusarium and the like, and has good specificity.
5.2 AHPND-3 specificity assay
Adopting an AHPND-3 primer group, respectively taking positive plasmids, water, grouper iridovirus, grouper nervous necrosis virus, white spot syndrome virus, prawn infectious hypodermal and hematopoietic necrosis virus, vibrio harveyi, prawn iridovirus, prawn enterocytozoon and fusarium as templates, and carrying out LAMP reaction for 60 minutes at 63 ℃ under the condition of adding FD. After the reaction was completed, the amplification result was analyzed by observing the amplification curve.
The results of the tests are shown in FIGS. 13-15: the AHPND-3 primer group can effectively detect positive plasmids, but can not detect water, grouper iridovirus, grouper nervous necrosis virus, white spot syndrome virus, prawn infectious hypodermal and hematopoietic necrosis virus, vibrio harveyi, prawn iridovirus, prawn enterocytozoon, fusarium and the like, and has good specificity.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention will still fall within the protection scope of the technical solution of the present invention.
Sequence listing
<110> Fujian province aquatic research institute (Fujian aquatic disease control center)
Primer group for detecting acute hepatopancreatic necrosis disease-vibrio parahaemolyticus of prawn and kit containing primer group
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1665
<212> DNA
<213> Vibrio parahaemolyticus consuming AHPND
<400> 1
atgagtaaca atataaaaca tgaaactgac tattctcacg attggactgt cgaaccaaac 60
ggaggcgtca cagaagtaga cagcaaacat acacctatca tcccggaagt cggtcgtagt 120
gtagacattg agaatacggg acgtggggag cttaccattc aataccaatg gggtgcgcca 180
tttatggctg gcggctggaa agtggctaaa tcacatgtgg tacaacgtga tgaaacttac 240
catttacaac gccctgataa tgcattctat catcagcgta ttgttgtaat taacaatggc 300
gctagtcgtg gtttctgtac aatctattac cactaagaag gtgctcacat gactaacgaa 360
tacgttgtaa caatgtcatc tttgacggaa tttaacccta acaatgctcg taaaagttat 420
ttatttgata actatgaagt tgatcctaac tatgctttca aagcaatggt ttcatttggt 480
ctttcaaata ttccttacgc gggtggtttt ttatcaacgt tatggaatat cttttggcca 540
aatacgccaa atgagccaga tattgaaaac atttgggaac aattacgtga cagaatccaa 600
gatttagtag atgaatcgat tatagatgcc atcaatggaa tattggatag caaaatcaaa 660
gagacacgcg ataaaattca agacattaat gagactatcg aaaacttcgg ttatgctgcg 720
gcaaaagatg attacattgg tttagttact cattacttga ttggacttga agagaacttt 780
aagcgcgagc tagacggtga tgaatggctt ggttatgcga tattgcctct attagcaaca 840
actgtaagtc ttcaaattac ttacatggct tgtggtctgg attataagga tgaattcggt 900
ttcaccgatt ctgatgtgca taagctaaca cgtaatattg ataagcttta tgatgatgta 960
tcgtcttaca ttacagaact cgctgcgtgg gctgataacg actcttacaa taatgcaaac 1020
caagataacg tgtatgatga agtgatgggt gctcgtagtt ggtgtacggt tcacggcttt 1080
gaacatatgc ttatttggca aaaaatcaaa gagttgaaaa aagttgatgt gtttgttcac 1140
agtaatttaa tttcatattc acctgctgtt ggttttccta gtggtaattt caactatatt 1200
gctacaggta cggaagatga aatacctcaa ccattaaaac caaatatgtt tggggaacgt 1260
cgaaatcgta ttgtaaaaat tgaatcatgg aacagtattg aaatacatta ttacaatcgc 1320
gtaggtcgac ttaaactaac ttatgaaaat ggggaagtgg tagaactagg caaggctcat 1380
aaatatgacg agcattacca atctattgag ttaaacggcg cttacattaa atatgttgat 1440
gttattgcca atggacctga agcaattgat cgaatcgtat ttcatttttc agatgatcga 1500
acatttgttg ttggtgaaaa ctcaggcaag ccaagtgtgc gtttgcaact ggaaggtcat 1560
tttatttgtg gcatgcttgc ggatcaagaa ggttctgaca aagttgccgc gtttagcgtg 1620
gcttatgaat tgtttcatcc cgatgaattt ggtacagaaa agtag 1665
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus using AHPND)
<400> 2
gtcgtggttt ctgtacaatc 20
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus using AHPND)
<400> 3
gccaaaagat attccataac gt 22
<210> 4
<211> 45
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus using AHPND)
<400> 4
agggttaaat tccgtcaaag atgactaaga aggtgctcac atgac 45
<210> 5
<211> 45
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus consuming AHPND)
<400> 5
ctatgctttc aaagcaatgg tttcaataaa aaaccacccg cgtaa 45
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus consuming AHPND)
<400> 6
tgataatgca ttctatcatc agc 23
<210> 7
<211> 22
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus using AHPND)
<400> 7
atttgaaaga ccaaatgaaa cc 22
<210> 8
<211> 47
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus using AHPND)
<400> 8
gtgagcacct tcttagtggt aatagttgta attaacaatg gcgctag 47
<210> 9
<211> 48
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus consuming AHPND)
<400> 9
tgacggaatt taaccctaac aatgcgcttt gaaagcatag ttaggatc 48
<210> 10
<211> 18
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus consuming AHPND)
<400> 10
caataccaat ggggtgcg 18
<210> 11
<211> 19
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus using AHPND)
<400> 11
tcgttagtca tgtgagcac 19
<210> 12
<211> 42
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus using AHPND)
<400> 12
cagggcgttg taaatggtaa gtctggaaag tggctaaatc ac 42
<210> 13
<211> 49
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus consuming AHPND)
<400> 13
gcattctatc atcagcgtat tgttgagtgg taatagattg tacagaaac 49
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus using AHPND)
<400> 14
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence (Vibrio parahaemolyticus using AHPND)
<400> 15
Claims (5)
1. A primer group for detecting prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus is characterized in that the sequence of the primer group is as follows:
F3：5’-TGATAATGCATTCTATCATCAGC-3’；
B3：5’-ATTTGAAAGACCAAATGAAACC-3’；
FIP：5’-GTGAGCACCTTCTTAGTGGTAATAGTTGTAATTAACAATGGCGCTAG-3’；
BIP：5’-TGACGGAATTTAACCCTAACAATGCGCTTTGAAAGCATAGTTAGGATC-3’；
or
F3：5’-CAATACCAATGGGGTGCG-3’；
B3：5’-TCGTTAGTCATGTGAGCAC-3’；
FIP：5’-CAGGGCGTTGTAAATGGTAAGTCTGGAAAGTGGCTAAATCAC-3’；
BIP：5’-GCATTCTATCATCAGCGTATTGTTGAGTGGTAATAGATTGTACAGAAAC-3’；
LF：5’-TTCATCACGTTGTACCACAT-3’
LB：5’-ATTAACAATGGCGCTAGTCG-3’。
2. The primer set of claim 1 is applied to the preparation of a prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus detection reagent.
3. A prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus detection kit, comprising at least one group of primer group of claim 1.
4. The detection kit for detecting acute hepatopancreatic necrosis disease-vibrio parahaemolyticus of prawn according to claim 3, further comprising reaction buffer, bst DNA polymerase, calcein, dNTPs and ddH 2 O (RNase-free) and a positive control.
5. The detection kit for detecting the acute hepatopancreatic necrosis disease-vibrio parahaemolyticus of the prawn as claimed in claim 4, wherein the positive control is a T cloning vector containing a nucleic acid sequence shown in SEQ ID No: 1.
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| CN113337626B (en) * | 2021-06-02 | 2024-03-12 | 江苏省海洋水产研究所 | Method for detecting acute strain and subacute strain of prawn VPAHPND |
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| CN110317792A (en) * | 2019-06-03 | 2019-10-11 | 台州学院 | Vibrio parahaemolyticus phage VP-HYP2 and its application |
| CN111172119A (en) * | 2020-03-10 | 2020-05-19 | 青岛诺安百特生物技术有限公司 | Novel vibrio parahaemolyticus phage with wide cracking spectrum, specific primer and application thereof |
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| CN105316422A (en) * | 2015-12-02 | 2016-02-10 | 中国科学院南海海洋研究所 | Kit for rapidly detecting pathogeny of acute hepatopancreatic necrosis disease of prawns and application of kit |
| WO2018056803A1 (en) * | 2016-09-21 | 2018-03-29 | Universiti Malaya | Rapid detection of prawn viruses, method and kit thereof |
| CN107523627B (en) * | 2017-09-21 | 2020-12-11 | 广州双螺旋基因技术有限公司 | LAMP kit for detecting acute hepatopancreas necrosis pathogen |
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| CN111172119A (en) * | 2020-03-10 | 2020-05-19 | 青岛诺安百特生物技术有限公司 | Novel vibrio parahaemolyticus phage with wide cracking spectrum, specific primer and application thereof |
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