CN112063750A - LAMP (Loop-mediated isothermal amplification) detection primer group of mandarin perch rhabdovirus, application of LAMP detection primer group and detection kit - Google Patents
LAMP (Loop-mediated isothermal amplification) detection primer group of mandarin perch rhabdovirus, application of LAMP detection primer group and detection kit Download PDFInfo
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Abstract
The invention discloses an LAMP (loop-mediated isothermal amplification) detection primer group of Siniperca chuatsi rhabdovirus, application thereof and a detection kit, wherein the primer group comprises a pair of outer primers, a pair of inner primers and a pair of loop primers; the outer primer comprises an L-F3 sequence and an L-B3 sequence, the L-F3 sequence of the outer primer is shown as SEQ ID NO. 1, and the L-B3 sequence of the outer primer is shown as SEQ ID NO. 2; the inner primer comprises an L-FIP sequence and an L-BIP sequence, wherein the L-FIP sequence of the inner primer is shown as SEQ ID NO. 3, and the L-BIP sequence of the inner primer is shown as SEQ ID NO. 4; the loop primer comprises an L-LF sequence and an L-LB sequence, wherein the L-LF sequence of the loop primer is shown as SEQ ID NO. 5, and the L-LB sequence of the loop primer is shown as SEQ ID NO. 6. And provides the application of the primer group in preparing a mandarin perch rhabdovirus detection kit and the kit. The primer group and the kit have the advantages of simple operation, quick detection, good specificity, high sensitivity, reliable result and the like.
Description
Technical Field
The invention relates to a group of LAMP detection primer groups and application thereof, and a detection kit, in particular to a group of LAMP detection primer groups of Siniperca chuatsi rhabdovirus, application thereof, and a detection kit.
Background
Rhabdoviruses (Rhabdoviruses) are enveloped negative strand RNA viruses, have strong pathogenicity and wide host range, and can infect various organisms including mammals, birds, reptiles, fishes, insects, plants and the like. Fish rhabdoviruses are one of the most abundant populations of teleostean viruses, and nearly twenty species of fish rhabdoviruses have been isolated and identified so far, such as: siniperca Chuatsi Rhabdovirus (SCRV), Micropterus Salmoides Rhabdovirus (MSRV), which are commonly associated with epidemics and significant economic losses in aquaculture.
The mandarin fish and the perch are important fresh water famous and excellent cultured varieties which are tender in meat quality, delicious in taste, high in protein content and deeply favored by consumers. However, the disease problem is increasingly highlighted due to the expansion of the culture scale and the increase of the culture density and the pollution of the water environment. Rhabdovirus disease is one of several common viral diseases in the process of mandarin fish and perch culture, mainly harms mandarin fish juvenile fish, and the death rate of fry can reach 30-50%; in recent years, the disease of the mandarin fish can be caused and mass death can occur frequently, which brings serious loss to the mandarin fish culture economy. Research finds that the Siniperca chuatsi rhabdovirus SCRV and the Micropterus salmoides rhabdovirus MSRV have a close relationship, and the sequence homology is up to more than 96%.
At present, the method for detecting rhabdovirus of mandarin fish and micropterus salmoides mainly depends on the traditional PCR method, and although the method can provide accurate and reliable detection results, expensive instruments and equipment, higher detection cost and higher technical requirements for detection personnel are required, so that the method is not suitable for large-scale screening, use and popularization of mandarin fish and micropterus salmoides culture sites.
Disclosure of Invention
The purpose of the invention is as follows: the first purpose of the invention is to provide a group of LAMP detection primer groups for rapidly and specifically detecting the presence of the L gene of the rhabdovirus of the Siniperca chuatsi, the second purpose of the invention is to provide application of the LAMP detection primer groups in preparation of a siniperca chuatsi rhabdovirus detection kit, and the third purpose of the invention is to provide a detection kit for the rhabdovirus of the Siniperca chuatsi.
The technical scheme is as follows: the LAMP detection primer group of the rhabdovirus of the mandarin perch comprises a pair of outer primers, a pair of inner primers and a pair of loop primers;
the outer primer comprises an L-F3 sequence and an L-B3 sequence, the L-F3 sequence of the outer primer is shown as SEQ ID NO. 1, and the L-B3 sequence of the outer primer is shown as SEQ ID NO. 2;
the inner primer comprises an L-FIP sequence and an L-BIP sequence, wherein the L-FIP sequence of the inner primer is shown as SEQ ID NO. 3, and the L-BIP sequence of the inner primer is shown as SEQ ID NO. 4;
the loop primer comprises an L-LF sequence and an L-LB sequence, wherein the L-LF sequence of the loop primer is shown as SEQ ID NO. 5, and the L-LB sequence of the loop primer is shown as SEQ ID NO. 6.
Furthermore, the molar ratio of the outer primer, the inner primer and the loop primer is 0.8-1.2: 6-9: 3-5.
The LAMP detection primer group of the rhabdovirus of the mandarin perch is applied to the preparation of a mandarin perch rhabdovirus detection kit.
The kit for detecting the rhabdovirus of the mandarin perch comprises a primer group.
Preferably, the kit further comprises reverse transcriptase, DNA polymerase, LAMP reaction solution, a positive control substance, a negative control substance and a color developing agent. The DNA polymerase is Bst DNA polymerase.
The LAMP reaction solution comprises: 10mM dNTP, 10 × ThermoPol reaction buffer and 150mM MgSO4The volume ratio of the three is 8:5: 2.
The positive control is plasmid obtained by connecting RNA-dependent RNA polymerase gene (L gene) fragment sequence to T vector, wherein the RNA polymerase gene fragment sequence is shown as SEQ ID NO: 7. The negative control was ultrapure water.
Has the advantages that: compared with the prior art, the primer group and the kit have the following advantages:
(1) the specificity is good: the DNA or RNA of aquatic pathogens such as siniperca chuatsi Infectious Spleen and Kidney Necrosis Virus (ISKNV), crucian hematopoietic necrosis virus (CyHV-2), leiocassis longirostris herpes virus (CCV), prawn White Spot Syndrome Virus (WSSV), Grass Carp Reovirus (GCRV), aeromonas hydrophila, Edwardsiella and the like are not amplified;
(2) the sensitivity is high: the lowest detection limit can reach 2x101 copies/mu l;
(3) the operation is simple: according to the technical scheme, complex instruments are not needed, special reagents are not needed, complicated steps such as denaturation of double-stranded DNA are not needed in advance, the reaction can be carried out only through a thermostat (heat-insulating barrel), an amplification result is judged through SYTO-9 fluorescent signals read by the thermostat fluorometer in real time in research and development, SYGR Green I dye can be used for being added into products for direct color development in popularization and application, technical requirements on operators are not high, and the method is suitable for being used in culture sites of siniperca chuatsi and micropterus salmoides.
Drawings
FIG. 1 is a diagram showing the results of Siniperca chuatsi rhabdovirus detection using a constant temperature fluorometer;
FIG. 2 is a result diagram of specificity of LAMP detection of Siniperca chuatsi rhabdovirus;
FIG. 3 is a diagram showing the result of LAMP-induced sensitivity detection of Siniperca chuatsi rhabdovirus, wherein the sequence from left to right is 2x106copies/μl～2x101copies/. mu.l and negative control;
FIG. 4 is a diagram showing the result of LAMP detection of rhabdovirus of Siniperca chuatsi.
Detailed Description
Both micropterus salmoides and the reagents used in this example are commercially available. The technical solution of the present invention is further illustrated by the following examples.
Example 1 kit establishment for detecting rhabdovirus of Siniperca chuatsi
The kit for detecting the rhabdovirus of the siniperca chuatsi, which is established based on the LAMP technology, comprises an LAMP primer group, AMV reverse transcriptase, Bst DNA polymerase, LAMP reaction liquid, positive control, negative control and color development agent SYTO-9.
(1) Designing LAMP primers: the LAMP primer is designed by taking an RNA dependent RNA polymerase gene (L gene) of the mandarin perch rhabdovirus as a target spot. The primer sequences are shown in Table 1. The molar ratio of the outer primer, the inner primer and the loop primer is 1:8: 4.
TABLE 1 primer sequence Listing
(2) LAMP reaction solution: containing 10mM dNTP, 10 XThermoPol reaction buffer, 150mM MgSO4The volume ratio of the aqueous solution to the three is 8:5: 2.
(3) the positive control is plasmid DNA containing rhabdovirus L gene segment, and the preparation method comprises the following steps: extracting genome RNA of mandarin fish rhabdovirus SCRV, performing RT-PCR amplification by using outer primers (SEQ ID NO:1 and SEQ ID NO:2) in a table 1, recovering an amplified fragment with the length of 411bp and the sequence shown as SEQ ID NO:7, and connecting the amplified fragment to a T vector by using a conventional method to obtain a plasmid, namely a positive control.
(4) The negative control was ultrapure water.
Example 2 method for establishing Siniperca chuatsi rhabdovirus detection by using constant temperature fluorometer
The method for detecting rhabdovirus of Micropterus salmoides by using the kit of example 1 comprises the following steps:
(1) preparation of micropterus salmoides rhabdovirus genome RNA: cutting tissue materials from tissues of Micropterus salmoides to be detected, placing the tissue materials into a sterilized mortar, adding liquid nitrogen, grinding, transferring into a centrifuge tube, adding 1ml of TRIzol reagent, and uniformly mixing; adding 0.2ml chloroform, shaking for 10-15s, standing at room temperature for 2-3 min; centrifuging at 4 deg.C for 15min at high speed (12000 Xg) to separate layers; slightly sucking out about 500ul of upper water phase, adding 500ul of isopropanol, mixing, standing at room temperature for 10min, centrifuging at 4 deg.C (12000 Xg) for 10min, discarding supernatant, and retaining RNA trace white precipitate; adding 1ml of 75% ethanol solution for RNA precipitation cleaning, mixing the sample lightly, and centrifuging at medium speed (7500 Xg) at 4 deg.C for 10 min; removing supernatant, standing at moderate room temperature (generally 5-10min), resuspending RNA in 30ul RNase-free water, and standing at 55-60 deg.C for 10 min; the RNA was kept at-80 ℃ until use.
(2) Isothermal gene amplification reaction: the isothermal gene amplification reaction is carried out according to the following reaction system: a25. mu.l reaction contained: ORF 77-F30.2. mu.M, ORF 77-B30.2. mu.M, ORF77-FIP 1.6. mu.M, ORF77-BIP 1.6. mu.M, ORF77-LF 0.8. mu.M, ORF77-LB 0.8. mu.M, LAMP reaction solution 12.5. mu.l, AMV reverse transcriptase 2.5U, Bst DNA polymerase 10U, 10 XSSYTO-90.5. mu.l, DNA to be detected 1-100 ng, and filling up to 25. mu.l with ultrapure water; setting a positive control and a negative control;
(3) and (5) judging a result: placing the reaction tube in a constant temperature fluorometer for reaction, observing the constant temperature fluorometer software to judge the amplification result, and identifying the result as shown in figure 1: if the S-shaped curve appears, the curve is positive, and if the S-shaped curve does not appear, the curve is negative.
Example 3 assay for specificity
The method of example 1 was used to detect positive RNA of rhabdovirus of mandarin fish and micropterus salmoides, and DNA or RNA of pathogens such as ISKNV, CyHV-2, CCV, WSSV, GCRV, Aeromonas hydrophila, Edwardsiella, etc.
The results of the identification are shown in FIG. 2: the LAMP primer group of the mandarin fish rhabdovirus is used for amplification reaction, positive RNA of the mandarin fish rhabdovirus and the micropterus salmoides rhabdovirus is normally amplified, negative water control and other pathogenic nucleic acids (DNA or RNA) are not amplified, and good specificity is shown.
Example 4 detection sensitivity experiment:
shrapnel containing mandarin perchPlasmid of the rhabdovirus L gene fragment was quantified, its concentration determined and plasmid copy number calculated from molecular weight diluted to 2X106 copies/μl、2x105 copies/μl、2x104 copies/μl、2x103 copies/μl、2x102 copies/μl、2x101copies/. mu.l. The diluted positive clones were each tested by the procedure of example 2. The results of the identification are shown in FIG. 3: the detection limit of the positive plasmid reaches 20 copies/mu l;
example 5 test repeatability experiments
RNA was prepared from rhabdovirus-positive Micropterus salmoides tissue according to example 2 and used as a template for detection; the results of the identification are shown in FIG. 4: in the same experiment, the amplification curves of 7 parallel samples are basically overlapped, which shows that the established LAMP method has better repeatability.
Sequence listing
<110> university of river and sea
NANJING FISHERIES Research Institute
<120> LAMP detection primer group of Siniperca chuatsi rhabdovirus, application thereof and detection kit
<160> 7
<170> SIPOSequenceListing 1.0
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<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
caatgatgac tcgtctcctc 20
<210> 3
<211> 44
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cagaatctcc aaggacttca cctggaccga caattacgac ttca 44
<210> 4
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcaagagtca cttaccgtcc agatctatgg ttgtgctctg ga 42
<210> 5
<211> 22
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgatctgtga gtggattagc ag 22
<210> 6
<211> 22
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ataagtggaa gccagaagga tc 22
<210> 7
<211> 411
<212> DNA/RNA
<213> Siniperca rhabdovirus (Mandarin fish and perch rhabdovirus)
<400> 7
aggatgatgt ccacgactga tacctttagg gacataggga ccgacaatta cgacttcatg 60
tttcagtccc tgctaatcca ctcacagatc acagcaggtg aagtccttgg agattctgat 120
gcaacaggaa cattgcattt tcatttggat tgcaaaggtt gcctgtccaa aatagaagag 180
gtagtcctag agtcaagagt cacttaccgt ccagaagaca gatctggatt acttaataag 240
tggaagccag aaggatcaga atggtcaaaa gaaagaacca gaccagaaat ccagagcaca 300
accatagagc ggttaaataa atcatccatt cattataaca tgggaagggc tcaaggattt 360
atttacggag atcagaagat gaccggaaga ggaggagacg agtcatcatt g 411
Claims (8)
1. A LAMP detection primer group of Siniperca chuatsi rhabdovirus is characterized in that: the primer group comprises a pair of outer primers, a pair of inner primers and a pair of loop primers;
the outer primer comprises a L-F3 sequence and a L-B3 sequence, the L-F3 sequence of the outer primer is shown as SEQ ID NO. 1, and the L-B3 sequence of the outer primer is shown as SEQ ID NO. 2;
the inner primer comprises an L-FIP sequence and an L-BIP sequence, the L-FIP sequence of the inner primer is shown as SEQ ID NO. 3, and the L-BIP sequence of the inner primer is shown as SEQ ID NO. 4;
the loop primer comprises an L-LF sequence and an L-LB sequence, the L-LF sequence of the loop primer is shown as SEQ ID NO. 5, and the L-LB sequence of the loop primer is shown as SEQ ID NO. 6.
2. The LAMP detection primer group for the rhabdovirus of siniperca chuatsi according to claim 1, which is characterized in that: the molar ratio of the outer primer, the inner primer and the loop primer is 0.8-1.2: 6-9: 3-5.
3. An application of the LAMP detection primer group of the rhabdovirus of siniperca chuatsi as claimed in claim 1 in preparation of a siniperca chuatsi rhabdovirus detection kit.
4. A detection kit for rhabdovirus of siniperca chuatsi is characterized in that: the kit comprises the primer set according to claim 1.
5. The detection kit for rhabdovirus of siniperca chuatsi as claimed in claim 4, wherein: the kit also comprises reverse transcriptase, DNA polymerase, LAMP reaction solution, a positive control substance, a negative control substance and a color developing agent.
6. The detection kit for rhabdovirus of siniperca chuatsi as claimed in claim 5, wherein: the DNA polymerase is Bst DNA polymerase.
7. The detection kit for rhabdovirus of siniperca chuatsi as claimed in claim 5, wherein: the positive control is a plasmid obtained by connecting an RNA-dependent RNA polymerase gene fragment sequence to a T vector, wherein the RNA polymerase gene fragment sequence is shown as SEQ ID NO. 7.
8. The detection kit for rhabdovirus of siniperca chuatsi as claimed in claim 5, wherein: the negative control is ultrapure water.
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Cited By (1)
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| CN113637802A (en) * | 2021-08-24 | 2021-11-12 | 中国水产科学研究院珠江水产研究所 | Kit and method for detecting rhabdovirus of weever by using digital PCR technology |
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Application publication date: 20201211 |