CN111019909A - Crustacean flavivirus and detection method and application thereof - Google Patents
Crustacean flavivirus and detection method and application thereof Download PDFInfo
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Abstract
The invention provides a crustacean flavivirus and a detection method and application thereof. The Infectious prematurity virus (Infectious precocity flavivirus) belongs to Infectious prematurity viruses and has a preservation number of CCTCC No. V201870. The shape is spherical, the diameter is 40-60 nm, and the macrobrachium rosenbergii can be infected. The invention also provides a kit of the infectious prematurity virus. Provides a new possibility for the detection, prevention and control of the infectious prematurity virus of crustacean aquatic products.
Description
Technical Field
The invention belongs to the field of aquaculture, and particularly relates to a flavivirus capable of infecting crustacean and a detection method thereof.
Background
Flaviviridae family (Flaviviridae) Is a single-stranded positive-strand RNA virus, and the flavivirus family is divided into 4 genera, flavivirus genus (flavivirus genus)Flavivirus) Hepatitis C virus genus (A), (B)Hepacivirus) And pestiviruses (b)Pestivirus) AndPegivirus. The inventor firstly found a virus of the flaviviridae family in macrobrachium rosenbergii, which belongs to a completely new class of viruses, and the virus is tentatively named as Infectious precocity virus (Infectious precocity flavivirus). Effective detection of this virus is a prerequisite for tracking, investigation, prevention and control of the virus and even other viruses of this genus, and it is therefore desirable to provide an effective method for detecting viruses.
Disclosure of Invention
The invention provides a kit for specifically detecting Infectious prematurity virus (Infectious precocity virus) aiming at the requirements of detection, prevention and control of Infectious prematurity virus infecting crustacean, the virus belongs to a new species of flaviviridae, and can infect crustacean.
In order to achieve the purpose, the invention adopts the following technical scheme.
An Infectious early virus (IPSFV), belonging to Flaviviridae family, with preservation number of CCTCC number V201870.
The infectious prematurity virus is spherical, is a single-stranded positive-strand RNA virus and has the diameter of 40-60 nm. Capable of infecting macrobrachium rosenbergii (Macrobrachium rosenbergii) And the like.
The infectious prematurity virus belongs to the infectious prematurity virus (I) (II)Infectious precocity flavivirus) Or viruses of the virus species named by other names. Since infectious precocious viruses are RNA viruses, their dependence on replicating their own genomic RNARNA polymerase in RNA lacks 3 '-5' exonuclease activity, so the enzyme has no error correction capability in the replication process, all or part of the genome has large variability, variation with homology of more than or equal to 82.7 percent may exist in fragments of all or part of specific sequences of the genome, and progeny viruses containing variant bases still have the capability of infecting and continuously replicating progeny in similar hosts. As a new virus, the International Committee for viral Classification (ICTV) may give other nomenclature rules, and thus Infectious precocity virus (Infectious precocity virus) is used as a generic term for the virus to refer to the same name virus protected by the present invention as well as all other names representing viruses that share gene or polypeptide sequence homology. The determined genome sequence of the infectious early-maturing virus CCTCC number V201870 is shown as SEQID NO. 1. The translated amino acid sequences of the genomic sequence include, but are not limited to: ORF1 has a 968 start site, a-1 programmed ribosomal frame shifting (PRF) at 5518, a 12090 stop site, and 3707 amino acids in total; ORF2 has a start site of 968 and a stop site of 5560 for a total of 1530 amino acids; ORF3 has a start position of 5686 and a stop position of 12090, with a total of 2134 amino acids.
The infectious prematurity virus is a single-stranded RNA virus, the synthesis of genome RNA is directly replicated by RNA-dependent RNA polymerase, the RNA-dependent RNA polymerase lacks 3 '-5' error correction function in the RNA replication process, the genome is easy to mutate during natural replication, the variation with homology of more than or equal to 82.7 percent may exist in the whole or partial specific sequence segment of the genome, and the virus with the variation of the genome sequence also belongs to the infectious prematurity virus. The genomic sequence of the infectious prematurity virus referred to in the present invention may therefore be:
(a) the method comprises the following steps An RNA sequence shown as SEQ ID NO. 1; or
(b) The method comprises the following steps RNA sequence with homology more than or equal to 82.7% with partial segment of (a); or
(c) The method comprises the following steps RNA sequence with homology more than or equal to 82.7% with the whole sequence of (a); or
(d) The method comprises the following steps A sequence formed by joining a plurality of partial fragments conforming to the characteristics of (a) or (b).
Also present in the infectious prematurity virus infected tissue is a nucleic acid sequence comprising the following sequence features:
(a) the method comprises the following steps A partial RNA sequence which is reverse complementary to the sequence shown in SEQ ID NO. 1; or
(b) The method comprises the following steps The complete RNA sequence which is reverse complementary to the sequence shown in SEQ ID NO. 1; or
(c) The method comprises the following steps An RNA sequence having a homology of 82.7% or more with the partial fragment of (a) or (b); or
(d) The method comprises the following steps RNA sequence with homology more than or equal to 82.7% with the whole sequence of (a) or (b); or
(e) The method comprises the following steps A sequence formed by joining a plurality of partial fragments conforming to the characteristics of (a) or (c).
The infectious precocious virus also contains a protein or polypeptide translated from its genomic nucleic acid sequence whose amino acid sequence meets the following characteristics:
(a) the method comprises the following steps 1, the amino acid sequence translated from all or part of the sequence of the genome of SEQ ID NO; or
(b) The method comprises the following steps An amino acid sequence translated from a sequence having at least 82.7% homology to all or part of (a).
A kit for detecting the infectious prematurity virus. The detection sequence of the kit is as follows:
(a) the method comprises the following steps 1, SEQ ID NO; or
(b) The method comprises the following steps (a) A converted DNA sequence; or
(c) The method comprises the following steps (a) And (b) an RNA or DNA sequence that is more than or equal to 82.7% homologous; or
(d) The method comprises the following steps An RNA or DNA sequence of (a), (b) or (c) comprising degenerate bases; or
(e) The method comprises the following steps An RNA or DNA sequence in which the degenerate base in (d) is replaced with hypoxanthine or other equivalent base; or
(f) The method comprises the following steps RNA and DNA sequences reverse complementary to (a) or (b) or (c) or (d) or (e); or
(g) The method comprises the following steps (a) Part of any of sequences (a) - (f).
Optionally, the length of the detection sequence is 12 nt to 3000 nt; more preferably 30 nt to 1200 nt; most preferably, the length is 40 nt to 600 nt.
A kit for detecting infectious prematurity virus, the detection sequence of which is the following amino acid sequence:
(a) the method comprises the following steps 1, a polypeptide sequence translated in whole or in part of the nucleic acid sequence of SEQ ID NO; or
(b) The method comprises the following steps A translated polypeptide sequence which is wholly or partially homologous to (a) a nucleic acid sequence having > 82.7% homology.
The kit takes the nucleic acid sequence or the polypeptide sequence provided by the invention as a detection target, and adopts a nucleic acid amplification method, a nucleic acid probe method, an immune reaction of antigen-antibody recognition or a method of antigen recognition by a nucleic acid aptamer for detection.
Further, the kit also comprises 1 pair of or more than 1 pair of primers.
The primer may be a nucleic acid specific sequence of defined bases or a nucleic acid sequence containing degenerate bases, and may also be a sequence comprising locked nucleic acids. In order to detect nucleic acid sequences that are more than or equal to 82.7% homologous, the simplest way is to treat a mutation site where single nucleotide diversity exists as a degenerate site of the corresponding base, so that a sequence of degenerate bases containing a plurality of mutated bases can be used as a primer sequence; hypoxanthine or other equivalent bases can also be substituted for this degenerate mode.
The length of the primer is 9 nt-45 nt; preferably, the length is 15 nt to 30 nt; more preferably, the length is 18nt to 25 nt.
Further, the primer is selected from any 2 or more than 2 nucleic acid sequences of the forward (F) and reverse (R) complementary sequences as shown in SEQ ID NO:2-SEQ ID NO: 45, or the nucleic acid sequences of SEQ ID NO: 1 or the reverse complementary nucleic acid sequences thereof between 2.
When 1 pair of primers is included, conventional PCR detection, SYBR Green I-stained real-time fluorescent quantitative PCR detection, digital PCR detection, or helicase-dependent isothermal amplification (HDA) detection may be employed.
When more than 1 pair of primers are contained, nested PCR or multiplex PCR detection can be adopted, the nested PCR adopts 2 pairs of nested primers, wherein the 1 pair of primers at the outer side are used as outer primers of the nested PCR for the first-step amplification, and the 1 pair of primers at the inner side are used as inner primers of the nested PCR for the second-step amplification; multiplex PCR uses 2 or more pairs of mutually independent primers to detect infectious precocity viruses or variants thereof at multiple sites or of different genotypes.
The method can also adopt the 1 pair of primers as F3 and B3 primers according to the design requirement of loop-mediated isothermal amplification (LAMP) primers, and selects 4 sequences on the inner side of the primers, and designs FIP and BIP primers by assembly for LAMP detection; and 1 pair of primers can be designed in the single-stranded loop region to serve as LF and LB primers for enhancing amplification and be used for rapid LAMP detection.
In the primer design, 3nt-50nt artificial sequence, anchoring sequence or other designed sequence can be connected to the 5' end of the primer, and other amplification detection reactions are carried out according to the anchoring primer multiple amplification detection or gene chip detection principle.
The detection method may further comprise a nucleic acid probe. The sequence of the nucleic acid probe is selected from the forward and reverse complementary nucleic acid specific sequences shown as SEQ ID NO. 2-SEQ ID NO. 45, or the nucleic acid specific sequence of SEQ ID NO. 1 among 2 or the reverse complementary nucleic acid specific sequence thereof.
The nucleic acid probe can be labeled by the incorporation method of all nucleotides or specific nucleotides, such as DIG-dUTP, with Digoxin (DIG), fluorescein or radioactive isotopes; the probe may also be labeled with DIG, fluorescein, a reporter group, a fluorescence quencher group, a radioisotope, or the like, for example, FAM, HEX, VIC, or the like, labeled at the 5 'end of the probe, TAMRA, or the like, labeled at the 3' end; the kit can directly carry out independent in-situ hybridization or dot hybridization detection on the genome nucleic acid of the infectious prematurity virus or other variant homologous viruses, and can also be combined with a real-time quantitative PCR technology to carry out fluorescence quantitative PCR detection on a TaqMan probe or a Beacon probe.
The detection method of the kit comprises but is not limited to conventional Polymerase Chain Reaction (PCR), constant-temperature convection PCR, nested PCR, real-time fluorescence PCR, constant-temperature convection real-time fluorescence PCR, digital PCR, dot hybridization, in situ hybridization, loop-mediated isothermal amplification (LAMP), rolling circle amplification technology (RCA), single-primer isothermal amplification, helicase-dependent isothermal amplification technology (HDA), cross primer amplification technology and nucleic acid express isothermal detection amplification technology; but also can include but not limited to multiplex PCR, multiplex real-time fluorescence PCR, gene chip and chip detection for 1 strain or more than 1 infectious precocious virus and homologous species or variant species thereof; but also can include, but is not limited to, the simultaneous detection of multiple PCR, multiple real-time fluorescence PCR, gene chip and chip for infectious prematurity virus and homologous species or variant species thereof.
The detection method of the kit also comprises a detection method adopting immune reaction. The detection object or detection target is:
(a) the method comprises the following steps 1 or the whole or part of the polypeptide or protein translated by the nucleic acid sequence with more than or equal to 82.7 percent of homology is antigen; or
(b) The method comprises the following steps (a) The antibody or aptamer of (a).
The antigen may be a viral strain whole, a split component, a capsid, a polypeptide, a genetically engineered protein or polypeptide. The antibody may be a polyclonal antibody, a hybridoma cell, a monoclonal antibody or a single chain antibody. The aptamer can be single-stranded RNA, single-stranded DNA or single-stranded locked nucleic acid sequence of one or more nucleic acid sequences obtained by screening one or more polypeptide specific sequences by SELEX technology, and the specific aptamer can be prepared by nucleic acid amplification, nucleic acid cloning or artificial synthesis. The antigen, antibody or aptamer may be prepared according to methods of the prior art.
The detection method of the kit can adopt competitive, indirect or sandwich type immunoreaction and also can adopt a solid support or an immunoprecipitation method. The antibodies or antigenic fragments of the infectious prematurity virus may be labeled using known techniques, such as fluorescent, chemiluminescent, radioactive or enzymatic labels. The signal of the amplified probe can be detected by methods of biotin and avidin, enzyme labeling and mediated immunoassay, such as ELISA test.
The scope of the present invention also includes antisera against the above-mentioned infectious prematurity virus, polyclonal antibodies, hybridoma cells, monoclonal antibodies, single-chain antibodies or strains or cell strains expressing single-chain antibodies, aptamers or strains expressing production aptamers cloned. The antiserum, the polyclonal antibody, the hybridoma cell, the monoclonal antibody, the single-chain antibody or the aptamer is prepared by using a virus strain of the infectious prematurity virus containing a polypeptide specific sequence, a splitting component of the virus strain, a genetic engineering protein or polypeptide of the virus strain as an immunogen according to a method in the prior art.
For example, where the antibody against the infectious precocious virus is a monoclonal antibody, the concentrated infectious precocious virus strain containing the specific sequence of the polypeptide or a specific antigenic fragment thereof, e.g., the antigenic proteins of ORF1, ORF2, ORF3, can be administered to an animal, e.g., a mouse, optionally with the addition of suitable adjuvants, e.g.: freund's complete adjuvant for primary immunization. After a suitable time interval, a second immunization is administered, optionally with an inactivated virus (or antigenic protein) and a suitable adjuvant. After appropriate time intervals, sera from immunized animals were collected to evaluate mice suitable for collecting spleen cells. Spleen cells and myeloma cells were collected from the suitable mice, such as: the FO cell line and the NS cell line were subjected to cell fusion with PEG. And (3) screening hybridoma cell strains with secretion capacity from the fusion cells to obtain fusion cell lines, wherein the fusion cell lines can secrete the monoclonal antibody against the infectious prematurity virus.
The invention has the following advantages:
the infectious prematurity virus is a new infectious prematurity virus and can infect macrobrachium rosenbergii. The invention provides a specific detection method based on the characteristics of infectious prematurity virus genome sequence, such as nucleic acid amplification, nucleic acid hybridization, antigen combination, nucleic acid aptamer recognition and the like, and a kit, detection equipment and application of detection analysis based on the method, and provides new possibility for detection and prevention and control of infectious prematurity virus of crustaceans aquatic products.
Biological preservation information
Infectious precocious virus (Infectious precocity flavivirus), the preservation number is CCTCC No. V201870, the preservation unit: china Center for Type Culture Collection (CCTCC), preservation address: the preservation center of Wuhan university in Wuhan, China has the following preservation date: 24/1/2019.
Drawings
FIG. 1 is an agarose gel electrophoresis image of the detection of infectious precocious virus genomes;
FIG. 2 is an electron microscope picture of Macrobrachium rosenbergii infected with infectious prematurity virus;
FIG. 3 is a phylogenetic evolutionary tree of infectious early maturing viruses;
FIG. 4 is a 2% agarose gel electrophoresis image of the detection of infectious precocious virus;
FIG. 5 is a 2% agarose gel electrophoresis of the detection of infectious precocious virus based on reverse complement sequences;
FIG. 6 shows the LAMP detection result of the infectious precocious virus;
FIG. 7 shows the result of fluorescence quantitative RT-qPCR detection of infectious prematurity virus;
FIG. 8 results of nucleic acid homology analysis of different infectious precocious virus isolates;
FIG. 9 results of amino acid homology analysis of different infectious precocious virus isolates.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
EXAMPLE 1 isolation, purification and characterization of infectious prematurity Virus
1.1 isolation and purification
(1) Adding 2-3 g of Macrobrachium rosenbergii cephalospora into a centrifuge tube, pre-cooling TNEP buffer solution and 200 mu L of PMSF isopropanol solution, and homogenizing for 10s at 10,000 rpm of a homogenizer under ice bath;
(2) centrifuging the homogenate at 10,000 g for 30 min at 4 deg.C in a high speed centrifuge, and retaining the supernatant;
(3) sequentially filtering out tissue debris, bacteria and other impurities by using filters with pore diameters of 0.45 mu m and 0.22 mu m; centrifuging the supernatant at 120,000 g for 240 min at 4 deg.C in an ultracentrifuge, soaking in TN buffer solution and slowly shaking to suspend the precipitate, and sucking 10 μ L for electron microscope observation;
(4) according to TEM scanning results, a concentration gradient of 20%, 31.5%, 43%, 54.5% and 66% of sucrose (W/W) is paved in an ultracentrifuge tube from top to bottom, after the ultracentrifuge tube is placed overnight at 4 ℃, supernatant is added to carry out centrifugation for 240 min at 120,000 g at 4 ℃, and after desugaring, viruses are obtained and are temporarily named as Infectious precocity viruses (Infectious precocity viruses);
(5) whole genome amplification of viral sequences: on the basis of obtaining a part of novel infectious precocious virus sequences by high-throughput sequencing, primers are designed on the basis of the obtained part of the novel infectious precocious virus sequences for Amplification and verification of a long fragment of the whole genome (FIG. 1), and then the 3 'end and the 5' end are amplified by using SMARTER RACE cDNA Amplification Kit to obtain the whole genome sequences, as shown in SEQ ID NO: 1.
TABLE 1 infectious precocious Virus genome sequencing primers
1.2 species identification
The picture of the infectious prematurity virus by electron microscope is shown in figure 2, the virus is spherical and has a diameter of 40-60 nm. Based on the amino acid sequence of RNA-dependent RNA polymerase (RdRp) of a virus belonging to the family Flaviviridae (genus), alignment was performed using Muscle software, a maximum similarity evolutionary tree was constructed using Mega software (FIG. 3), and a phylogenetic evolutionary tree based on RNA-dependent RNA polymerase (RdRp) (FIG. 3) showed that the virus belongs to a new species of the family Flaviviridae. It is preserved with the preservation number of CCTCC number V201870.
Example 2 infection of Macrobrachium rosenbergii with infectious precocious Virus
Taking a diseased macrobrachium rosenbergii sample to prepare homogenate, carrying out an artificial infection experiment on the diseased macrobrachium rosenbergii sample through feeding infection and dipping bath infection after aseptic filtration and ultracentrifugation, wherein the macrobrachium rosenbergii can be infected by two modes, and the main symptoms of the infected macrobrachium rosenbergii are shown as late growth and early sexual maturity. An electron micrograph of macrobrachium rosenbergii infected with the infectious prematurity virus is shown in fig. 2. As can be seen from the pictures, the virus particles of the infectious prematurity virus can be seen in the macrobrachium rosenbergii samples.
EXAMPLE 3 PCR kit for detecting infectious precocious Virus
Primers were synthesized according to the sequences in table 3, and PCR kits for detecting infectious prematurity virus were prepared with the components at the concentrations in table 2:
TABLE 2 PCR kit composition for detecting infectious precocious viruses
TABLE 3 primer sequences for detection of infectious precocious viruses
Detection was performed with reference to the following methods:
taking a diseased macrobrachium rosenbergii sample 20 mg and samples with tembusu virus (TMUV), macrobrachium rosenbergii nodavirus (MrNV), yellowhead virus genotype 8 (YHV-8), dying nodavirus (CMNV) and healthy macrobrachium rosenbergii in 1.5mL of RNase-free EP tubes respectively, grinding, and adding 0.75mL of TRIzolTMFully shaking and uniformly mixing the reagents, and standing at room temperature for 5 min; centrifuging at 4 deg.C and 12000 rpm for 5min, and collecting supernatant and placing into a new microcentrifuge tube without RNase; adding 1/5 volumes of chloroform, shaking to mix vigorously for 15sec, and standing at room temperature for 5 min; centrifuge at 12000 rpm for 15min at 4 ℃. Carefully sucking the upper aqueous phase, and respectively transferring the upper aqueous phase into a new microcentrifuge tube without RNase; adding isopropanol with the same volume, turning the centrifuge tube upside down, mixing thoroughly, and standing at room temperature for 10 min; the mixture was centrifuged at 12000 rpm for 10min at 4 ℃ and the supernatant was decanted. Adding 1 mL of 75% ethanol without RNase, washing the precipitate by turning upside down, centrifuging at 7000 rpm at 4 ℃ for 5min, and carefully pouring off the supernatant; the precipitate was air dried at room temperature. Adding 20 μ L RNase-free water to dissolve RNAThe pellet was used immediately for RT-PCR or stored at-80 ℃ until use. Just before use, the concentration of the RNA template is adjusted to be 500 ng/muL. Reverse transcription into cDNA, followed by nested PCR amplification using the systems shown in tables 4 and 5:
TABLE 4 first round PCR amplification System
TABLE 5 second round PCR amplification System
The specific reaction system and conditions are as follows:
a large volume premix was prepared except for Taq DNA polymerase, and stored at-20 ℃ in aliquots. Before detection, sucking the enzyme-free premixed solution, adding Taq DNA polymerase with a corresponding volume in proportion, uniformly mixing, and subpackaging 24 mu L/tube. First round amplification procedure: denaturation at 94 deg.C for 2 min; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 55 s; extension at 72 ℃ for 10 min. The second round of amplification procedure was: denaturation at 94 deg.C for 2 min; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 30 s; extension at 72 ℃ for 10 min.
The PCR product was electrophoresed on a 2% agarose gel, and the results are shown in FIG. 4. In FIG. 4 (top), a primary amplified band of 871nt was observed. A319 nt secondary amplification band was observed in FIG. 4 (bottom). In FIG. 4, TMUV (1), MrNV (2), YHV (3), CMNV (4), normal Macrobrachium rosenbergii (6) and water (7) did not show amplified bands in one and two rounds, while diseased Macrobrachium rosenbergii (5) respectively amplified a 871nt band in one round and a 319 nt band in two rounds.
Example 4 PCR kit for detecting infectious precocious Virus designed based on reverse complementary sequence
Primers were synthesized according to the sequences in table 5, and PCR kits for detecting infectious prematurity virus were prepared with the components at the concentrations in table 6:
TABLE 6 PCR kit composition for detecting infectious precocious viruses
TABLE 7 primer sequences for detection of infectious precocious viruses
Detection was performed with reference to the following methods:
taking a diseased macrobrachium rosenbergii sample 20 mg and a sample with TMUV, MrNV, YHV-8, CMNV and healthy macrobrachium rosenbergii, respectively, adding into a 1.5mL RNase-free EP tube, grinding, and adding 0.75mL TRIzolTMFully shaking and uniformly mixing the reagents, and standing at room temperature for 5 min; centrifuging at 12000 rpm at 4 deg.C for 5min, and respectively taking supernatant into a new microcentrifuge tube without RNase; adding 1/5 volumes of chloroform, shaking to mix vigorously for 15sec, and standing at room temperature for 5 min; centrifuge at 12000 rpm for 15min at 4 ℃. Carefully sucking the upper aqueous phase, and respectively transferring the upper aqueous phase into a new microcentrifuge tube without RNase; adding isopropanol with the same volume, turning the centrifuge tube upside down, mixing thoroughly, and standing at room temperature for 10 min; the mixture was centrifuged at 12000 rpm for 10min at 4 ℃ and the supernatant was decanted. Adding 1 mL of 75% ethanol without RNase, respectively, reversing the solution to wash the precipitate, centrifuging at 4 ℃ and 7000 rpm for 5min, and carefully pouring off the supernatant; the precipitate was air dried at room temperature. Adding 20 mu L of RNase-free water to dissolve the RNA precipitate, and immediately using for RT-PCR or storing at-80 ℃ for later use. Just before use, the concentration of the RNA template is adjusted to be 500 ng/muL. Reverse transcription into cDNA, followed by nested PCR amplification using the systems shown in tables 8 and 9:
TABLE 8 first round PCR amplification System
TABLE 9 second round PCR amplification System
The specific reaction system and conditions are as follows:
a large volume premix was prepared except for Taq DNA polymerase, and stored at-20 ℃ in aliquots. Before detection, sucking the enzyme-free premixed solution, adding Taq DNA polymerase with a corresponding volume in proportion, uniformly mixing, and subpackaging 24 mu L/tube. First round amplification procedure: denaturation at 94 deg.C for 2 min; 30 cycles of 94 ℃ for 30s, 59 ℃ for 30s and 72 ℃ for 65 s; extension at 72 ℃ for 10 min. The second round of amplification procedure was: denaturation at 94 deg.C for 2 min; 30 cycles of 94 ℃ for 30s, 59 ℃ for 30s and 72 ℃ for 30 s; extension at 72 ℃ for 10 min.
The PCR product was electrophoresed on a 2% agarose gel, and the results are shown in FIG. 3. In FIG. 5 (top), a primary amplified band of 1038nt was observed. A secondary 395 nt amplified band was observed in FIG. 5 (bottom). In FIG. 5, TMUV (1), MrNV (2), YHV (3), CMNV (4), normal Macrobrachium rosenbergii (5) and water (6) did not show amplified bands in either round, while diseased Macrobrachium rosenbergii (7) amplified 1038nt of the band of interest in one round and 395 nt of the band of interest in the second round, respectively.
Example 5 LAMP detection of specific sequences of infectious precocious Virus
Primers shown in SEQ ID NO: 38-43 in Table 10 were first synthesized, and then the detection of infectious precocious viruses was performed according to the following procedure:
TABLE 10 LAMP primers for infectious precocious viruses
(1) Preparation of template RNA: taking 20 mg of macrobrachium rosenbergii sample into a 1.5mL RNA enzyme-free EP tube, grinding, and adding 0.75mL TRIzolTMFully shaking and uniformly mixing the reagents, and standing at room temperature for 5 min; centrifuging at 12000 rpm at 4 deg.C for 5min, and collecting supernatant to a new microcentrifuge tube without RNase; adding 1/5 volumes of chloroform, shaking to mix vigorously for 15sec, and standing at room temperature for 5 min; centrifuge at 12000 rpm for 15min at 4 ℃. Carefully sucking the upper aqueous phase, and transferring the upper aqueous phase into a new microcentrifuge tube without RNase; adding isopropanol with the same volume, turning the centrifuge tube upside down, mixing well,standing at room temperature for 10 min; the mixture was centrifuged at 12000 rpm for 10min at 4 ℃ and the supernatant was decanted. Adding 1 mL of 75% ethanol without RNase, washing the precipitate by turning upside down, centrifuging at 7000 rpm at 4 ℃ for 5min, and carefully pouring off the supernatant; the precipitate was air dried at room temperature. Adding 20 mu L of RNase-free water to dissolve the RNA precipitate, and immediately using for RT-PCR or storing at-80 ℃ for later use. Adjusting the concentration of the RNA template to be 500 ng/mu L before use;
(2) preparing a reaction system: 0.2 mu M each of the primer HE-F3 and the primer HE-E3, 0.8 mu M each of the primer HE-FIP and the primer HE-BIP, 1.6 mu M each of the primer HE-LF and the primer HE-LB, 1.4 mM each of dNTP, MgCl 26 mM, betaine 1.2M, Tris-HCl20 mM, KCl 10 mM, MgSO 42 mM，(NH4)2SO410 mM, triton X-1000.1%,Bstand 8U of DNA polymerase, and adding sterile double distilled water to enable the final volume of each tube to be 25 muL. After the reaction system is prepared, uniformly mixing, and subpackaging into a sterile EP (EP) tube (the specification is 200 mu L);
(3) the gene amplification reaction was carried out according to the following reaction procedure: keeping the temperature at 63 ℃ for 40 min, and keeping the temperature at 80 ℃ for 5 min;
(4) judging a detection result: after the reaction is finished, adding a nucleic acid dye GeneFinder into the reaction systemTMAnd 0.5 muL, then observing the color of the reaction product of the detected sample in the sun, wherein if the color is green fluorescence, the result of the detection of the infectious precocity virus of the sample is positive, and if the color is light orange, the result of the detection of the infectious precocity virus of the sample is negative.
By using the method, macrobrachium rosenbergii samples with infectious prematurity viruses, samples with tembusu virus (TMUV) and penaeus vannamei samples with White Spot Syndrome Virus (WSSV) from different sources are detected, and sterile water is additionally arranged as a negative control, so that the detection result shows that the macrobrachium rosenbergii sample tube with the infectious prematurity viruses as a template shows green fluorescence, and the templates and the sterile water tubes from other sources all show light orange (figure 6).
EXAMPLE 6 fluorescent quantitative RT-PCR assay kit for detecting infectious prematurity Virus
Primers and probes were synthesized according to the sequences in table 12, and the fluorescent quantitative RT-PCR assay kit for detecting infectious prematurity viruses was prepared according to the concentrations in table 11 with the components:
TABLE 11 fluorescent quantitative RT-PCR kit composition for detecting infectious prematurity virus
TABLE 12 fluorescent quantitative RT-PCR primer sequence and probe sequence for detecting infectious prematurity virus
Taking a diseased macrobrachium rosenbergii sample 20 mg and a sample with TMUV respectively in a 1.5mL RNase-free EP tube, grinding, and adding 0.75mL TRIzolTMFully shaking and uniformly mixing the reagents, and standing at room temperature for 5 min; centrifuging at 12000 rpm at 4 deg.C for 5min, and respectively taking supernatant into a new microcentrifuge tube without RNase; adding 1/5 volumes of chloroform, shaking to mix vigorously for 15sec, and standing at room temperature for 5 min; centrifuge at 12000 rpm for 15min at 4 ℃. Carefully sucking the upper aqueous phase, and respectively transferring the upper aqueous phase into a new microcentrifuge tube without RNase; adding isopropanol with the same volume, turning the centrifuge tube upside down, mixing thoroughly, and standing at room temperature for 10 min; the mixture was centrifuged at 12000 rpm for 10min at 4 ℃ and the supernatant was decanted. Adding 1 mL of 75% ethanol without RNase, respectively, reversing the solution to wash the precipitate, centrifuging at 4 ℃ and 7000 rpm for 5min, and carefully pouring off the supernatant; the precipitate was air dried at room temperature. Adding 20 mu L of RNase-free water to dissolve the RNA precipitate, and immediately using for fluorescent quantitative RT-PCR or storing at-80 ℃ for later use. Just before use, the concentration of the RNA template is adjusted to be 500 ng/muL, and then the primers shown in Table 12 are used for carrying out fluorescent quantitative RT-PCR amplification.
The specific reaction system and conditions are as follows:
TABLE 13 fluorescent quantitative RT-PCR reaction system for detecting infectious prematurity virus
The volume of each component of the reaction system is calculated according to the quantity of the samples, the reaction system is added into a 1.5mL RNA-free enzyme centrifuge tube under the condition of keeping out of the sun, one sample is made into three parallel samples, and the three parallel samples are fully inverted, uniformly mixed and quickly subpackaged into eight rows of tubes. And adding the RNA of the sample into the eight rows of tubes in sequence, centrifuging and carrying out quantitative PCR. The specific reaction conditions are as follows: reverse transcription at 55 deg.C for 10min, and denaturation at 95 deg.C for 1 min; then 95 ℃ for 10s, 60 ℃ for 30s, 40 cycles.
The detection results showed that TMUV and negative control did not peak, positive control and positive test samples did peak (fig. 7).
Example 7 ELISA detection kit for detecting infectious prematurity Virus antigen
The kit comprises the following components: 10:1 rabbit anti-infectious prematurity virus polyclonal antibody coated 96-well plate 1 block, bovine serum albumin confining liquid 10 mL, 160:1 mouse anti-infectious prematurity virus monoclonal antibody 0.5 mL, 160:1 horse radish peroxidase goat anti-mouse IgM secondary antibody enzyme conjugate 0.5 mL, TMB color development liquid, 2M sulfuric acid, tissue lysate 5mL, washing liquid (PBST, NaCl 0.8 g, KH)2PO40.02 g,Na2HPO4·12H20.29 g of O, 0.02 g of KCl, 200.05 mL of Tween200.05 mL of sodium azide, 100 mL of double distilled water with pH 7.4), 5mL of PBS sample diluent, 1 positive control and 1 negative control. The detection is carried out according to the following steps:
(1) coating and sealing: diluting the antibody to a proper concentration, adding 100 muL into each hole, and placing at 37 ℃ for 4 h; discard the liquid in the wells (to avoid evaporation, the plates should be capped or placed flat in a metal wet box with wet gauze at the bottom); 5% calf serum was blocked at 37 ℃ for 40 min. When the reaction is sealed, the sealing liquid is filled in each reaction hole, air bubbles in each hole are removed, and after the sealing is finished, the holes are filled with the washing liquid for 3 times, and each time is 3 min. The washing method comprises the following steps: sucking reaction liquid in the holes, filling the hole with washing liquid, placing for 2min for slight shaking, sucking liquid in the holes, pouring out the liquid, and patting on absorbent paper to dry;
(2) the sample to be tested is added (a suitable concentration gradient is established): the dilution of 1:50-1:400 is generally adopted during detection, and the sample suction amount is guaranteed to be more than 20 muL. And adding the diluted samples into enzyme-labeled reaction holes, adding at least two holes in each sample, wherein each hole is 100 mu L, and incubating for 60min at 37 ℃. Washing with washing solution for 3 times, each time for 3 min;
(3) adding an enzyme-labeled antibody: according to the reference working dilution provided by the enzyme conjugate supplier. Incubate at 37 ℃ for 60 min. Adding 100 mu L to each hole. Washing the mixture as before;
(4) adding substrate solution (prepared as used): adding 100 muL of TMB-hydrogen peroxide urea solution into each hole, and placing the hole at 37 ℃ in a dark place for 3-5 minutes;
(5) and (3) terminating the reaction: adding 50 muL of stop solution into each hole to stop the reaction, and measuring the experimental result within 20 min;
(6) and (5) judging a result: and detecting the absorbance value of each hole under the wavelength of 450 nm, and judging the result, wherein the result is shown in a table 14:
TABLE 14 ELISA test results
Example 8 high throughput sequence analysis of infectious precocious Virus
The infected macrobrachium rosenbergii in the example 2 is determined to be positive by the infectious prematurity virus through an electron microscope and the example 3, and then 6 positive samples are selected. Meanwhile, 2 infectious prematurity virus positive samples obtained by epidemiological sampling using the methods of example 3, example 5 and example 6 were used. After 8 samples were RNA-extracted, host ribosomal RNA was removed using a commercial kit, a library was created, Illumina Hiseq was used for sequencing, sequencing data was obtained and assembled, and Genious (version 11.1.5) and DNAstar biological software were used for analysis, which revealed that the infectious prematurity virus had the lowest nucleic acid homology of 82.7% (FIG. 8) and the lowest amino acid homology of 84.2% (FIG. 9) among the 8 samples.
Sequence listing
<110> research institute for aquatic products in yellow sea of China institute for aquatic science
<120> crustacean flavivirus, and detection method and application thereof
<130>20191105
<160>1
<170>PatentIn version 3.5
<210>1
<211>12630
<212>RNA
<213>Infectious precocity flavivirus
<400>1
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cucuccuugg ccgccaccca guuucucaug caucgaugga ucggcuuggc ugguuaugug 9600
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gcuggggagg agucuuaugu auccacgucu acaucaaugg aucucucacu cuuggauggu 10020
ccucaaaccc ucuuccgccu ccaugccgcc cuggacccaa gagucuugga ccuaauggaa 10080
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gaccugagag cuucuuuuuc uuggcacaag augggaucaa auucugaggu agucaacaga 10440
guaauagcuu ucuucaccuc uaaguucgcu ccuuauuuua gguacuauca aacucuuuac 10500
agagccuaca ccuuaacuuc aacaaaaauu ugggaugugu gguacauguu uaaguugaag 10560
uaugauaggc caaauaaagu acuuacugag cagcaauaug aggagaucuc agcuauauau 10620
gaugaccugu cccggaaucu gccaagcaug ccccugcuua cagagcagga gauuggagca 10680
gcccucaacc gcaaaggagc cagcggaaug cccccuccgu augacaacaa aaccuugggg 10740
gaacucuggg acgaugacau cuucagggcu ggcuuuuggg auuuuguuuc agagguuaga 10800
ucuggaauug ucaccggccu cgaguuuuuc cauacgaugg gcaaaagaga gaagaaagau 10860
cuccucuaca aaccaagaag aaguucccga cugauagccu aucuuccugc uuacuauaga 10920
cuucucgagu uacacguuuu cggaucaacg auacguugga cucgagauga uuugccuucu 10980
gcaguaaccc acauaccuuu gguagauuau ggcgaucuga uggcuggcuu ugaagcauac 11040
gaagccaaug auguggaggc augggauaca aaggucggua gucaauuucu agauuuggag 11100
gaucauuucu uuagaggucu cucgcaugau ccucuucuag uugagagaau auaugagcuu 11160
uauaggagcc cccuuauacu ucuuccaaua uaugaggggg gggacuacaa agcucauuug 11220
auucaaggcc uugggaagag aaugaguggg acuuacguca cguacauugc caacacuguc 11280
accaauacug uccuuaacug cuacagggcc aaaagagccu uuuccugcac cacacaagag 11340
gucuuuagua aacucucuuu uuucauauca ggagaugacc ugguuauagc aggaacucuu 11400
gaaaauaugc agcgccuggc agaacaggac guccaugaug aguuaggcuu caaucuuaaa 11460
ggaggcuaca aacccauaac ugagaacuuu gaagagauag acuuuuguuc acaccauuau 11520
accaagguug ugaagaagga uaagaaaagu caugagaucc ucuuuucaaa guggaugcca 11580
guccgcccca uccccgagau auuugggaga gcccgucuuc uugugggugc ucuggguggc 11640
gaguugaugg ggcguuggac uucgguagaa agagcucacg cagcaacugu gggccgccag 11700
cuuuuguuca acuacuggca ccuaagagau gucaggcacu uggccuuagg ucuaugcagu 11760
guagccccag aaaacauguu uuugcuuggu aaacaaucag uugcuaccac cccuaaacca 11820
uggcuuaacc aagaaaaggu ggacgagaug augcuucgcu gguuuucacu cccucuaaau 11880
gagcucccau augugcgcca ucgaguggac auggaacuug gcaguacuau ucacgucaaa 11940
gaagcuuuaa cagcaucuag gcgagcugcc augcuugauu auuaugagga gaucugcagu 12000
gaguguuauc ucguuggugg gcgcaacugg cucgaugcua uggcaaagua ucgcuacggg 12060
ucgguuggga gagcccccuc cuucuuuuag aagggacucu ccauccugua caguguaaau 12120
auuugugucg cacucacuuu guaaauguaa gcacgcuuuu gggcaggcau gcccuuacug 12180
uaagaaaaug ccccaaauuc cucucuuagg caaggagguu ucugguuugc acuaucgccu 12240
guucgugaug cuaugggguu agcauugcgu ucuguauuua ccccccgaug agacguugag 12300
cgaaucucau uguacauagc ucagaccuug aaaguuuuga gacucccuuc aaggccuaug 12360
uauuagaguc ucguuuuuaa guuuacacac cgaaggucgu aagaccuucg gcuaauuggc 12420
cacagacaua cagauuaaac cauuggcuua aagccacucu acuguggcuc auuucaaauu 12480
gaauccaaau uugacaugua uacaccaccu ccaccugggg uuuaacaggu ggggaguacu 12540
acucacgcaa ccacagcgca cuaaugaguc cgaaaaauuu cggugcgcug gggaugugag 12600
aguaacauuc caaaaaaaaa aaaaaaaaaa 12630
Claims (10)
1. An Infectious precocious flavivirus (Infectious precocity virus) with the preservation number of CCTCC No. V201870.
2. The infectious precocity virus of claim 1, having a genomic sequence homology of not less than 82.7% to the sequence shown in SEQ ID NO. 1; or
The homology of the amino acid sequence of the polypeptide with the translated amino acid sequence of the sequence shown in SEQ ID NO. 1 is not less than 84.2%.
3. Use of a virus according to any one of claims 1 to 2 in the preparation of a reagent for its detection.
4. A kit for detecting the virus of any one of claims 1-2, wherein the detection sequence is:
(a) the method comprises the following steps 1, or a part or all of the RNA sequence shown in SEQ ID NO; or
(b) The method comprises the following steps (a) A converted DNA sequence; or
(c) The method comprises the following steps (a) And (b) an RNA or DNA sequence that is more than or equal to 82.7% homologous; or
(d) The method comprises the following steps An RNA or DNA sequence of (a), (b) or (c) comprising degenerate bases; or
(e) The method comprises the following steps An RNA or DNA sequence in which the degenerate base in (d) is replaced with hypoxanthine or other equivalent base; or
(f) The method comprises the following steps Part or all of the RNA and DNA sequences reverse complementary to (a) or (b) or (c) or (d) or (e).
5. The kit of claim 4, wherein the method comprises at least 1 pair of primers comprising DNA or RNA fragments extracted from the viral genomic sequence of claim 2;
preferably, the primer is selected from a sequence shown in SEQ ID NO. 2-SEQ ID NO. 45 and a fragment in a sequence between any 2 sequences in SEQ ID NO. 2-SEQ ID NO. 45.
6. The kit according to claim 4, wherein the kit comprises 2 pairs of primers, the sequences of the outer primers are shown in SEQ ID NO 2 and 3 or SEQ ID NO 6 and 7, and the sequences of the inner primers are shown in SEQ ID NO 4 and 5 or SEQ ID NO 8 and 9.
7. The kit according to claim 4, wherein the detection method of the kit is selected from polymerase chain reaction, loop-mediated isothermal amplification, helicase-dependent isothermal amplification or nucleic acid hybridization;
preferably, the kit adopts a loop-mediated isothermal amplification method, wherein the F3 primer is shown as SEQ ID NO. 38, the B3 primer is shown as SEQ ID NO. 39, the FIP primer is shown as SEQ ID NO. 40, the BIP primer is shown as SEQ ID NO. 41, the LF primer is shown as SEQ ID NO. 42, and the LB primer is shown as SEQ ID NO. 43.
8. The kit according to claim 5, further comprising 1 or more than 1 probe;
preferably, the primer sequence of the kit is shown as SEQ ID NO. 44 and SEQ ID NO. 45, the fluorescent probe sequence is shown as SEQ ID NO. 46, the fluorescent group is FAM, and the quenching group is TAMRA.
9. A kit for detecting the virus of any one of claims 1-2, wherein the detection agent is:
(a) the method comprises the following steps 1, a polypeptide translated in whole or in part of the sequence shown as SEQ ID NO; or
(b) The method comprises the following steps A translated polypeptide in whole or in part which has a nucleic acid sequence which is more than or equal to 82.7% homologous to (a); or
(c) The method comprises the following steps (a) The antibody of (a) or (b); or
(d) The method comprises the following steps (a) The aptamer of (a) or (b).
10. The kit of claim 9, wherein the kit is characterized by using enzyme-linked immunosorbent assay, fluoroimmunoassay, immunochromatographic strip or specific amplification of aptamer sequence or other immunological or immunology-like detection established by polyclonal antibody, monoclonal antibody, single-chain antibody or aptamer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111588836A (en) * | 2020-05-21 | 2020-08-28 | 广东海洋大学深圳研究院 | Application of CD40 protein in preparation of product for improving immunity of tilapia to streptococcus |
| CN116376848A (en) * | 2022-12-13 | 2023-07-04 | 中国水产科学研究院黄海水产研究所 | Artemia virus and detection method and application thereof |
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| CN101671748A (en) * | 2009-10-21 | 2010-03-17 | 中山大学 | Primer for detecting infectious bursal disease viruses and method and kit thereof |
| CN109628638A (en) * | 2018-12-21 | 2019-04-16 | 中国水产科学研究院黄海水产研究所 | Detection method and its application based on prawn east virus genome sequence |
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| CN101671748A (en) * | 2009-10-21 | 2010-03-17 | 中山大学 | Primer for detecting infectious bursal disease viruses and method and kit thereof |
| CN109628638A (en) * | 2018-12-21 | 2019-04-16 | 中国水产科学研究院黄海水产研究所 | Detection method and its application based on prawn east virus genome sequence |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111588836A (en) * | 2020-05-21 | 2020-08-28 | 广东海洋大学深圳研究院 | Application of CD40 protein in preparation of product for improving immunity of tilapia to streptococcus |
| CN111588836B (en) * | 2020-05-21 | 2023-03-21 | 广东海洋大学深圳研究院 | Application of CD40 protein in preparation of product for improving immunity of tilapia to streptococcus |
| CN116376848A (en) * | 2022-12-13 | 2023-07-04 | 中国水产科学研究院黄海水产研究所 | Artemia virus and detection method and application thereof |
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