CN114369681A - Product and method for detecting bicistronic virus-3 - Google Patents

Abstract

The invention discloses a product and a method for detecting bicistronic virus-3. The invention discovers a novel bicistronic virus from the macrovirome sequencing of the Roche chinensis infected with the 'iron-shell shrimp' disease, and the novel bicistronic virus is tentatively named as bicistronic virus-3. On the basis of the obtained bicistronic virus genome sequence, a corresponding nested PCR and fluorescent quantitative PCR detection method is constructed by designing primers. The established nested PCR detection method has strong specificity and high sensitivity, and the minimum detection limit is 1 copy/mu L; the minimum detection limit of the established fluorescent quantitative PCR detection method is also 1 copy/. mu.L, the variation coefficients in groups and among groups do not exceed 5 percent, the repeatability is good, the detection result is reliable, and the method is suitable for detecting the bicistronic virus-3.

Description

Product and method for detecting bicistronic virus-3

Technical Field

The invention belongs to the technical field of aquatic virus detection. And more particularly to a product and method for detecting dicistronic virus-3.

Background

With the intensive research on viral aquatic diseases, more and more evidences indicate that more than one pathogen may cause viral aquatic diseases. For example, the pathogens of the crab green "lethargy" include two types of crab reovirus (McRV) and crab bicistronic virus (McDV). The mud crab bicistronic virus (McDV) is one of three bicistronic viruses occurring in aquatic animals, and comprises Taura Syndrome Virus (TSV) causing Taura syndrome of prawns and macrobrachium rosenbergii bicistronic virus (MrDV) causing macrobrachium rosenbergii juvenile syndrome in addition to McDV. The aquatic bicistronic virus brings a huge impact to the development of the aquaculture industry of China, particularly the breeding industry of crustaceans.

The occurrence of the disease of the macrobrachium rosenbergii, namely macrobrachium rosenbergii, brings a great attack to the culture of the macrobrachium rosenbergii, and the shrimp cannot die after being infected by the disease, but the growth of the shrimp is slow or the growth of the shrimp is stopped due to the phenomenon of precocious puberty, so that the waste of feed and the large-area serious yield reduction are caused. Establishing pathogeny is the key point of viral aquatic disease research, and if the pathogeny of the 'iron shell shrimp' disease can be established and a corresponding detection method is established, the infected Luo shrimp can be removed as soon as possible in the screening process of the seed shrimp or shrimp larvae and the like, the transmission of virus is inhibited, and the loss is reduced. The invention discovers a new dicistronic virus by enriching the virus in the siderophores and sequencing a macrovirus group, and the sequence comparison discovers that the virus is different from the known dicistronic virus (MrTV and McDV-2), so that the virus is named as the dicistronic virus-3 (MrDV-3). Chinese patent CN 103243180A discloses a method for detecting blue crab bicistronic mRNA virus-1 real-time fluorescence quantitative RT-PCR, a primer group, a probe and a kit thereof, but at present, no product and method for detecting MrDV-3 exist.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of primers, kits or methods for detecting MrDV-3, namely bicistronic mRNA virus-3 in the Luo shrimp in the prior art, and provides a product and a method for detecting the bicistronic mRNA virus-3.

It is a first object of the present invention to provide a target gene for detecting bicistronic virus-3.

The second purpose of the invention is to provide a primer group for detecting the bicistronic virus-3.

The third purpose of the invention is to provide a nested PCR primer for detecting bicistronic virus-3.

The fourth purpose of the invention is to provide a fluorescent quantitative PCR primer for detecting the bicistronic virus-3.

The fifth purpose of the invention is to provide the application of the target gene and the primer group in the preparation of products for detecting the bicistronic virus-3.

It is a sixth object of the present invention to provide a kit for detecting dicistronic virus-3.

The seventh purpose of the invention is to provide a nested PCR method for detecting bicistronic virus-3.

The eighth purpose of the invention is to provide a fluorescent quantitative PCR method for detecting the bicistronic virus-3.

The above purpose of the invention is realized by the following technical scheme:

the invention discloses a novel bicistronic virus through enrichment and macrovirome sequencing of viruses in the body of the siderophorus ferrugineus, the sequence alignment shows that the bicistronic virus is different from the known bicistronic viruses (MrTV and McDV-2), and the phylogenetic analysis result also shows that the bicistronic virus is far away from the genetic relationship between the MrTV and the McDV-2 and is named as the bicistronic virus-3 (MrDV-3).

According to the invention, a product and a method for detecting the bicistronic virus-3 in the shrimp roentgen are designed and developed by taking the nucleotide sequence of 4754-5524 th site in the genome as a target gene according to the genome sequence (shown as SEQ ID NO. 1) of MrDV-3 obtained by sequencing.

The invention firstly provides a target gene for detecting bicistronic mRNA virus-3 (MrDV-3), and the sequence of the target gene is shown as SEQ ID NO. 2. Experimental results show that the bicistronic virus-3 in a sample can be successfully detected by designing a primer aiming at the target gene shown in SEQ ID NO.2, so that the application of the invention protects the application of the target gene in preparing a product for detecting the bicistronic virus-3.

The invention also provides a primer group for detecting the bicistronic virus-3, which comprises a primer capable of amplifying the target gene shown in SEQ ID NO. 2.

Preferably, the primer group is nested PCR primers shown in SEQ ID NO. 3-6.

Preferably, the primer group is a fluorescent quantitative PCR primer shown in SEQ ID NO. 7-8.

The invention simultaneously applies and protects the application of the primer group in preparing products for detecting the bicistronic virus-3.

The invention also provides a kit for detecting the bicistronic virus-3, and the kit comprises a reagent for detecting the target gene shown in SEQ ID NO. 2.

Preferably, the reagent contains a primer group for detecting the bicistronic virus-3.

Preferably, the reagent contains nested PCR primers shown in SEQ ID NO. 3-6.

Preferably, the reagent contains a fluorescent quantitative PCR primer shown in SEQ ID NO. 7-8.

The invention also provides a recombinant plasmid, which contains a target gene sequence shown in SEQ ID NO. 2.

Preferably, the vector of the recombinant plasmid is pMD-19T, see example 2.

Preferably, the kit for detecting the bicistronic virus-3 contains the recombinant plasmid as a positive control.

The invention also applies to protect the application of the recombinant plasmid in preparing products for detecting the bicistronic virus MrDV-3.

The invention also provides a nested PCR method for detecting the bicistronic virus-3, which comprises the following steps:

s1, extracting tissue RNA of a sample to be detected and carrying out reverse transcription on the tissue RNA into cDNA;

s2, performing PCR amplification by using the cDNA obtained in the step S1 as a template and using nested PCR primers shown in SEQ ID NO. 3-6;

s3, detecting a PCR result through gel electrophoresis, wherein if a specific band with the size of 771bp appears in the first round or a specific band with the size of 449bp appears in the second round, the detected sample contains the bicistronic virus-3.

The invention also provides a fluorescent quantitative PCR method for detecting the bicistronic virus-3, which comprises the following steps:

s1, extracting tissue RNA of a sample to be detected and carrying out reverse transcription on the tissue RNA into cDNA;

s2, taking the cDNA obtained in the step S1 as a template, and carrying out qPCR reaction by using fluorescent quantitative PCR primers shown in SEQ ID NO. 7-8, wherein if an amplification curve appears in the qPCR result and the Ct value corresponding to the curve is less than 35, the result is regarded as a positive result; if no amplification curve exists or the Ct value corresponding to the curve is more than or equal to 40, the result is regarded as negative; if the Ct value corresponding to the curve is between 35 and 40, the experiment is recommended to be repeated, if the Ct value of the repeated result is less than 40 and the amplification curve has obvious peaks, the result is regarded as a positive result, otherwise, the result is negative.

Preferably, when the tissue sample is selected for RNA extraction, the tissue under the shell of the young shrimp, or the gill and muscle of the prawn, or the swimming foot of the parent shrimp is selected, as shown in example 3.

Preferably, the annealing temperature of the primers in the first round of nested PCR is 50 ℃ to 60 ℃, see example 3.

More preferably, the annealing temperature of the primers in the first round of nested PCR is 60 ℃ as shown in example 3.

Preferably, the annealing temperature of the primers in the second round of nested PCR is 50 ℃ to 60 ℃, see example 3.

More preferably, the annealing temperature of the primers in the second round of nested PCR is 60 ℃ as shown in example 3.

Specifically, the amplification system of the first round of nested PCR reaction is as follows: 2 × Accurate Taq Master Mix10 μ L, 5 μ M forward and reverse primers 0.5 μ L each, 1 μ L cDNA template, 8 μ L sterile water.

Specifically, the amplification procedure of the nested PCR first round reaction is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; then further extension at 72 ℃ for 10min, and finally storage at 4 ℃.

Specifically, if no positive band is detected in the first round of nested PCR amplification, diluting the product by 50-100 times with sterile water, and then using the diluted product as a template to perform the second round of PCR amplification.

Specifically, the amplification procedure of the nested PCR second round reaction is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; then further extension at 72 ℃ for 10min, and finally storage at 4 ℃.

Specifically, the reaction system of the fluorescent quantitative PCR is as follows: 1 μ L cDNA, primer F0.2 μ L primer R0.2 μ L2 × SYBR GREEN I MIX 5 μ L ddH₂Make up to 10. mu.L of O.

Specifically, the reaction procedure of the fluorescent quantitative PCR is: 5min at 95 ℃; at 95 ℃ for 10s, at 60 ℃ for 10s, at 72 ℃ for 10s, for 45 cycles; melting curve 95 deg.C for 5s, 65 deg.C for 1 min.

The invention has the following beneficial effects:

the invention designs and screens out a primer for detecting MrDV-3 on the basis of a target gene sequence of MrDV-3 shown in SEQ ID NO.2, and establishes a nested and fluorescent quantitative PCR method for detecting MrDV-3. The nested PCR detection method has the advantages of strong specificity, high sensitivity, good repeatability and reliable detection result, and the minimum detection limit is 1 copy/mu L. The fluorescence quantitative PCR detection method has high sensitivity, the lowest detection limit is also 1 copy/mu L, and the variation coefficients in groups and among groups are lower than 5 percent, which shows that the method has good repeatability and reproducibility and stable and reliable results. The PCR detection method can be used for detecting the bicistronic mRNA virus MrDV-3 in the culture process of the Luo shrimp, is used for screening the seed shrimp without toxicity, reduces the loss in the culture process, and is suitable for being popularized to basic aquaculture units.

Drawings

FIG. 1 shows the phylogenetic analysis of MrDV-3.

FIG. 2 shows the amplification results of nested PCR-based primers.

FIG. 3 shows the amplification results of nested PCR primers, wherein the left panel shows the amplification results of one amplification primer and the right panel shows the amplification results of two amplification primers.

FIG. 4 shows the result of detecting the optimal annealing temperature of nested PCR-amplified primers, which is repeated three times, wherein the annealing temperatures of lanes 1-6 are 50 deg.C, 50.9 deg.C, 53.3 deg.C, 55.7 deg.C, 56.8 deg.C and 59.9 deg.C, and lane 7 is a negative control.

FIG. 5 shows the result of detecting the optimal annealing temperature of nested PCR secondary amplification primers, which is repeated three times, wherein the annealing temperatures of lanes 1-6 are 50 deg.C, 50.9 deg.C, 53.3 deg.C, 55.7 deg.C, 56.8 deg.C and 59.9 deg.C, and lane 7 is a negative control.

FIG. 6 shows the results of the sensitivity detection of nested PCR-one amplification primers, which were repeated three times, and the template concentrations in lanes 1-8 were 10⁷copies/μL、10⁶copies/μL、10⁵copies/μL、10⁴copies/μL、10³copies/μL、10²copies/. mu.L, 10 copies/. mu.L and 1 copy/. mu.L.

FIG. 7 shows the results of the sensitivity detection of nested PCR primers, which were repeated three times, and the template concentrations in lanes 1-8 were 10⁷copies/μL、10⁶copies/μL、10⁵copies/μL、10⁴copies/μL、10³copies/μL、10²copies/. mu.L, 10 copies/. mu.L and 1 copy/. mu.L.

FIG. 8 shows the result of specific detection of nested PCR-amplified primers, lanes 1-4 correspond to positive samples of Infectious specificity virus, Microbrachiumrosenbrigeriporiornavirus 1, Microbrachiumrosenbergiidicistrovirus 3, mu crab dicostrirus and mu crab reovir, respectively, lane 5 is a positive control, and lane 6 is a negative control.

FIG. 9 shows the specific detection results of nested PCR secondary amplification primers, wherein lanes 1-4 correspond to positive samples of Infectious specificity virus, Microbrachiumrosenbrigeriporiornavirus 1, Microbrachiumrosenbergiidicistrovirus 3, mu crab dicostrirus and mu crab reovirus, respectively, lane 5 is a positive control, and lane 6 is a negative control.

FIG. 10 shows the detection results of nested PCR-PCR primers on 20 gill samples of Lobster rosenbergii in Huzhou, Zhejiang, where N is a negative control.

FIG. 11 shows the detection results of 20 Roche shrimp gills samples from Huzhou, Zhejiang with nested PCR double amplification primers, where N is a negative control.

FIG. 12 is a fluorescent quantitative PCR standard curve.

FIG. 13 shows the amplification curve of the fluorescent quantitative PCR primers, with the concentration of the A-H positive recombinant plasmids being 10⁸copies/μL～10copies/μL。

FIG. 14 shows the result of specific detection of fluorescent quantitative PCR, in which A is an Infectious specificity virus positive sample, B is a Microbrachiumrosenbergiiicrnavirus 1 positive sample, C is a Microbrachiumresenbergiidicistrovirus 3 positive sample, D is a mud crab Dicystrovirus and mud crab reovirus positive sample, E is a positive control, and F is a negative control.

FIG. 15 shows the results of fluorescence quantitative PCR primers on 20 Luo-shi shrimp gill samples from Huzhou city, Zhejiang province.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

EXAMPLE 1 design of nested PCR primers

1. Design of nested PCR primers

The invention enriches the viruses in the bodies of the decapterus ferrugineus shrimps, and finds a new bicistronic virus through macrovirus group sequencing, the sequence alignment finds that the viruses are different from the known bicistronic viruses (MrTV and McDV-2), and the phylogenetic analysis result (shown in figure 1) also shows that the viruses have a far relative relationship with the MrTV and the McDV-2, so the viruses are temporarily named as the bicistronic virus-3 (MrDV-3).

According to the invention, a PCR primer is designed by taking the nucleotide sequence of 4754-5524 th site in the genome as a target gene according to the genome sequence of MrDV-3 obtained by the macro virus to be detected. The genome sequence of MrDV-3 is shown as SEQ ID NO.1, and the target gene sequence is shown as SEQ ID NO. 2. Suitable nested PCR amplification primers MrDV-3-1-F/R and MrDV-3-2-F/R were obtained by designing nested PCR primers with Primer Premier 6 and strictly screening the designed primers by Primer select of Lasergene 7.1, which are shown in Table 1.

TABLE 1 nested PCR primers for MrDV-3 detection

The sizes of the fragments to be amplified by the nested PCR primer are 771bp and 449bp respectively. Wherein the primer MrDV-3-1-F is 24bp long and is positioned at 4754-4777 th site from the 5' end of the genome, and the primer MrDV-3-1-R is 22bp long and is positioned at 5503-5524 th site; the primer MrDV-3-2-F is 23bp long and is positioned at the 4935-4957 th site, and the primer MrDV-3-2-R is 23bp long and is positioned at the 5361-5383 th site.

EXAMPLE 2 construction of the Positive recombinant plasmid pMD19-T-MrDV-3

1. Cloning of the target Gene

Extracting, filtering and enriching viruses from the Luo shrimp showing the 'siderosis', extracting tissue RNA and reversely transcribing the tissue RNA into cDNA. The obtained virus cDNA is used as a template, and the nested PCR external primer MrDV-3-1-F/R in the table 1 is used for amplifying a target sequence. The PCR reaction used a 20. mu.L system: 2 × Accurate Taq Master Mix10 μ L (2 × Accurate Taq premix (containing dye, product No. AG11019, Ex.) 5 μ M forward and reverse primers 0.5 μ L each, 1 μ L cDNA template, 8 μ L sterile water.

The PCR reaction procedure is shown in table 2:

TABLE 2 cloning of the target genes PCR program

Sequencing the PCR product with the band with the size consistent with the expected size shown by the electrophoresis result, comparing the sequencing with the sequence spliced by the macrovirus group, and finding that the sequences of the two are consistent, thereby indicating that the MrDV-3 target gene is successfully cloned.

2. Construction of pMD19-T-MrDV-3 positive recombinant plasmid

The gel was recovered and purified by using TaKaRa pMD according to the instruction of TaKaRa gel recovery kit (9762)^TMThe 19-T Vector Cloning Kit (6013) ligated the gel recovery product with the pMD-19T Vector. The ligation products were transferred into DH 5. alpha. E.coli competent cells by heat shock method, plated on Amp-resistant LB medium with appropriate shaking, incubated overnight at 37 ℃ in a constant temperature incubator, and single colonies were selected the next day.

3. Identification of Positive recombinant plasmids

PCR detection is carried out on the bacterial liquid of the screened single colony by using a primer MrDV-3-1-F/R, the bacterial liquid with a target strip is screened and sent to Tianyihui company for sequencing, and the sequencing result is consistent with the sequence spliced by the macrovirus group, which indicates that the pMD19-T-MrDV-3 positive recombinant plasmid is successfully constructed.

4. Extraction of recombinant plasmid

After the identified bacterial liquid is propagated, plasmid extraction is carried out by using a TaKaRa plasmid extraction kit (9760).

EXAMPLE 3 establishment of nested PCR detection method

1. Extraction of MrDV-3 viral RNA

(1) Extraction of tissue total RNA

When selecting the tissue sample, the tissue under the shell of the shrimp seed, the branchia and muscle of the prawn or the swimming foot of the parent prawn are preferably selected. Using Shanghai Promega

The Super total RNA extraction kit (cat # LS1040) extracts the total RNA of the sample tissue, and the specific experimental steps are carried out according to the kit specification.

(2) Reverse transcription of RNA into cDNA

The obtained tissue total RNA was reverse-transcribed into cDNA using Evo M-MLV reverse transcription premix type kit (cat # AG11728) of Exkory corporation, and the specific experimental procedures were performed with reference to the kit instructions.

The reverse transcription adopts a 20-microliter reaction system, template RNA (500-1000 ng) is added into the system, 4 microliter of 5 XEvo M-MLV RT MASTER MIX is added, the 20 microliter is supplemented by water, the water bath is carried out at 37 ℃ for 15min, the water bath is carried out at 85 ℃ for inactivation for 5s, and the frozen storage is carried out for standby application at-20 ℃.

2. Nested PCR amplification

(1) First round nested PCR amplification

The cDNA synthesized by reverse transcription is used as a template to carry out nested PCR detection, and meanwhile, sterilized water is used as a negative control. The first round of PCR amplification uses a primer MrDV-3-1-F/R, and the PCR reaction adopts a 20 mu L system: 2 × Accurate Taq Master Mix10 μ L, 5 μ M forward and reverse primers 0.5 μ L each, 1 μ L cDNA template, 8 μ L sterile water.

The PCR amplification procedure was: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1 min; preserving at 72 deg.C for 10min and 4 deg.C.

After the PCR reaction, 8. mu.L of the PCR product was detected by 1% agarose gel electrophoresis, and the first round of nested PCR amplification results are shown in FIG. 2. The amplified fragment size of the primer MrDV-3-1-F/R is 771bp, and as can be seen from FIG. 2, a positive band with the size consistent with the expected size is amplified by the first round of PCR, and no band exists in a negative control, which indicates that the MrDV-3 virus detection result of the sample is positive and the virus carrying amount of the sample is high.

(2) Second round nested PCR amplification

If the first round of nested PCR amplification product does not detect a positive strip, diluting the first round of nested PCR amplification product by 50-100 times with sterilized water as a template, and performing second round of nested PCR amplification, wherein a primer used in the second round of amplification is MrDV-3-2-F/R, the PCR reaction system is as above, and the amplification procedure is as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1 min; preserving at 72 deg.C for 10min and 4 deg.C.

And (3) taking 8 mu of LPCR amplification product to carry out gel electrophoresis detection, wherein the result is shown in figure 3, the left figure uses one amplification primer, the right figure is obtained by diluting the one amplification product by 50 times and then amplifying the one amplification product by using two amplification primers, and the two amplified electrophorograms have positive bands with the sizes consistent with the expected sizes and no bands of negative controls, which indicates that the MrDV-3 detection result of the sample is positive, but the toxic quantity of the sample is low.

3. Exploration of optimal annealing temperature of primers

The first round amplification primer MrDV-3-1-F/R (hereinafter referred to as first amplification) and the second round amplification primer MrDV-3-2-F/R (hereinafter referred to as second amplification) of nested PCR are respectively used, the positive recombinant plasmid of the embodiment 2 is used as a template, amplification is carried out by adopting different annealing temperatures (50 ℃ -60 ℃), and the annealing temperature corresponding to the brightest band is selected as the optimal annealing temperature. The PCR reaction system was as above, and the experiment was repeated three times with sterile water as a negative control. The result of detecting the optimal annealing temperature of the first amplification primer is shown in FIG. 4, wherein Lane 7 is the negative control, the annealing temperatures corresponding to Lanes 1-6 are 50 deg.C, 50.9 deg.C, 53.3 deg.C, 55.7 deg.C, 56.8 deg.C and 59.9 deg.C, respectively, and the band corresponding to Lane 6 is brightest, so 60 deg.C is selected as the optimal annealing temperature for the first amplification. The results of the detection of the optimal annealing temperature of the second amplification primer are shown in FIG. 5, and the annealing temperature corresponding to the lane is the same as that shown in FIG. 4, which shows that 60 ℃ is the optimal annealing temperature of the second amplification primer. The modified one-and two-amplification PCR reaction procedures are shown in Table 3:

TABLE 3 nested PCR reaction procedure

4. Sensitive detection of nested PCR primers

In order to detect the sensitivity and the repeatability of the nested PCR primer, the positive recombinant plasmids with different concentrations are used as templates, the first amplification primer and the second amplification primer are respectively used for carrying out PCR amplification on the templates, each experiment is repeated for 3 times, and the amplification product is detected by using 1% agarose gel. Diluting the positive recombinant plasmid by 10 times for 8 gradients to ensure that the concentration of the recombinant plasmid is 10 in sequence⁷copies/μL、10⁶copies/μL、10⁵copies/μL、10⁴copies/μL、10³copies/μL、10²copies/. mu.L, 10 copies/. mu.L and 1 copy/. mu.L, the reaction system is as above, and the reaction procedure is as in Table 3.

The detection result of the first amplification is shown in FIG. 6, and the template concentrations corresponding to lanes 1-8 are 10 in sequence⁷copies/μL、10⁶copies/μL、10⁵copies/μL、10⁴copies/μL、10³copies/μL、10²coThe amplification results of three replicates were consistent with the same pies/. mu.L, 10 copies/. mu.L and 1 copy/. mu.L, with the lowest detection limit of one amplification being 10³copies/μL。

The sensitivity detection results of the second amplification are shown in FIG. 7, the template concentrations corresponding to lanes 1-8 are consistent with those of FIG. 6, the amplification results of the third repetition are consistent, the lowest detection limit of the second amplification is 1 copy/. mu.L, and the number of copies of the second amplification can be accurately obtained.

5. Specific detection of nested PCR primers

In order to detect the specificity of the nested PCR primer, the invention respectively extracts the RNA of positive samples of Infectious Precocity Virus (IPV), macrobrachium rosenbergii small RNA-like virus-1 (MrPV-1), macrobrachium rosenbergii bicistronic virus-3 (MrPV-3), mud crab bicistronic virus (McDV) and mud crab reovirus (McRV) and carries out reverse transcription to obtain cDNA. The obtained cDNA is taken as a template, a first amplification primer MrDV-3-1-F/R is used for amplification, then a second round of PCR amplification is carried out by taking an amplification product of the first amplification as the template and using a second amplification primer MrDV-3-2-F/R, and the amplification system and the reaction procedure are the same as above.

The specific detection results of the nested PCR primers are shown in FIGS. 8 and 9, respectively, and lanes 1-4 in FIGS. 8 and 9 correspond to the positive samples of Infectious precocity virus, Microbrachiumrosenbergiiporiornavirus 1, Microbrachiumrosenbergiidicistro virus 3, mumcrabdivistrovirus and mumcrab reovirus (because the mumcrabdivistrovirus is the satellite virus of the mumcrab reovirus, the two viruses are in the same positive sample in the actual detection process), lane 5 is the positive control, and lane 6 is the negative control. As can be seen from FIG. 8, there was no band in all the groups except the positive control, indicating that the specificity of one amplification primer was good. As can be seen from FIG. 9, the bands of the other groups except the MrDV-3 positive sample and the positive control were not found, indicating that the specificity of the second amplification primer is good. The lack of a band in the MrDV-3 positive sample in FIG. 8 may be due to the fact that the sample concentration is too low to detect.

Example 4 actual nested PCR detection of samples of Luo's shrimp in Huzhou, Zhejiang

The nested PCR primers were used to detect 20 gill samples of Luo shrimp collected from Huzhou city, Zhejiang, with the specific experimental procedures in example 3. As shown in FIG. 10, the amplification result of the first round of amplification by nested PCR showed no bands, and thus the first round of PCR product was diluted 50-fold to be used as a template for the second round of PCR detection. The amplification results of the secondary amplification are shown in FIG. 11, and specific bands appear in 9 samples, which indicates that the nested PCR detection method for MrDV-3 virus established by the invention is suitable for actual detection.

Example 5 design and detection of fluorescent quantitative PCR primers

1. Primer design for fluorescent quantitative PCR

Using the same target gene as in example 1, design of fluorescent quantitative PCR primers was performed using Primer 6.0 and stringent screening of the primers was performed using Primer select from Lasergene 7.1, resulting in 1 pair of suitable qPCR amplification primers MrDV-3-q-F/R with a fragment size to be amplified of 247bp, as shown in Table 4. The MrDV-3-q-F primer is 24bp long and is positioned at 4827-4850 th sites of a MrDV-3 virus genome from the 5' end, and the MrDV-3-q-R primer is 25bp long and is positioned at 5049-5073 th sites of the genome.

TABLE 4 fluorescent quantitative PCR amplification primers

2. Establishment of fluorescent quantitative PCR method

RNA extraction and reverse transcription As in example 3, the cDNA synthesized by reverse transcription was used as a template, and qPCR detection was performed using MrDV-3-q-F/R. The real-time fluorescence quantification system used was: roche LightCycler480 II; the qPCR kit is of Ekery

Green Pro Taq HS premix type qPCR kit II (AG 11702).

The qPCR reaction system is as follows: 1 μ L cDNA, primer F0.2 μ L primer R0.2 μ L2 × SYBR GREEN I MIX 5 μ L ddH₂O make up to 10. mu.L.

The qPCR reaction program is shown in table 5:

TABLE 5 qPCR reaction procedure

If an amplification curve appears in the qPCR result and the corresponding Ct value is less than 35, the result is regarded as a positive result; if no amplification curve exists or the Ct value is more than or equal to 40, the result is regarded as a negative result; if the Ct value is between 35 and 40, the experiment is recommended to be repeated, if the Ct value of the repeated result is less than 40 and the amplification curve has obvious peaks, the corresponding sample can be judged to be positive, and if not, the corresponding sample is negative.

3. Drawing of standard curve and qPCR detection

The positive recombinant plasmid pMD19-T-MrDV-3 described in example 2 was diluted by 10-fold decrease, and 10 clones were selected²copies/μL～10⁷qPCR amplification was performed at 6 dilutions total copies/μ L, taking the logarithm of the copy number of the recombinant plasmid as the X-axis and Ct as the Y-axis, and a standard curve was established, as shown in fig. 12, and the equation of the obtained standard curve was: y-3.3122 x + 34.058. According to the determination, the correlation coefficient (r2) of the method is 0.9987, and the amplification efficiency (E) is 100.4%, which indicates that the primers designed by the method are good and the reaction system is normal.

The positive recombinant plasmid is diluted by 10 times and decreased progressively, and 10 copies/mu L-10 are selected⁸qPCR amplification was performed at 8 dilutions copies/. mu.l, and the fluorescent quantitative PCR amplification curves for the different concentrations of positive recombinant plasmid are shown in fig. 13. As can be seen, S-shaped amplification curves were observed except for the negative control, which did not have S-shaped amplification curves, wherein A was present at a concentration of 10⁸copies/. mu.L, B is 10⁷copies/. mu.L, C10⁶copies/. mu.L, D is 10⁵copies/. mu.L, E is 10⁴copies/. mu.L, F is 10³copies/. mu.L, G is 10²The results show that the drugs are in a dosage of 10 copies/. mu.LThe fluorescent quantitative PCR method has wide applicable concentration range.

4. Sensitivity detection

Respectively diluting the positive plasmid standard substance to 10⁰copies/μL～10³copies/mu L, 4 dilutions in total, amplifying the dilutions by using a qPCR primer, repeating the dilutions by 20 times, and taking the minimum dilution with the variation coefficient less than 5% and the positive detection rate greater than 95% as the minimum detection limit in the reaction system and the procedure. As shown in Table 6, it is understood that when the dilution is not less than 1 copy/. mu.L, the variation coefficient is less than 5% and the positive detection rate is more than 95%, so that the minimum detection limit is 1 copy/. mu.L, and the sensitivity can be accurate to the one-bit copy number.

TABLE 6 sensitivity detection of primers

5. Specificity detection

The cDNA of the sample used for the specific detection described in example 3 was used as a template, and qPCR detection was performed on the cDNA of the above virus using a fluorescent quantitative primer MrDV-3-q-F/R, respectively. The results of the specific detection of the primers are shown in FIG. 14, in which A is an Infectious precocity virus positive sample, B is a Microbrachium rosenbergiiicrobiosis virus1 positive sample, C is a Microbrachium rosenbergii virus 3 positive sample, D is a mud crabdivirus and a mud crab reovirus positive sample, E is a positive control, and F is a negative control. As can be seen from the figure, only the positive sample and the positive control of MrDV-3 have an S-type amplification curve, and the result is consistent with the nested PCR, which indicates that the specificity of the fluorescent quantitative PCR primer is good.

6. Repeatability detection

After 10 times of degressive dilution is carried out on the positive recombinant plasmid, 5 dilutions of plasmid standard substance are selected for detection, and the concentration is 10³copies/μL、10⁴copies/μL、10⁵copies/μL、10⁶copies/. mu.L and 10⁷copies/. mu.L, was tested in triplicate and the intra-group coefficient of variation was calculated from the Ct values. In addition, the reaction system and the procedure for performing the batch-to-batch repeatability tests at 3 different time points, calculating the batch-to-batch variation coefficient according to the Ct value, evaluating the stability of the established method by utilizing the batch-to-batch variation coefficient, and detecting the repeatability of the primer are the same as above. The repeatability detection results of the fluorescent quantitative PCR primers are shown in Table 7, the intra-group variation coefficient is 0.42-2.67%, the inter-group variation coefficient is 0.77-4.05%, and the intra-group variation coefficient and the inter-group variation coefficient are both below 5%, which shows that the method has good repeatability and reproducibility and stable and reliable results.

TABLE 7 repeatability tests

Example 6 actual qPCR detection of samples of Luo's shrimp in Huzhou, Zhejiang

The constructed fluorescent quantitative PCR method is utilized to detect 20 gill samples of the Luo-shrimps taken from Huzhou city of Zhejiang, and the specific steps refer to example 5.

The detection result of the fluorescent quantitative PCR is shown in FIG. 15, and the Ct values of 20 Luo shrimp gill samples amplified by the qPCR primer MrDV-3-q-F/R are shown in Table 8. As can be seen from the figure, 11 samples in total have S-type amplification curves, and the Ct value detection result of qPCR in Table 8 shows that the Ct value of 9 samples is less than 35, which is a positive detection result; the rest samples have no Ct value and amplification curve, and the detection result is completely consistent with the nested PCR. The Ct values of the positive detection results are all between 25.94 and 32.1, are 282 copies/muL to 4 copies/muL after being converted into copy number, and are positioned in the nested PCR first amplification (10)³copies/. mu.L) and two limits of detection (1 copy/. mu.L).

Table 820 Ct values for qPCR detection of gill samples of Lobster rosenbergii

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Zhongshan university

<120> a product and method for detecting bicistronic virus-3

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8891

<212> DNA

<213> Bicistronic Virus-3 (Macrobrachium rosenbergii Dicistro virus 3)

<400> 1

gccatacggc tctatttaaa tatccccgat ctatctcgtt ctgggccacc cacttcggct 60

gggagtctgg agacgatgtc ctccgtgcac caacgtttgc gtgctgtacg atggttagga 120

tctcggaacc ctttcacttt agtacttcat aatcatttca taatcattat ttttattaaa 180

gtggctcgtt cccgatggcg agtcagcctt gtagtaaccg accggttgct gatcgtgtct 240

agcgatacac atgataatca attgaataag caatagctct gcaacgaata gcattcggaa 300

ttcgcaaaag ttgtatctat tgttgaagct gttgagtaag tgtgtagtag gtatttttcg 360

gaccagcctt atggcgcccc ttaattgggt tttgtcagtc gcgtaatctc ttgtgacgag 420

aggttcggat gcgcgatgta gtagaatgta acctccgtca cgacccccca tggatcattt 480

aactaattca atttcttttg tttcacttga ggatgagagc aagcgtatga gagaggtgac 540

gtgtacgtac aagttgtacg tggactttga tgtttggcgc gagttagact ggaagtacgc 600

cggtgatgat gccacccaag cttgtcttga tgagtattat acccggaagc tggagttgtt 660

ggacatgatg aatcgtgagg atgagaccac agatctggca ccttacgtta attttattgt 720

cgatttgagg aagatgaaag ttgaaggcaa gcttttcttg ctttccctag attctgaaga 780

cgacgacgat cgggactacg gttctgaaga aggctttgct cagttcttgc tgagcttgat 840

ggacaatgaa gctgctgaag acttaggttt tgctgaggat ttaacagagg aaggcgtgga 900

acccaaccct ggccccgttg tttgcagcac tttgcgagat aatcgcttaa gcaaagtgac 960

tccaagcagc gtgagagagt tgaagaagta cgccaagaaa tgtgcccccg agctgaggaa 1020

gatcgcggcg cgagctgcga aacgaggaaa gaaggaagtc gaaatacgtg ctcaaattgg 1080

cgcggaatac ttccaagtag acaatccctt agtcaacccc ccgaaacacg aaacccccgc 1140

cgacaaagac cctcctaaac ctgattgccc gaattgccac aaagaacatt gcaattgttt 1200

tgccaagcga gcgagtgttg tcgcttcaat agctagtgtg atcgcttctc taatccggat 1260

tgcggaaacc cttacgggac atgcgcagat cggaggttcc ctcttcaaat caatgtttag 1320

cggtttggct gatggcacca aggagacggt agtgcaagct attaaagaga cagtgaatga 1380

agccctagac gcccaactcc cgtacatccc tattactgtg cgtggtgcgc tgcaagtgtg 1440

tgtagcagcg gcctttcttc atgctttatg tgggcttggt atcattttgg cggatgtcgc 1500

aagatgtctg ctaggattga tattaggttc aaacaaacag tacgaagctg tagcacagat 1560

tggtaatgaa tggatggaca tagatggccc gaagagtgat gacgcaaccg aaagtataat 1620

gagatacatc ccaataggtg tgtctgttgt gctctctgga ttggtggcct ttgctgtagg 1680

caagcttcct ggtcgagata attctcccga agcttggatg agaaagattg cagcgttccc 1740

aagagcgtgt tctgcgttca gcgatatatc caaatatatc caaacgatag tgaacccctt 1800

gtggagcaag ttccaaatgt gtgtgttagg ctacgaccgt gacctacttg gcgacgcgat 1860

accggagatt ggtaagtgga tggaagaagt tgaaagatac tccttcaaag ctgcgtgcga 1920

tgagatgatg aagacgcgtg acggacgatt cctgatttct acgctttata tgaaaggaca 1980

caagttgatg cttaagtatc aacctgccct gactcctgag tatagatcag caatgcaacg 2040

atgtctggtg atggctgcga agctaaagac ccacgtcgag acgaacttcc ctgaagttaa 2100

gagtgtgcgc acaacccccc tagggttgtg gctagtagga gagtcacaga ttggtaaatc 2160

gagattgcaa tatttgattg ctacgcattt gtgcgcggta gcgggtatta gtgacttgaa 2220

gaaccagatc tacatgagga ataccgccca ggagttctgg gacgcataca acggccaatt 2280

tgtgtgcgtg tacgatgact tcggacaaca aaaggatgcg gttagtagcc ccaaccttga 2340

gtttatggag atcatccgat cgattggccc ctttcccttt ccccttcaca tggctgacat 2400

cagtgagaaa agttccgcca gatttacctc tggtgttctg atgtgcagca cgaacaaccg 2460

ctacctcaag gttgaatctt tgacttacga cgatgccgtt tggaatcgtt ttccacagtc 2520

ttacatcgtg gagttgaaag atgagtataa gattgaggag accggcccgg acggtaaacg 2580

acgaacccga cttaacattc caaaagctag agacgcgcgc cctggtatgg agataaaccc 2640

ttatatttat acatttaaaa gatttgatgc gagagcccgt ctcagtagaa atgctgagac 2700

tggcgaagtg tatgaatggg atgagtttat ttccgtgctg gagcgcgatt tgcaagatcg 2760

catgcaagac ggaaacgcct tggataactg gttggattct tacgccaaag agctatgtgc 2820

caagaaaggt gtggctcaaa ttggtgtcgg tggtgagcaa ttgccaacat ccgatgccat 2880

tagagagtgc acccaagagg gacccgatca agtcacaaac cctaaatata acttaggcga 2940

gttccttgat tgggctgtag cgttaaagtc caacccggta gaccccaatg atgattcgta 3000

caataaatac gtgatctatg cgagcgcgga gaagcctcat gttaaaggag acccttacga 3060

gattagcctc tatgactatg ccccagtgat gatggatgag cacgtcttta gacaattgtt 3120

ggtagctttc tcgcgagaga aagatggatt caagtctaga gttggtgaca cgatgaggat 3180

gtgcaagagt ctgtgtgacg acgcatatga gaggttgcct gagaaagtga ggattcttta 3240

cacgactgta aaaggttttg tgaaggacgc gatcgatggt ctgtggtctt ttgtgaaaga 3300

ccacaagttg atggccacta tgttggtagc gataccagcg ctggcggcca tgatgcgagc 3360

gaggaagaac aagcaggatg acgacgagtg tgactgtgag tggaagtgcg agagtgaccc 3420

acgcgttttg caaccgcgct ctcgtcccac cgtgaaagca gtgggcagga cgcgaattgc 3480

gagaggcgga gcggagcaag gacagagcca gaaccaactg gacatcatag agattgtacg 3540

aagaggacag tacttgatag ttgcgcagta tgaagatgga accgaaatta acctcggttg 3600

tgtgacgcaa ttaataggta ctgtgtttat gatgcccaga cacttcttga cctacctgtc 3660

ggaccgaccc cccaataaga tcatatttta tcactcccat agtagccagt tagttattaa 3720

ccgtcagtat gatgggttgt ttcaggacgt tgtatgtgtt gaacaggaaa tagctgatgg 3780

caccgcggga gggatggatg tggtcttctt cacgatcccc caatttatga gaggaaaaga 3840

catcaccaag cattttgcca ccgagtcaga gatggctaag atggctggta ggcggatagt 3900

tggaaccttg tctggagtcg atattgacaa aaaaaatgaa gcctctttca taacgaaaag 3960

tggagagttg gagctgcagt taaacaaaca cgttaattat actttggaag gtaagattga 4020

acgtaatgtt gtgtcaactt cgatatgcca atatcgaatc ccgaccaaat tcggagactg 4080

tggcaagatc gtgacactga acacagatgc tataagagga aagatcgttg gcattcatgt 4140

cagcggcacc accatcggag tgaattacgc ccaggtagtt agttatgaga ccatacagac 4200

tgccttggag ggtttgccca aagtggcgca gataggatta gcattgaatt cagtgaatga 4260

aggagcaggt gaaccgatag acgcaggatt cattcacctt ggcaccatta aggtgcccgt 4320

cgcccaaagt tccaagaccg tgataggccc aagtaagctc cacaacaaaa tcacgccagc 4380

aacaacgcga cctgcgttgc tcaagcctat cacaatagat ggcgtgtatc acgatcccct 4440

aattgaagga gcaaagaagg ccgggattcc ctgtgggtat gtacctccag atattcttga 4500

ccaagcgacc cgggatgttt ttgtcaacat ctctaagaaa aacccagaca gtgtggaaaa 4560

ccatgtgttg gactacgaac aagccataga gggtatccct ggagatgagt tctttcaacc 4620

aattaataga acgacatctc ccggataccc atatatgact gaaacgccag caaaaggaca 4680

cagaggtaaa accaaatgga tgggtaagca cgactacgac tacgattctg aatttgccct 4740

acgactacga caagatacga tggagttgat tgagaagtgc cgaaacaatg agcctttcga 4800

ggtgatttgg gttgacactc tcaaggatga gaggagagca gaggagaagg tgagagccgg 4860

taagaccagg gtcatttcga atggcccaat gcattttaat atcgctttta gaatgtattt 4920

catgactgcg ctagtcaacc ttagactagg acgcatatac aatgggatcg cagtaggcat 4980

aaatgtttgg agcgcagagt gggacgctct ggccaaacat ttattatcga actctcccta 5040

tgtattagat ggtgatttcc gcttgtttga tgggtcactg atagatacgg ttatgtggaa 5100

gatctttgag atattagacg cgcagtacga cgacggaaat accaacttac ggagaaatct 5160

gtggtatcac gtcgtttatg ctgtgcgtct atgtagaaac cgagtctatc aatgcaccca 5220

ttcccttcca agcggtttcg ttgcaacagc agaggtcaat agcctctatg tgaacatcat 5280

atttagatgc gcctacttgg tgcttgcaag gaaaagcggc tatgttggcg acaacatgga 5340

tgctttcaac cagaatgtga aacttgtggc ttatggagat gacaacatct actctgtgag 5400

ccccaacatc attcacttct ttaatatgaa cacaataact gaagcaatga gacagtttgg 5460

gatggattac accccagcgg acaaaagcga agacacaaga ccctacaaga ctatcaccga 5520

ggtctctttc ttgaaaagga acttcaagag agtggacact gcgcatggga tcagtcccct 5580

gtatatgtgc cccgccgact tggagagccg cctggagatg ctcaattgga caaaatccaa 5640

aggaatagat tcaggacctg aagaggccat ggtgataaca gacgttttga aagagttgtc 5700

gatgcacgga tgccgcatct acaacgagta cgcaccaaag atcattcggt gtgctgtcga 5760

agaaggcatc actggtttta gggacgaagg cccgacctat taccacatga aagtcatagc 5820

gggcaatggt gtacctcggc cgtgtgatct tgcgagacct atacaaaatt ccaatgtcaa 5880

taaaggtctc tgcactgctg cagccgaaag tggcgtgagt atttactctt ataccctagg 5940

atcgccagtg gcagccccac attatccaag ggaacattgg tgcgacgcgc agcctgagct 6000

agctcgcgtt taaggaacag cttactgatc aacaaaattt taacgctaat cccgatataa 6060

ccctgacgac ctctgctatg acaacggaca cgattacttt gagagatgat ggatcgacgg 6120

cggtggacaa gtatgtttcg cgagaagcgg atttgccggc cgttatgtat gattctatca 6180

ctgagacgga tgaacatacg atcaaggatt tccttggacg catggtcatc attgaccagg 6240

gcacgtggtc ttctacccaa gccgctggtg ccactttggc aaatttaaca tttccttctg 6300

ctttgtttaa aaccgggtct actaattata atcaaaatgt caataaactt gatggttttg 6360

ctgcgatgaa agccaaggtc agagttcgca ttgaggtcaa ttcccagccc ttccaggcgg 6420

gagcgttgct gttgcattat gttccatatt cggaatacat gaactcgcac gcaaaatggt 6480

acaccaacac caccactgat ttggtggccg cgtctgggtg ccctcatgtg gttatgaact 6540

tggccaacac cgctagtatg gcctttgtta ccccttacat ttccccttat ttgttttaca 6600

atttgccgca aggccagggt tcatttggta atgttgtcat ttcagttttg gcgcctttgt 6660

cgtccgctgc tgcgaattct tgcaattata ccatttgggc tgcatttgag gacgtggagc 6720

tcagatatcc cactgacgcc ccgctttcaa cggtgtacgc tcaggtgggc aaggagatac 6780

agaagatgga gggcaggggt tctatatccg gggtggtgcg atcggttggc actgcagtgt 6840

cagacgtgtt gccatgggtt gggctggggt ggttatcaga gcctgctagg tttttgactg 6900

atgcgggcga atcggtgctt aaaatgctgg ggttttccaa acccagtgtc gaggcgccgg 6960

taactcgtgt taagcaatcg ccgacgcagt tttttttgaa tgctgatggt gcggacacat 7020

cgcacaagtt gggtttgagt gcagctaatg ccctggctac tttgccgggc tgggctggga 7080

ctgatgagga tgagatgaga ttggattaca tatgttctcg acctaattat tacaacaagt 7140

tctcttggac caccgagtct accgcggaca cttctctgta tattcaggcg aattccccgc 7200

tatggacaca atcgttggat gctttggctg caggtaacta cgcgcaaacc gtgtcgttgc 7260

ctctcattgc caaagtcgcc tcgcaattcg gaacttggcg tggtactatg gtttatactt 7320

tccatgttgt taagacccag ttccattctg ggcgcctccg tgtttctttt cgcccctttt 7380

cttaccccgc tggagcagct gcttcagatg ttcagtttgt gaatcaaccg ggttacgctt 7440

acaccgacga gatagatctc agtagtggaa ccacttttac ttttgaggtc ccgttcgttt 7500

cggtgcgacc gtggatgcat tgctattacg acgcaaagac cgcatatccg ggtggagaca 7560

tcaggaatag cgctactggc atcgtgcagt tgtccgtcat caatccattg gtggcggcga 7620

gcacagtaaa tagcagcgtg gacatcctgg tgtttgtgag tatgaaagaa gcgcagtttg 7680

ctagtcctgt gaactcgtca tatctgccat ttggtatccc caacgttgcg cagatcgggc 7740

gggcgaggat tgtgccgact aagcagagtt cggatcagat gagtgagagg agcgagcttt 7800

cgatgttacc gtatgcggtg tgcatgggtg agaatgtgac ttcggttcgt cagttgttga 7860

agcgttactc gtatttgggt agagtgtcac cttctgtgtt ggcagccacc gccacgcaga 7920

tggggtcttc cgggagggga ttcgtcattt ttccttgggc gcctgtaact ccgcagaacg 7980

gtgcaatatc gaacaccttg ggtgtgcaaa caccaaagtt tattagtact tacaaaacgg 8040

ttagcggtgc tgcgactacc actattgcac aatacgtgga tacgtattcc caattttatc 8100

cgttgtatgc tttttacaga ggctccatgc gttttaaagt ggtggtcgcc gtgaagggcc 8160

cgaattatga cgcatctttg cccatcaatg tatatatcca cttgacgaac ccagcttctg 8220

ctggcaacat gaacccgatt atgagttctg tggtcgcaga tggtgcgggc gcgagctcaa 8280

atctgggttc tggaccgttg cagtgcttgt ttgacacacc tattgccact ggtgccggtt 8340

tgaccaaagt tggttttgcg taccagccgg agcttggggc ttacaagtgt gctgtcattc 8400

ctggttttga aggggccatt gagtttgagg tacctttcca ttgtactggc catatggtac 8460

ccaccaatta cggtattttc gatcagacca gtgctcgctc cattttcttt cccttcccta 8520

ttgtctccat tatgggttca actacccctg ctggagtccc tatcctcagc aagtgcgaat 8580

tcgatgttta tcgagctgtt ggggacgatt tttccttcgg tggtctcatt gggtcgcctc 8640

agcatgcgct gtggcaatcc actatagacc ccatttagac ctcgcccgtt acgcggtcga 8700

gctatcttct ttttacacca cctgttgcaa agcaatgcag gtgctggcac cattttggtg 8760

ttatttaaca atcacgtaaa cccgtggacc ggagctttcg cagtcggttt agcatatcac 8820

attcttagtg aagtggtgct atggtcaagg cgtttttgtt tttgtttaat tggttcaaaa 8880

aaaaaaaaaa a 8891

<210> 2

<211> 771

<212> DNA

<213> Bicistronic Virus-3 (Macrobrachium rosenbergii Dicistro virus 3)

<400> 2

gatacgatgg agttgattga gaagtgccga aacaatgagc ctttcgaggt gatttgggtt 60

gacactctca aggatgagag gagagcagag gagaaggtga gagccggtaa gaccagggtc 120

atttcgaatg gcccaatgca ttttaatatc gcttttagaa tgtatttcat gactgcgcta 180

gtcaacctta gactaggacg catatacaat gggatcgcag taggcataaa tgtttggagc 240

gcagagtggg acgctctggc caaacattta ttatcgaact ctccctatgt attagatggt 300

gatttccgct tgtttgatgg gtcactgata gatacggtta tgtggaagat ctttgagata 360

ttagacgcgc agtacgacga cggaaatacc aacttacgga gaaatctgtg gtatcacgtc 420

gtttatgctg tgcgtctatg tagaaaccga gtctatcaat gcacccattc ccttccaagc 480

ggtttcgttg caacagcaga ggtcaatagc ctctatgtga acatcatatt tagatgcgcc 540

tacttggtgc ttgcaaggaa aagcggctat gttggcgaca acatggatgc tttcaaccag 600

aatgtgaaac ttgtggctta tggagatgac aacatctact ctgtgagccc caacatcatt 660

cacttcttta atatgaacac aataactgaa gcaatgagac agtttgggat ggattacacc 720

ccagcggaca aaagcgaaga cacaagaccc tacaagacta tcaccgaggt c 771

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gatacgatgg agttgattga gaag 24

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gacctcggtg atagtcttgt ag 22

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcaaccttag actaggacgc ata 23

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtcatctcca taagccacaa gtt 23

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgagaggag agcagaggag aagg 24

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccatcaaaca agcggaaatc accat 25

Claims (10)

1. A target gene for detecting a dicistronic virus-3 is characterized in that the nucleotide sequence of the target gene is shown as SEQ ID NO. 2.

2. A primer set for detecting a dicistronic virus-3, which comprises a primer capable of amplifying a target gene shown in SEQ ID NO. 2.

3. The primer group of claim 2, wherein the primer group is nested PCR primers shown in SEQ ID No. 3-6.

4. The primer group of claim 2, wherein the primer group is a fluorescent quantitative PCR primer shown in SEQ ID No. 7-8.

5. Use of the target gene of claim 1 or the primer set of any one of claims 2 to 4 for the preparation of a product for detecting bicistronic virus-3.

6. A kit for detecting dicistronic virus-3, which comprises a reagent for detecting the target gene of claim 1.

7. The kit according to claim 6, wherein the reagent contains the primer set according to any one of claims 2 to 4.

8. A nested PCR method for detecting bicistronic virus-3, which is characterized by comprising the following steps:

s1, extracting tissue RNA of a sample to be detected and carrying out reverse transcription on the tissue RNA into cDNA;

s2, performing PCR amplification by using the cDNA obtained in the step S1 as a template and using nested PCR primers shown in SEQ ID NO. 3-6;

s3, detecting a PCR result through gel electrophoresis, wherein if a specific band with the size of 771bp appears in the first round or a specific band with the size of 449bp appears in the second round, the detected sample contains the bicistronic virus-3.

9. A fluorescent quantitative PCR method for detecting bicistronic virus-3 is characterized by comprising the following steps:

s1, extracting tissue RNA of a sample to be detected and carrying out reverse transcription on the tissue RNA into cDNA;

s2, taking the cDNA obtained in the step S1 as a template, and carrying out qPCR reaction by using fluorescent quantitative PCR primers shown in SEQ ID NO. 7-8, wherein if an amplification curve appears in the qPCR result and the Ct value corresponding to the curve is less than 35, the result is regarded as a positive result; if no amplification curve exists or the Ct value corresponding to the curve is more than or equal to 40, the result is regarded as negative; if the Ct value corresponding to the curve is between 35 and 40, the experiment is recommended to be repeated, if the Ct value of the repeated result is less than 40 and the amplification curve has obvious peaks, the result is regarded as a positive result, otherwise, the result is negative.

10. The method according to claim 8 or 9, wherein the step of extracting RNA from the sample tissue in step S1 is performed by selecting the tissue under the shell of young shrimp, or the gill and muscle of prawn, or the swimming leg of parent prawn.

Images