CN115896341A - qRT-PCR kit applied to juvenile freshwater crab reovirus detection - Google Patents

Abstract

The invention relates to the field of aquatic pathogen detection, in particular to a qRT-PCR kit applied to the detection of a juvenile reovirus of a blue crab, and the kit is used for detecting an ORF (open reading frame) region of a genomic fragment VP11 of the reovirus of the blue crab. The invention provides a high-sensitivity kit for detecting reovirus of juvenile blue crabs, which has the advantages of high sensitivity and strong specificity of detection technology, and can be used for effectively detecting flea larvae in different periods. The method is particularly suitable for detecting the juvenile crabs with low reovirus content and the fertilized egg virus of the juvenile crabs, and can be applied to the investigation of the infection conditions of the juvenile crabs.

Description

qRT-PCR kit applied to juvenile freshwater crab reovirus detection

Technical Field

The invention relates to the technical field of aquatic animal pathogen detection and disease control, in particular to a qRT-PCR kit and a detection method applied to the detection of reovirus of scylla paramamosain fries.

Background

Early studies showed that infection with Mud Crab Reovirus (MCRV) can cause nearly 100% mortality in crabs. Recent researches show that the virus infection is very common, almost all blue crab culture ponds can detect the virus, and the virus poses a great threat to the healthy culture of the blue crabs.

Researches show that MCRV can infect young blue crabs in various ways to cause the young blue crabs to be toxic. Therefore, pathogen detection is carried out at the young stage of the blue crabs and is a key point for cutting off MCRV transmission. In the blue crab breeding process, multiple development stages from fertilized egg hatching to young crab development are needed, the MCRV carrying condition of the fertilized egg and the hatched young crab is known, and reliable guarantee can be provided for healthy offspring seed production. Earlier researches show that after the fertilized eggs and the flea larvae are infected with MCRV, the virus content is generally low, and a high-sensitivity diagnosis technology is required to be used for detection, so that a false negative detection result can be avoided. Therefore, the development of a high-sensitivity MCRV detection method is a precondition for obtaining healthy seedlings.

TaqMan probe fluorescent quantitative PCR is one of the high-sensitivity pathogen detection technologies which are generally adopted at present. Compared with SYBR Green fluorescent quantitative PCR, the fluorescent quantitative PCR has lower background signal and stronger sensitivity. The novel TaqMan-MGB probe further improves the signal-to-noise ratio of the technology, has more accurate experimental result and higher resolution, and is one of the most sensitive fluorescence quantitative PCR technologies at present.

Disclosure of Invention

The invention discovers that the expression level difference of different genes of MCRV is obvious, and VP11 is the gene with the highest virus expression quantity. The invention aims to further design a PCR primer and a fluorescent MGB probe based on a high-expression VP11 sequence, establish a MCRV detection method with higher sensitivity and meet the requirement of MCRV detection of young blue crabs.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention creates a high-sensitivity TaqMan probe qRT-PCR detection kit for detecting micro-pathogens of young blue crabs. Early researches show that MCRV infection phenomena exist in young blue crab seeds and even fertilized eggs, and the fertilized eggs and the larvae generally have very low virus carrying capacity, so that a highly sensitive detection technology needs to be developed. Among the current conventional fluorescent quantitative PCR detection methods, the TaqMan MGB probe method has the highest sensitivity although it lacks a dissolution curve analysis program. Therefore, on the basis of the MCRV high expression gene VP11 sequence, a specific primer and a specific probe are designed in a conserved region, so that a TaqMan MGB probe qRT-PCR detection kit and a method are established. The MCRV detection method is high in sensitivity, can accurately and quantitatively detect 10 copies/reaction, and has the lower limit of qualitative detection of 2.5 copies/reaction. In addition, the detection method has strong specificity, and has no specific amplification with 5 common crustacean pathogen (MCDV, WSSV, DIV1, EHP and vibrio) nucleic acid samples. By using the detection method, the flea larvae in different periods are detected, and effective detection is realized. The results show that the method can be applied to the investigation of MCRV infection conditions of the young blue crabs.

The kit can specifically detect MCRV without cross reaction with common crustacean pathogens. In the invention, the nucleic acid samples of the most common pathogens (such as MCDV, WSSV, DIV1, EHP and vibrio parahaemolyticus) in the blue crabs and the crustaceans are detected, and no cross reaction occurs, which shows that the detection method has strong specificity and can be used for the specific detection of MCRV.

The sensitivity of the kit is higher than that of the conventional MCRV detection method. The specific reason is 2 points. Firstly, the sensitivity of quantitative detection of the TaqMan MGB probe qRT-PCR detection method is 10 copies/reaction, the lower limit of qualitative detection is 2.5 copies/reaction, and the detection capability reaches the upper limit of the detection capability of the TaqMan probe fluorescence quantitative detection method. Secondly, the detection method is established based on the high-expression gene VP11, and has obvious advantages compared with the detection method established based on VP1 and VP6 (the expression level of the two is lower than that of VP 11). In addition, the sensitivity of the detection of the ordinary RT-PCR and nested RT-PCR is lower than that of the quantitative PCR. Therefore, the detection method has higher sensitivity than the existing MCRV detection method in general.

The invention provides a high-sensitivity MCRV detection kit, which has high detection technology sensitivity and is particularly suitable for detecting blue crab seedlings and fertilized egg viruses with low MCRV content. The early researches of the invention find that MCRV is a conditioned pathogen, and the individual blue crabs, fertilized eggs and flea larvae all easily carry the pathogen, but only when the external environment is suitable, the MCRV can cause the virus to multiply greatly, so that the death of the blue crabs and the larvae is caused. Under the normal condition, the virus content of the germ cells and the larvae of the germ cells of the blue crabs is low, and a high-sensitivity detection method is needed to avoid the occurrence of false negative. In addition, the seedlings are infected, so that virus transmission is easy in thousands of miles, and loss which is difficult to estimate is caused, so that the requirement on whether the seedlings carry MCRV is very strict, the detection limit of the detection technology reaches 2.5 copies/reaction, and the requirement on the detection of the pathogeny of the seedlings can be well met.

Based on the technical scheme, the invention provides a qRT-PCR kit applied to blue crab seedling reovirus detection, the kit detects the ORF region of a blue crab reovirus genome segment 11 (VP 11), primers are designed to refer to a VP11 segment sequence (GenBank number HQ 414137.1) of an MCRV genome and VP11 segment sequences (SsrV, genBank number HQ414137.1; MCRV-NH, SEQ ID NO: 4) of other two separated strains, and quantitative primers are designed in a conserved region in the ORF region of a VP11 gene.

The optimal target sequence for MCRV detection is the ORF region of the viral genome fragment 11 (VP 11). MCRV expresses 13 genes together, relative expression levels of the 13 genes in the MCRV are compared through experiments, the VP11 gene expression level is the highest, the VP12 gene is the second, and the experiment result is shown in figure 1. The quantitative primer is designed based on the high expression gene, and the sensitivity of pathogen detection can be improved from the background level. The MCRV detection method reported in the literature is mainly based on VP1 or VP6, and the expression levels of the two genes are significantly lower than that of VP11, which means that the starting point of the detection method established based on VP11 is high.

Further, the kit comprises a pair of specific primers designed based on the ORF region of VP11, and the sequences of the upstream primer and the downstream primer are respectively as follows: the upstream primer MCRVRF sequence is 5.

Furthermore, the kit also comprises a specific TaqMan Probe based on VP11, and the Probe sequence VP11-Probe:5'-FAM-CTG ATG CGT TCG ATT-MGB-3' (SEQ ID NO: 3).

Further, the kit may further comprise a Taq enzyme Premix reagent (e.g., 2 × Premix Ex Taq (Probe qPCR)) containing a fluorescence Probe, a reverse transcriptase Premix reagent (e.g., primescript RT Master Mix containing Random primer Random 6), a standard plasmid diluted in a gradient, a positive control, and a negative control (sterilized double distilled water).

The Taq enzyme Premix reagent and the reverse transcription reagent contained in the kit are both commercially available reagents, and 2 XPremix Ex Taq (Probe qPCR) and Primescript RT Master Mix are the reagents recommended to be used in the invention, and can be used but are not limited to the two reagents. The standard plasmid is a constructed double-stranded DNA vector containing the full length of the ORF region of the VP11 gene and is pMD19T-VP11. The standard plasmid concentration range (1X 10) ⁸ 、 1×10 ⁷ 、1×10 ⁶ 、1×10 ⁵ 、1×10 ⁴ 、1×10 ³ 、1×10 ² 、1×10 ¹ Copy/. Mu.L). The positive control was a diluted standard plasmid (1X 10) ⁵ Copy/. Mu.L), negative control is ddH ₂ O。

Further, the PCR reaction system of the kit is as follows: 2 × Premix Ex Taq (Probe qPCR) 10. Mu.L, VP11-F and VP11-R each 0.4. Mu.L (10. Mu.M), VP11-Probe 0.4. Mu.L (10. Mu.M), ROX 0.4. Mu.L (added or not according to the requirement of the apparatus), DNA template 2. Mu.L, ddH ₂ O6.8. Mu.L, total reaction volume 20. Mu.l. Where ROX reference dye addition is dependent on instrument type, some instrument devices do not require reference dye addition.

Further, the PCR reaction program of the kit is as follows: pre-denaturation at 95 ℃ for 30s; then denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s,40 cycles.

Further, the method for detecting the virus by using the kit comprises the following steps:

(a) Extracting total RNA of fertilized eggs or flea larvae according to the kit instruction, and detecting the quality of the RNA; adding DNase I into the RNA sample with qualified quality to remove DNA pollution;

(b) During reverse transcription, a reverse transcription system needs to add Random primer Random6 in addition to reverse transcriptase and RNase inhibitor to ensure the reverse transcription effect, and is placed in water bath at 42 ℃ for 30min;

(c) In the prepared fluorescent quantitative RT-PCR reaction system, except that the reaction system contains 10 mu L of 2 XPimmix Ex Taq (Probe qPCR), 0.4 mu L (10 mu M) of each of VP11-F and VP11-R, 0.4 mu L (10 mu M) of VP11-Probe, 0.4 mu L (optional) of ROX, 2 mu L of DNA template (nucleic acid of a sample to be detected, a positive or negative control sample), 6.8 mu L of ddH2O and 20 mu L of total reaction volume;

(d) The reaction conditions of the fluorescent quantitative RT-PCR are as follows: pre-denaturation at 95 ℃ for 30s; then, denaturation at 95 ℃ for 5s, annealing extension at 60 ℃ for 30s,40 cycles.

In the second aspect of the invention, a pair of specific primers based on the ORF region of the genomic fragment 11 (VP 11) of the scylla serrata is provided, and the sequences of the upstream primer and the downstream primer are respectively as follows: the upstream primer MCRVRF sequence is 5.

In a third aspect of the invention, a TaqMan Probe based on the ORF region of the genomic fragment 11 (VP 11) of the scylla reovirus is provided, wherein the Probe sequence VP11-Probe:5'-FAM-CTG ATG CGT TCG ATT-MGB-3' (SEQ ID NO: 3).

In a fourth aspect of the invention, the application of the specific primers and/or the probes in the preparation of the kit for detecting the reovirus of the juvenile crab is provided.

Further, the tissue type to be sampled of the kit is the individual scylla, hemolymph or gill tissue (when hemolymph is difficult to sample), and the gill tissue is taken.

The invention has the advantages that:

1. the pathogen detection method has high sensitivity, and the detection sensitivity of the detection kit is improved from two layers:

(1) The detection method has the advantage of high expression level of the target gene. The blue crab reovirus co-expresses 13 protein genes, and the expression level analysis shows that the VP11 gene expression level is the highest. The existing blue crab reovirus detection method is mostly established based on VP1 or VP6 gene design primers, and the expression levels of the two genes are obviously lower than that of VP11. In the same detection sample, the primer is designed based on the high expression gene for pathogen detection, which means that the number of virus templates is more, so that the existence of the virus is easier to detect, and the detection sensitivity is improved.

(2) The detection kit has high sensitivity of the fluorescence quantitative PCR detection technology. Compared with the common RT-PCR, nested RT-PCR and colloidal gold technologies, the fluorescence quantitative PCR detection technology has higher sensitivity and is a commonly accepted and used high-sensitivity pathogen detection technology at present. The lower limit of the MCRV detection technology established by the invention is 10 copies/reaction, and the lower limit of the pathogen detection is 2.5 copies/reaction, which reaches the limit level of the fluorescent quantitative PCR detection technology.

Therefore, the sensitivity of the MCRV detection kit established based on the 2 points is obviously higher than that of the MCRV detection method reported at present.

2. The detection method has high sensitivity and specificity

When the detection method is used for detecting common pathogens of the blue crabs and prawns, no visible amplification curve appears in samples containing MCDV, WSSV, SHIV, EHP and vibrio parahaemolyticus nucleic acid, which indicates that the detection method has strong specificity and does not influence the MCRV detection result due to mixed infection or existence of the pathogens (figure 5).

3. The invention provides a high-sensitivity MCRV fluorescent quantitative PCR detection kit and a use method thereof, and the kit has very good application value in MCRV detection of green crab seedlings and fertilized eggs and virus infection mechanism research.

Firstly, the virus detection method has very attractive application prospect in the MCRV detection aspect of the young blue crab. MCRV is a conditioned pathogen, which is easily carried by the germ cell and the larva of the blue crab, and the virus content is low, so that the virus can be greatly proliferated only when the external environment is proper, and the death of the blue crab and the larva can be caused. The characteristic that seedlings carry MCRV needs a high-sensitivity detection method to avoid false negative. In addition, the seedlings are infected, so that virus transmission is easy in thousands of miles, and loss which is difficult to estimate is caused, so that the requirement on whether the seedlings carry MCRV is very strict, the detection limit of the detection technology reaches 2.5 copies/tube, and the requirement on the detection of the pathogeny of the seedlings can be well met.

Secondly, the virus detection method has good application in the aspect of pathogen infection mechanism. The MCRV infection mechanism research relates to the pathogen proliferation rule research, the virus content in the early virus infection stage is very low, the detection kit is used for analyzing the infection condition and the proliferation rule of the virus in the blue crab body, and the result reliability is higher.

Drawings

FIG. 1 shows the relative expression of 13 predicted genes of MCRV in blue crab hemolymph.

FIG. 2.MCRV standard curve with sample copy number on abscissa and C on ordinate _T The value is obtained.

FIG. 3 shows that the concentrations of No. 1-8 standard plasmid in the qRT-PCR amplification curve chart are 1 × 10 ⁸ ，1×10 ⁷ ，1×10 ⁶ ，1×10 ⁵ ，1×10 ⁴ ，1×10 ³ ，1×10 ² And 1X 10 ¹ Copies/. Mu.L.

FIG. 4 shows that the plasmid concentrations of standard samples No. 1-4 of the qRT-PCR amplification curves in the sensitivity experiment are 1 × 10 ¹ ，0.5×10 ¹ ，0.25×10 ¹ And 1X 10 ⁰ Copies/. Mu.L.

FIG. 5. Specificity assay; 1, positive control; 2-3, MCRV positive samples; 4,MCDV;5,WSSV;6,DIV1;7,EHP;8, vibrio parahaemolyticus; 9, negative control.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example 1: of 13 genes of the blue crab reovirus, VP11 gene has the highest expression level

(1) RNA extraction blue crab after ice bath for 5 minutes, a small amount of hemolymph was extracted by a sterile syringe to extract total RNA according to the procedure of a total RNA extraction Kit (for example, transzol UP Plus RNA Kit from Korea).

(2) And after the total RNA extracted by reverse transcription is qualified through inspection, performing reverse transcription after the total RNA is detected to meet the requirements. The reverse transcription was performed according to the instructions of the reverse transcription kit (e.g., TAKARA Primescript RT Master Mix). During reverse transcription, a reverse transcription system contains reverse transcriptase, an RNase inhibitor and an RNA template, in order to ensure the reverse transcription effect, a Random primer Random6 is added and placed in a water bath at 42 ℃ for 30min, and the synthesized cDNA is used for subsequent PCR amplification.

(3) In order to analyze the relative expression level of each gene of MCRV, corresponding specific primers were designed and synthesized for 13 predicted virus genes respectively (see Table 1). Relative expression levels of the respective genes were analyzed by qRT-PCR using 18S rRNA as an internal reference gene.

(4) The qRT-PCR qRT-PCR reaction system takes virus infected blue crab hemolymph cDNA as a template, and simultaneously adds 10 mu L of 2x SYBR Premix Ex Taq, 0.4 mu L (10 nM) of upstream primer and downstream primer respectively, 6.8 mu L of sterile water, 2 mu L of cDNA template and 0.4 mu L of ROX dye, and the total volume is 20 mu L. Reaction parameters are as follows: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 60s, and reading the plate once; a total of 40 cycles; finally, the temperature was increased from 60 ℃ to 95 ℃ and the fluorescence was measured every 0.5 ℃ increase. The relative content of 13 viral genes was expressed according to the formula (relative expression = 2) ^18sCT-MCRVCT ) And (4) carrying out analysis. The results show that there are significant differences in the expression of each gene during MCRV infection (figure 1). The average value of the relative expression amount of the VP11 gene is the highest, and the relative expression amount is obviously higher than that of other most genes. Research results suggest that virus detection can be performed by designing a primer according to the VP11 gene sequence, so that the detection sensitivity can be obviously improved.

TABLE 1 primer sequence information

Example 2: standard quality grain construction

(1) RNA extraction and reverse transcription the cDNA synthesized was used for quantitative analysis according to the procedure of example 1.

(2) Nucleic acid fragment amplification PCR reaction System: primeSTAR HS Taq Fidelity enzyme premix 25. Mu.L, upstream and downstream primers (MCRVVP 11F1 and MCRVVP11R 1) 2. Mu.L each (10 nM), template 1. Mu.L, sterile water to 50. Mu.L. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 50 sec, for a total of 35 cycles, and extension at 72 ℃ for 10 min.

(3) And (3) cloning the gene fragment and recovering a PCR positive product by using a sequencing gel, connecting a pMD19-T vector, transferring into DH5a competent cells, coating and inoculating on an LB solid culture dish containing benzyl amine, selecting bacterial colonies, shaking the bacteria for culture, identifying by PCR, and sending at least 3 positive strains to Shanghai biological engineering Limited company for sequencing.

(4) After the determination of the standard plasmid concentration and dilution confirm the success of plasmid construction, the OD260 and OD280 values (1 OD absorbance value is equivalent to 50 mu g/mL dsDNA) are measured by an ultramicro spectrophotometer, thereby determining the plasmid purity and calculating the total plasmid mass. The calculation formula is as follows: copy number of plasmid per microliter (copies/. Mu.L) = total mass of plasmid (μ g/. Mu.L)/plasmid molecular weight; plasmid molecular weight =2 × 330 × nt (where nt is the number of bases of the plasmid). Standard plasmid was serially diluted 10-fold until Nx 10 ¹ Copies/. Mu.L were stored at-20 ℃ until use.

Example 3: establishment of TaqMan probe qRT-PCR reaction system and conditions

(1) RNA extraction and reverse transcription the cDNA synthesized was used for quantitative analysis according to the procedure of example 1.

(2) Primer and probe primer design reference MCRV genome VP11 segment sequence (GenBank accession number HQ 414137.1) and VP11 segment sequence of two other isolated strains (SsrV, genBank accession number HQ414137.1; MCRV-NH, SEQ ID NO: 4), quantitative primers and probes are designed in the conserved region inside the ORF region of the VP11 gene: a pair of quantitative primers (VP 11-F:5'-GTC AGA ATG TCG TTC ATA CTT TGT-plus 3'; VP11-R:5'-ATT CAG GAG TTC CGG ACA GAT-plus 3') and a TaqMan Probe (VP 11-Probe:5'-FAM-CTG ATG CGT TCG ATT-MGB-plus 3').

(3) TaqMan Probe qRT-PCR reaction in the prepared fluorescent quantitative RT-PCR reaction system, standard plasmids with different concentration gradients are added except 2 XPremix Ex Taq (Probe qPCR), ROX, a forward primer VP11-F, a reverse primer VP11-R and a Probe VP11-Probe, and sterilized double distilled water is supplemented, and the total volume is 20 mu l. The specificity and feasibility of the primers were determined by analyzing the melting curve of the qRT-PCR product, using a pre-experiment with the recommended standard procedure. On the basis, the optimal proportioning concentration of the upstream primer and the downstream primer (the minimum CT value) is obtained) is determined through an orthogonal test; by changing the PCR reaction temperature, the optimal annealing temperature and reaction time are searched, and finally the reaction parameters are determined.

(4) The qRT-PCR reaction system and condition establishment experimental results show that when the final concentration of the primers is 2 mu mol/L and the annealing and reaction temperature is 60 ℃, the detection sample can obtain smaller C _T Values and stronger fluorescence signal intensities. The optimal reaction overall was determined by screening assays for primer and probe concentrations to be 20 μ L:2 XPrimix Ex Taq (Probe qPCR) 10. Mu.L, VP11-F and VP11-R each 0.4. Mu.L (10. Mu.M), VP11-Probe 0.4. Mu.L (10. Mu.M), ROX 0.4. Mu.L, DNA template 2. Mu.L, ddH ₂ O6.8. Mu.L. Optimized TaqMan qRT-PCR reaction program: pre-denaturation at 95 ℃ for 3min; the mixture was left at 95 ℃ for 10s,60 ℃ for 30s, and subjected to 40 cycles.

Example 4: calculation of amplification efficiency of qRT-PCR

The above dilution series of standards (1X 10) ⁸ 、1×10 ⁷ 、1×10 ⁶ 、1×10 ⁵ 、1×10 ⁴ 、1×10 ³ 、1×10 ² 、 1×10 ¹ Copy/. Mu.L) as a reaction template, 5 replicates per gradient, a fluorescent quantitative PCR reaction was performed according to the method established in example 3, a standard curve was automatically drawn by the instrument and a linear equation and a correlation coefficient R were generated ² . Calculating the amplification efficiency of the primers according to the slope of the linear equation, and calculating each gradient C by using a statistical method _T Coefficient of Variation (CV) of the values, analysis of their reproducibility and stability. The experimental results are shown in fig. 2: the linear equation: c _T = 3.242X +40.442 (where X is the logarithm of the plasmid number), the correlation coefficient R of the linear equation ² 0.998, indicating a good linear relationship of the individual point values on the standard curve and a primer amplification efficiency of 103.416% calculated based on the slope of the standard curve, which is in the optimal range for this type of reaction (95-105%). Statistical analysis to obtain C _T The value variation Coefficient (CV) is between 0.2% and 1.4%, indicating the detection methodThe method has good stability and repeatability (table 2).

TABLE 2 CT values and coefficient of variation of MCRV standard plasmids

Example 5: qRT-PCR sensitivity analysis

On the basis of the aforementioned 10-fold serial dilution of the standard plasmid, 10, 5, 2.5 and 1 copies/. Mu.L of the standard plasmid were further prepared. Fluorescent quantitative PCR reaction using the plasmid diluted in multiple ratios as a template, and analysis C _T A value variation coefficient determining a detection limit value of the method; and (4) judging whether the infection degree and the content of the virus exist or not by analyzing the Ct value corresponding to the amplification curve. As can be seen from FIG. 3, when the standard plasmid is used as the template, the plasmid quantity is 10, 5 and 2.5 copies/reaction, and the amplification can be effectively performed, so that a good S-shaped amplification curve can be obtained, the repeatability among samples is good when the plasmid concentration is 10 copies/μ L, and the detection value is still in the linear range of the standard curve, which indicates that the detection method can be used for the absolute quantification of pure virus particles, and the sensitivity is as high as 10 copies/μ L. When the plasmid concentration was 5 or 2.5 copies/. Mu.L, the measurement was already nonlinear and was not suitable for absolute quantification of the sample. Nevertheless, both gradient plasmid samples gave "S" type amplification curves, indicating that they were still useful for qualitative detection of viruses with a sensitivity of 2.5 copies/. Mu.L (FIG. 4).

Example 6: qRT-PCR specificity analysis

To analyze the specificity of the detection method, fluorescent quantitative PCR reaction is performed by using MCRV-carrying branchial tissue sample as a positive control, healthy scylla branchial tissue cDNA sample as a negative control, and crustacean common pathogenic nucleic acid sample such as cDNA of MCDV (mu Crab Dicystrovirus) and DNA of WSSV (White Spot Syndrome Virus), DIV1 (Decapod Iridescen Virus 1), EHP (Enterobacter hepatopenai) or Vibrio parahaemolyticus. The experimental results show that no visible amplification curve appears in the samples containing MCDV, WSSV, DIV1, EHP and Vibrio parahaemolyticus nucleic acids, which indicates that the method has strong specificity and does not influence the MCRV detection result due to mixed infection or existence of the pathogens (figure 5).

Example 7: application of blue crab reovirus detection kit in blue crab larva MCRV detection

To further verify the practicability of the detection method and understand the MCRV infection condition of the blue crab larvae, the detection method established in example 3 is used for detecting the blue crab larvae collected from a blue crab fingerling farm in Ninghai, zhejiang, including 11 flea larvae in the III stage, 30 megalops larvae and 18 young crabs for MCRV detection. The test results show that all three stages of larvae have different degrees of infection, and the test results are shown in Table 3, and the infection rates of the flea larvae, the megalopas and the juvenile crabs are 18.18 percent, 30.00 percent and 33 percent respectively. The detection method can be used for detecting the young blue crabs.

TABLE 3 blue crab infection investigation

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

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Claims (9)

1. A qRT-PCR kit applied to the detection of the reovirus of the young blue crab is characterized in that the kit detects the ORF area of the genome segment VP11 of the reovirus of the blue crab; the kit comprises a pair of specific primers, and the sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.

2. The kit of claim 1, further comprising a specific TaqMan probe, wherein the probe sequence is shown in SEQ ID NO. 3.

3. The kit of claim 2, further comprising a Taq enzyme premix reagent containing a suitable fluorescent probe and a reverse transcriptase premix reagent containing a Random primer Random 6.

4. The kit according to claim 3, wherein the PCR reaction system of the kit is: 2 XPrimix Ex Taq (Probe qPCR) 10. Mu.L, VP11-F and VP11-R each 0.4. Mu.L (10. Mu.M), VP11-Probe 0.4. Mu.L (10. Mu.M), ROX 0.4. Mu.L (added depending on the need of the instrument), DNA template 2. Mu.L, ddH ₂ O6.8. Mu.L, total reaction volume 20. Mu.l.

5. The kit according to claim 4, wherein the PCR reaction program of the kit is: pre-denaturation at 95 ℃ for 30s; then denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s,40 cycles.

6. A pair of specific primers based on ORF region of the genomic segment VP11 of the scylla serrata reovirus is characterized in that the sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.

7. A TaqMan probe based on an ORF region of a genomic fragment VP11 of a scylla serrata reovirus is characterized in that a probe sequence is shown as SEQ ID NO. 3.

8. The application of the specific primer of claim 6 in preparing a kit for detecting the reovirus of the juvenile blue crab.

9. The use of the probe of claim 7 in the preparation of a kit for detecting reovirus in juvenile crab seedlings.

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