A qRT-PCR kit for detecting reovirus in blue crab seedlings

Abstract

The present invention relates to the field of aquatic pathogen detection, and specifically to a qRT-PCR kit for detecting blue crab seed reovirus, wherein the kit detects the ORF region of the blue crab seed reovirus genome fragment VP11. The present invention provides a highly sensitive blue crab seed reovirus detection kit, the detection technology of which has high sensitivity and strong specificity, and can effectively detect zoeae at different stages. It is particularly suitable for the detection of blue crab seed and fertilized egg viruses with low blue crab seed reovirus content, and can be applied to the investigation of the infection status of blue crab larvae.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic animal pathogen detection and disease prevention and control, and in particular to a qRT-PCR kit and a detection method for detecting reovirus in mud crab seedlings. Background Art

[0002] Early studies have shown that infection with Mud Crab Reovirus (MCRV) can lead to nearly 100% mortality in mud crabs. Studies in recent years have shown that the virus infection is very common and can be detected in almost all mud crab breeding ponds, posing a huge threat to the healthy breeding of mud crabs.

[0003] Studies have found that MCRV can infect blue crab larvae in a variety of ways, causing the seedlings to be infected. Therefore, pathogen detection at the blue crab seedling stage is a key point to cut off the spread of MCRV. In the blue crab breeding process, it needs to go through multiple developmental stages from hatching of fertilized eggs to crablets. Understanding the MCRV status of fertilized eggs and hatched larvae can provide reliable protection for the production of healthy seedlings. Previous studies have found that after fertilized eggs and their zoeae are infected with MCRV, the virus content is usually low, and high-sensitivity diagnostic technology must be used for detection to avoid false negative test results. Therefore, the development of a highly sensitive MCRV detection method is a prerequisite for obtaining healthy seedlings.

[0004] TaqMan probe fluorescent quantitative PCR is one of the most commonly used high-sensitivity pathogen detection technologies. Compared with SYBRGreen fluorescent quantitative PCR, it has lower background signal and higher sensitivity. The new TaqMan-MGB probe further improves the signal-to-noise ratio of this technology, making the experimental results more accurate and the resolution higher. It is one of the most sensitive fluorescent quantitative PCR technologies currently. Summary of the invention

[0005] The present invention found that the expression levels of different genes of MCRV were significantly different, and VP11 was the gene with the highest viral expression. The purpose of the present invention is to further design PCR primers and fluorescent MGB probes based on the highly expressed VP11 sequence, establish a more sensitive MCRV detection method, and meet the needs of MCRV detection in blue crab seedlings.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention has created a highly sensitive TaqMan probe qRT-PCR detection kit for detecting trace pathogens in blue crab seedlings. Previous studies have found that blue crab seedlings and even fertilized eggs are infected with MCRV. Considering that the amount of virus carried by fertilized eggs and larvae is usually very low, it is necessary to develop a highly sensitive detection technology. Among the current conventional fluorescent quantitative PCR detection methods, the TaqMan MGB probe method lacks a melting curve analysis program, but it has the highest sensitivity. To this end, the present invention designs specific primers and specific probes in its conserved region based on the sequence of the MCRV highly expressed gene VP11, and thus establishes a TaqMan MGB probe qRT-PCR detection kit and method. The MCRV detection method has high sensitivity, can accurately quantify 10 copies / reaction, and the lower limit of qualitative detection is 2.5 copies / reaction. In addition, the detection method has strong specificity, and there is no specific amplification with nucleic acid samples of 5 common crustacean pathogens (MCDV, WSSV, DIV1, EHP and Vibrio). Using this detection method, zoeae larvae at different periods are detected, and effective detection is achieved. The above results indicate that this method can be applied to investigate the MCRV infection status of blue crab larvae.

[0008] The kit of the present invention can specifically detect MCRV without cross-reaction with common pathogens of crustaceans. In the present invention, nucleic acid samples of several most common pathogens in mud crabs and crustaceans (such as MCDV, WSSV, DIV1, EHP and Vibrio parahaemolyticus) were detected, and no cross-reaction occurred, indicating that the detection method has strong specificity and can be used for specific detection of MCRV.

[0009] The sensitivity of the kit of the present invention is higher than that of the currently common MCRV detection method. There are two specific reasons. First, the sensitivity of the quantitative detection of the TaqMan MGB probe qRT-PCR detection method is 10 copies / reaction, and the lower limit of the qualitative detection is 2.5 copies / reaction. This detection capability has reached the upper limit of the detection capability of the TaqMan probe fluorescence quantitative detection method. Secondly, the detection method is based on the highly expressed gene VP11, and has obvious advantages over the current detection method based on VP1 and VP6 (both of which have lower expression levels than VP11). In addition, the sensitivity of ordinary RT-PCR and nested RT-PCR detection is lower than that of quantitative PCR. Therefore, on the whole, the sensitivity of this detection method is higher than that of the currently existing MCRV detection method.

[0010] The present invention provides a highly sensitive MCRV detection kit, the detection technology of the detection kit is highly sensitive, and is particularly suitable for the detection of viruses in blue crab seedlings and fertilized eggs with low MCRV content. The preliminary study of the present invention found that MCRV is a conditional pathogen, and blue crab individuals, fertilized eggs and zoeae are all prone to carry the pathogen, but only when the external environment is suitable, will the virus multiply in large quantities, causing the death of blue crabs and larvae. Under normal circumstances, the virus content of blue crab fertilized eggs and their larvae is low, and a highly sensitive detection method is required to avoid the occurrence of false negatives. In addition, seedlings are infected and can easily spread the virus thousands of miles away, causing inestimable losses. Therefore, it is very demanding to determine whether the seedlings carry MCRV. The detection limit of this detection technology has reached 2.5 copies / reaction, which can well meet the needs of seedling pathogen detection.

[0011] Based on the above technical scheme, the first aspect of the present invention provides a qRT-PCR kit for detecting blue crab seedling reovirus, the kit detects the ORF region of blue crab reovirus genome segment 11 (VP11), and the primers are designed with reference to the MCRV genome VP11 segment sequence (GenBank No. HQ414137.1) and the VP11 segment sequences of two other isolated strains (SsRV, GenBank No. HQ414137.1; MCRV-NH, SEQ ID NO: 4), and quantitative primers are designed in the conserved region inside the ORF region of the VP11 gene.

[0012] The optimal target sequence for MCRV detection is the ORF region of viral genome segment 11 (VP11). MCRV expresses a total of 13 genes. The relative expression levels of the 13 genes in MCRV were compared experimentally and it was found that the expression level of VP11 gene was the highest, followed by VP12 gene. The experimental results are shown in Figure 1 Designing quantitative primers based on highly expressed genes can improve the sensitivity of pathogen detection from the background level. The MCRV detection methods reported in the literature are mainly based on VP1 or VP6. The expression levels of these two genes are significantly lower than VP11, which means that the starting point of the detection method based on VP11 is high.

[0013] Furthermore, the kit includes a pair of specific primers designed based on the ORF region of VP11, and the upstream and downstream primer sequences are: the upstream primer MCRVRF sequence is 5′-GTC AGA ATG TCG TTC ATA CTT TGT-3′ (SEQ ID NO: 1), and the downstream primer MCRVRR sequence is 5′-ATT CAG GAG TTC CGG ACA GAT-3′ (SEQ ID NO: 2).

[0014] Furthermore, the kit also includes a specific TaqMan probe based on VP11, and the probe sequence is VP11-Probe: 5′-FAM-CTG ATG CGT TCG ATT-MGB-3′ (SEQ ID NO: 3).

[0015] Furthermore, the kit also includes a Taq enzyme premix reagent suitable for a fluorescent probe (e.g., 2×Premix Ex Taq (Probe qPCR)), a reverse transcriptase premix reagent (e.g., Primescript RT Master Mix, containing random primer Random6), a gradient dilution standard plasmid, a positive control, and a negative control (sterile double distilled water).

[0016] The Taq enzyme premix reagent and reverse transcription reagent contained in the kit are commercially available reagents. 2×PremixEx Taq (Probe qPCR)) and Primescript RT Master Mix are the reagents recommended for use in the present invention. Both of them can be used but are not limited to these two reagents. The standard plasmid is a constructed double-stranded DNA vector containing the full length of the VP11 gene ORF region, which is pMD19T-VP11. The concentration range of the standard plasmid (1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 The positive control was a diluted standard plasmid (1×10 5 copies / μL), and the negative control was ddH 2 O.

[0017] Furthermore, the PCR reaction system of the kit is: 2×Premix Ex Taq (Probe qPCR) 10 μL, VP11-F and VP11-R 0.4 μL (10 μM), VP11-Probe 0.4 μL (10 μM), ROX 0.4 μL (added or not depending on the instrument), DNA template 2 μL, ddH 2 O 6.8μL, the total reaction volume is 20μl. The addition of ROX reference dye depends on the instrument type, and some instruments do not require the addition of reference dye.

[0018] Furthermore, the PCR reaction program of the kit is: pre-denaturation at 95°C for 30s; then denaturation at 95°C for 5s, annealing and extension at 60°C for 30s, and 40 cycles.

[0019] Furthermore, the method for detecting viruses using the kit comprises the following steps:

[0020] (a) Extracting total RNA from fertilized eggs or zoea according to the kit instructions and testing the RNA quality; adding DNase I to RNA samples with qualified quality to remove DNA contamination;

[0021] (b) During reverse transcription, in addition to reverse transcriptase and RNase inhibitor, the reverse transcription system needs to add random primer Random6 to ensure the reverse transcription effect and place it in a 42°C water bath for 30 min;

[0022] (c) In the prepared fluorescence quantitative RT-PCR reaction system, in addition to 10 μL of 2×Premix Ex Taq (ProbeqPCR), 0.4 μL of VP11-F and VP11-R (10 μM), 0.4 μL of VP11-Probe (10 μM), 0.4 μL of ROX (optional), 2 μL of DNA template (sample nucleic acid to be tested, positive or negative control sample), and 6.8 μL of ddH2O, the total reaction volume is 20 μL;

[0023] (d) The reaction conditions of fluorescence quantitative RT-PCR were as follows: pre-denaturation at 95°C for 30 s, followed by denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, for 40 cycles.

[0024] The second aspect of the present invention provides a pair of specific primers based on the ORF region of the blue crab reovirus genome fragment 11 (VP11), and the upstream and downstream primer sequences are: the upstream primer MCRVRF sequence is 5′-GTC AGA ATG TCG TTCATA CTT TGT-3′ (SEQ ID NO: 1), and the downstream primer MCRVRR sequence is 5′-ATT CAG GAG TTC CGG ACAGAT-3′ (SEQ ID NO: 2).

[0025] In a third aspect of the present invention, a TaqMan probe based on the ORF region of the genomic segment 11 (VP11) of the blue crab reovirus is provided, wherein the probe sequence is VP11-Probe: 5′-FAM-CTG ATG CGT TCG ATT-MGB-3′ (SEQ ID NO: 3).

[0026] The fourth aspect of the present invention provides the use of the specific primers and / or probes described above in the preparation of a blue crab seed reovirus detection kit.

[0027] Furthermore, the sampled tissue types of the kit are blue crab zoa, hemolymph or gill tissue (gill tissue is taken when hemolymph sampling is difficult).

[0028] The advantages of the present invention are:

[0029] 1. The pathogen detection method has high sensitivity. The present invention improves the detection sensitivity of the detection kit from two aspects:

[0030] (1) The detection method has the advantage of high expression levels of target genes. Blue crab reovirus expresses a total of 13 protein genes. Through expression level analysis, we found that the VP11 gene has the highest expression level. Most of the existing blue crab reovirus detection methods are based on the design of primers for the VP1 or VP6 genes. The expression levels of these two genes are significantly lower than VP11. In the same test sample, designing primers based on highly expressed genes for pathogen detection is equivalent to a larger number of viral templates, which makes it easier to detect the presence of the virus and improves the sensitivity of the test.

[0031] (2) The fluorescence quantitative PCR detection technology of the detection kit itself has high sensitivity. Compared with ordinary RT-PCR, nested RT-PCR, and colloidal gold technology, the fluorescence quantitative PCR detection technology has higher sensitivity and is also a currently recognized and commonly used high-sensitivity pathogen detection technology. The lower limit of pathogen quantification of the MCRV detection technology established by the present invention is 10 copies / reaction, and the lower limit of pathogen detection is 2.5 copies / reaction, which has reached the limit level of fluorescence quantitative PCR detection technology.

[0032] Therefore, the MCRV detection kit established in the present invention based on the above two points has a significantly higher sensitivity than the currently reported MCRV detection method.

[0033] 2. The detection method has strong sensitivity and specificity

[0034] The detection method of the present invention was used to detect common pathogens of blue crabs and shrimps. It was found that no visible amplification curves appeared in samples containing nucleic acids of MCDV, WSSV, SHIV, EHP and Vibrio parahaemolyticus, indicating that the detection method has strong specificity and will not affect the MCRV detection results due to the mixed infection or presence of these pathogens ( Figure 5 ).

[0035] 3. The present invention provides a highly sensitive MCRV fluorescent quantitative PCR detection kit and a method for using the kit, which has very good application value in the detection of MCRV in blue crab seedlings and fertilized eggs, and the study of viral infection mechanisms.

[0036] First of all, this virus detection method has a very attractive application prospect in the detection of MCRV in blue crab seedlings. MCRV is a conditional pathogen. Blue crab fertilized eggs and their larvae are easily carried by the pathogen, and the virus content is very low. Only when the external environment is suitable will the virus proliferate in large quantities, causing the death of blue crabs and larvae. The characteristic of seedlings carrying MCRV requires a highly sensitive detection method to avoid false negatives. In addition, seedlings are infected and can easily spread the virus thousands of miles away, causing immeasurable losses. Therefore, whether the seedlings carry MCRV is very demanding. The detection limit of this detection technology has reached 2.5 copies / tube, which can well meet the needs of seedling pathogen detection.

[0037] Secondly, the virus detection method also has a good application in pathogen infection mechanism. The study of MCRV infection mechanism involves the study of pathogen proliferation law. The virus content is very low in the early stage of virus infection. Using this detection kit to analyze the infection status and proliferation law of the virus in the blue crab body, the results are more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 .Relative expression levels of 13 predicted genes of MCRV in the hemolymph of blue crab.

[0039] Figure 2 The horizontal axis of the MCRV standard curve is the copy number of the sample, and the vertical axis is C T value.

[0040] Figure 3 In the standard plasmid qRT-PCR amplification curve, the concentrations of standard plasmids No. 1 to No. 8 were 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 and 1×10 1 copies / μL.

[0041] Figure 4 .Sensitivity experiment qRT-PCR amplification curve The concentrations of standard plasmids No. 1 to 4 were 1×10 1 , 0.5×10 1 , 0.25×10 1 and 1×10 0 copies / μL.

[0042] Figure 5 .Specificity experiment; 1, positive control; 2-3, MCRV positive samples; 4, MCDV; 5, WSSV; 6, DIV1; 7, EHP; 8, Vibrio parahaemolyticus; 9, negative control. DETAILED DESCRIPTION

[0043] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0044] Example 1: Among the 13 genes of blue crab reovirus, VP11 gene has the highest expression level

[0045] (1) RNA Extraction After the blue crab is ice-bathed for 5 minutes, a small amount of hemolymph is extracted with a sterile syringe for RNA extraction. Total RNA is extracted according to the steps of a total RNA extraction kit (eg, Transzol UP Plus RNA Kit from Quanshijin Company).

[0046] (2) After the total RNA extracted by reverse transcription is inspected and tested to be qualified, reverse transcription is performed after it meets the requirements. The specific steps of reverse transcription are carried out according to the operating instructions of the reverse transcription kit (for example, TAKARA Primescript RT Master Mix). During reverse transcription, in addition to reverse transcriptase, RNase inhibitor and RNA template, the reverse transcription system needs to add random primer Random6 to ensure the reverse transcription effect, and is placed in a 42°C water bath for 30 minutes. The synthesized cDNA is used for subsequent PCR amplification.

[0047] (3) Primers To analyze the relative expression levels of each MCRV gene, specific primers were designed and synthesized for the 13 predicted viral genes (see Table 1). The relative expression of each gene was analyzed by qRT-PCR using 18S rRNA as the internal reference gene.

[0048] (4) qRT-PCR The qRT-PCR reaction system used the hemolymph cDNA of the virus-infected blue crab as a template, and added 10 μL of 2×SYBR Premix Ex Taq, 0.4 μL of upstream and downstream primers (10 nM), 6.8 μL of sterile water, 2 μL of cDNA template, and 0.4 μL of ROX dye, with a total volume of 20 μL. Reaction parameters: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s, annealing at 60°C for 60 s, and plate reading once; a total of 40 cycles; finally, the temperature was raised from 60°C to 95°C, and the fluorescence value was detected every 0.5°C increase. The relative content of the 13 viral genes was calculated according to the formula (relative expression = 2 18sCT-MCRVCT ) were analyzed. The results showed that there were significant differences in the expression of each gene during MCRV infection ( Figure 1 ). Among them, the average relative expression of VP11 gene was the highest, which was significantly higher than most other genes. The research results suggest that designing primers based on VP11 gene sequence for virus detection can significantly improve the sensitivity of detection.

[0049] Table 1 Primer sequence information

[0050]

[0051] Example 2: Standard plasmid construction

[0052] (1) RNA extraction and reverse transcription were performed according to the steps in Example 1, and the synthesized cDNA was used for quantitative analysis.

[0053] (2) PCR reaction system for nucleic acid fragment amplification: 25 μL of PrimeSTAR HS DNA Taq fidelity enzyme premix, 2 μL of upstream and downstream primers (MCRVVP11F1 and MCRVVP11R1) (10 nM), 1 μL of template, and sterile water to 50 μL. PCR reaction conditions: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 50 s, a total of 35 cycles, and extension at 72°C for 10 min.

[0054] (3) Gene fragment cloning and sequencing The PCR-positive products were recovered from the gel, connected to the pMD19-T vector and transferred into DH5a competent cells. The products were inoculated on LB solid culture dishes containing ampicillin. The colonies were selected for shake culture and identified by PCR. At least 3 positive strains were sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

[0055] (4) Determination and dilution of standard plasmid concentration After confirming that the plasmid was successfully constructed, the OD260 and OD280 values ​​were measured by ultra-micro spectrophotometer (1 OD absorbance value is equivalent to 50 μg / mL of dsDNA) to determine the plasmid purity and calculate the total mass of the plasmid. The calculation formula is as follows: number of plasmid copies per microliter (copies / μL) = total mass of plasmid (μg / μL) / molecular weight of plasmid; molecular weight of plasmid = 2×330×nt (where nt is the number of bases in the plasmid). The standard plasmid was diluted 10 times in series until N×10 1 copies / μL, stored at -20℃ for future use.

[0056] Example 3: Establishment of TaqMan probe qRT-PCR reaction system and conditions

[0057] (1) RNA extraction and reverse transcription were performed according to the steps in Example 1, and the synthesized cDNA was used for quantitative analysis.

[0058] (2) Primer and probe design The primer design was based on the VP11 segment sequence of the MCRV genome (GenBank No. HQ414137.1) and the VP11 segment sequences of two other isolated strains (SsRV, GenBank No. HQ414137.1; MCRV-NH, SEQ ID NO: 4). Quantitative primers and probes were designed in the conserved region within the ORF region of the VP11 gene: a pair of quantitative primers (VP11-F: 5′–GTC AGA ATG TCG TTC ATA CTT TGT–3′; VP11-R: 5′–ATT CAG GAG TTC CGG ACAGAT–3′) and a TaqMan probe (VP11-Probe: 5′–FAM-CTG ATG CGT TCG ATT-MGB–3′).

[0059] (3) TaqMan probe qRT-PCR reaction In the prepared fluorescent quantitative RT-PCR reaction system, in addition to 2×Premix Ex Taq (Probe qPCR), ROX, forward primer VP11-F, reverse primer VP11-R and probe VP11-Probe, standard plasmids of different concentration gradients were added, and sterilized double distilled water was added to a total volume of 20 μl. First, a preliminary experiment was performed using the recommended standard procedure, and the specificity and feasibility of the primers were determined by analyzing the melting curve of the qRT-PCR product. On this basis, the optimal ratio concentration of the upstream and downstream primers was determined by orthogonal experiments (to obtain the minimum CT value (cycle threshold)); by changing the PCR reaction temperature, the optimal annealing temperature and reaction time were found, and the reaction parameters were finally determined.

[0060] (4) The experimental results of establishing the qRT-PCR reaction system and conditions showed that when the final concentration of the primers was 2 μmol / L and the annealing and reaction temperature was 60°C, a smaller C T The optimal total reaction system was determined to be 20 μL by screening the primer and probe concentrations: 10 μL 2×Premix Ex Taq (ProbeqPCR), 0.4 μL (10 μM) each of VP11-F and VP11-R, 0.4 μL (10 μM) of VP11-Probe, 0.4 μL (10 μM) of ROX, 2 μL of DNA template, and ddH 2 O 6.8 μL. Optimized TaqMan qRT-PCR reaction program: 95°C pre-denaturation for 3 min; 95°C for 10 s, 60°C for 30 s, 40 cycles.

[0061] Example 4: Calculation of qRT-PCR amplification efficiency

[0062] The above serial dilution standard (1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 1000 copies / μL) as the reaction template, 5 replicates were set for each gradient, and the fluorescence quantitative PCR reaction was carried out according to the method established in Example 3. The instrument automatically drew the standard curve and generated the linear equation and correlation coefficient R 2 The amplification efficiency of the primers was calculated based on the slope of the linear equation, and the C value of each gradient was calculated using statistical methods. T The coefficient of variation (CV) of the value was calculated to analyze its repeatability and stability. Figure 2 Shown: Linear equation: C T =-3.242X+40.442 (where X is the logarithm of the number of plasmids), the correlation coefficient of the linear equation is R 2 The value of the standard curve is 0.998, indicating that the point value on the standard curve has a good linear relationship, and the primer amplification efficiency calculated based on the slope of the standard curve is 103.416%, which is within the optimal range of this type of reaction (95-105%). T The coefficient of variation (CV) of the values ​​was between 0.2% and 1.4%, indicating that the detection method had good stability and repeatability (Table 2).

[0063] Table 2 CT values ​​and coefficients of variation of MCRV standard plasmids

[0064]

[0065]

[0066] Example 5: qRT-PCR sensitivity analysis

[0067] Based on the 10-fold serial dilution of the aforementioned standard plasmid, 10, 5, 2.5 and 1 copy / μL standard plasmids were further prepared. The diluted plasmids were used as templates for fluorescence quantitative PCR reaction. T The coefficient of variation of the value is used to determine the detection limit of the method; by analyzing the Ct value corresponding to the amplification curve, the degree of virus infection and content are determined. Figure 3It can be seen that when the standard plasmid is used as a template, the plasmid numbers of 10, 5, and 2.5 copies / reaction can all be effectively amplified, and a good "S"-shaped amplification curve can be obtained. When the plasmid concentration is 10 copies / μL, the repeatability between samples is very good, and the detection value is still within the linear range of the standard curve, indicating that this detection method can be used for the absolute quantification of pure virus particles, with a sensitivity of up to 10 copies / μL. When the plasmid concentration is 5 or 2.5 copies / μL, the detection value is no longer linear and is no longer suitable for absolute quantification of samples. Despite this, both gradient plasmid samples can obtain "S"-shaped amplification curves, indicating that they can still be used for qualitative detection of viruses, with a sensitivity of 2.5 copies / μL ( Figure 4 ).

[0068] Example 6: qRT-PCR specificity analysis

[0069] To analyze the specificity of the detection method, samples of gill tissue carrying MCRV were used as positive controls, and cDNA samples of gill tissue of healthy blue crabs were used as negative controls. Common crustacean pathogen nucleic acid samples, such as MCDV (Mud Crab Dicistrovirus) cDNA and WSSV (White Spot Syndrome Virus), DIV1 (Decapod Iridescent Virus 1), EHP (Enterocytozoon hepatopenaei) or Vibrio parahaemolyticus DNA, were used for fluorescence quantitative PCR reactions. The experimental results showed that no visible amplification curves appeared when using samples containing nucleic acids of MCDV, WSSV, DIV1, EHP and Vibrio parahaemolyticus, indicating that the detection method has strong specificity and will not affect the MCRV detection results due to the mixed infection or presence of these pathogens ( Figure 5 ).

[0070] Example 7: Application of the Blue Crab Reovirus Detection Kit in Detection of MCRV in Blue Crab Larvae

[0071] In order to further verify the practicality of the detection method and understand the MCRV infection of blue crab larvae, the detection method established in Example 3 was used to detect blue crab seedlings collected from a blue crab seedling farm in Ninghai, Zhejiang, including 11 zoeae in stage III, 30 megalopa larvae and 18 crablets for MCRV detection. The test results showed that the larvae in the three stages were all infected to varying degrees. The test results are shown in Table 3. The infection rates of stage III zoeae, megalopa and crablets were 18.18%, 30.00% and 33%, respectively. This shows that the detection method can be used for blue crab seedling detection.

[0072] Table 3 Survey on infection status of blue crab

[0073]

[0074] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application. Sequence Listing <110> East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences <120> A qRT-PCR kit for detecting reovirus in blue crab seedlings <130> / <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty four <212> DNA <213> Artificial <400> 1 gtcagaatgt cgttcatact ttgt 24 <210> 2 <211> twenty one <212> DNA <213> Artificial <400> 2 attcaggagt tccggacaga t 21 <210> 3 <211> 15 <212> DNA <213> Artificial <400> 3 ctgatgcgtt cgatt 15 <210> 4 <211> 612 <212> DNA <213> Mud Crab Reovirus <400> 4 atgaataggt caaaagcaat aaacttccaa ccttttatgt tagaaactcg gccacccccta 60 accaccatcc ctataatgga ccagttggtt gaaattggag aacgttctaa tcaaaagtgg 120 agcatgaccg accggttgtt ctttgcgatt aggaagatca atcctatatt cgtcacttcg 180 agccagatac cttcaaaatt tgattacacc attctccaga tgcccactca gctaattgcc 240 tcattgaaag agacactttt gttcttagcc ttctcatatt acctaagaga atatcaagat 300 aaggttggtc aaatgaaatt ttacccagta gccatgaaaa acatgattcc tattgtcaac 360 tatctcaaag atcgtgttca taacaacttt gacactactt tggaacaggc atatcgtcag 420 aatgtcgttc atactttgtt tgcttctgat gcgttcgatt tactttccgg catgatcgct 480 actactagac ttgatctgat tcagaggacc aggatctgtc cggaactcct gaatgtactt 540 aacaaaatgt cctttattct catttatgca ccaaatcgac catctatact ctcttggaaa 600 aaccaaagtt ga 612

Claims

1. A qRT-PCR kit for detecting reovirus in blue crab seedlings, characterized in that: The kit detects the ORF region of the blue crab reovirus genome fragment VP11; the kit includes a pair of specific primers, and the upstream and downstream primer sequences are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively; the kit also includes a specific TaqMan probe, and the probe sequence is shown in SEQ ID NO:

3.

2. The kit according to claim 1, characterized in that The kit also includes a Taq enzyme premix reagent suitable for a fluorescent probe and a reverse transcriptase premix reagent containing a random primer Random6.

3. Specific primers and TaqMan probes based on the ORF region of the VP11 genome fragment of the blue crab reovirus, characterized in that: The upstream and downstream primer sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the TaqMan probe sequence is shown in SEQ ID NO:

3.

4. Use of the specific primers and TaqMan probes as claimed in claim 3 in the preparation of a kit for detecting reovirus in blue crab seedlings.

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