A fluorescent quantitative RT-PCR detection kit and detection method for screening MCRV-free crabs

Abstract

The present invention relates to the field of aquatic pathogen detection, specifically a fluorescent quantitative RT-PCR detection kit and detection method for screening blue crab reovirus-free crab breeders, wherein the kit detects the ORF region of the blue crab reovirus genome fragment VP11. The detection primers are designed based on the conserved region of the gene in the current epidemic strain and the early strain, avoiding the gene mutation site and ensuring the universality of the detection method. The sensitivity of the kit of the present invention is significantly higher than the currently reported blue crab reovirus detection method, and it has strong specificity. It has no specific amplification for 5 common crustacean pathogens, reduces the detection cost, improves the detection efficiency, and can meet the needs of screening blue crab reovirus-free crab breeders under minimally invasive conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic animal pathogen detection and disease prevention and control, in particular to a fluorescent quantitative RT-PCR detection kit and a detection method for screening MCRV-free crabs. Background Art

[0002] Scylla paramamosain, commonly known as mud crab, is an important marine aquaculture species in my country. Diseases pose a major threat to the healthy aquaculture of mud crabs, and mud crab reovirus (MCRV) is the primary pathogen of aquacultured mud crabs. MCRV is a double-stranded, non-enveloped RNA virus with a 12-segment genome and 13 predicted open reading frames encoding 13 proteins. Early studies showed that infection with this virus can cause nearly 100% mortality. Recent studies have shown that infection with this virus is highly prevalent, with the virus being detected in nearly all mud crab aquaculture ponds. Therefore, screening for MCRV-free seed crabs, purifying the pathogen during the breeding stage, and obtaining MCRV-free seed (SPF) are imperative.

[0003] Currently, there are several MCRV detection methods, such as conventional RT-PCR, RT-LAMP, nested RT-PCR, and fluorescent quantitative RT-PCR. Most of these methods rely on primer design based on the VP1 gene. Considering that this gene is not expressed at high levels, this limits the sensitivity of the detection technique. Therefore, it is necessary to design primers for target genes with higher expression levels to improve the sensitivity of the detection method. Furthermore, current MCRV detection methods all involve sampling gill tissue for testing. This method of killing blue crabs for virus detection is clearly unsuitable for screening MCRV-free crabs in breeding farms. Therefore, it is necessary to develop a sampling method that causes minimal damage to blue crabs.

[0004] Fluorescence quantitative PCR technology has been widely used in research such as pathogen quantitative detection due to its high sensitivity, strong specificity, and accuracy and reliability. Compared with TaqMAN probe methods, molecular beacon methods, and LUX primer methods, the SYBR Green fluorescence quantitative detection method offers simple primer design, low cost, and a unique melting curve analysis procedure, making it one of the most widely used pathogen detection technologies. Summary of the Invention

[0005] The purpose of the present invention is to design primers based on viral genes with higher expression levels, and with the help of the sensitivity and practicality of the SYBR Green fluorescent quantitative detection method, develop a MCRV detection method with less trauma to blue crabs, so as to meet the needs of MCRV purification in blue crab species.

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

[0007] The present invention has created a highly sensitive MCRV fluorescence quantitative detection kit that can meet the needs of screening MCRV-free crab breeders under minimally invasive conditions. To maximize the sensitivity of detection, the present invention discovered through research that the viral load in the hemolymph of blue crabs is the highest, and that the VP11 gene has the highest expression level among all 13 viral genes. Therefore, the present invention designed a pair of specific primers based on the conserved region of the VP11 gene sequence and established a SYBR Green qRT-PCR detection method, which has a quantitative detection limit of 50 copies / reaction and a qualitative detection limit of 12.5 copies / reaction. Considering that this detection method also has the advantages of tissue and target gene selection, its sensitivity is significantly higher than other detection methods currently reported. This detection method does not specifically amplify any of the five common crustacean pathogens. Using this kit to detect the virus content in the hemolymph of blue crabs, it was found that the positive rates of 22 breeding crabs and 20 commercially available blue crabs were 54.44% and 85.00%, respectively. In summary, the present invention provides a highly sensitive and practical MCRV detection kit and method, which can meet the needs of screening MCRV-free crab species.

[0008] Based on the above technical solution, the first aspect of the present invention provides a pair of specific primers, wherein the sequence of the upstream primer MCRVRF is: 5'-CAC CCC TAA CCA CCA TCC CTA T-3' (SEQ ID NO: 2), and the sequence of the downstream primer MCRVRR is: 5'-CTT CCT AAT CGC AAA GAA CAA CC-3' (SEQ ID NO: 3).

[0009] Furthermore, the specific primers are used in detecting blue crab reovirus.

[0010] Furthermore, the specific primers are used in the preparation of a fluorescent quantitative RT-PCR detection kit for screening crabs free of blue crab reovirus.

[0011] The second aspect of the present invention provides a fluorescent quantitative RT-PCR detection kit for screening crabs without blue crab reovirus, wherein the kit detects the ORF region of the blue crab reovirus genome segment 11 (VP11). The optimal target sequence for MCRV pathogen detection is the ORF region of the viral genome segment 11 (VP11). By comparing the expression levels of 13 genes predicted in blue crab reovirus, it was found that the expression level of VP11 gene was the highest, followed by VP12 gene. The experimental results are shown in FIG. Figure 2 .

[0012] The kit can specifically detect MCRV and does not cross-react with common crustacean pathogens MCDV, WSSV, DIV1, EHP and Vibrio parahaemolyticus.

[0013] Furthermore, the kit includes a pair of specific primers designed based on the ORF region of VP11, the sequence of the upstream primer MCRVRF is: 5'-CAC CCC TAA CCA CCA TCC CTA T-3' (SEQ ID NO: 2), and the sequence of the downstream primer MCRVRR is: 5'-CTT CCT AAT CGC AAA GAA CAA CC-3' (SEQ ID NO: 3).

[0014] Furthermore, the kit also includes a Taq enzyme premix containing SYBR Green dye (e.g., 2×SYBR Premix Ex Taq), a reverse transcriptase premix containing a random primer Random6 (composed of four bases randomly arranged and 6 bases in length) (e.g., Primescript RT Master Mix), a serially diluted standard plasmid (containing the ORF11 sequence), a positive control (a certain concentration of standard plasmid), a negative control (sterile double-distilled water), and a ROX reference dye.

[0015] Among them, the Taq enzyme premix reagent and reverse transcriptase premix reagent used in the above quantitative analysis are commercial reagents. Different brands of products are expected to have similar effects. 2×SYBR Premix Ex Taq and Primescript RT Master Mix are reagents suitable for verifying the effects of the present invention. The constructed standard plasmid contains the ORF region sequence of the VP11 gene, which is the target sequence of the quantitative primer. After the plasmid is constructed, it is linearized and the concentration is measured, and it is diluted into standards of different concentrations (1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 5×10 1 copies / μL). The positive control was 5×10 1 ~1×10 9 For plasmids with any concentration between copies / μL, intermediate concentration plasmids are recommended.

[0016] Furthermore, the PCR reaction system of the kit is: 10 μl of 2×SYBR Premix Ex Taq premix, 0.4 μl of ROX dye, 0.4 μl of upstream and downstream primers (10 μmol / L), 2 μl of cDNA template, 6.8 μl of sterile double-distilled water, and a total reaction volume of 20 μl.

[0017] Furthermore, the PCR reaction program of the kit is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and plate reading once, for a total of 40 cycles; finally, the temperature is increased from 60°C to 95°C, and the fluorescence value is detected every 0.5°C increase.

[0018] Furthermore, the sampling tissue of the kit is blue crab hemolymph.

[0019] By comparing the virus content in various tissues of blue crabs infected with blue crab reovirus, it was found that the virus content in hemolymph was the highest ( Figure 3 MCRV detection methods currently reported in the literature usually use gill tissue for pathogen detection. Compared with extracting RNA from hemolymph for pathogen detection, under the same detection conditions, the latter is more likely to detect samples with low levels of viral infection. In addition, using hemolymph to detect pathogens does not require killing the crabs, which can also reduce production costs.

[0020] The kit only needs to extract a small amount (~50μl) of hemolymph for MCRV detection, and can detect whether the blue crab is infected with MCRV under minimally invasive conditions. It is particularly suitable for screening MCRV-free crabs.

[0021] Furthermore, the specific method of the kit for detecting viruses includes the following steps:

[0022] (a) Total RNA was extracted from hemolymph and RNA quality was tested. Qualified RNA samples were treated with DNase I to remove DNA contamination.

[0023] (b) During reverse transcription, the template RNA was first denatured at 70°C for 5 minutes and quickly placed on ice for 3 minutes. Random primer Random6, reverse transcriptase, and RNase inhibitor were then added, and the reaction was incubated at 42°C for 30 minutes.

[0024] (c) In the prepared fluorescent quantitative RT-PCR reaction system, in addition to 2×SYBR Premix Ex Taq, forward primer MCRVRF, and reverse primer MCRVRR, the nucleic acid sample to be tested, a standard, and a positive or negative control should be added. Finally, sterile double-distilled water should be added to a total volume of 20 μl.

[0025] (d) Fluorescence quantitative RT-PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and plate reading once, for a total of 40 cycles; finally, the temperature was increased from 60°C to 95°C, with fluorescence values ​​measured every 0.5°C increase.

[0026] (e) Determine the specificity of the amplified product by analyzing the peak shape and melting temperature of the PCR product melting curve; determine the presence and extent of viral infection by analyzing the Ct value corresponding to the amplification curve, and calculate the viral content.

[0027] The third aspect of the present invention provides a method for screening reovirus-free crabs under minimally invasive conditions, which comprises extracting a small amount of blue crab hemolymph (~50 μl / crab) and performing virus detection using the above-mentioned kit.

[0028] Simply extracting RNA from a small amount of mud crab hemolymph and using the previously established fluorescent quantitative RT-PCR detection kit can successfully select MCRV-free broodstock crabs. This procedure causes minimal damage to the broodstock crabs and does not affect subsequent broodstock reproduction. It can provide healthy MCRV-free broodstock crabs for mud crab hatchery farms and has great potential for application in the MCRV purification and production of mud crab hatchery crabs.

[0029] The virus detection method can also be used for mud crab pathogen detection and pathogen infection related research. Compared with taking gill tissue for pathogen detection, extracting hemolymph to detect MCRV and analyze MCRV proliferation patterns is more convenient and quick.

[0030] In a fourth aspect, the present invention provides an ORF sequence of the VP11 genome segment of the current epidemic strain of blue crab reovirus (MCRV-NH), the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0031] Furthermore, compared with the ORF11 of the early strains (the Genbank numbers of the corresponding segments of MCRV and SsRV are HQ414137.1 and JQ287709.1, respectively), 4 bases in the ORF11 sequence of MCRV-NH were mutated, including a mutation of nucleotide 7 from T to A (T 7 →A), the nucleotide at position 27 mutated from T to C (T 27 →C), the nucleotide at position 259 mutated from C to T (C 259 →T), and the 440th nucleotide mutated from C to T (C 440 →T), see Figure 1 The detection primers of the present invention are designed based on the conserved region of the gene in the current epidemic strains and early strains, avoiding the gene mutation sites and ensuring the universality of the detection method.

[0032] The advantages of the present invention are:

[0033] 1. High sensitivity of pathogen detection method; the present invention improves the detection sensitivity of the detection kit from three aspects:

[0034] (1) The detection method of the present invention has the advantage of high target gene expression level. Blue crab reovirus expresses a total of 13 protein genes. The present invention found that the expression level of VP11 gene was the highest through expression level analysis. Most of the existing blue crab reovirus detection methods are based on the design of primers for VP1 or VP6 genes, and the expression levels of these two genes are significantly lower than VP11. In the same test sample, pathogen detection based on primers designed for highly expressed genes 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 detection.

[0035] (2) The fluorescence quantitative PCR detection technology used in the detection kit of the present invention is highly sensitive. Compared with conventional RT-PCR, nested RT-PCR, and colloidal gold techniques, fluorescence quantitative PCR detection technology is more sensitive and is currently recognized as a commonly used high-sensitivity pathogen detection technology. The MCRV detection technology established in the present invention has a lower limit of 50 copies / reaction for pathogen quantification and 12.5 copies / reaction for pathogen qualitative detection, which has reached the upper level of fluorescence quantitative PCR detection technology.

[0036] (3) The detection method of the present invention also has the advantage of tissue selection for pathogen detection. During the development of the present invention, experiments confirmed that in MCRV-infected blue crabs, the virus content is highest in the hemolymph, followed by the gills. Most of the MCRV detection methods currently reported in the literature use gill tissue extraction for virus detection. In comparison, based on our experimental results, virus detection by extracting hemolymph is more sensitive.

[0037] Therefore, the MCRV detection kit established in the present invention based on the above three reasons has a significantly higher sensitivity than the currently reported MCRV detection method.

[0038] 2. The detection method of the present invention has strong specificity;

[0039] The detection method was used to detect common pathogens of blue crabs and shrimps. It was found that no visible amplification curves appeared in samples containing MCDV, WSSV, SHIV, EHP and Vibrio parahaemolyticus nucleic acids. This shows that the detection method of the present invention has high specificity and will not affect the MCRV detection results due to the mixed infection or presence of these pathogens.

[0040] 3. The present invention provides a highly sensitive blue crab reovirus fluorescence quantitative PCR detection kit and its use method, which has great application value in the selection of MCRV-free crab species, MCRV detection and infection mechanism research.

[0041] First, this virus detection method holds great promise for selecting MCRV-free blue crab fry. Selecting MCRV-free blue crab fry is a prerequisite for producing virus-free seedlings. Currently, this method is primarily performed through random sampling and assessment of the virus prevalence in the population. This involves killing some blue crabs and taking their gill tissue for testing. This test kit allows for virus testing by extracting a small amount of hemolymph, allowing the blue crabs to continue to be farmed. This method reduces costs and improves efficiency.

[0042] Secondly, this virus detection method offers advantages in pathogen detection and epidemiological research, particularly during the mud crab on-growing stage, from snap crabs to mud crabs. Hemolymph extraction during this stage makes detection more convenient and more sensitive. Of course, this method can also be used to detect pathogens by extracting gill tissue.

[0043] Again, this virus detection method also has good applications in pathogen infection mechanisms and can be used to analyze the infection status and proliferation patterns of viruses in blue crabs. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Comparison of ORFs sequences of VP11 fragments of MCRV-NH, MCRV, and SsRV isolates; four bases were mutated in the former compared with the latter two.

[0045] Figure 2 Relative expression levels of the 13 predicted MCRV genes in the hemolymph of mud crabs. VP11 gene expression was the highest, followed by VP12.

[0046] Figure 3 .Tissue distribution of MCRV: 1, hemolymph; 2, gills; 3, intestine; 4, heart; 5, stomach; 6, hepatopancreas; 7, muscle.

[0047] Figure 4 PCR product melting curve and standard curve; (A) Melting curve analysis of quantitative PCR amplification products, showing a single peak near 81.5°C; (B) Standard curve generated using MCRV standard as template, showing good linear relationship among all sample points.

[0048] Figure 5 Fluorescence quantitative PCR amplification curve and sensitivity analysis; (A) The concentrations of standard plasmids No. 1 to No. 9 in the figure are 1×10 9 , 1×108 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 and 5×10 1 (B) The concentrations of standard plasmids No. 1 to 3 in the figure are 50, 25, and 12.5 copies / μL, and the detection limit of this detection method can reach 12.5 copies / μL.

[0049] Figure 6 .MCRV specific detection test results 1, MCRV positive sample; 2, MCDV; 3, WSSV; 4, DIV1; 5, EHP; 6, Vibrio parahaemolyticus.

[0050] Figure 7 .MCRV proliferation trend in blue crab hemolymph (n=6) After MCRV infects healthy blue crabs, the virus proliferates exponentially and the virus can be detected within 24 hours; the virus proliferation enters the plateau phase after 120 hours. DETAILED DESCRIPTION

[0051] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0052] Example 1: Obtaining the ORF11 sequence of the VP11 genome segment of the current epidemic strain of blue crab reovirus (MCRV-NH2020)

[0053] (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 procedures of a total RNA extraction kit (e.g., Transzol UP Plus RNA Kit from Quanshijin Company).

[0054] (2) After the total RNA extracted by reverse transcription is inspected and tested to meet the requirements, reverse transcription is performed. 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. Finally, the reaction is placed at 42℃ for 30 minutes. The synthesized cDNA is used for subsequent PCR amplification.

[0055] (3) Primers: Using the MCRV genome VP11 gene sequence (Genbank accession number HQ414137.1) as a reference sequence, a pair of specific primers (VP11F: 5'-CAG AGG TGA TTG ACT TCT CT-3', SEQ ID NO: 4; VP11R: 5'-CAC TTC TAC GGT GAA CTG AT-3', SEQ ID NO: 5) were designed to amplify the genomic fragment.

[0056] (4) The PCR reaction system used for nucleic acid fragment amplification was as follows: 25 μL of PrimeSTAR HS DNA Taq Fidelity Enzyme premix, 2 μL of upstream and downstream primers (10 nM), 1 μL of template, and sterile water to 50 μL. PCR reaction conditions were: 35 cycles of pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 50 seconds, followed by extension at 72°C for 10 minutes.

[0057] (5) Gene Fragment Cloning and Sequencing: PCR-positive products were recovered from gels, ligated to the pMD19-T vector, and transformed into DH5a competent cells. The cells were then plated onto LB solid culture plates containing ampicillin. Colonies were selected for shake culture and identified by PCR. At least three positive strains were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The ORF sequence of VP11 (SEQ ID NO: 1) was obtained using online software based on the sequencing results.

[0058] (6) ORF sequence and similarity analysis of VP11. Similarity comparison revealed that the ORF sequences of the early strains MCRV and SsRV were identical. However, compared with the ORF region of MCRV-NH, four bases in the ORF11 sequence of MCRV-NH were mutated, including a mutation of the 7th nucleotide from T to A (T 7 →A), the nucleotide at position 27 mutated from T to C (T 27 →C), the nucleotide at position 259 mutated from C to T (C 259 →T), and the 440th nucleotide mutated from C to T (C 440 →T), see Figure 1 This indicates that MCRV-NH is different from the early strains.

[0059] Example 2: Among the 13 genes of blue crab reovirus, VP11 gene has the highest expression level

[0060] (1) RNA extraction and reverse transcription were performed according to the steps in Example 1, and the synthesized cDNA was used for quantitative analysis.

[0061] (2) Primers To analyze the relative expression levels of MCRV genes, specific primers were designed and synthesized for each of the 13 predicted viral genes (Table 1). The relative expression levels of each gene were analyzed by qRT-PCR using 18S rRNA as the internal reference gene.

[0062] (3) qRT-PCR The qRT-PCR reaction system used the virus-infected blue crab hemolymph cDNA as a template, and added 2×SYBR Premix Ex Taq 10μL, ROX dye 0.4μL, upstream and downstream primers 0.4μL each (10nM), cDNA template 2μL, sterile water 6.8μL, and a total volume of 20μL. Reaction parameters: 95℃ pre-denaturation for 3min; 95℃ denaturation for 10s, 60℃ annealing for 30s, plate reading once, a total of 40 cycles; finally, the temperature was increased from 60℃ to 95℃, and the fluorescence value was detected every 0.5℃ 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 various genes during MCRV infection ( Figure 2 VP11 gene expression levels were the highest, significantly higher than most other genes. The results suggest that designing primers based on the VP11 gene sequence for virus detection can significantly improve detection sensitivity.

[0063] Table 1 Primer sequence information

[0064]

[0065]

[0066] Example 3: Comparison of the Content of Scylla Reovirus in Various Tissues of Scylla

[0067] (1) RNA Extraction and Reverse Transcription Six blue crabs were selected for analysis of the tissue distribution of virus infection. After hemolymph extraction, the blue crabs were dissected and the gills, hepatopancreas, heart, stomach, intestine, and muscle tissues were obtained. Referring to the RNA extraction and reverse transcription steps in Example 1, total RNA from each tissue was obtained and cDNAs were synthesized for quantitative analysis.

[0068] (2) Primers: Using the MCRV genome VP11 gene ORF sequence as a reference sequence, a pair of specific primers, MCRVVP11RF and MCRVVP11RR, were designed. 18S rRNA was used as an internal reference gene, and the internal reference gene primers were 18SRF and 18SRR (Table 1).

[0069] (3) qRT-PCR fluorescence quantitative PCR reaction system and procedure refer to Example 2. The relative content of virus in each tissue is calculated according to the formula (relative virus content = 218sCT-MCRVCT The relative amount of MCRV in infected blue crabs was analyzed by qRT-PCR. Figure 3 As shown, the virus was distributed in all tissues tested, with significantly higher levels in hemolymph and gills compared to other tissues. The order of average relative viral content was hemolymph, gills, intestine, heart, stomach, hepatopancreas, and muscle. These results suggest that using hemolymph as a sampling tissue for MCRV detection can improve the sensitivity of the detection method.

[0070] Example 4: Establishment of qRT-PCR reaction system and conditions

[0071] (1) Construction of standard plasmids The steps of gene fragment cloning and sequencing were carried out in accordance with the relevant steps in Example 1. After confirming that the plasmid construction was successful, the OD260 and OD280 values ​​were measured by ultra-micro spectrophotometer (1OD absorbance value is equivalent to 50μg / mL of dsDNA) to determine the purity of the plasmid 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 the plasmid (μg / μL) / molecular weight of the plasmid; molecular weight of the plasmid = 2×330×nt (where nt is the number of bases in the plasmid). The standard plasmid was serially diluted 10 times until N×10 1 copies / μL, stored at -20℃ for future use.

[0072] (2) Optimization and establishment of fluorescence quantitative RT-PCR reaction parameters

[0073] In the prepared fluorescent quantitative RT-PCR reaction system, in addition to 2×SYBR Premix Ex Taq, forward primer MCRVRF and reverse primer MCRVRR, standard plasmids of different concentration gradients were added, and sterilized double-distilled water was supplemented to a total volume of 20μl. A preliminary experiment was first 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 through 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 finally the reaction parameters were determined. The experimental results showed that the PCR product only had a single peak near 81.5°C, indicating that the primer was a specific amplification ( Figure 4 A) When the final concentration of primers is 2 μmol / L, the annealing and reaction temperature is 60°C, the detection sample can obtain a smaller C TThe results were optimized to achieve a higher fluorescence signal intensity. After experimental optimization, the final fluorescence quantitative PCR reaction system was determined to be 20 μL: 10 μL of 2×SYBR Premix Ex Taq, 0.4 μL of ROX reference dye, 0.4 μL of each upstream and downstream primer (10 μM), 2 μL of cDNA template, and 6.8 μL of sterile water. The reaction parameters were: 95°C pre-denaturation for 5 minutes; 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and a single plate read. Finally, the temperature was increased from 60°C to 95°C, with fluorescence readings taken every 0.5°C increase.

[0074] (3) Analysis of primer amplification efficiency

[0075] The above serial dilution standards (1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 5×10 1 100 copies / μL) as the reaction template, 5 replicates were set for each gradient, and the fluorescence quantitative PCR reaction was performed according to the established fluorescence quantitative qRT-PCR reaction system and parameters. 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 values ​​of each gradient were calculated using statistical methods. T The coefficient of variation (CV) of the values ​​was used to analyze their repeatability and stability. Figure 4 B shows: the linear equation is C T =-3.346X+37.12 (X is the logarithm of the plasmid copy number), correlation coefficient R 2 =0.997, indicating that the standard curve has a good linear relationship. The primer amplification efficiency calculated based on the standard curve is 99.0%, which is within the optimal range (95-105%). T The coefficient of variation (CV) of the values ​​was between 0.2% and 1.5%, indicating that the detection method had good repeatability and high stability (Table 2).

[0076] Table 2 CT values ​​and coefficients of variation of MCRV standard plasmids

[0077]

[0078]

[0079] (4) Sensitivity analysis of detection methods

[0080] Based on the 10-fold serial dilution of the aforementioned standard plasmid, 50, 25, and 12.5 copies / μL standard plasmids were further prepared. The serially 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; the specificity of the amplified product is determined by analyzing the peak shape and melting temperature of the PCR product melting curve; the degree of virus infection and content are determined by analyzing the corresponding Ct value of the amplification curve. Figure 5 As can be seen from A, when the standard plasmid is used as a template, all reactions can be effectively amplified, and a good "S"-shaped amplification curve is obtained. When the plasmid concentration is 50 copies / μL, the reproducibility 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 50 copies / μL. When the plasmid concentration is 25 and 12.5 copies / μL, the detection value is nonlinear and is no longer suitable for absolute quantification of samples. Despite this, both gradient plasmid samples can obtain an "S"-shaped amplification curve, indicating that it can still be used for qualitative detection of viruses, with a sensitivity of 12.5 copies / μL ( Figure 5 B).

[0081] (5) Specificity analysis of detection methods

[0082] To analyze the specificity of the detection method, a gill tissue sample carrying MCRV was used as a positive control, and a cDNA sample of gill tissue from a healthy blue crab was used as a negative control. Fluorescence quantitative PCR reactions were performed using nucleic acid samples from common crustacean pathogens, such as MCDV cDNA and DNA from WSSV, DIV1, EHP, or Vibrio parahaemolyticus. The experimental results showed that no visible amplification curves were observed when using samples containing nucleic acid from MCDV, WSSV, DIV1, EHP, and Vibrio parahaemolyticus, indicating that the detection method has high specificity and that the presence or absence of mixed infection with these pathogens will not affect the MCRV detection results. Figure 6 ).

[0083] Example 5: Application of the Blue Crab Reovirus Detection Kit in Screening MCRV-Free Crabs and Pathogen Detection

[0084] In order to evaluate the practicality of the detection method and understand the MCRV infection status, 22 breeding crabs collected from Hainan in May 2021 and 20 commercial blue crabs collected from Zhejiang in September 2021 were tested for MCRV infection. The MCRV detection method established in Example 4 was used for pathogen detection, and the results are shown in Table 3. By extracting a trace amount of hemolymph, 22 breeding crabs from Hainan were tested, of which 12 were MCRV positive, with an infection rate of 54.55%; 10 healthy breeding crabs without MCRV infection were cultivated, and all achieved egg holding and hatching in the later stage. The above results show that the detection method can be used to screen MCRV-free breeding crabs, and extracting a small amount of hemolymph from breeding crabs does not affect the later egg holding and hatching. In addition, 20 commercial crabs purchased from a farm in Zhejiang were tested, and the infection rate was 85.00% (Table 3). This shows that the detection method can meet the needs of investigating the pathogen infection of blue crabs.

[0085] Table 3 Survey on infection of blue crab

[0086]

[0087] Example 6: Application of the Blue Crab Reovirus Detection Kit in Analysis of Blue Crab Infection Status

[0088] Six healthy, roughly uniform-sized mud crabs (~250 g each) were purchased from a Shanghai farm and tested for MCRV infection. The experimental water temperature was set at 21°C. A crude viral extract was prepared according to the literature (Xiong Juan. Determination of the partial gene sequence of the reovirus in Scylla serrata and establishment of an RT-LAMP assay [D]. Wuhan: Huazhong Agricultural University, 2011). 200 μL of the crude viral extract was injected into the experimental group of mud crabs at the base of the legs. A small amount of hemolymph was extracted at 0, 24, 48, 72, 96, 120, 144, 168, and 192 hours after infection for RNA analysis. During the infection period, mud crab mortality was recorded. The experimental results showed that after MCRV infection of healthy mud crabs, the virus proliferated exponentially. Virus was detectable after 24 hours, and by 120 hours, the virus proliferation entered a plateau phase, slowing the rate of viral proliferation. Thereafter, the viral content increased slightly before beginning a downward trend. Figure 7 In addition, the present invention found that no blue crab died during the entire infection cycle. This shows that the detection method can be used to analyze the MCRV infection status.

[0089] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from 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 fluorescent quantitative RT-PCR detection kit and detection method for screening MCRV-free crabs <130> / <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 612 <212> DNA <213> Mud Crab Reovirus <400> 1 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 <210> 2 <211> twenty two <212> DNA <213> Artificial <400> 2 cacccctaac caccatccct at 22 <210> 3 <211> twenty three <212> DNA <213> Artificial <400> 3 cttcctaatc gcaaagaaca acc 23 <210> 4 <211> 20 <212> DNA <213> Artificial <400> 4 cagaggtgat tgacttctct 20 <210> 5 <211> 20 <212> DNA <213> Artificial <400> 5 cacttctacg gtgaactgat 20

Claims

1. A pair of specific primers, characterized in that, The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

2. Use of the specific primers according to claim 1 in preparing a fluorescent quantitative RT-PCR detection kit for screening crabs free of blue crab reovirus.

3. A fluorescent quantitative RT-PCR detection kit for screening crabs free of blue crab reovirus, 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 designed based on the ORF region of VP11, the sequence of the upstream primer MCRVRF is shown in SEQ ID NO: 2, and the sequence of the downstream primer MCRVRR is shown in SEQ ID NO:

3.

4. The kit according to claim 3, wherein The kit also includes a Taq enzyme premix reagent containing SYBR Green dye and a reverse transcriptase premix reagent containing random primer Random6.

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