A kit and method for rapid detection of Macrobrachium rosenbergii infectious precocious virus

Abstract

The present invention discloses a kit for rapid detection of Macrobrachium rosenbergii infectious precocious virus and a method for detecting Macrobrachium rosenbergii infectious precocious virus using the kit, belonging to the technical field of virus detection. The kit comprises PCR Mix, enzyme Mix, negative quality control product, and positive quality control product, wherein the PCR Mix comprises upstream and downstream primers and fluorescent probes, and the specific nucleotide sequence is shown in SEQ ID NO.1 to SEQ ID NO.9. The kit of the present invention can not only rapidly and accurately detect Macrobrachium rosenbergii infectious precocious virus, but also has the advantages of strong specificity, low detection limit, high sensitivity, and simple operation, and is suitable for large-scale application.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a kit and a detection method for rapidly detecting infectious precocious virus of Macrobrachium rosenbergii based on fluorescence quantitative PCR. Background Art

[0002] Infectious Precocity Virus (IPV) is an RNA virus associated with precocious puberty in Macrobrachium rosenbergii, the so-called iron shrimp syndrome (IPS), which can cause slow growth and precocious puberty in Macrobrachium rosenbergii. In addition, IPV is highly contagious. Once it occurs, all shrimp in the pond will show symptoms such as precocious puberty and slow growth. Before the shrimp fry are put into the pond, they will not show any symptoms. They will only be discovered when they grow to 5-6 cm or even when they are harvested, causing huge economic losses to the shrimp farming industry. For this highly contagious and harmful disease, only a prevention-oriented strategy can be adopted. Early and accurate detection of whether Macrobrachium rosenbergii is infected with IPV and targeted treatment will help reduce economic losses and prevent the spread of pathogens. At present, there are few studies on infectious precocity virus of Macrobrachium rosenbergii, and its detection is also based on traditional virus detection methods, such as high-throughput sequencing. However, due to the particularity and complexity of IPV virus, these detection methods have long detection time, high cost, complex operation, high detection difficulty, and are not conducive to large-scale promotion and application.

[0003] Taqman probe is a fluorescent labeled probe, which is widely used in fluorescent quantitative PCR technology. During the PCR amplification process, by adding specific Taqman fluorescent probes, the amplification of the virus can be monitored in real time. The two ends of the Taqman probe are labeled with a reporter fluorescent group and a quencher fluorescent group respectively. When the probe binds to the target DNA, the quencher group absorbs the fluorescence emitted by the reporter group, thereby achieving synchronization of the accumulation of fluorescent signals and the formation of PCR products. This method can not only detect viruses quickly and accurately, but also has high sensitivity and high specificity. At present, there is still a lack of effective means for the detection of Macrobrachium rosenbergii infectious precocious virus, which is difficult to meet the actual needs of the shrimp farming industry. Therefore, if a kit that can quickly and accurately detect Macrobrachium rosenbergii infectious precocious virus can be designed, it will be possible to strengthen the monitoring of Macrobrachium rosenbergii infectious precocious virus, facilitate early diagnosis and early warning, and promote the healthy development of the Macrobrachium rosenbergii industry. Summary of the invention

[0004] In view of the defects of the prior art, the present invention designs a kit for rapidly detecting infectious precocious virus of Macrobrachium rosenbergii, and establishes a method for detecting infectious precocious virus by using the kit.

[0005] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions:

[0006] In a first aspect of the present invention, a kit for detecting infectious precocious virus of Macrobrachium rosenbergii is provided, characterized in that the kit comprises PCR Mix, enzyme Mix, negative quality control product, and positive quality control product; wherein the PCR Mix comprises at least one group selected from the following three groups of upstream and downstream primers and fluorescent probes:

[0007] The nucleotide sequences of the first group of upstream and downstream primers and fluorescent probes are shown in SEQ ID NO.1 to SEQ ID NO.3;

[0008] The nucleotide sequences of the second group of upstream and downstream primers and fluorescent probes are shown in SEQ ID NO.4 to SEQ ID NO.6;

[0009] The nucleotide sequences of the third group of upstream and downstream primers and fluorescent probes are shown in SEQ ID NO.7 to SEQ ID NO.9.

[0010] Preferably, the upstream primer concentration is 50-500 mM, the downstream primer concentration is 50-500 mM, and the fluorescent probe concentration is 25-250 mM.

[0011] Preferably, the fluorescent probe is a Taqman probe, the 5' end of which is connected to a fluorescent light-emitting group, and the 3' end of which is connected to a fluorescent quenching group.

[0012] Preferably, the fluorescent light-emitting group is one of FAM, VIC, HEX, JOE, ROX, Texas-Red, and Cy5; and the fluorescent quenching group is one of DABCYL, DABSYL, TAMRA, BHQ-1, BHQ-2, and BHQ-3.

[0013] Preferably, the PCR Mix also includes PCR Probe Buffer; the PCR Probe Buffer consists of 20mM~80M Tris-HCl with a pH of 7.5~8.9, 10mM~50mM U-containing dNTP, 100mM~4M KCl, 1mM~50mM MgCl2, 0.01~0.3% by volume of Tween-20, 0.01~0.1% by volume of tetrahydrothiophene 1-oxide, 10mM~5M tetramethylammonium chloride, 10mM~100mM betaine, 0.01~0.1% by volume of DMSO, and 0.01~0.3% by volume of Triton X-100.

[0014] Preferably, the enzyme Mix comprises 0.5-2U hot start Taq enzyme, 0.5-5U reverse transcriptase, 0.1U-10U UDG enzyme, 1U-10U RNase inhibitor, 1%-50% glycerol by volume, 10mM-200mM potassium ions, and 0.01-1mM EDTA.

[0015] Preferably, the negative quality control product is nuclease-free water; and the positive quality control product comprises a plasmid solution synthesized using the IPV target gene sequence.

[0016] In a second aspect of the present invention, a method for detecting infectious precocious virus of Macrobrachium rosenbergii using the above-mentioned kit is provided, comprising the following steps:

[0017] Step S1: extracting the nucleic acid solution in the sample to be tested;

[0018] Step S2: using the nucleic acid solution of step S1 as a template, performing a PCR amplification reaction on the template using the kit for detecting infectious precocious virus of Macrobrachium rosenbergii described in claim 1, and detecting the concentration of the product.

[0019] Preferably, in step S2, a 25 μL PCR reaction system is used, including 19 μL PCR Mix, 1 μL enzyme Mix, and 5 μL template.

[0020] Preferably, the PCR amplification reaction program is: 50°C for 1 min; 95°C for 2 min; 93°C for 10 s, 55-65°C for 30 s, and 40 cycles.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a kit capable of detecting infectious precocious virus based on fluorescent quantitative PCR technology, wherein the kit includes PCR Mix, enzyme Mix, negative quality control product, and positive quality control product, wherein the PCR Mix includes upstream primers, downstream primers, Taqman fluorescent probe, and PCR Probe Buffer; the enzyme Mix contains hot start Taq enzyme, reverse transcriptase, UDG enzyme, RNase inhibitor, glycerol, potassium ions, and EDTA. At the same time, the present invention also establishes a method for detecting infectious precocious virus with the kit, which is simple to operate. Compared with traditional virus detection methods, the kit and detection method of the present invention have high sensitivity and high specificity, and its detection limit is as low as 5 copies / mL, which can not only quickly and accurately detect IPV virus, but also reduce costs and is suitable for large-scale use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 is a schematic diagram of Taqman oligonucleotide fluorescent probe;

[0025] Figure 2 It is a diagram of the IPV detection results of the kit composed of three groups of primers and probes in Example 2, in which: IPV-F1R1P1 is the first group, IPV-F2R2P2 is the second group, and IPV-F3R3P3 is the third group;

[0026] Figure 3 This is a graph showing the accuracy test results of the kit for detecting infectious precocious virus of Macrobrachium rosenbergii in Example 4;

[0027] Figure 4 This is a sensitivity test result diagram of the kit for detecting infectious precocious virus of Macrobrachium rosenbergii in Example 5. In the figure, E7 is 1×10 7 copies / mL, E6 is 1×10 6 copies / mL, E5 is 1×10 5 copies / mL, E4 is 1×10 4 copies / mL, E3 is 1×10 3 copies / mL, E2 is 1×10 2 copies / mL, E1 is 10 copies / mL, 0.5E7 is 5 copies / mL, 0.25E1 ​​is 2.5 copies / mL, and NTC is H2O;

[0028] Figure 5 This is a schematic diagram of a standard curve of a kit for detecting infectious precocious virus of Macrobrachium rosenbergii in Example 5;

[0029] Figure 6 This is a graph showing the specific detection results of the kit for detecting infectious precocious virus of Macrobrachium rosenbergii in Example 6. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0032] Example 1: A kit for detecting infectious precocious virus of Macrobrachium rosenbergii

[0033] 1. Design and synthesize primers and probes

[0034] According to the conserved region sequence (SEQ ID NO. 10) of the IPV virus (GenBank: ON382579.1) on the NCBI platform, three sets of oligonucleotide upstream and downstream primers and Taqman oligonucleotide fluorescent probes with nucleotide sequences as shown in Table 1 were designed. The primers and probes were synthesized by Sangon Biotechnology Co., Ltd.

[0035] Table 1 Sequence list of oligonucleotide upstream primer, downstream primer and Taqman fluorescent probe

[0036]

[0037] In Table 1, IPV-F represents the upstream primer, IPV-R represents the downstream primer, and IPV-P represents the Taqman fluorescent probe.

[0038]

[0039] 2. Kit Composition

[0040] The kit includes: PCR Mix, enzyme Mix, negative quality control product, and positive quality control product.

[0041] like Figure 1 As shown, the PCR Mix contains 300mM of oligonucleotide upstream primer, 300mM of oligonucleotide downstream primer, and 100mM of Taqman oligonucleotide fluorescent probe. The 5' end of the Taqman oligonucleotide fluorescent probe is connected to a fluorescent luminescent group, and the 3' end is connected to a fluorescent quenching group, and the fluorescent luminescent group is one of FAM, VIC, HEX, JOE, ROX, Texas-Red, and Cy5; the fluorescent quenching group is one of DABCYL, DABSYL, TAMRA, BHQ-1, BHQ-2, and BHQ-3.

[0042] The PCR Mix further comprises a PCR Probe Buffer, which is composed of 20 M Tris-HCl with a pH of 8.0, 30 mM U-containing dNTP, 1 M KCl, 30 mM MgCl2, 0.1% (V / V) Tween-20, 0.05% (V / V) tetrahydrothiophene 1-oxide, 2 M tetramethylammonium chloride, 50 mM betaine, 0.05% (V / V) DMSO, and 0.1% (V / V) Triton X-100.

[0043] The enzyme mix contains hot start 1U Taq polymerase, 2U reverse transcriptase, 5U UDG enzyme, 5U RNase inhibitor, 30% (V / V) glycerol, 120mM potassium ion, and 0.2mM EDTA.

[0044] The positive quality control product is a plasmid solution synthesized using the IPV target gene sequence (SEQ ID NO.11), and the negative quality control product is nuclease-free water.

[0045]

[0046] 3. Fluorescence quantitative PCR detection method

[0047] Shrimp tissue samples were selected as samples to be tested, and fluorescent quantitative PCR detection was performed using the above kit. The specific steps included:

[0048] 1. Use a commercial RNA extraction kit, such as a nucleic acid extraction reagent based on a silica gel membrane centrifugal column method or a nucleic acid extraction reagent based on a magnetic bead method, and extract the sample to be tested according to the kit instructions. Finally, collect 80 μL of nucleic acid solution for direct subsequent testing or store at -80°C. Both negative and positive quality control products need to be extracted.

[0049] 2. Preparation of PCR reaction system

[0050] According to the total number of reactions N required for the test, add 19 μL PCR Mix and 1 μL enzyme Mix to each PCR reaction tube. Calculate the total amount required, mix well, and then dispense into N specially prepared PCR reaction tubes; then add 5 μL each of the negative control, positive control, and nucleic acid solution of the sample to be tested extracted in step 1 to the PCR reaction tube, cover the tube tightly, mix well, and then centrifuge at 400r / min for 3~5s to collect the solution and place it at the bottom of the tube. The PCR amplification reaction procedure is shown in Table 2 below:

[0051] Table 2 PCR amplification reaction schedule

[0052]

[0053] 3. After the reaction is completed, the fluorescence signal is collected, and the detection channel amplification curve and the amplification cycle number Ct value are recorded. The detection result is judged according to the amplification curve and the amplification cycle number. The result judgment criteria are shown in Table 3 below:

[0054] Table 3 Test result judgment criteria

[0055]

[0056] Example 2: Different primer probe tests

[0057] Using the three sets of IPV upstream and downstream primers and Taqman fluorescent probes in Table 1 (the first set: IPV-F1 upstream primer, IPV-R1 lower primer and IPV-P1 probe, the second set: IPV-F2 upstream primer, IPV-R2 lower primer and IPV-P2 probe; the third set: IPV-F3 upstream primer, IPV-R3 lower primer and IPV-P3 probe), three RT-qPCR reagents were prepared, and IPV positive nucleic acid was used as the PCR test sample. The three RT-qPCR reagents prepared by the three sets of primers and probes were respectively composed of a kit for detection. The other components of the kit and the detection method are the same as in Example 1.

[0058] Test results such as Figure 2 As shown, IPV-F1R1P1 is the first group, IPV-F2R2P2 is the second group, and IPV-F3R3P3 is the third group. These three groups of RT-qPCR reagents can all detect IPV.

[0059] Example 3: IPV RT-qPCR reagent primer probe ratio test

[0060] The concentrations of 21 sets of primers and probes are set, that is, the primer concentration is 50~500mM, and the probe concentration is 25~250mM. The 21 sets of primers and probes are respectively composed of a kit for detection. The other components of the kit and the detection method are the same as in Example 1. The specific primer probe test data are shown in Table 4 (taking IPV-F1 upstream primer, IPV-R1 lower primer and IPV-P1 probe as examples, the data group with a probe concentration of 100nM and the data group with a primer concentration of 300nM are selected for display). In Table 4, the concentrations of the upstream and downstream primers are equal each time they are tested, which is convenient for effective amplification. The PCR Mix primer concentration is between 50nM and 500mM, and the probe concentration is between 25~250mM, and the RT-qPCR amplification effect is good.

[0061] Table 4 IPV primer-probe ratio screening table

[0062]

[0063] Example 4: Accuracy Test

[0064] Using shrimp tissue nucleic acid samples with known IPV positive and negative results, the known samples were tested using the kit and detection method described in Example 1.

[0065] The results are as follows Figure 3 As shown, amplification occurred in IPV-positive samples, while amplification did not occur in IPV-negative samples, indicating that the kit and detection method of the present invention can detect the corresponding targets, with a positive detection rate of 100% and a negative compliance rate of 100%, without false positives or missed detections.

[0066] Example 5: Sensitivity test

[0067] The IPV plasmid was diluted with TE buffer and quantified using Nanodrop and Qubit. The result was 1×10 11 copies / μL, and then converted to copies / mL and performed gradient dilution. Nine concentration gradients were set, namely E7: 1×10 7 copies / mL, E6: 1×10 6 copies / mL, E5: 1×10 5 copies / mL, E4: 1×10 4 copies / mL, E3: 1×10 3 copies / mL, E2: 1×10 2 The sensitivity test was performed using the kit and detection method of Example 1, using 9 gradient concentrations of IPV plasmid as templates, 10 copies / mL, 0.5E1: 5 copies / mL, and 0.25E1: 2.5 copies / mL.

[0068] Test results such as Figure 4 As shown, the IPV plasmid concentration was 1×10 7 The detection range of the test kit and the established test method of the present invention is high in sensitivity to IPV, and the detection limit is as low as 5 copies / mL. Figure 5 As shown, the concentration of this kit is 1.0×10 7 copies / mL~1.0×10 1 There is a good linear relationship between copies / mL. The linear equation fitted by the kit is: Ct=-3.5788lgC+37.99, and the correlation coefficient R 2 =0.9969, where the y-axis represents the Ct value and the x-axis represents the logarithmic value of the concentration of the positive reference, i.e., lgC.

[0069] Example 6: Specificity test

[0070] The positive samples of Macrobrachium rosenbergii Nodavirus (MrNV), Macrobrachium rosenbergii dicistrovirus (MrDV), shrimp yellow head virus (YHV) and IPV were used as specific detection samples, and the kit and detection method for detecting infectious precocious virus of Macrobrachium rosenbergii in Example 1 were used for detection.

[0071] Test results such as Figure 6As shown, IPV-positive samples showed specific amplification; while Macrobrachium rosenbergii Nodavirus, Macrobrachium rosenbergii dicistrovirus, and shrimp yellowhead virus, that is, MrNV-positive samples, MrDV-positive samples, and YHV-positive samples in the figure did not show specific amplification, indicating that the kit of the present invention and the established detection method have good specificity for IPV.

[0072] In summary, the kit and detection method provided by the present invention can quickly and accurately detect the infectious precocious virus of Macrobrachium rosenbergii, without false positives and missed detections, are simple to operate, highly practical, and have high sensitivity and specificity. The detection limit is as low as 5 copies / mL, and is suitable for the early diagnosis and identification of the infectious precocious virus of Macrobrachium rosenbergii.

[0073] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A kit for detecting infectious precocious virus of Macrobrachium rosenbergii, characterized in that: The kit includes PCR Mix, enzyme Mix, negative quality control product, and positive quality control product; wherein the PCR Mix includes at least one group selected from the following three groups of upstream and downstream primers and fluorescent probes: The nucleotide sequences of the first group of upstream and downstream primers and fluorescent probes are shown in SEQ ID NO.1 to SEQ ID NO.3; The nucleotide sequences of the second group of upstream and downstream primers and fluorescent probes are shown in SEQ ID NO.4 to SEQ ID NO.6; The nucleotide sequences of the third group of upstream and downstream primers and fluorescent probes are shown in SEQ ID NO.7 to SEQ ID NO.9; The PCR Mix also includes 20mM to 80M Tris-HCl with a pH of 7.5 to 8.9, 10mM to 50mM U-containing dNTP, 100mM to 4M KCl, 1mM to 50mM , PCR Probe Buffer composed of 0.01~0.3% Tween-20 by volume, 0.01~0.1% tetrahydrothiophene 1-oxide by volume, 10mM~5M tetramethylammonium chloride, 10mM~100mM betaine, 0.01~0.1% DMSO by volume, and 0.01~0.3% Triton X-100 by volume; The enzyme mix comprises 0.5-2U hot start Taq enzyme, 0.5-5U reverse transcriptase, 0.1U-10U UDG enzyme, 1U-10U RNase inhibitor, 1%-50% glycerol by volume, 10mM-200mM potassium ion, and 0.01-1mM EDTA; The negative quality control product is nuclease-free water; the positive quality control product contains a plasmid solution synthesized using the IPV target gene sequence.

2. The kit for detecting infectious precocious virus of Macrobrachium rosenbergii according to claim 1, characterized in that: The concentration of the upstream primer is 50-500 mM, the concentration of the downstream primer is 50-500 mM, and the concentration of the fluorescent probe is 25-250 mM.

3. The kit for detecting infectious precocious virus of Macrobrachium rosenbergii according to claim 1, characterized in that: The fluorescent probe is a Taqman probe, the 5' end of which is connected to a fluorescent luminescent group, and the 3' end of which is connected to a fluorescent quenching group.

4. The kit for detecting infectious precocious virus of Macrobrachium rosenbergii according to claim 3, characterized in that: The fluorescent light-emitting group is one of FAM, VIC, HEX, JOE, ROX, Texas-Red, and Cy5; the fluorescent quenching group is one of DABCYL, DABSYL, TAMRA, BHQ-1, BHQ-2, and BHQ-3.

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