Primer, kit and method for detecting IHHNV virus based on RPA (recombinase polymerase amplification) and CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats)

Abstract

The invention discloses a primer, a kit and a method for detecting an IHHNV virus based on RPA (recombinase polymerase amplification) and CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats). According to the method, a recombinase polymerase amplification technology (RPA) and a CRISPR technology are combined, firstly, in the PRA process, target DNA is recognized through specific primer combination, strand displacement reaction is carried out under the action of single-stranded DNA binding protein and DNA polymerase, and mass amplification is carried out under the constant-temperature condition; according to the present invention, the RPA amplification product is amplified by using the CRISPR-Cas12a protein, the CRISPR-Cas12a protein is combined with the RPA amplification product under the mediation of the specific crRNA, the complex can efficiently cut the fluorescently-labeled single-stranded DNA in the system so as to release the fluorescence reporter group, the target DNA can be detected by collecting the fluorescence signal, and the IHHNV fluorescence detection kit and the IHHNV fluorescence detection strip can be developed so as to adapt to different detection scenes and requirements.

Description

Technical Field

[0001] The present invention belongs to the field of virus detection, and in particular relates to a primer, a kit and a method for detecting IHHNV virus based on RPA and CRISPR-Cas12a. Background Art

[0002] With the expansion of aquaculture scale, shrimp diseases have shown a diversification of infectious pathogens, which has become an important problem that plagues the green development of shrimp aquaculture. In the past decade, the main pathogens commonly found in shrimp aquaculture include infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) strains, decapod iridescent virus 1 (DIV1) strains, Enterocytozoon hepatopenaei (EHP) and Acute hepatopancreatic necrosis disease causing Vibrio (Vp AHPND).

[0003] IHHNV was first discovered in the shrimp Penaeus stylirostris in Hawaii, USA in 1981. [1] , has spread rapidly around the world and is now widely distributed in various wild and farmed shrimps around the world. [2,3] , commonly found in Southeast Asian shrimp (Penaeus monodon) and wild American shrimp (Penaeus vannamei, Penaeus cyaneus and other wild shrimps along the Pacific coast) [4] The first signs of IHHNV infection in shrimp are lethargy, followed by immobility, sinking to the bottom upside down, muscle opacity, inactivity, or death. [5] In addition, some shrimp infected with IHHNV do not show obvious clinical symptoms, but can exist in the population as a source of infection. [6] . The infected population will also develop Runt-deformity syndrome (RDS), the main effects of which are slow growth and epidermal deformities, but no mortality. Based on the above pathological characteristics, farmed shrimps infected with IHHNV often have problems such as high individual deformity rates, slowed growth rates and poor farming performance, causing huge economic losses to the shrimp farming industry. According to statistics, in 2020, the prevalence of IHHNV in the positive detection rate of major and emerging disease pathogens in major shrimp farming areas was still high, at around 10%. [7] , so IHHNV deserves our continued and in-depth attention.

[0004] As one of the most harmful shrimp viruses in the world, IHHNV has a huge negative impact on shrimp farming. [8] The World Organization for Animal Health (WOAH) has listed IHHNV as one of the important diseases of crustaceans that must be reported. The symptoms of different species infected with IHHNV vary, but most of them are chronic infections, which can cause death in severe cases, with a mortality rate of up to 90%. [9] Currently, there is no effective treatment for IHHNV infection. Once a large-scale infection occurs, it is difficult to take effective measures to control it in the first place, which can easily cause huge economic losses and chain reactions. Therefore, regular testing and early diagnosis are crucial.

[0005] The current detection methods mainly adopt biological methods, and traditional microscopic examination, bacterial culture and other methods have been gradually replaced. At present, the detection of IHHNV mainly adopts the common PCR method in GB / T 25878-2010, and the diagnosis and quarantine of IHHNV in imported and exported shrimp mostly use the fluorescent PCR method in SN / T 1673-2013. LAMP technology is also gradually being used as a new type of room temperature detection technology. Ge Hui et al. developed a visual loop-mediated isothermal amplification (LAMP) technology method with a minimum detection limit of 10.3 copies / μL

[10] However, the design of visualized LAMP primers is complex, with many false positives, and the visual judgment is very likely to cause subjective errors in human interpretation, which has certain defects in actual detection.

[0006] Based on the advantages of CRISPR technology in detection cost, efficiency, portability and specificity, it has gradually been applied in animal disease detection, but this method is currently less commonly used in aquatic disease detection. In recent years, studies have reported that shrimp disease co-infection is common. In 2014, Hu Wenjuan et al. tracked, detected and artificially infected some freshwater shrimp Penaeus vannamei in Shanghai for WSSV and IHHNV infection and co-infection. The test results showed that the average single infection rates of WSSV and IHHNV were 42.6% and 38.5%, respectively, and the double virus co-infection rate was 20.5%.

[11] Therefore, the identification and detection of IHHNV is widely used in front-line farms. Summary of the invention

[0007] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a primer for detecting IHHNV virus based on RPA and CRISPR-Cas12a.

[0008] A second object of the present invention is to provide a fluorescence detection kit for detecting IHHNV virus based on RPA and CRISPR-Cas12a.

[0009] The third object of the present invention is a test strip for detecting IHHNV virus based on RPA and CRISPR-Cas12a.

[0010] The fourth object of the present invention is to provide the application of primers, fluorescence detection kits and test strips for detecting IHHNV virus based on RPA and CRISPR-Cas12a.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] A primer for detecting IHHNV virus based on RPA and CRISPR-Cas12a, comprising a pair of RPA primers and cRNA, the nucleotide sequence of which is as follows:

[0013] RPA2-F: 5′-GTCATCCCGACGACGAAGAATGGACAGAA-3′ (SEQ ID NO. 1);

[0014] RPA2-R: 5′-TTCTGGTGTTGAAGTTCTTGGAGGAGTTTGA-3′ (SEQ ID NO. 2);

[0015] crRNA1: 5′-UAAUUUCUACUAAGUGUAGAUGUUCGUGCGUUCUCGAAGCA-3′ (SEQ ID NO. 3).

[0016] A fluorescence detection kit for detecting IHHNV virus based on RPA and CRISPR-Cas12a, comprising the above-mentioned primers for detecting IHHNV virus based on RPA and CRISPR-Cas12a.

[0017] The kit also includes at least one of RPA Buffer, NEBuffer r2.1, Cas 12a protein, RNase inhibitor, ssDNA, positive control and negative control.

[0018] The sequence of the ssDNA is as follows: 5'-FAM-TTATT-BHQ1-3'.

[0019] The positive control is the IHHNV standard positive plasmid.

[0020] The negative control is DEPC water.

[0021] A test strip for detecting IHHNV virus based on RPA and CRISPR-Cas12a, comprising the above-mentioned primers for detecting IHHNV virus based on RPA and CRISPR-Cas12a.

[0022] The test strip also includes at least one of a CRISPR Cas12 / 13 HybriDetect test strip, NEBuffer r2.1, Cas12a protein, an RNase inhibitor, ssDNA, RPA Buffer, a positive control, and a negative control.

[0023] The sequence of the ssDNA is as follows: 5'-FAM-TTTTTTT-Biotin-3'.

[0024] The use of at least one of the primers, fluorescence detection kit and test strips for detecting IHHNV virus based on RPA and CRISPR-Cas12a in detecting IHHNV virus for non-disease diagnosis and treatment purposes.

[0025] A method for detecting IHHNV virus for non-disease diagnosis and treatment purposes based on RPA and CRISPR-Cas12a, comprising the following steps:

[0026] (1) extracting nucleic acid from the sample to be tested as a DNA template;

[0027] (2)RPA amplification

[0028] Prepare RPA amplification system (50 μL): 29.4 μL of Buffer A, 2 μL of 10 μmol / L upstream and downstream primers RPA2-F and RPA2-R, 11.1 μL of DEPC water, 2.5 μL of Buffer B, 3 μL of DNA template, perform RPA amplification at 39±1°C to obtain RPA amplification products; wherein the nucleotide sequences of the upstream and downstream primers RPA2-F and RPA2-R are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively;

[0029] (3)RPA-Cas12a detection:

[0030] ①CRISPR-Cas12a fluorescence detection

[0031] Prepare CRISPR-Cas12a fluorescence detection system (20 μL): NEBuffer r2.1 2 μL, 1 μmol / L Cas 12a protein 0.5 μL, 250-500 nmol / L crRNA 1 μL, 200 nmol / L-5 μmol / L ssDNA 1 μL, 40 U / μL RNase inhibitor RRI 0.25 μL, RPA amplification product 3 μL, DEPC water is added to 20 μL; then the prepared reaction tube is centrifuged and mixed instantaneously, reacted at 37 ± 1 ° C for 40-60 min, the fluorescence value is measured, and the amplification curve is observed; wherein the nucleotide sequence of crRNA is shown in SEQ ID NO.2, and the nucleotide sequence of ssDNA is shown in SEQ ID NO.4;

[0032] and / or

[0033] ②CRISPR-Cas12a test strip detection

[0034] Prepare CRISPR-Cas12a test strip detection system (100 μL): NEBuffer r2.1 2 μL, 1 μmol / L Cas12a protein 0.5 μL, 250-500 nmol / L crRNA 1 μL, 200 nmol / L-10 μmol / L ssDNA 2 μL, 40 U / μL RNase inhibitor RRI 0.25 μL, RPA amplification product 3 μL, DEPC water to 20 μL; react at 37±1°C for 40 min-60 min, after the reaction is completed, add 80 μL of DEPC water, blow evenly and immerse the sample detection end of the test strip in the reaction solution, and observe the results of the control band and the detection band; wherein the nucleotide sequence of crRNA is shown in SEQ ID NO.2, and the nucleotide sequence of ssDNA is shown in SEQ ID NO.5;

[0035] (4) Result judgment

[0036] If the test result in step ① shows a fluorescent signal and gradually increases, and the fluorescence curve is S-shaped, it means that the sample to be tested is an IHHNV-positive sample (IHHNV virus is present); conversely, if the test result in step ① has no obvious fluorescent signal, it means that the sample to be tested is an IHHNV-negative sample;

[0037] If red bands appear in both the control band and the test band in the test results in step ②, it means that the sample to be tested is an IHHNV-positive sample (IHHNV virus is present); if red bands appear in the control band in the test results in step ②, but no red bands appear in the test band, it means that the sample to be tested is an IHHNV-negative sample; if no red bands appear in both the control band and the test band in the test results in step ②, it means that the test result is invalid and needs to be retested.

[0038] The time for RPA amplification in step (2) is 10 to 30 minutes, preferably 10 minutes.

[0039] The preparation method of the RPA amplification system described in step (2) is as follows: Buffer A, upstream and downstream primers RPA2-F, RPA2-R and DEPC water are pre-prepared and mixed and then transferred to the basic reaction unit to fully and evenly dissolve the lyophilized powder of the basic reaction unit in the RPA nucleic acid amplification kit, and then Buffer B is added to the inner side of the reaction unit tube cap, and finally a DNA template is added into the tube to obtain the RPA amplification system.

[0040] The RPA amplification described in step (2) also includes the step of verifying the obtained amplification product by agarose gel electrophoresis.

[0041] The final concentration of crRNA described in steps ① and ② is preferably 250nmol / L.

[0042] The final concentration of ssDNA1 in steps ① and ② is preferably 200 nmol / L.

[0043] The reaction time in steps ① and ② is preferably 40 min.

[0044] Compared with the prior art, the present invention has the following advantages and effects:

[0045] 1. The present invention combines recombinase polymerase amplification (RPA) technology with CRISPR (Clustered regularly interspaced short palindromic repeat) technology to develop a set of IHHNV isothermal rapid detection methods: First, the PRA process recognizes the target DNA through specific primer binding, and a strand displacement reaction occurs under the action of single-stranded DNA binding protein and DNA polymerase, and amplifies a large number of IHHNV under constant temperature conditions.

[12] ; Then, the CRISPR-Cas12a protein binds to the RPA amplification product under the mediation of specific crRNA. The complex can efficiently cut the fluorescently labeled single-stranded DNA in the system to release the fluorescent reporter group, and the target DNA can be tested by collecting the fluorescent signal

[13] .

[0046] 2. Based on RPA and CRISPR-cas12a technologies, the present invention develops two sets of constant temperature detection methods for IHHNV, namely the fluorescence method and the test strip method, which can perform rapid and convenient instant detection of a small amount of DNA in clinical samples, so as to provide new means for the early detection and diagnosis of IHHNV for different detection scenarios and needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a graph showing the results of RPA amplification of an IHHNV-positive plasmid (in the graph, M: DL2000 DNA Maker; lane 1: RPA1 product (297 bp); lane 2: RPA2 product (272 bp); lane 3: RPA3 product (289 bp); lane 4: RPA4 product (253 bp); N: negative control).

[0048] Figure 2 This is the screening result diagram of RPA primers and crRNA.

[0049] Figure 3 It is a graph showing the optimization results of the RPA-Cas12a detection system; wherein A is a fluorescence curve graph showing the optimization of the crRNA concentration in the fluorescence detection system; and B is a graph showing the optimization results of the ssDNA concentration in the test strip detection system (1 to 5 in the graph represent ssDNA final concentrations of 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, and 800 nmol / L, respectively).

[0050] Figure 4 It is a graph of specificity test results; wherein A is a curve graph of the fluorescence detection system; and B is a test strip detection system (in the graph, 1 to 5 represent IHHNV, DIV1, WSSV, EHP and blank control, respectively).

[0051] Figure 5 is the result of the sensitivity test (error bars represent mean ± difference, n = 3 replicates; * indicates significant difference (P < 0.01)); A is the fluorescence value of RPA-Cas12a detection; B is the curve of RPA-Cas12a fluorescence detection system; C is the RPA-Cas12a test strip detection system (1 to 6 in the figure represent 10 4 , 10 3 , 10 2 , 10 1, 10 0 , blank control); D is real-time fluorescence quantitative PCR method.

[0052] Figure 6 It is a sample test result diagram; among them, A and B are the RPA-Cas12a fluorescence test results of 2 actual clinical samples respectively; C is the RPA-Cas12a test strip test result of actual clinical samples (1 to 10 in the figure represent 10 IHHNV positive samples respectively); D is the RPA-Cas12a fluorescence test result of mixed samples; E is the RPA-Cas12a test strip test result of mixed samples (1 to 5 in the figure represent IHHNV and WSSV co-infected sample 1, IHHNV and WSSV co-infected sample 2, IHHNV and EHP co-infected sample 1, WSSV sample, EHP sample respectively). DETAILED DESCRIPTION

[0053] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0054] Example 1

[0055] 1. Materials

[0056] 1.1 Viruses, nucleic acids and samples

[0057] The conservative sequence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp (Genbank AF218266.2) was selected, and the IHHNV-positive plasmid was synthesized by Shanghai Sangon Biotechnology Co., Ltd.; 10 positive nucleic acid samples of infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp, 1 nucleic acid sample of white spot syndrome virus (WSSV) of shrimp, 1 nucleic acid sample of Decapod iridescent virus1 (DIV1), 1 nucleic acid sample of Enterocytozoon hepatopenaei (EHP), 2 positive nucleic acid samples of IHHNV and WSSV co-infection, and 1 positive nucleic acid sample of IHHNV and EHP co-infection were nucleic acid samples stored in the Animal Quarantine Laboratory of Gongbei Customs Technical Center. These samples were determined to be positive by fluorescent PCR and nested PCR methods. The detection methods were derived from the 2021 edition of the WOAH "Aquatic Animal Disease Diagnostic Manual" and Chinese Fisheries Standards; the primers, probes and detection methods involved are shown in Table 1.

[0058] Table 1

[0059]

[0060] In the table, FAM: fluorescent dye modification; TAMRA: fluorescent dye tetramethylrhodamine modification.

[0061] 1.2 Main instruments and reagents

[0062] Viral DNA / RNA extraction kit (4.0) was purchased from Xi'an Tianlong Technology Co., Ltd.; RPA nucleic acid amplification kit was purchased from Anpu Future Biotechnology Co., Ltd.; Agarose H was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; Cas12a protein was purchased from NEB Co., Ltd., USA; NEBuffer r2.1 (10X) was purchased from NEB Co., Ltd., USA; RNase inhibitor (Murine RNase Inhibitor RRI) was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; CRISPR Cas12 / 13HybriDetect test strips were purchased from Nanjing Wobo Biotechnology. The fully automatic rotary nucleic acid extractor (model: GeneRotex) was purchased from Xi'an Tianlong Technology Co., Ltd.; the gel imager (model: AlphaImager HP) was purchased from Alpha Corporation of the United States; the isothermal real-time fluorescence detector (model: Genchek-1) was purchased from Hangzhou Zhongce Biotechnology Co., Ltd.; the real-time fluorescence PCR instrument (model: QuantStudio 5) was purchased from Applied Biosystems of the United States; the high-speed refrigerated centrifuge (model: 5424R) was purchased from Eppendorf AG of Germany; and the spectrohotometer ultraviolet spectrophotometer (model: ND-1000) was purchased from Thermo Fisher of the United States.

[0063] 2. Methods

[0064] 2.1RPA primer and cRNA design and synthesis

[0065] The conserved gene fragments in the IHHNV gene sequence (NC_002190.2) found in GenBank were used to design specific crRNAs, and two crRNAs (crRNA1 and crRNA2) with higher comprehensive scores were selected for subsequent experiments. Two sets of RPA primers (RPA1-F / R and RPA2-F / R) were designed in the gene sequence region where crRNA1 and crRNA2 were located, and two sets of RPA primers (RPA3-F / R and RPA4-F / R) were designed in the gene sequence region where crRNA2 was located. ssDNA was designed according to different experimental methods, fluorescence method: 5'-FAM-TTTTTTT-BHQ1-3' (FAM: fluorescent dye modified; BHQ1: black hole quencher-1 modified) (SEQ ID NO.26); test strip method: 5'-FAM-TTTTTTT-Biotin-3' (FAM: fluorescent dye modified; Biotin: biotin modified) (SEQ ID NO.27). The RPA primers and crRNA used in this study are shown in Table 1. The above RPA primers, crRNA and ssDNA were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0066] Table 1 IHHNV RPA primers, crRNA sequences

[0067]

[0068] 2.2 Sample nucleic acid extraction

[0069] The nucleic acids of the 10 shrimp IHHNV-positive samples in 1.1 were extracted according to the instructions of the viral DNA / RNA extraction kit (4.0). The extracted nucleic acid templates were detected by UV spectrophotometer. The template OD 260 / OD 280 The ratio was between 1.8 and 2.0 for subsequent tests. At the same time, the nucleic acids of the positive samples of WSSV, DIV1, EHP, IHHNV and WSSV co-infection, and IHHNV and EHP co-infection in 1.1 were extracted in the same way for subsequent tests.

[0070] 2.3 Establishment of RPA-Cas12a detection method

[0071] 2.3.1 Detection was performed using the RPA nucleic acid amplification kit. RPA amplification system (50 μL): 29.4 μL of Buffer A, 2 μL of upstream and downstream primers (10 μmol / L), and 11.1 μL of DEPC water were pre-prepared and mixed, and then transferred to the basic reaction unit to fully and evenly dissolve the lyophilized powder of the basic reaction unit in the RPA nucleic acid amplification kit. Add 2.5 μL of Buffer B to the inside of the reaction unit tube cap, add 3 μL of the DNA template extracted in 2.2 above to the tube, centrifuge briefly, and amplify at 39°C for 10 minutes. After the RPA amplification is completed, take 5 μL of the amplified product for 1% agarose gel electrophoresis to determine the amplification results. The experiment was repeated 3 times.

[0072] 2.3.2CRISPR-Cas12a fluorescence detection system (20μL): NEBuffer r2.1 2μL, Cas 12a protein (1μmol / L) 0.5μL, crRNA (250nmol / L) 1μL, ssDNA (5'-FAM-TTATT-BHQ1-3') (5μmol / L) 1μL, RNase inhibitor RRI (40U / μL) 0.25μL, RPA amplification product 3μL, DEPC water supplemented to 20μL; the prepared reaction tube was centrifuged and mixed instantly, placed in a Genchek-1 real-time fluorescence detector, reacted at 37°C for 40min, the fluorescence value was measured, and the amplification curve was observed. The experiment was repeated 3 times.

[0073] 2.3.3 Detection using CRISPR Cas12 / 13HybriDetect test strips. CRISPR-Cas12a test strip detection system (100μL): NEBuffer r2.1 2μL, Cas 12a protein (1μmol / L) 0.5μL, crRNA (200nmol / L) 1μL, ssDNA (5'-FAM-TTTTTTT-Biotin-3') (10μmol / L) 2μL, RNase inhibitor RRI (40U / μL) 0.25μL, RPA amplification product 3μL, DEPC water to 20μL, react at 37°C for 40min. After the reaction is completed, add 80μL of DEPC water, blow evenly, immerse the sample detection end of the test strip in the reaction solution, and observe the results of the control band and the detection band. The experiment was repeated 3 times.

[0074] 2.4 Screening of RPA primers and crRNA

[0075] 2.4.1 According to the method in 2.1, the conserved gene fragments in the IHHNV gene sequence (NC_002190.2) were searched in GenBank, and 4 sets of RPA-specific primers were designed. Then, RPA amplification was performed according to the method in 2.3.1 to screen out the primers with a single specificity for the amplification product. The experiment was repeated 3 times.

[0076] 2.4.2 Use the CRISPR-Cas12a fluorescence detection method in 2.3.2, use the selected RPA primers and corresponding crRNA for detection, and determine the optimal RPA primer-crRNA combination based on the end point fluorescence value and amplification curve. The experiment was repeated 3 times.

[0077] 2.5 Optimization of CRISPR-Cas12a detection system

[0078] 2.5.1 Optimize the crRNA concentration in the CRISPR-Cas12a fluorescence detection system. According to the CRISPR-Cas12a fluorescence detection method in 2.3.2, set the final crRNA concentration to 5 concentrations: 25, 50, 100, 250, and 500 nmol / L, and keep other conditions unchanged. Use DEPC water as a blank control, observe the fluorescence values ​​and amplification curves under different conditions, and explore the optimal crRNA concentration.

[0079] 2.5.2 Optimize the ssDNA concentration in the CRISPR-Cas12a test strip detection system. According to the CRISPR-Cas12a test strip detection method in 2.3.3, set the final ssDNA concentration in 100μL reaction solution to 5 concentrations of 50, 100, 200, 400, and 800nmol / L, and keep other conditions unchanged. Use DEPC water as a blank control, observe the fluorescence value and amplification curve under different conditions, and explore the optimal concentration of ssDNA.

[0080] 2.6 Specificity test

[0081] The IHHNV positive sample nucleic acid (one of the 10 samples), WSSV, DIV1, and EHP nucleic acid extracted in step 2.2 were used as templates, and DEPC water was used as a blank control. The method established in step 2.3 was used for detection to verify the specificity of the detection method. Three repeated tests were set up, and the fluorescence data were collected and analyzed by the fluorescence method, and the results of the control band and the test band were observed by the test strip method.

[0082] 2.7 Sensitivity test

[0083] The IHHNV positive plasmid was diluted 10-fold in a 10-fold gradient. 4 10 3 10 2 10 110 0 There are 5 gradients in total, with DEPC water as the blank control, and the method established in step 2.3 is used for detection to test the sensitivity of the method. Three repeated tests are set up, and the fluorescence method collects and analyzes the fluorescence data, and the test strip method observes the results of the control strip and the test strip.

[0084] The above samples were tested using the real-time fluorescence quantitative PCR method in the "Technical Specifications for Quarantine of Infectious Subcutaneous and Hematopoietic Necrosis" (SN / T 1673-2013) (primer and probe sequences are shown in Table 1 above: SEQ ID NO.4 to SEQ ID NO.6) to compare and verify the sensitivity of this detection method.

[0085] 2.8 Clinical sample verification

[0086] The nucleic acid of the 10 IHHNV-positive samples extracted in step 2.2 was tested using the method established in step 2.3 to verify the actual sample detection of this research method. Three replicates were set up.

[0087] The nucleic acids of 2 IHHNV and WSSV co-infection positive samples, 1 IHHNV and EHP co-infection positive sample, 1 WSSV positive sample, and 1 EHP positive sample extracted in step 2.2 were used as templates and tested using the method established in step 2.3 to verify the mixed sample detection of this research method. Three repeated tests were set up.

[0088] 2.9 Statistical analysis of data

[0089] This study was conducted using the statistical analysis software Graphpad Prism 8. All data were expressed as mean ± standard deviation, and the collected fluorescence values ​​were analyzed for significant differences using the t-test. P < 0.05 is indicated by *, and the statistical value represents a significant difference; ns represents no significant difference.

[0090] 3. Results and Analysis

[0091] 3.1 Screening of RPA primers and crRNA

[0092] The synthesized IHHNV standard positive plasmid was used as a template, and the four designed primers were used for RPA amplification. The amplified products were subjected to 1% agarose gel electrophoresis, and DEPC water was used as a negative control. Figure 1 As shown: The results showed that the four sets of RPA primers could successfully amplify DNA fragments that matched the size of the target band. The amplification products of RPA2 (RPA2-F, RPA2-R) and RPA3 (RPA3-F, RPA3-R) primers were single and specific.

[0093] The RPA primers were matched with crRNA: RPA2-crRNA1 and RPA3-crRNA2 were tested, and DEPC water was used as negative control (Negative). The peak time and fluorescence curve of the RPA2-crRNA1 group were better than those of the RPA3-crRNA2 group ( Figure 2 ), so the RPA2-crRNA1 group was selected in subsequent experiments.

[0094] 3.2 Optimization of RPA-Cas12a detection system

[0095] The RPA-Cas12a detection system was constructed using IHHNV positive plasmid. The optimization results of the fluorescence detection system showed that the higher the crRNA concentration, the stronger the fluorescence intensity. When the final crRNA concentration reached 250nmol / L, the fluorescence intensity was significantly enhanced; when the final crRNA concentration was 500nmol / L, the fluorescence curve was S-shaped ( Figure 3 A). When the final crRNA concentration reached 250 nmol / L or above, the fluorescence value and the fluorescence curve were clearly distinguished. Considering the cost, the final crRNA concentration of 250 nmol / L was selected for subsequent experiments. The results of 3.1 and 3.2 showed that the fluorescence intensity of the RPA-Cas12a fluorescence detection system increased with time. When the fluorescence acquisition time was 40 min, the fluorescence detection intensity was high and reached a plateau. In order to meet the needs of rapid detection, 40 min was used as the subsequent experimental conditions.

[0096] Optimize the ssDNA concentration in the test strip detection system. It was found that when the ssDNA concentration was 50nmol / L, a detection band appeared on the negative test strip, indicating that false positive results are easily generated when the ssDNA concentration is low. The higher the ssDNA concentration, the darker the color of the detection band. When the ssDNA concentration reached 200nmol / L, the color difference of the detection band was not much ( Figure 3 B). Therefore, a final concentration of 200 nmol / L of the test strip reporter molecule was selected as the concentration for subsequent experiments.

[0097] 3.3 Specificity test

[0098] The RPA2-crRNA1 group was used to detect the nucleic acid of IHHNV positive samples (one of the 10 samples), WSSV, DIV1, and EHP nucleic acid samples using the optimized fluorescence method (crRNA final concentration of 250 nmol / L, ssDNA final concentration of 200 nmol / L, reaction time of 40 min, and the rest was the same as the method in 2.3). IHHNV detection showed obvious fluorescence signals, while no specific amplification curves were seen for other viruses ( Figure 4 A). The test strip test results showed that the IHHNV test strip was positive, and there was no detection band in the other groups of viruses and the blank control ( Figure 4 B) The results show that the primers in this detection system have no cross-reaction with other viral nucleic acids and have good specificity.

[0099] 3.4 Sensitivity test

[0100] The IHHNV positive plasmid was diluted 10-fold, and 5 dilution gradients (10 4 copies / μL~10 0 The fluorescence values ​​collected by the CRISPR-Cas12a fluorescence method were statistically analyzed. The plasmid concentration was 10 4 ~10 1 The fluorescence value can be detected in the range of copies / μL, among which 10 1 copies / μL and 10 0 The difference in the detection fluorescence value of template copies / μL was very significant (P=0.0085) ( Figure 5 A and 5B). The test strip method results showed 10 4 ~10 1 The detection bands can be observed in the range of 10 copies / μL. 0 copies / μL template no bands appear ( Figure 5 C), so the minimum concentration detected by the test strip detection system and the fluorescence detection system is the same, which is 10 copies / μL.

[0101] At the same time, the fluorescence PCR method was used to verify the sensitivity. The results showed that 10 4 ~10 2 The copies / μL group had a specific amplification curve, and the limit Ct value was 39 (see Figure 5 D); 10 1 There is no amplification curve starting from 100 copies / μL. By comparison, the CRISPR-Cas12a method established in the present invention is 10 times more sensitive than the industry standard fluorescent PCR method.

[0102] 3.5 Clinical sample testing

[0103] The optimized fluorescence method and test strip method established in this study (crRNA final concentration of 250nmol / L, ssDNA final concentration of 200nmol / L, reaction time of 40min, and the rest of the method is the same as in 2.3) were used to detect the 10 IHHNV positive samples in 2.2. The fluorescence detection results are shown in Figure 6A and 6B, the test results of the test strip are as follows Figure 6 As shown in C: The results showed that all were positive, which was consistent with the detection results of the real-time fluorescence quantitative PCR method, and the conformity rate of the detection results was 100%. The method established in this study can be applied to the detection of actual clinical samples.

[0104] Mixed sample testing Figure 6 As shown in D and 6E: The results showed that the nucleic acids of 2 IHHNV and WSSV co-infection positive samples and 1 IHHNV and EHP co-infection positive sample were positive, while the WSSV and EHP positive samples were negative, indicating that this research method can be used to detect mixed infection samples.

[0105] In summary, the method of the present invention can detect mixed samples and improve the efficiency of IHHNV detection. Compared with the current national standard method, the detection method established by the present invention shortens the detection time to within 50 minutes, improves the detection efficiency and time, and has equivalent or higher sensitivity than the entry and exit standard method. The fluorescence method uses a portable fluorescence detector to quickly read the test results, which can be used for detection in breeding bases and laboratories. The detection line of the paper strip method is shallow when the sample virus concentration is low, and false negative results are prone to occur if the operation is improper, but based on its convenience in operation, it can be used for rapid initial screening at the front line such as farms and ports.

[0106] Both detection methods in the present invention can read the test results quickly and easily under constant temperature conditions, get rid of the limitations of large instruments such as fluorescent PCR instruments, and adapt to the growing demand for on-site rapid detection and screening or epidemic monitoring. They are suitable for regular monitoring and preliminary screening of IHHNV in shrimp farms or grassroots laboratories, so as to take effective control measures in a timely manner and reduce economic losses.

[0107] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0108] References

[0109] [1] DV Lightner, RM Redman, TA Bell. Infectious hypodermal and hematopoietic necrosis, a newly recognized virus disease of penaeidshrimp. Journal of Invertebrate Pathology, 1983, 42(1): 62-70.

[0110] [2] JA Brock.Diseases of crustacea:Diseases caused by microorganisms.1990.

[0111] [3] L Owens, IG Anderson, M Kenway. Infectious hypodermal andhematopoietic necrosis virus (IHHNV) in ahybrid penaeid prawn from tropical Australia. Diseases of Aquatic Organisms, 1992, 14 (3): 219-228.

[0112] [4] CL Browdy, D Bratvold, AD Stokes. The New Wave: Proceedings of the Special Session on Sustainable Shrimp Culture, Aquaculture 2001.2001.

[0113] [5] Zhang Na, Huang Lei, Si Ze En. Establishment and clinical application analysis of dual LAMP detection method for white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus in shrimp. Fisheries Science and Technology Information, 2020, 47(02): 68-72.

[0114] ZHANG N, HUANG L, SI Z.2020,47(02):68-72.(in Chinese).

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[0116] [7] Li Yingxia. Evaluation of three rapid and highly sensitive detection kits for shrimp pathogens. 2021, Master: 76.

[0117] LI Y.2021, Master:76.(in Chinese).

[0118] [8] Yang Bing, Song Xiaoling, Huang Jie, Lei Zhiwen. Research progress on epidemiology and detection technology of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV). Chinese Fisheries Science, 2005, (04): 519-524.

[0119] YANG B.2005,(04):519-524.(in Chinese).

[0120] [9] Yan D, Chen B. Research progress on pathogenicity of infectious hypodermal and hematopoietic necrosis virus. Progress in Fisheries Science, 2018, 39(03): 167-172. Yan D, Chen B. 2018, 39(03): 167-172. (in Chinese).

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[10] Ge Hui, Wu Jianshao, Yang Zhangwu, Wu Liyun, Zhang Zhe, Du Xiuping, Hong Wei, Lin Kebing, Lin Qi, Lin Jiaming, Zhou Chen. Optimization and application of visual loop-mediated isothermal amplification (LAMP) technology for infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei. Fisheries Research, 2021, 43(04): 357-365.

[0122] GE H, WU J, YANG Z, WU L, ZHANG Z, DU X, HONG W, LIN K, LIN Q, LIN J, ZHOUC.2021,43(04):357-365.(in Chinese).

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[12] J Li, J Macdonald, F von Stetten. Review: a comprehensive summary of a decade development of the recombinase polymerase amplification. Analyst, 2018, 144(1):31-67.

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[13] J S Chen,E Ma,L B Harrington.CRISPR-Cas12a target bindingunleashes indiscriminate single-stranded DNase activity.Science,2018,360(6387):436-439.

Claims

1. A primer for detecting IHHNV virus based on RPA and CRISPR-Cas12a, characterized in that: It includes a pair of RPA primers and cRNA, and its nucleotide sequence is as follows: RPA2-F: 5′-GTCATCCCGACGACGAAGAATGGACAGAA-3′; RPA2-R: 5′-TTCTGGTGTTGAAGTTCTTGGAGGAGTTTTGA-3′; crRNA1: 5′-UAAUUUCUACUAAGUGUAGAUGUUCGUGCGUUCUCGAAGCA-3′.

2. A fluorescence detection kit for detecting IHHNV virus based on RPA and CRISPR-Cas12a, characterized in that: Including the primers for detecting IHHNV virus based on RPA and CRISPR-Cas12a as described in claim 1.

3. The kit according to claim 2, characterized in that: The kit further comprises at least one of RPA Buffer, NEBuffer r2.1, Cas 12a protein, RNase inhibitor, ssDNA, positive control and negative control; The sequence of the ssDNA is as follows: 5'-FAM-TTATT-BHQ1-3'.

4. A test strip for detecting IHHNV virus based on RPA and CRISPR-Cas12a, characterized in that: Including the primers for detecting IHHNV virus based on RPA and CRISPR-Cas12a as described in claim 1.

5. The kit according to claim 4, characterized in that: The test strip further comprises at least one of a CRISPR Cas12 / 13 HybriDetect test strip, NEBuffer r2.1, Cas 12a protein, an RNase inhibitor, ssDNA, RPA Buffer, a positive control, and a negative control; The sequence of the ssDNA is as follows: 5'-FAM-TTTTTTT-Biotin-3'.

6. Use of at least one of the primers for detecting IHHNV virus based on RPA and CRISPR-Cas12a according to claim 1, the fluorescent detection kit for detecting IHHNV virus based on RPA and CRISPR-Cas12a according to any one of claims 2 to 5, and the test strip for detecting IHHNV virus based on RPA and CRISPR-Cas12a according to any one of claims 4 to 5 in detecting IHHNV virus for non-disease diagnosis and treatment purposes.

7. A method for detecting IHHNV virus for non-disease diagnosis and treatment purposes based on RPA and CRISPR-Cas12a, characterized in that, The steps include: (1) extracting nucleic acid from the sample to be tested as a DNA template; (2)RPA amplification Prepare RPA amplification system: 29.4 μL of Buffer A, 2 μL of 10 μmol / L upstream and downstream primers RPA2-F and RPA2-R, 11.1 μL of DEPC water, 2.5 μL of Buffer B, 3 μL of DNA template, perform RPA amplification at 39±1°C to obtain RPA amplification products; wherein the nucleotide sequences of the upstream and downstream primers RPA2-F and RPA2-R are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; (3)RPA-Cas12a detection: ①CRISPR-Cas12a fluorescence detection Prepare CRISPR-Cas12a fluorescence detection system: NEBuffer r2.1 2μL, 1μmol / L Cas 12a protein 0.5μL, 250-500nmol / L crRNA 1μL, 200nmol / L-5μmol / L ssDNA 1μL, 40U / μL RNase inhibitor RRI 0.25μL, RPA amplification product 3μL, DEPC water is added to 20μL; then the prepared reaction tube is centrifuged and mixed instantaneously, reacted at 37±1°C for 40-60min, the fluorescence value is measured, and the amplification curve is observed; wherein the nucleotide sequence of crRNA is shown in SEQ ID NO.3, and the nucleotide sequence of ssDNA is shown in SEQ ID NO.4; and / or ②CRISPR-Cas12a test strip detection Prepare CRISPR-Cas12a test strip detection system: NEBuffer r2.1 2μL, 1μmol / L Cas 12a protein 0.5μL, 250-500nmol / L crRNA 1μL, 200nmol / L-10μmol / L ssDNA 2μL, 40U / μL RNase inhibitor RRI 0.25μL, RPA amplification product 3μL, DEPC water to 20μL; react at 37±1℃ for 40min-60min, after the reaction is completed, add 80μL of DEPC water, blow evenly and immerse the sample detection end of the test strip in the reaction solution, and observe the results of the control band and the detection band; wherein, the nucleotide sequence of crRNA is shown in SEQ ID NO.3, and the nucleotide sequence of ssDNA is shown in SEQ ID NO.5; (4) Result judgment If the test result in step ① shows a fluorescent signal and gradually increases, and the fluorescence curve is S-shaped, it means that the sample to be tested is an IHHNV-positive sample; conversely, if the test result in step ① shows no obvious fluorescent signal, it means that the sample to be tested is an IHHNV-negative sample; If red bands appear in both the control band and the test band in the test results in step ②, it means that the sample to be tested is an IHHNV-positive sample; if red bands appear in the control band in the test results in step ②, but no red bands appear in the test band, it means that the sample to be tested is an IHHNV-negative sample; if no red bands appear in both the control band and the test band in the test results in step ②, it means that the test results are invalid and need to be retested.

8. The method according to claim 1, characterized in that: The preparation method of the RPA amplification system described in step (2) is as follows: Buffer A, upstream and downstream primers RPA2-F, RPA2-R and DEPC water are pre-prepared and mixed and then transferred to the basic reaction unit to fully and evenly dissolve the lyophilized powder of the basic reaction unit in the RPA nucleic acid amplification kit, and then Buffer B is added to the inner side of the reaction unit tube cap, and finally a DNA template is added into the tube to obtain the RPA amplification system.

9. The method according to claim 1, characterized in that: The time of RPA amplification in step (2) is 10 to 30 minutes; The final concentration of crRNA described in steps ① and ② is 250nmol / L; The final concentration of ssDNA1 described in steps ① and ② is 200 nmol / L; The reaction time described in steps ① and ② is 40 min.

10. The method according to claim 1, characterized in that: The RPA amplification described in step (2) also includes the step of verifying the obtained amplification product by agarose gel electrophoresis.

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