Primer probe combination for rpa detection of cold temperature of big-headed turtle arteritis virus and application thereof

Abstract

The application discloses a Chinese broad-headed pond turtle arterivirus (CBHPTAV) normal-temperature detection primer probe combination based on an RPA amplification technology and application thereof. The primer probe combination provided by the application comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO. 3, a reverse primer with a nucleotide sequence as shown in SEQ ID NO. 4 and a probe with a nucleotide sequence as shown in SEQ ID NO. 23, and after modification, the primer probe combination can quickly and accurately detect whether a to-be-detected sample contains the Chinese broad-headed pond turtle arterivirus under room temperature conditions, has the advantages of high sensitivity, strong specificity, simple operation and the like, can detect a sample with a concentration as low as 1.089 copies / uL, and the detection time is only 20 min, which is greatly shortened compared with PCR, and a temperature control instrument is not needed, so that the primer probe combination is very suitable for animal pathogen detection in a breeding site.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the room-temperature RPA detection primer and probe combination for giant turtle arteritis virus (CBHPTAV) and its application. Background Technology

[0002] As turtle and tortoise farming develops towards large-scale and intensive operations, the incidence and mortality rates are rising, posing a significant threat to the industry. A group of turtles exhibited skin ulcers, scab formation on their bodies, loss of appetite, liver and lung hemorrhage, and subsequently died within a short period. Mauremys reevesii In juvenile turtles, a virus was identified as the main pathogenic factor. The gene sequence of this virus is highly homologous (98% similarity) to Chinese broad-headed pondturtle arterivirus (CBHPTAV) in the order Nidovirales, and it possesses cross-species transmission capabilities, infecting various turtle species such as the Chinese pondturtle and the yellow-margined box turtle. CBHPTAV is a newly discovered virus in recent years. Initially, it was classified in the family Arteriviridae, but after genomic sequence analysis, the International Committee on Taxonomy of Viruses (ICTV) reclassified it along with Chinese soft-shelled turtle hemorrhagic fever virus (TSHSV) into the newly established family Cremegaviridae. Currently, detection methods and commercially available kits for this virus have not yet been established. Developing rapid and accurate detection techniques for CBHPTAV, especially room-temperature detection techniques, will greatly facilitate early detection, real-time monitoring, and epidemiological investigations, and is of great significance for disease control.

[0003] Currently, the commonly used pathogen detection technology is polymerase chain reaction (PCR). However, this technology requires specialized molecular biology laboratories and sophisticated instruments, and is time-consuming, failing to meet the needs of on-site diagnosis in aquaculture. While LAMP technology can achieve isothermal amplification, its amplification temperature is 60-65°C, making room-temperature amplification impossible. Recombinase polymerase amplification (RPA) is a relatively new isothermal nucleic acid amplification technology that does not require sophisticated temperature control equipment like PCR instruments. Its optimal amplification temperature is approximately 39-42°C, offering the potential for nucleic acid detection at room temperature; however, reports on room-temperature detection are currently limited. Primers and probes are the core components of RPA detection technology, determining its effectiveness. However, there is no mature professional software for RPA primer and probe design; the detection effect depends on the primer designer's experience and extensive experimental verification. Sometimes, even from dozens of candidate primer-probe combinations, it is difficult to select usable detection primers. Therefore, primer design is crucial to the success of RPA detection methods.

[0004] In aquaculture operations, there is an urgent need to establish rapid and visualized detection methods. While traditional PCR technology is commonly used, its visualization relies on agarose gel electrophoresis, requiring specialized and expensive equipment, making it inconvenient for non-professionals in aquaculture. Lateral flow immunochromatographic strips (LFD), however, can achieve visualized detection without complex equipment and are typically used for antibody detection. With appropriate modification of nucleic acid primers and probes, this technology can also be used for nucleic acid detection. By carefully designing combinations of RPA primers and probes, visualized detection of RPA amplification products can be achieved. Summary of the Invention

[0005] As a newly discovered virus in recent years, no detection method for CBHPTAV has been reported to date, especially the challenge of room temperature detection remains unresolved. To achieve rapid detection of CBHPTAV at room temperature and overcome instrument limitations, this invention aims to provide a primer-probe set for room temperature detection of the giant turtle arteritis virus and its application. Specifically, it involves a primer or primer-probe combination based on the RPA method and its detection method, and visualizes the detection results using lateral flow chromatography (LFD), enabling rapid detection of CBHPTAV at room temperature.

[0006] The first objective of this invention is to provide a pair of RPA primers for detecting the large-headed turtle arteritis virus CBHPTAV, wherein the RPA primers include a forward primer and a reverse primer, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0007] The second objective of this invention is to provide a primer-probe set for detecting arteritis virus in giant turtles, comprising a forward primer, a reverse primer, and a probe; the nucleotide sequence of the forward primer is shown in SEQ ID NO.3, the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4, and the nucleotide sequence of the probe is shown in SEQ ID NO.23.

[0008] Preferably, the 5' end of the reverse primer is modified with a special marker; the 5' end of the probe is modified with a fluorescent group, the 3' end is modified with a polymerase extension blocking group, and the A base is replaced by dSpacer at the 33 nt position.

[0009] Preferably, the special marker is biotin or digoxigenin, the fluorescent group is a 6-FAM group, and the polymerase extension blocking group is a C3 Spacer group.

[0010] A third objective of this invention is to provide a kit for detecting arteritis virus in giant turtles, the kit comprising the aforementioned primer and probe set and RPA amplification reagent.

[0011] Preferably, the RPA amplification reagent includes reverse transcriptase, recombinase, single-stranded DNA binding protein, DNA polymerase, amplification buffer containing dNTPs, and magnesium acetate.

[0012] The fourth objective of this invention is to provide a kit for detecting arteritis virus in giant turtles, the kit comprising the primer and probe set, RPA amplification reagent, and LFD nucleic acid test strip; the nucleic acid test strip comprises a control area, a detection area, and a sample application well, wherein the detection area is immobilized with anti-nucleic acid marker antibody, and the reagent pad is labeled with anti-nucleic acid marker ligand latex complex.

[0013] A fifth object of the present invention is to provide the application of the said primers or the said primer-probe set in the preparation of a kit or kit for detecting arteritis virus in giant turtles.

[0014] A sixth object of the present invention is to provide the application of the said primers, the said primer-probe set, the said kit, or the said kit in the detection of arteritis virus in turtles for non-disease diagnostic purposes.

[0015] The seventh objective of this invention is to provide a method for detecting arteritis virus in turtles (not for disease diagnosis), comprising the following steps:

[0016] S1. Extract RNA from the sample to be tested or extract RNA from the sample to be tested and reverse transcribe it into cDNA;

[0017] S2. Using the aforementioned kit and the RNA or cDNA obtained in step S1 as a template, perform RPA amplification to obtain the amplification product; the RPA amplification reaction program is 25℃-42℃ for 20 min;

[0018] S3. Mix the amplification product obtained in step S2 with nuclease-free water at a volume ratio of 1:7, and drop the mixture into the sample well of the nucleic acid test strip. Read the test result. If both the test line and the control line of the nucleic acid test strip show bands, the sample to be tested contains the giant turtle arteritis virus. If only the control line shows a band and the test line does not show a band, the sample to be tested does not contain the giant turtle arteritis virus.

[0019] Preferably, the reaction program for RPA amplification is 25°C for 20 min.

[0020] The beneficial effects of this invention are:

[0021] The primer and probe set and detection method for CBHPTAV provided by this invention have the advantages of high sensitivity, strong specificity, and simple operation. It can detect samples with a concentration as low as 1.089 copies / µL, and the detection time is only 20 min, which is much shorter than that of PCR. Most importantly, the primer and probe set and detection method provided by this invention can detect CBHPTAV at room temperature without the need for temperature control equipment, making it very suitable for animal pathogen detection in breeding fields. Attached Figure Description

[0022] Figure 1 This is the result of RPA primer screening for CBHPTAV. Primers 1-12 correspond to RPA1-12 primers, respectively, with cDNA containing CBHPTAV virus as the template; NC is the negative control, with water as the template; M is the DNA scale bar DL2000.

[0023] Figure 2 These are the results of detecting CBHPTAV using RPA2 primers at different reaction temperatures. NC1-NC2 are negative controls, and water is the template. The reaction temperatures for T1-T5 are 25.0℃, 30.0℃, 35.1℃, 38.2℃, and 42.0℃, respectively. M is the DNA scale bar DL2000.

[0024] Figure 3 This is the detection result of cDNA or RNA containing CBHPTAV using the RPA2 primer and probe combination under reaction conditions of 42℃; NC is the negative control, and water is the template.

[0025] Figure 4 This is the detection result of RPA2 primer and probe combination on cDNA or RNA containing CBHPTAV at room temperature; NC is the negative control, and water is the template.

[0026] Figure 5 The results are the specificity test results of RPA2 primer and probe combination for detecting CBHPTAV; 1 uses grouper iridovirus (SGIV) DNA as template, 2 uses red spot grouper nerve necrosis virus (RGNNV) cDNA as template, 3 uses mandarin fish sand grain virus (MFAV) cDNA as template, NC uses water as template (negative control), and Y uses CBHPTAV cDNA as template (positive control).

[0027] Figure 6These are the test results for the detection limit of CBHPTAV using the RPA2 primer and probe combination; 1-5 are the results of using CBHPTAV cDNA with concentrations of 1089 copies / µL, 108.9 copies / µL, 10.89 copies / µL, 1.089 copies / µL, and 0.1089 copies / µL as templates, respectively, while NC uses water as a template (CBHPTAV concentration of 0). Detailed Implementation

[0028] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0029] Example 1: RPA primer screening for CBHPTAV detection

[0030] (1) Design and screening of RPA primers and sample preparation

[0031] Primers were designed based on the CBHPTAV genome sequence obtained through sequencing. These primers were longer than conventional primers, exceeding 25 nt, and did not form primer dimers. The designed primers were compared with different CBHPTAV strains, and primer sequences consistent across different strains were selected. Furthermore, the sequences were compared with the NCBI online database to screen for primers that were unlikely to form non-specific amplifications in other viruses or turtle / tortoise genomes.

[0032] After preliminary screening, 12 pairs of primers (RPA1-12) were obtained as shown in Table 1. The oligonucleotides were synthesized by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd., and the primer concentration was configured to be 10 μM.

[0033] Table 1. Primer information for RPA1-12

[0034]

[0035] (2) Preparation of viral RNA in the test sample

[0036] Viral nucleic acid was extracted from turtle anal swabs (dissolved in PBS solution) or internal organs (crushed with liquid nitrogen or tissue ball mill) containing known CBHPTAV using the NPA-32P fully automated nucleic acid extraction and purification system and the MagaBio plus Viral DNA / RNA Purification Kit III (Hangzhou Bori Technology Co., Ltd., catalog number BSC86S1EX). The final step was to dissolve the nucleic acid in nuclease-free water.

[0037] (3) Preparation of viral cDNA in the sample to be tested

[0038] To facilitate the preservation of nucleic acids, cDNA was used as the template for the amplification reaction in this embodiment. The prepared RNA was reverse transcribed into cDNA using the reverse transcription reagent MaximaH Minus cDNA Synthesis Master Mix (Thermo Fisher Scientific Inc., catalog number M1681).

[0039] (4) Primer screening

[0040] The reagents used in this embodiment for the RPA reaction contain reverse transcriptase, recombinase, single-stranded DNA-binding protein, DNA polymerase, DNA amplification buffer containing dNTPs, and magnesium acetate. The kit used in this embodiment was purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd. as the RNA Isothermal Rapid Amplification Kit (Colloidal Gold Test Strip Type) Type II (Catalog No. WLRN8209KIT).

[0041] Prepare 50 μL of reaction solution according to the reaction system shown in Table 2 below, where primer F and primer R are the forward and reverse primers of RPA1-12, respectively. The negative control is to replace the cDNA of CBHPTAV with nuclease-free water.

[0042] Table 2 Primer screening reaction system for detecting CBHPTAV

[0043]

[0044] When preparing the solution, add B buffer last, and add it to the tube wall without touching the reaction solution. After all the reagents in each tube are prepared, cap the tubes, mix them thoroughly, and centrifuge to ensure that the reaction starts simultaneously in each tube.

[0045] After preparing the reaction system, place the reaction tube at 42℃ for 20 min.

[0046] After the reaction was complete, the amplification product was extracted with an equal volume (i.e., 50 μL) of a Tris-saturated phenol / chloroform / isoamyl alcohol mixture (volume ratio 25:24:1) to remove the enzyme from the reaction system. The reaction solution was then inverted to mix and centrifuged until the layers separated.

[0047] Take 10 μL of supernatant, add 2 μL of loading buffer (6× Loading buffer), mix well to obtain the loading sample, and take 8 μL of the loading sample for electrophoresis on a 2% agarose gel at 120 V for 30 min. After electrophoresis, take a picture using a gel imaging system. The results are as follows. Figure 1 As shown, only the amplification product of the RPA2 primer had a clear target band (166 bp), and the product was confirmed to be the CBHPTAV sequence by sequencing comparison.

[0048] Therefore, RPA2 primers are the most suitable RPA primers for detecting CBHPTAV, and RPA2 primers were used as RPA amplification primers in subsequent experiments.

[0049] Example 2: Optimization of reaction temperature for RPA2 detection of CBHPTAV

[0050] To verify whether the RPA2 primers can be used without temperature control instruments and to detect CBHPTAV at room temperature, this embodiment lowers the detection temperature to 25°C.

[0051] According to the reaction system in Table 2, primers F and R are the forward and reverse primers of RPA2 primers, respectively. Following the operating steps in Example 1, the reaction temperatures were set to 25.0℃, 30.0℃, 35.1℃, 38.2℃, and 42.0℃, respectively, and the reaction time was 20 min.

[0052] The results are as follows Figure 2 As shown, the RPA2 primers were able to detect the target band (166 bp) of CBHPTAV in the temperature range of 25.0℃–42.0℃. Therefore, the RPA2 primers can detect CBHPTAV at room temperature as low as 25.0℃ without the need for temperature control instruments, which can meet the requirements for rapid detection in aquaculture environments.

[0053] Example 3: Establishment of RPA-LFD Joint Detection Method

[0054] (1) Probe sequence design

[0055] Based on the primer sequence of RPA2, a probe sequence compatible with the primers is designed, and the probe sequence and primer sequence should avoid forming a dimer.

[0056] (2) Primer and probe sequence modification

[0057] Biotin was modified at the 5' end of the RPA2-R primer and labeled as RPA2-Rbiotion (5' Biotin-TGCTCCCGGATGGCAAAATTAATTATAG-3'). Biotin can be replaced with other specific markers such as digoxigenin.

[0058] The probe sequence (RPA2-probe: 5'-TTTTCTTCTTTTTGTTAATCAAGAGGAATTGTATATCCGTCTTGCCTGT-3', SEQ ID NO.23) is modified with a 6-FAM group at the 5' end, a C3 Spacer group at the 3' end, and a dSpacer in the middle of the sequence. The dSpacer is 32 nt from the 5' end, replacing the A base at the 33 nt position. The resulting modified probe sequence is as follows: 5' 6-FAM-TTTTCTTCTTTTTGTTAATCAAGAGGAATTGT / idSp / TATCCGTCTTGCCTGT-C3Spacer-3'.

[0059] (3) Preparation of LFD nucleic acid test strips

[0060] In this embodiment, a chromatography double antibody sandwich method LFD test strip is used for detection. The test strip that can detect FAM-labeled probes is selected, specifically the Diamond single nucleic acid test strip (item number B110202) purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0061] (4) Detection

[0062] RNA and cDNA of the sample to be tested were prepared according to the steps in Example 1. A 50 μL reaction mixture was prepared according to the table below (Table 3).

[0063] Table 3 Reaction system for RPA-LFD combined detection of CBHPTAV

[0064]

[0065] Add buffer B to the tube wall without touching the reaction solution. After all the reagents in each tube are prepared, cap the tubes, mix them thoroughly, and centrifuge to ensure that the reaction starts simultaneously in all tubes.

[0066] React at 42℃ for 20 min.

[0067] Take 10 μL of the reaction product and add 70 μL of nuclease-free water. Mix well and then drop the entire mixture into the sample well (S) of the nucleic acid test strip.

[0068] Read the test results within 15 minutes after spotting. Samples with bands on both the T line (test line) and the C line (control line) are positive. Samples with only a band on the C line and no band on the T line are negative. If there are no bands on either the T line or the C line, the nucleic acid test strip product is invalid.

[0069] Test results ( Figure 3 The results show that the RPA-LFD combined detection method established in this embodiment can detect RNA or cDNA samples containing CBHPTAV.

[0070] Example 4: RPA-LFD combined detection of CBHPTAV under room temperature conditions

[0071] The reaction system and steps of Example 3 were followed for testing, with the reaction conditions adjusted to room temperature (25°C) for 20 min.

[0072] Test results ( Figure 4 The results showed that the RPA-LFD combined detection method can detect RNA or cDNA samples containing CBHPTAV at room temperature (25℃).

[0073] Example 5: Detection Specificity Test

[0074] This embodiment uses nucleic acids from other viruses found in aquatic farms as the detection target to test the detection specificity of the RPA-LFD combined detection method.

[0075] DNA from grouper iridovirus (SGIV), cDNA from red-spotted grouper neuronecrosis virus (RGNNV), cDNA from mandarin fish sandworm virus (MFAV), and cDNA from CBHPTAV were used as reaction templates.

[0076] Prepare a 50 μL reaction system according to Table 3. The nucleic acid amplification templates for each tube are SGIV DNA, RGNNV cDNA, MFAV cDNA, CBHPTAV cDNA (positive control), and nuclease-free water (negative control). Perform detection according to the steps in Example 3.

[0077] Test results as follows Figure 5 As shown, the RPA2 primer and probe combination has strong specificity, can accurately detect CBHPTAV positive samples, and has no cross-reactivity with other viruses.

[0078] Example 6: Detection Limit Test

[0079] A CBHPTAV cDNA sample with a known concentration of 1089 copies / µL was taken and serially diluted 10-fold to obtain five different concentrations: 1089 copies / µL, 108.9 copies / µL, 10.89 copies / µL, 1.089 copies / µL, and 0.1089 copies / µL.

[0080] Prepare a 50 μL reaction system according to Table 3. The nucleic acid amplification templates for each tube are cDNA of different concentrations of CBHPTAV and nuclease-free water (negative control), respectively. Perform detection according to the steps in Example 3.

[0081] Test results as follows Figure 6 As shown, the RPA2 primer and probe combination exhibits high sensitivity in detecting CBHPTAV, capable of detecting samples with concentrations as low as 1.089 copies / uL.

Claims

1. A pair of RPA primers for detecting arteritis virus in giant-headed turtles, characterized in that, The RPA primers include a forward primer and a reverse primer, and the nucleotide sequences of the forward primer and the reverse primer are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. A primer and probe set for detecting arteritis virus in giant-headed turtles, characterized in that, It includes a forward primer, a reverse primer, and a probe; the nucleotide sequence of the forward primer is shown in SEQ ID NO.3, the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4, and the nucleotide sequence of the probe is shown in SEQ ID NO.

23.

3. The primer-probe set according to claim 2, characterized in that, The 5' end of the reverse primer is modified with a special marker; the 5' end of the probe is modified with a fluorescent group, the 3' end is modified with a polymerase extension blocking group, and the A base is replaced by dSpacer at the 33 nt position.

4. The primer-probe set according to claim 3, characterized in that, The special marker is biotin or digoxigenin, the fluorescent group is 6-FAM, and the polymerase extension blocking group is C3 Spacer.

5. A kit for detecting arteritis virus in giant-headed turtles, characterized in that, The kit comprises the primer and probe set and RPA amplification reagent as described in any one of claims 2-4.

6. The reagent kit according to claim 5, characterized in that, The RPA amplification reagents include reverse transcriptase, recombinase, single-stranded DNA binding protein, DNA polymerase, amplification buffer containing dNTPs, and magnesium acetate.

7. A kit for detecting arteritis virus in giant-headed turtles, characterized in that, The kit includes the primer and probe set, RPA amplification reagent, and LFD nucleic acid detection test strip as described in any one of claims 2-4.

8. The use of the primers of claim 1 or the primer-probe set of any one of claims 2-4 in the preparation of a kit or kit for detecting arteritis virus in giant turtles.

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