Primer pair, probe, kit and detection method for bovine enterovirus RT-RAA fluorescent rapid detection

Abstract

The invention relates to a primer pair, a probe, a kit and a detection method for bovine enterovirus RT-RAA fluorescent rapid detection, and particularly relates to a primer pair, a probe, a kit and a detection method for rapidly and efficiently detecting the bovine enterovirus 5 'UTR end fluorescent RT-RAA through fluorescent RT-RAA. The primer comprises an upstream primer and a downstream primer, the sequences of the upstream primer and the downstream primer are shown as SEQ.ID NO.3 and SEQ.ID NO.4 respectively, and the sequence of the probe is shown as SEQ.ID NO.1. The primer and the probe are high in sensitivity, strong in specificity and good in repeatability, and have a better linear relationship in a standard plasmid concentration range of 1.10 * 10 < 1 >-1.10 * 10 < 4 > copies / microliter, and the lowest detection limit can reach 1.10 * 10 < 1 > copies / microliter. And rapid and efficient detection of the bovine enterovirus can be realized in 20 minutes in a Genchek fluorescence detector.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer pair, a probe, a kit and a detection method for rapid detection of bovine enterovirus RT-RAA fluorescence type. Background Art

[0002] Bovine enterovirus (BEV) is prevalent in spring and summer and is one of the important factors endangering the development of cattle breeding industry. The disease is a newly emerging infectious disease, and its main symptoms include fever, dry cough, dyspnea, diarrhea and reproductive disorders. The virus often co-infects with other pathogens, causing respiratory and digestive system related diseases, and even leading to the death of livestock, posing a direct threat to the healthy development of animal husbandry.

[0003] Since BEV was first isolated in 1959, BEV infection has been reported successively. Currently, the main methods for detecting bovine enterovirus are RT-PCR, RT-qPCR and LAMP, but since these methods are time-consuming and costly, it is urgent to establish a rapid and efficient detection method to provide an effective way for the monitoring and prevention of bovine enterovirus.

[0004] Recombinase-aided amplification assay (RAA) is a new type of constant temperature nucleic acid amplification detection technology. The reaction system consists of Escherichia coli recombinase UvsX, single-stranded DNA binding protein (SSB), DNA polymerase buffer, etc. The principle is that under constant temperature conditions, the recombinase UvsX binds to specific primers on the template DNA to search for homologous sequences and bind, and with the assistance of single-stranded binding protein, the double-stranded DNA of the template DNA is opened and a stable single-stranded structure is formed. Then the DNA polymerase starts the template synthesis from the 3 ends of the primer to form two double-stranded DNAs. This cycle is repeated to achieve amplification. The reaction can be completed at a constant temperature of 39°C for 20 minutes.

[0005] Therefore, it is urgent to establish a rapid and efficient detection method combined with recombinase-mediated amplification technology to provide an effective way for the monitoring and prevention of bovine enterovirus. Summary of the invention

[0006] The main methods for detecting bovine enterovirus in the prior art are RT-PCR, RT-qPCR and LAMP, but these methods are time-consuming and labor-intensive. The present invention provides a primer pair, a probe, a kit and a detection method for RT-RAA fluorescent rapid detection of bovine enterovirus. The purpose of the present invention is to provide an RT-RAA primer and probe for detecting bovine enterovirus, and to perform reverse transcription recombinase-mediated isothermal amplification (RT-RAA) on bovine enterovirus by using the primer and probe to achieve rapid, specific, sensitive, visual and simple detection of bovine enterovirus.

[0007] To achieve the above purpose, the technical solution of the present invention is as follows:

[0008] An RT-RAA primer and probe for detecting bovine enterovirus, comprising a forward primer BEV-5'UTR-F2, a reverse primer BEV-5'UTR-R1 and a probe BEV-5'UTR-Probe for identifying the 5'UTR gene of bovine enterovirus;

[0009] The sequences of the forward primer BEV-5'UTR-F2, the reverse primer BEV-5'UTR-R1 and the probe BEV-5'UTR-Probe are as follows:

[0010] Forward primer BEV-5'UTR-F: 5'-TCACTGTACGTAGCCAAGAGCCTATTGTGC-3' (SEQ ID NO. 3),

[0011] Reverse primer BEV-5'UTR-R: 5'-CGGAGTACCGAAAGTAGTCTGTTCCGCCTC-3' (SEQ ID NO. 4),

[0012] Probe BEV-5'UTR-Probe: 5'-CCTCCGAGCGTGTGCGCACAATCCAGTGTTGCTACGTCGTAACGCGTAAG-3' (SEQ ID NO. 1);

[0013] The modified site of the probe BEV-5'UTR-Probe is labeled with a FAM fluorescent group at the 30th base T from the 5'UTR end, the 32nd base C is replaced by THF tetrahydrofuran, the 33rd base T is labeled with a BHQ1 quenching group, and the 3' end is modified with a C3 spacer blocking.

[0014] The present invention also provides a kit for detecting bovine enterovirus, the kit comprising the above-mentioned RT-RAA primers and probes for detecting bovine enterovirus;

[0015] The kit for detecting bovine enterovirus provided by the present invention further comprises a reaction reagent, a reaction buffer, RNase Free ddH2O, a positive quality control product and a negative quality control product, and the reaction buffer comprises B buffer.

[0016] The present invention also provides an application of the above-mentioned kit for detecting bovine enterovirus, specifically an application of the kit for detecting bovine enterovirus in preparing a preparation for detecting bovine enterovirus, wherein the application is for non-diagnostic purposes and comprises the following steps:

[0017] 1) Extracting RNA from the sample to be tested;

[0018] 2) RT-RAA reaction system amplification: The extracted RNA is used as a template to add the RT-RAA primers and probes for detecting bovine enterovirus in the kit and the reagents containing reverse transcriptase recombinase-mediated isothermal amplification to form an RT-RAA reaction system for RT-RAA amplification;

[0019] The total RT-RAA reaction system is 50 L: A buffer 25 μL, 10 μM BEV-5'UTR-F and BEV-5'UTR-R 2.5 μL each, 10 μM BEV-5'UTR-Probe 0.6 uL, RNase Free ddH20 11.9 μL and the sample RNA template (or standard plasmid) to be tested 5 μL, B buffer 2.5 μL;

[0020] Result judgment:

[0021] Quality control standard: If the negative control has no amplification curve or the peak time is ≥20min (Ct value ≥40), it is a valid result; if the positive control has an obvious amplification curve or the peak time is ≤15min (Ct value ≤30), it is a valid result; otherwise, the experimental result is invalid;

[0022] Result description and judgment:

[0023] The sample to be tested is negative when there is no amplification curve or the peak time is greater than 18 minutes (Ct value greater than 36), and is positive when a typical amplification curve appears or the peak time is ≤18 minutes (Ct value ≤36); the template in the negative control is RNaseFree ddH20, and the template in the positive control is a constructed standard plasmid or positive strain of bovine enterovirus.

[0024] In the application, the final concentration of the primer in the reaction system is 0.5 mM, and the final concentration of the probe in the reaction system is 0.12 mM.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] 1. The present invention provides a primer for RT-RAA fluorescence detection of bovine enterovirus. The primer pair can specifically amplify bovine enterovirus, and has no cross reaction with the nucleic acid of bovine diarrhea virus (BVDV), bovine rotavirus (BRV), bovine astrovirus (BAstV), bovine kub virus (BKV) and Shiga toxin-producing Escherichia coli (STEC), and has strong specificity. The primer pair can be used to detect bovine enterovirus, overcoming the shortcomings of existing conventional methods.

[0027] 2. The present invention also provides a kit for detecting bovine enterovirus. The kit prepared using the primer pair can complete the detection in 20 minutes under a constant temperature of 39°C. It has the characteristics of simple operation, low cost, strong specificity, high sensitivity, good repeatability, etc., which are not possessed by traditional methods.

[0028] 3. The present invention also provides a method for detecting bovine enterovirus, which uses the above-mentioned kit and primer probe for detection, can complete amplification in a very short time, reduces the difficulty of operation, and can also improve the sensitivity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a diagram showing the screening results of the bovine enterovirus RT-RAA primer pair in Example 2 of the present invention;

[0030] Figure 2 The figure is a graph showing the agarose electrophoresis result of the PCR amplification product of the bovine enterovirus primer pair in Example 2 of the present invention (1-9 are primer combinations, and 10-18 are corresponding negative controls);

[0031] Figure 3 The sensitivity test result of the kit of the present invention in Example 5 of the present invention (1-6 is 1.10×10 4 —1.10×10 -1 copies / μL, 7 as negative control);

[0032] Figure 4 This is a diagram of the specific detection results of the kit of the present invention in Example 7 of the present invention (1-7 are nucleic acids of 7 pathogens, namely BEV, BCoV, BVDV, BKV, BRV, BAstV, and STEC, and 8 is a negative control). DETAILED DESCRIPTION

[0033] The technical scheme in the embodiment of the present invention is described clearly and in detail in conjunction with the following drawings. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0034] The specific embodiments of the present invention are described in detail, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0035] Embodiment 1:

[0036] Primer pairs and probes for RT-RAA fluorescent rapid detection of bovine enterovirus:

[0037] According to the bovine enterovirus nucleic acid sequence published on NCBI, 15 bovine enterovirus genomes (including type E and type F) were selected to analyze their homology. It was found that only the 5'UTR end was the most conserved, so this region was selected to design primer probes; the design of RT-RAA primers follows the following basic principles: RAA primers are between 30-35bp, can be longer than 35bp but not shorter than 30bp, so general PCR primers are not suitable for RAA; the length of the amplified product should be between 100-200bp, and try not to exceed 500b p, if the amplification product is too long, the sensitivity and reaction time of the reaction will increase and affect the experimental effect; secondly, for the sequence of the primer, avoid repeated G at the 5' end (3-5bp), preferably C or T, and preferably G and C at the 3' end (the last 3 bases); the GC content is between 30% and 70%; avoid forming secondary structures and primer dimers between primers; the probe length of RAA is about 46-50bp, and tetrahydrofuran residues are added in the middle of the probe, at least 30bp from the 5' end to the tetrahydrofuran residue, and at least 15bp from the tetrahydrofuran residue to the 3' end.

[0038] According to the above design principles, three pairs of primers and probes were designed as shown in Table 1:

[0039]

[0040] The modified site of the probe BEV-5'UTR-Probe is labeled with a FAM fluorescent group at the 30th base T from the 5' end, the 32nd base C is replaced by THF tetrahydrofuran, the 33rd base T is labeled with a BHQ1 quenching group, and the 3' end is modified with a C3spacer blocking group;

[0041] RNA extraction: Take fecal samples from diseased cattle in the farm, add them into a 1.5mL EP tube at a ratio of 1:5 between sample and saline, shake and mix well, freeze and thaw repeatedly for 3 times, and extract RNA from the fecal samples according to the instructions;

[0042] Construction of standard plasmid: RVA virus was amplified using RT-PCR primers, the amplified product was recovered from gel and connected to the pMD18-T vector, transformed into Escherichia coli DH5a competent cells and cultured, the plasmid was extracted and the concentration was measured; the constructed recombinant plasmid was stored at -20°C after sequencing verification and named 18-T-BEV-5'UTR.

[0043] Embodiment 2:

[0044] Screening of primer pairs: The above three pairs of primers were combined in pairs to obtain the following nine combinations: F1R1, F2R2, F3R3, F1R2, F1R3, F2R1, F2R3, F3R1, F3R2, and a recombinant plasmid named 18-T-BEV-5'UTR was constructed. Its concentration was 1.10×10 11 copies / , and the recombinant plasmid was diluted 10-fold to 1.10×10 6 The primer pairs were screened (Genchek fluorescence detector reaction program: 39°C 20min), and the 9 primer pairs were verified by conventional RT-PCR. Figure 2 As shown, under the same conditions, the primer pair F2R1 had the smallest Ct value and the highest amplification efficiency, and the conventional RT-PCR verified that the band position was correct, clear and bright, and there was no dragging of mixed bands. Therefore, the primer pair F2R1 was selected as the optimal primer pair for subsequent experiments.

[0045] Embodiment 3:

[0046] A reaction system of RT-RAA nucleic acid amplification kit (fluorescence type) is shown in Table 2:

[0047] Reagents concentration Volume (total volume 50 μL) BufferA - 25μL Upstream primer 10μM 2.5μL Downstream primer 10μM 2.5μL Probe 10μM 0.6μL <![CDATA[ddH2O]]> - 11.9μL template - 5μL Buffer B - 2.5μL

[0048] Embodiment 4:

[0049] Amplification reaction conditions: Genchek series fluorescence detector: 39°C for 20 min, or fluorescence quantitative PCR instrument: 39°C for 60 s, 39°C for 30 s, 40 cycles to collect fluorescence;

[0050] Result analysis and judgment

[0051] Genchek fluorescence detector: can automatically interpret the test results;

[0052] Other fluorescent quantitative PCR instruments: Positive control: a typical amplification curve appears, and the peak time is ≤15min (Ct value ≤30), which is a valid result; negative control: no amplification curve appears, or the peak time is ≥20min (Ct value ≥40), which is a valid result;

[0053] Test samples: A typical amplification curve appears, and it is positive when the peak time is ≤18min (Ct value ≤36); the peak time is >18min (Ct value >36).

[0054] Embodiment 5:

[0055] RT-RAA sensitivity test for bovine enterovirus

[0056] Using the primer pair screened in Example 2, 1.10×10 4 Copies / μL—1.10×10 -1 5 μL of each of the recombinant plasmids with 6 concentrations of 1,000 copies / μL was used as a template, ddH2O was used as a negative control, the reaction solution was prepared according to the system in Example 3, and the specific amplification of RT-RAA was performed according to the reaction procedure in Example 4.

[0057] The sensitivity test results are as follows Figure 3 As shown, the minimum detection limit is 1.10×10 1 copies / μL, with good sensitivity.

[0058] Embodiment 6:

[0059] Bovine enterovirus RT-RAA specificity test

[0060] Using the primer pairs screened in Example 2, BEV virus nucleic acid was used as a template, six pathogenic nucleic acids of BCoV, BVDV, BKV, BRV, BAstV, and STEC were used as controls for the specific test, ddH2O was used as a negative control, the reaction solution was prepared according to the system in Example 3, and the specific amplification of RT-RAA was performed according to the reaction procedure in Example 4.

[0061] Specific test results such as Figure 4 As shown, only BEV showed an amplification curve, and there was no cross-reaction with the six pathogens including BCoV, BVDV, BKV, BRV, BAstV, and STEC, indicating good specificity.

[0062] Embodiment 7:

[0063] Repeatability test of RT-RAA of bovine enterovirus

[0064] Select 1.10×10 4 copies / μL, 1.10×10 3 copies / μL, 1.10×10 2 The three concentrations of 1000 copies / μL were repeated three times for each concentration, ddH2O was used as a negative control, the reaction solution was prepared according to the system in Example 3, and the specific amplification of RT-RAA was performed according to the reaction procedure in Example 4; the coefficient of variation of the intra-group and inter-group repeatability tests was calculated;

[0065] The results of the repeatability test are shown in Table 3 below. The coefficients of variation within and between groups are both less than 5%, indicating that the repeatability and stability of the present invention are good;

[0066]

[0067]

[0068] Embodiment 8:

[0069] Clinical sample detection: 108 samples of cattle diarrhea feces were taken to extract RNA, and 5 μL was taken as a template. The primers screened in Example 2 were used to prepare the reaction solution according to the system in Example 3, and the specific amplification of RT-RAA was performed according to the reaction procedure in Example 4;

[0070] The results of clinical sample testing are shown in Table 4 below. Comparison and verification with fluorescent quantitative PCR and common RT-PCR showed that 52 positive samples were detected at the same time, with a compliance rate of 100%, indicating that the method has good applicability;

[0071] Detection Methods Number of tests Number of positive Positive rate RT-PCR 108 52 47.22％ RT-qPCR 108 52 47.22％ RT-RAA 108 52 47.22％

[0072] In summary, the application of bovine enterovirus RT-RAA fluorescence detection method has high sensitivity, strong specificity, good repeatability, simple operation, low cost, short time consumption, saving time and labor. Amplification can be completed in 20 minutes at a constant temperature of 39°C using this primer pair and kit.

[0073] The embodiments described above are some embodiments of the present invention, rather than all embodiments. They are not intended to limit the scope of the invention claimed for protection, but merely represent selected embodiments of the present invention. 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.

Claims

1. An RT-RAA primer and probe for detecting bovine enterovirus, characterized in that: The method comprises a forward primer BEV-5'UTR-F, a reverse primer BEV-5'UTR-R and a probe BEV-5'UTR-Probe for identifying the 5'UTR gene of bovine enterovirus; The sequences of the forward primer BEV-5'UTR-F, the reverse primer BEV-5'UTR-R and the probe BEV-5'UTR-Probe are as follows: Forward primer BEV-5'UTR-F: 5'-TCACTGTACGTAGCCAAGAGCCTATTGTGC-3', SEQ ID NO.3, Reverse primer BEV-5'UTR-R: 5'-CGGAGTACCGAAAGTAGTCTGTTCCGCCTC-3', SEQ ID NO. 4; Probe BEV-5'UTR-Probe:5'-CCTCCGAGCGTGTGCGCACAATCCAGTGTTGCTACGTCGTAACGCGTAAG-3', SEQ ID NO.1; The modified site of the probe BEV-5'UTR-Probe is labeled with a FAM fluorescent group at the 30th base T from the 5'UTR end, the 32nd base C is replaced by THF tetrahydrofuran, the 33rd base T is labeled with a BHQ1 quenching group, and the 3' end is modified with a C3spacer blocking group.

2. A kit for detecting bovine enterovirus, characterized in that: The kit comprises the RT-RAA primer and probe for detecting bovine enterovirus according to claim 1; The kit for detecting bovine enterovirus further comprises a reaction reagent, a reaction buffer, RNase FreeddH2O, a positive quality control product and a negative quality control product, and the reaction buffer comprises B buffer.

3. The use of a kit for detecting bovine enterovirus according to claim 2, characterized in that: Specifically, the kit for detecting bovine enterovirus is used in the preparation of a preparation for detecting bovine enterovirus, and the application is for non-diagnostic purposes and comprises the following steps: 1) Extracting RNA from the sample to be tested; 2) RT-RAA reaction system amplification: The extracted RNA is used as a template to add the RT-RAA primers and probes for detecting bovine enterovirus in the kit and the reagents containing reverse transcriptase recombinase-mediated isothermal amplification to form an RT-RAA reaction system for RT-RAA amplification; The total RT-RAA reaction system is 50L: A buffer 25μL, 10μM BEV-5'UTR-F and BEV-5'UTR-R 2.5μL each, 10μM BEV-5'UTR-Probe 0.6uL, RNase Free ddH2011.9μL and the sample RNA template or standard plasmid to be tested 5μL, B buffer 2.5μL; Result judgment: Quality control standard: If the negative control has no amplification curve or the peak time is ≥20min, and the Ct value is ≥40, it is a valid result; if the positive control has an obvious amplification curve or the peak time is ≤15min, and the Ct value is ≤30, it is a valid result; otherwise, the experimental result is invalid; Result description and judgment: The sample to be tested is negative when there is no amplification curve or the peak time is greater than 18 minutes, and the Ct value is greater than 36. It is positive when a typical amplification curve appears or the peak time is ≤18 minutes, and the Ct value is ≤36. Among them, the template added to the negative control is RNase FreeddH20, and the template in the positive control is the constructed standard plasmid or positive strain of bovine enterovirus.

4. The use of a kit for detecting bovine enterovirus according to claim 3, characterized in that: The final concentration of the primer in the reaction system is 0.5 mM, and the final concentration of the probe in the reaction system is 0.12 mM.

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