Real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection primer, probe and detection kit for sheep enterovirus TaqMan

Abstract

The invention provides a sheep enterovirus TaqMan real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection primer and a probe. The nucleotide sequences of the primer and the probe are as follows: an upstream primer CEV-qF: 5 '-TCCTCCGGRGCCGTGAATGC-3', a downstream primer CEV-qF: 5 '-TCCTCCGGRGCCGTGAATGC-3', a downstream primer CEV-qF: 5 '- The downstream primer CEV-qR is 5 '-CCGAAAGTAGTCTGTTCCGCC-3', and the downstream primer CEV-qR is 5 '- And the probe CEV-P is 5 '-Cy5-AACCTCCGAGCGTGTGCGCA-BHQ2-3', and the probe has the following structural formula shown in the description. The sheep enterovirus TaqMan real-time fluorescent quantitative RT-PCR detection method established by utilizing the primer and the probe is rapid, time-saving, high in specificity, good in repeatability and high in sensitivity, and the lowest detection limit is 23 copies / mu L.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a TaqMan real-time fluorescence quantitative RT-PCR detection primer, a probe and a detection kit for sheep enterovirus. Background Art

[0002] Enterovirus (EV) is widely present in humans and various animals, and can cause various diseases in humans and animals. EV can cause a variety of diseases in humans, including nonspecific febrile diseases, hand, foot and mouth disease, neonatal sepsis-like disease, encephalitis, acute flaccid paralysis, etc. The impact of EV on livestock is more about respiratory and digestive system problems. Although it rarely causes death, its existence directly damages the healthy growth of livestock. Caprine enterovirus (CEV) was first discovered in sheep feces by Hungarian scholars in 2012. At present, 12 CEV whole genome sequence information can be retrieved from the GenBank database, among which the TB4-OEV strain reported by Hungarian scholars is classified as the representative strain of EV-G5, the CEV-JL14 reported by Chinese scholar Wang Mingyue in 2014 is classified as the representative strain of EV-G20, and the 990 / UK-NI strain determined by British scholars in 2018 is classified as the representative strain of EV-G7. The remaining 9 CEV whole genome sequences have not yet been classified by ICTV. The understanding of CEV molecular epidemiology is far from enough and needs extensive and in-depth research.

[0003] The real-time fluorescence quantitative PCR method is developed from the common PCR detection technology. It designs specific primers based on the conserved regions of the genome and introduces probes or fluorescent dyes to quantitatively detect the virus. This method takes less time and has better specificity and sensitivity than the traditional PCR method. The CEV RT-PCR detection method established by Dong Kun et al. (2021) can quickly diagnose samples suspected of CEV infection, with a minimum detection limit of 1.13×10 3 copies / μL; Xiao Dai et al. (2022) established a fluorescent quantitative PCR detection method for detecting porcine enterovirus G type, and the positive rate of EV-G in pig herds in Sichuan was 31.1%; Wu Dan et al. (2012) designed and synthesized primers and probes for amplifying the 5′ UTR sequence of bovine enterovirus (BEV) and established a universal TaqMan fluorescent quantitative RT-PCR rapid detection method for detecting BEV. The minimum detection amount of BEV by this method is 0.1TCID 50 / 0.1 mL, which is 10 times more sensitive than the virus isolation method. At present, there is no report on the TaqMan real-time fluorescence quantitative RT-PCR detection method and kit for CEV.

[0004] In order to further understand the molecular epidemiology of CEV, it is necessary to establish a more sensitive and high-throughput molecular biological detection method and create a kit for promotion and application. Based on this, the present invention establishes a CEV TaqMan real-time fluorescence quantitative RT-PCR detection method, through sensitivity, specificity and repeatability testing, and compares the detection ability with ordinary RT-PCR, evaluates the detection ability of this method, and finally assembles it into a kit for application. This is of great significance for the prevention and control of CEV. Summary of the invention

[0005] The purpose of the present invention is to provide a TaqMan real-time fluorescence quantitative RT-PCR detection primer, a probe and a detection kit for sheep enterovirus. The TaqMan real-time fluorescence quantitative RT-PCR detection method for sheep enterovirus established by the present invention using the primers and probes is fast and time-saving, has strong specificity, high sensitivity and good repeatability.

[0006] The present invention discloses a TaqMan real-time fluorescence quantitative RT-PCR detection primer and probe for sheep enterovirus, wherein the nucleotide sequences of the primer and probe are as follows: Upstream primer CEV-qF: 5′-TCCTCCGGRGCCGTGAATGC-3′ Downstream primer CEV-qR: 5′-CCGAAAGTAGTCTGTTCCGC-3′ Probe CEV-P: 5'-Cy5-AACCTCCGAGCGTGTGCGCA-BHQ2-3' Cy5 stands for Cyanine 5; BHQ2 stands for Black Hole Quencher-2.

[0007] The CEV TaqMan real-time fluorescence quantitative RT-PCR detection method of the present invention is as follows: (1) Extract nucleic acid from the sample to be tested, and use the above primers and probes to perform TaqMan real-time fluorescence quantitative RT-PCR detection on sheep enterovirus. The PCR amplification system for detection is: 20 μL of total reaction system, including 2×One Step RT-PCR Buffer Ⅲ 10 μL, ExTaq HS 0.4 μL, PrimeScript RT Enzyme Mix Ⅱ 0.4 mL, probe 0.8 μL, Rox Ⅱ 0.4 μL, upstream and downstream primers 0.4 μL each, RNA template 2 μL, RNase Free H2O 5.2 μL. The amplification program is to use a two-step method for amplification, 42°C 5 min, 95°C 10 s; another 95°C 5 s, 58°C 34 s, a total of 42 cycles.

[0008] (2) After the reaction is completed, the Ct value is read, with a Ct value of 34 as the cutoff. When the sample Ct value is <34, it is judged as positive; when the sample Ct value is ≥34, it is judged as negative.

[0009] The present invention also provides the use of the primers (CEV-qF, CEV-qR) and the probe (CEV-P) in preparing a TaqMan real-time fluorescence quantitative RT-PCR detection reagent or a detection kit for ovine enterovirus.

[0010] The present invention designs primer pairs and probes, establishes a Taqman method for detecting CEV in real-time fluorescence quantitative RT-PCR, has no cross-reaction to five common sheep infection viruses including foot-and-mouth disease virus (FMDV), peste des petits ruminants virus (PPRV), sheep pox virus (SPV), orf virus (ORFV) and bovine viral diarrhea virus (BVDV), and has good specificity; the lowest sensitivity detection limit is 23 copies / μL, and the sensitivity is good; the repeatability evaluation shows that the CV within and between groups is less than 2.5%; 598 samples are tested, and the CEV positive rate detected by the common RT-PCR method is 5.02%, and the CEV positive rate detected by the TaqMan real-time fluorescence quantitative RT-PCR is 13.88%. It can be seen that the CEV TaqMan real-time fluorescence quantitative RT-PCR detection method established by the present invention is more sensitive and has stronger detection ability, which provides a molecular detection method and kit with high sensitivity and good specificity for CEV molecular epidemiological investigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Amplification of target fragment; M: DNA molecular mass standard; 1, 2: both are CEV / JSNT / 1 / 2022 strain.

[0012] Figure 2 Identification of positive clones; M: DNA molecular quality standard; 1: pGEM-T vector; 2, 3, 4: all are recombinant plasmids.

[0013] Figure 3 Identification by linearization treatment; M: DNA molecular mass standard; 1, 3, 5, 7: plasmids after linearization treatment, of which 1 is an empty vector, and 3, 5, 7 are recombinant plasmids; 2, 4, 6, 8: circular plasmids without linearization treatment, of which 2 is an empty vector, and 4, 6, 8 are recombinant plasmids.

[0014] Figure 4 PCR amplification of each virus to verify the established method; M: DNA molecular mass standard; 1: FMDV; 2: PPRV; 3: BVDV; 4: SPV; 5: OrfV.

[0015] Figure 5CEV real-time fluorescence quantitative RT-PCR specificity test results; 1: standard; 2: FMDV; 3: PPRV; 4: BVDV; 5: SPV; 6: OrfV; 7: negative control.

[0016] Figure 6 CEV real-time fluorescence quantitative RT-PCR sensitivity test results; 1-8: 2.3×10 7 ~2.3×10 0 copies / μL standard; 9: negative control.

[0017] Figure 7 CEV real-time fluorescence quantitative RT-PCR amplification kinetic curve; 1~5: 2.3×10 6 ~2.3×10 2 copies / μL of standard.

[0018] Figure 8 CEV real-time fluorescence quantitative RT-PCR standard curve.

[0019] Fig. 9 PCR amplification of positive samples; M: DNA molecular mass standard; 1~9: positive samples. M: DNA Marker. DETAILED DESCRIPTION

[0020] The present invention is specifically described below through specific implementation modes. Example

[0021] 1. Materials and Methods 1.1 Cells and strains Baby hamster kidney cells (BHK-21), caprine enterovirus (CEV), foot-and-mouth disease virus (FMDV), capripox virus (CPV), orofacial sore virus (OrfV), peste des petits ruminants virus (PPRV), and bovine viral diarrhea virus (BVDV) were all preserved and supplied by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0022] 1.2 Field samples A total of 598 samples of sheep anal swabs, feces, etc. were collected from Gansu, Qinghai, Jiangsu and other regions.

[0023] 1.3 Main Reagents EZNA®Total RNA Kit I was purchased from Omega BIO-TEK; HiScript II One StepRT-PCR Kit2 was purchased from Nanjing Novozymes Biotech Co., Ltd.; DNA Marker, DNA fragment gel recovery kit, and One Step PrimeScript™RT-PCR Kit (Perfect Real Time) were all purchased from Takara Biotech (Beijing) Co., Ltd.

[0024] 1.4 Design and synthesis of primers and probes The complete gene sequences of 12 CEV strains in the Genbank database (ID: JQ277724, KU297674, MG958646, MN598038, MN598033, MN598034, MN598035, MN598036, MN598040, MN598041, OV176440, MZ679284) and this research group obtained a strain of JSNT / 1 / 2022 (patent application publication number: CN202211479502.8). After BLAST analysis, relatively conservative regions were selected to design RT-PCR primer pairs and TanMan real-time fluorescence quantitative RT-PCR primer pairs and probes for detection, and commissioned Sangon Biotech (Shanghai) Co., Ltd. to synthesize them. The concentrations of primers and probes were diluted to 10 μmol / L for use. The primer and probe sequences are shown in Table 1.

[0025] 1.4 Preparation of standards The target gene region amplified by RT-PCR method includes the range of real-time fluorescence quantitative RT-PCR amplification. Therefore, the JSNT / 1 / 2022 strain was used as a template, and the target fragment was amplified by RT-PCR and cloned into the pGEM-T vector. After sequencing was correct, the plasmid was named pGEM-T-CEV plasmid and restriction endonuclease was used. N I was linearized, and RNA was transcribed using a T7 promoter in vitro transcription kit. Finally, the in vitro transcribed RNA product was digested with RNase-free DNase to eliminate the DNA used as a transcription template. The RNA concentration was measured and the copy number was calculated, which was used as a standard for sensitivity analysis of the detection method.

[0026] 1.5 Establishment and optimization of one-step fluorescence quantitative RT-PCR reaction system The total reaction system was 20 μL, including 10 μL of 2× One Step RT-PCR Buffer, 0.4 μL of ExTaq HS, 0.4 μL of Prime Script RT Enzyme Mix, 0.8 μL of probe, 0.4 μL of Rox, 0.4 μL of upstream and downstream primers, 2 μL of RNA template, and 5.2 μL of RNase Free H2O. The reaction conditions were 42 °C for 5 min; 95 °C for 10 s; and 42 cycles of 95 °C for 5 s and 58 °C for 34 s.

[0027] Using the standard as template, the primer concentration (0.1-0.35 μmol / L), probe concentration (0.35-0.5 μmol / L) and annealing temperature (56.0, 57.0, 58.0, 59.0, 60.0°C) were optimized to determine the optimal reaction conditions.

[0028] 1.6 Specificity test The nucleic acids of FMDV, PPRV, BVDV, CPV, and OrfV were used as templates and ddH2O as a negative control to verify the specificity of the established CEV real-time fluorescence quantitative RT-PCR detection method to prevent cross-reaction with common susceptible viruses.

[0029] 1.7 Sensitivity test The standard template was diluted in a 10-fold gradient series with a concentration gradient of 2.3×10 7 ~2.3×10 0 copies / μL, and ddH2O was used as a negative control to determine the detection limit (LOD).

[0030] 1.8 Establishment of standard curve According to the optimized best reaction system and conditions, the standard was diluted 10 times and 2.3×10 2 ~2.3×10 6 The dilution solution of the standard sample with a concentration gradient of copies / μL was used as the template, and ddH2O was used as the negative control. After amplification, the standard curve and its linear equation and correlation coefficient (R 2 ) and amplification efficiency are automatically generated by the machine.

[0031] 1.9 Repeatability test Select three dilution standards (2.3×10 7 , 2.3×10 6 , 2.3×10 5copies / μL) was used as a template to perform intra-group repeated tests on the established CEV real-time fluorescence quantitative RT-PCR detection method, with each concentration repeated 3 times. At different time periods, the above-mentioned standard samples with the same concentration were taken for inter-group repeated tests, with each concentration repeated 3 times, with an interval of 5 days each time. The intra-group and inter-group coefficients of variation were calculated based on the Ct values ​​obtained from the test results to evaluate the repeatability of the method.

[0032] 1.10 Field sample testing From March 2023 to January 2024, 598 samples of feces, anal swabs, oral swabs, lymph nodes, etc. were collected from sheep farms in some areas of Lanzhou, Zhangye, Qinghai, etc., as shown in Table 2. Viral RNA was extracted using EZNA®Total RNA Kit I and tested using the established CEV real-time fluorescence quantitative RT-PCR detection method. The reaction system is shown in Table 3. The CEV-positive samples were verified using the conventional RT-PCR method. The reaction system is shown in Table 4. The reaction conditions for CEV real-time fluorescence quantitative RT-PCR were 42 °C for 5 min, 95 °C for 10 s, 95 °C for 5 s, and 58 °C for 34 s for a total of 42 cycles. After amplification, the positive and negative results were determined based on the Ct value and the curve.

[0033] The common RT-PCR reaction conditions were as follows: 50℃ for 30 min, 94℃ for 3 min; 94℃ for 30 sec, 57℃ for 30 sec, 72℃ for 1 min, for a total of 35 cycles; and finally 72℃ for 5 min. The PCR products were analyzed by electrophoresis on 2% agarose gel.

[0034] result 2.1 Preparation of Standards 2.1.1 Amplification of target fragment Using RNA extracted from CEV / JSNT / 1 / 2022 strain as a template, RT-PCR amplification was performed using the CEV detection primer pair (F379 / R667), and the expected target band of approximately 288 bp was obtained ( Figure 1 ), and the target fragment was recovered by gel purification.

[0035] Identification of positive clones The purified target fragment was connected to the pGEM-T vector and transformed. A single colony was picked for expansion culture. When the culture medium became turbid, the plasmid was extracted. The cloned plasmid was identified by PCR using the universal M13 primer (see Figure 2), the positive plasmid was sequenced, and the plasmid with accurate sequencing was named pGEM-T-CEV plasmid for future use.

[0036] 2.1.3 Linearization Use restriction enzymes N Ⅰ The pGEM-T-CEV plasmid was linearized and the same plasmid was used as a control during electrophoresis. The results showed that the recombinant plasmid after linearization showed a clear single band with the expected size, while the recombinant plasmid that was not linearized showed a fuzzy and diffuse band due to its circular shape, which was consistent with the expected experimental results ( Figure 3 ).

[0037] 2.1.4 Calculation of standard plasmid copy number The formula for calculating RNA concentration is: RNA concentration (copies / μL) = 6.02×1023×RNA concentration (ng / μL)×10-9 / (length of in vitro transcribed RNA)×340. N The total length of the Ⅰ restriction site is 385 bp, so the total length of the transcription product is 385 bp, and the copy number of the final standard is 2.3×10 10 copies / μL.

[0038] Determination of reaction system and conditions After optimization (see Table 5 and Table 6), the optimal amplification system for CEV real-time fluorescence quantitative RT-PCR detection method was determined to be: buffer 2×One Step RT-PCR Buffer Ⅲ 10μL, Ex Taq HS 0.4 μL, PrimeScript RTEnzyme Mix Ⅱ 0.4 ΜL, probe 0.8 μL, Rox Ⅱ 0.4μL, upstream and downstream primers 0.4 μL each, RNA template 2 μL, RNase Free H2O 5.2μL; the optimal amplification program was: 42℃ 5 min, 95℃ 10 s, another 95℃ 5 s, 58℃34s for a total of 42 cycles.

[0039] Specificity test First, the nucleic acid samples of FMDV, PPRV, BVDV, CPV, and OrfV were amplified by PCR using the corresponding specific primers, and then CEV RT-qPCR was performed simultaneously with the standard. The PCR products were electrophoresed by gel electrophoresis to analyze the specificity of the method. The results showed that the selection of common viruses on sheep was accurate, and the results are shown in Figure 4; CEVRT-qPCR does not react with the above common viruses, indicating that this method has high specificity and can be used for laboratory diagnosis of CEV. The results are shown in Figure 5 .

[0040] Sensitivity test With a concentration gradient of 2.3×10 7 ~2.3×10 0 The standard dilution of 1000 copies / μL was used as the template, and ddH2O was used as the negative control. The results showed that the lowest detection limit of the established CEV real-time fluorescence quantitative RT-PCR detection method was 23 copies / mL. Figure 6 .

[0041] Establishment of standard curve The standard was diluted 10-fold to 2.3 × 10 2 ~2.3×10 6 The amplification standard curve was obtained by using the established CEV real-time fluorescence quantitative RT-PCR detection method. The results showed that the template concentration was 2.3×10 2 ~2.3×10 6 There is a good linear relationship between copies / μL and Ct value (see Figure 7 ), derive the standard curve R 2 =0.0.995. (See Figure 8 ) 2.5 Repeatability test The results of the repeatability test showed that the intra-group coefficient of variation CV was in the range of 0.34% to 0.7%, and the inter-group CV was in the range of 1.75% to 2.27% (see Table 7). In short, the CV was small.

[0042] Test result determination The Ct value of 34 was used as the cutoff point. When the sample Ct value was <34, it was judged as positive; when the sample Ct value was ≥34, it was judged as negative.

[0043] Field sample test results A total of 598 field samples were tested, with an overall CEV positive rate of 13.88%. The CEV positive rates in different regions ranged from 1.79% to 35.19%. The detailed results are shown in Table 8. Nine positive nucleic acids were randomly selected for further verification using ordinary RT-PCR, and the results were consistent (see Fig. 9 ).

[0044] 2.7 Kit Preparation The primers and probes used in the kit were commissioned to be synthesized by a professional biological gene synthesis company, and then diluted to a concentration of 10mM for use. The probes were stored in brown tubes and light-proof packaging boxes. Other reaction reagents were purchased from Nanjing Novozymes Biotech Co., Ltd. and quantitatively packaged and labeled under sterile conditions. Each kit detects 20 samples and is stored below -20°C. The kit components are shown in Table 9: Table 9 Kit components

Claims

1. A TaqMan real-time fluorescence quantitative RT-PCR detection primer and probe for ovine enterovirus, characterized in that: The nucleotide sequences of the primers and probes are as follows: Upstream primer CEV-qF: 5'-TCCTCCGGRGCCGTGAATGC-3' Downstream primer CEV-qR: 5'-CCGAAAGTAGTCTGTTCCGC-3' Probe CEV-P: 5′-Cy5-AACCTCCGAGCGTGTGCGCA-BHQ2-3′.

2. Use of the primers and probes as claimed in claim 1 in the preparation of TaqMan real-time fluorescence quantitative RT-PCR detection reagents or detection kits for ovine enterovirus.

3. A TaqMan real-time fluorescence quantitative RT-PCR detection kit for ovine enterovirus, characterized in that: The kit comprises the primers and probes according to claim 1.

4. A TaqMan real-time fluorescence quantitative RT-PCR detection method for ovine enterovirus based on the primers and probes of claim 1, comprising the following steps: (1) Extract nucleic acid from the sample to be tested, and use the above primers and probes to perform TaqMan real-time fluorescence quantitative RT-PCR detection on sheep enterovirus. The PCR amplification system for detection is: the total reaction system is 20 μL, including buffer 2×One Step RT-PCRBuffer Ⅲ 10 μL, Ex Taq HS 0.4 μL, PrimeScript RT Enzyme Mix Ⅱ 0.4 mL, probe 0.8 μL, Rox Ⅱ 0.4 μL, upstream and downstream primers 0.4 μL each, RNA template 2 μL, RNase Free H2O 5.2 μL; the amplification program is to use a two-step method for amplification, 42°C 5 min, 95°C 10 s; another 95°C 5 s, 58°C 34 s, a total of 42 cycles; (2) After the reaction is completed, read the Ct value. When the sample Ct value is <34, it is judged as positive; when the sample Ct value is ≥34, it is judged as negative.

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