Primer probe combination, kit and method for detecting ENTV-2

Abstract

The invention belongs to the technical field of biology. The invention provides a cdPCR primer probe combination for detecting ENTV-2. The primer probe combination is composed of an upstream primer, a downstream primer and a probe, wherein the sequence of the upstream primer is 5 '-ACCTCTGATTTCCTGTGACACAT-3', and the sequence of the downstream primer is 5 '-ACCTCTGATTGACACAT-3'; the sequence of the downstream primer is 5 '-TTCCTGCACCAGCTGTTGTG-3', and the sequence of the probe is 5 '-fluorescent reporter group-CGATCAGTGCCCTAACAAGAGA-fluorescent quenching group-3'. The method for detecting the ENTV-2, established by the invention, has the advantages that the detection limit can reach 0.541 copies / mu L, the specificity and the repeatability are excellent, the accuracy is excellent, the coincidence rate with the detection result of an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method is 100%, and the application prospect is good.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a primer-probe combination, a kit and a method for detecting ENTV-2. Background Art

[0002] Enzootic nasal adenocarcinoma (ENA) is a chronic, progressive, contagious disease caused by the enzootic nasal tumor virus of goats (ENTV-2). Similar to Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus of sheep (ENTV-1), ENTV-2 belongs to the genus β-retrovirus and is a linear, single-stranded, positive-sense RNA virus with a genome size of approximately 7500 base pairs. Its structure resembles that of eukaryotic mRNA, with a methylated cap (mGpppGmp) at the 5' end and a polyadenylation (polyA) at the 3' end. The genome is flanked by noncoding regions and an overlapping coding region consisting of the gag-pro-pol-env gene. ENTV-2 virions are round, 90-110 nm in diameter, and their outermost layer is the envelope, which is covered with numerous 9 nm-diameter spikes. The tumor tissue and nasal fluid of sick sheep contain a large number of virus particles.

[0003] Researchers have conducted extensive research on the in vitro culture of ENTV-2, but to date, no cell lines or mature, infectious clones capable of stable viral culture have been found. The open reading frame (ORF) of the ENTV-2 env gene begins within the pol gene and encodes a polypeptide precursor approximately 617 amino acid residues long with a predicted molecular weight of 68 ku. The env ORF encodes a polyprotein precursor with two cleavage sites: one protease cleavage site that cleaves the gene into a surface protein (Su) and a transmembrane protein (Tm). The other site is located after the signal peptide sequence. Su contains a site that binds to the surface receptor Hyal2 on target cells.

[0004] Research has shown that env is an oncogene, but it lacks tissue-specific factors for tumor formation. Its tissue tropism is determined by both the 3′-specific element (U3) and env. Clinical manifestations of infection include serous nasal discharge, significant respiratory distress, and emaciation. The proliferation of tumor tissue can cause facial and even cranial deformation, with exophthalmos. The disease has a long incubation period, ranging from several months to 2-4 years in naturally infected animals. Death is the typical outcome, and early clinical diagnosis is difficult, posing a serious threat to the goat industry. The disease was first reported in Germany in 1939 and has since been reported in other countries and regions. Since its initial report in Inner Mongolia in my country in 1995, cases of ENA in goats have been reported in Hunan, Sichuan, Anhui, Shaanxi, Guizhou, Fujian, and Chongqing. Currently, there are no effective medications or vaccines to prevent and treat the disease, resulting in significant economic losses for the goat industry. Therefore, the development of rapid, accurate, and efficient detection methods is crucial for the diagnosis and prevention of this disease.

[0005] As a rapid and sensitive diagnostic method, fluorescent quantitative PCR has been widely used in pathogen detection, molecular diagnosis, molecular biology research, animal and plant quarantine, and food safety testing. It has obvious advantages over traditional RT-PCR detection methods.

[0006] Chip-based digital PCR (cdPCR) is a new absolute quantitative nucleic acid detection technology based on DNA in vitro amplification technology and droplet technology. The key to this technology lies in template dilution. During operation, the sample is evenly distributed to each microwell of the chip to form tens of thousands of different reaction units. Each microwell may or may not contain one or several target DNA molecules. The target molecules in each microwell are amplified separately. Finally, the reader reads the fluorescent markers in the PCR droplets to measure whether there are target bands, and calculates the concentration or copy number of the target molecules through the Poisson distribution method, thereby achieving absolute quantification of the sample. Therefore, the sensitivity of cdPCR technology is twice as accurate as that of ordinary fluorescent quantitative PCR, and it greatly reduces the impact of PCR amplification efficiency.

[0007] So far, all technologies for ENTV-2 detection have had problems with poor detection limits. For example, the method reported in the literature "EvaGreen-based real-time PCR assay for sensitive detection of enzootic nasal tumor virus 2" (DOI: 10.1016 / j.mcp.2019.02.003) has a detection limit of only 55 copies / μL; another example is the method reported in the literature "Establishment of a TaqMan fluorescent quantitative RT-PCR method for detection of endemic intranasal tumor virus in goats" (Acta Animal Husbandry and Veterinary Sinica, 2024, 55(5): 2259-2266.) with a detection limit of only 10.19 copies / μL. Therefore, there is still a certain distance between the existing technologies and the actual application needs of the industry.

[0008] In addition, to the best of the inventor's knowledge, there has been no report on the use of cdPCR technology to detect and identify ENTV-2. Summary of the Invention

[0009] In view of the shortcomings of the prior art, the object of the present invention is to provide a detection method for goat endemic intranasal tumor virus (i.e., ENTV-2) with a detection limit as low as 0.541 copies / μL and a related primer-probe combination.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] A cdPCR primer-probe combination for detecting ENTV-2, the primer-probe combination consisting of an upstream primer, a downstream primer and a probe; wherein,

[0012] The sequence of the upstream primer is: 5'-ACCTCTGATTCTCCTGTGACACAT-3' (see SEQ ID NO. 1);

[0013] The sequence of the downstream primer is: 5'-TTCCTGCACCAGCTGTTGTG-3' (see SEQ ID NO. 2);

[0014] The sequence of the probe is: 5'-fluorescent reporter group-CGATCAGTGGCCCCTAACACAAGAGA-fluorescent quencher group-3'.

[0015] Specifically, the fluorescent reporter group includes a FAM group.

[0016] Specifically, the fluorescence quenching group includes a BHQ1 group.

[0017] A kit for detecting ENTV-2, comprising the aforementioned primer-probe combination.

[0018] Use of the aforementioned primer-probe combination or the aforementioned kit in detecting ENTV-2 for non-disease diagnosis purposes.

[0019] A method for detecting ENTV-2 for non-disease diagnosis purposes, comprising the aforementioned primer-probe combination and conducting a reaction by constructing a cdPCR reaction system.

[0020] Preferably, the cdPCR reaction system is: 2.0 μL of 10×dPCR Mix, 1.0 μL of upstream primer and downstream primer each with a concentration of 10 μmol / L, 0.5 μL of probe primer with a concentration of 10 μmol / L, 2.0 μL of template cDNA, and DEPC water is added to make up to 20.0 μL.

[0021] Preferably, the reaction procedure of the reaction is: 95°C for 10 min, 95°C for 30 s, 60°C for 45 s, for a total of 40 cycles.

[0022] Beneficial effects of the present invention:

[0023] The method for detecting ENTV-2 established by the present invention has a detection limit of up to 0.541 copies / μL; it also has excellent specificity and repeatability; and it also has excellent accuracy, with a 100% coincidence rate with the detection results of the RT-PCR method, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the cdPCR sensitivity test result of the present invention;

[0025] Figure 2 is the cdPCR standard curve of the present invention;

[0026] Figure 3 This is the cdPCR specificity test result of the present invention. DETAILED DESCRIPTION

[0027] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0028] Example 1

[0029] 1.1 Instruments, Equipment and Main Reagents

[0030] The sample preparation instrument, cdPCR PCR amplifier, biochip reader, digital PCR 20K chip kit, and cdPCR 10× premix are all products of Zhenzhun Biotechnology (Shanghai) Co., Ltd.; primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0031] 1.2 Sample Source

[0032] The nucleic acid samples of ENTV-2, foot-and-mouth disease virus (FMDV), goatpox virus (GTPV), orfvirus (ORFV), peste despetits ruminants virus (PPRV), and Mycoplasma caprico-pneumoniae (Mccp) were all stored in the Guizhou Provincial Institute of Animal Husbandry and Veterinary Medicine.

[0033] 1.3 Preparation of Standards

[0034] RNA templates were extracted from ENTV-2-positive samples stored in our laboratory and amplified using primers F1 and R1 using the RT-PCR method. The primer sequences are shown in Table 1. Amplification conditions: 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 68°C for 45 s, 30 cycles; 68°C for 5 min. After the reaction, the PCR product was excised and purified by gel extraction. The recovered product was ligated into the ClonExpress II One Step Cloning Vector pUC-19 (2686 bp) and subsequently transformed into DH5α competent cells. Clonal recombinant strains positive for the ENTV-2 gene fragment were screened. Plasmids were extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and verification. The plasmid mass concentration was determined, the copy number was calculated, and the plasmid was stored in a -80°C freezer for future use.

[0035] 1.4 Detection method construction

[0036] 1.4.1 Primer and probe design and synthesis

[0037] Based on the ENTV-2SWP gene sequence published in GenBank (accession number KU258870.1), a pair of specific primers and a TaqMan probe were designed using PrimerPremier 5 software (Table 1). A FAM fluorescent reporter group was attached to the 5' end of the probe, and a BHQ1 fluorescent quencher group was added to the 3' end. All primers and probes used in the experiment were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0038] Table 1 Sequence information of primers and probes used in the experiment

[0039]

[0040] 1.4.2 Establishment of detection method

[0041] The cdPCR reaction system and protocol were optimized by Zhenzhun Biotechnology (Shanghai) Co., Ltd. The optimal reaction system consisted of 2.0 μL of 10× dPCR Mix, 1.0 μL each of F2 / R2 (10 μmol / L), 0.5 μL of P2 (10 μmol / L), and 2.0 μL of template cDNA, which was then made up to 20.0 μL with DEPC water. The optimal reaction protocol was 95°C for 10 min, followed by 40 cycles of 95°C for 30 s and 60°C for 45 s.

[0042] 1.4.3 Reading and Analysis of Test Results

[0043] The digital chip after PCR amplification is placed in a reader analyzer and the chip is read to obtain the gene copy number per microliter of reaction solution. The nucleic acid copy number concentration in the original sample is then calculated according to the following formula: Sample copy number concentration (copies / μL) = Gene copy number per microliter of reaction solution (copies / μL) × Total reaction volume (μL) / Added template volume (μL).

[0044] 1.5 Sensitivity Verification

[0045] The initial nucleic acid concentration of the prepared ENTV-2 plasmid standard was determined to be 18.6 ng / μL (approximately 5.41×10 9 copies / μL, and the sample was diluted 10-fold into 11 gradients (5.41×10 9 ~5.41×10 -2 copies / μL, and take the last 4 diluted samples (5.41×10 2 ~5.41×10 -2 The minimum detection limit (CLD) of the cdPCR assay was determined using the theoretical copy number of the plasmid standard as the ordinate and the actual copy number as the abscissa to plot a standard curve and verify the linearity of the cdPCR results.

[0046] 1.6 Repeatability Verification

[0047] Three ENTV-2 positive nucleic acid samples with different concentration gradients were selected for simultaneous cdPCR detection, with three replicates for each sample to verify the intra-batch reproducibility of the method. One of the samples was tested by cdPCR three times, with three replicates each time, to verify the inter-batch reproducibility of the method.

[0048] 1.7 Specificity Verification

[0049] cdPCR detection was performed using the nucleic acid samples of sheep FMDV, GTPV, ORFV, PPRV, and Mccp stored in our laboratory to verify the specificity of the method.

[0050] 1.8 Clinical Sample Testing

[0051] Nucleic acid was extracted from 20 samples of dead sheep tissues stored in our laboratory and tested using the cdPCR method established in this study. The results were also compared with those of the RT-PCR method.

[0052] 2 Results and Analysis

[0053] 2.1 Sensitivity test

[0054] ENTV-2 plasmid standard (5.41×10 2 ~10 -2 The cdPCR method established in this study was used to amplify the actual copy number of the amplified product (copies / μL). A standard curve was then constructed to correlate the actual copy number of the amplified product with the copy number of the standard template. The linear relationship was: y = 1.096x - 4.6577, with a correlation coefficient R² = 0.9919. This demonstrated good linearity and high sensitivity of the cdPCR method, with a minimum detection limit of 0.43 copies / μL and an actual detection limit of 0.541 copies / μL.

[0055] 2.2 Specificity test

[0056] cdPCR amplification was performed using nucleic acid samples from ENTV-2, FMDV, GTPV, ORFV, PPRV, and Mccp. The results showed that only the ENTV-2-positive nucleic acid template was amplified positively, while the nucleic acids from other pathogens were negative, indicating the high specificity of this method.

[0057] 2.3 Repeatability test

[0058] Three ENTV-2-positive nucleic acid samples with varying concentrations were amplified as directed. The coefficients of variation for both intra- and inter-batch replicates were less than 13%, demonstrating good reproducibility. The intra- and inter-batch means, variances, and coefficients of variation are shown in Table 2.

[0059] Table 2 cdPCR repeatability test results

[0060]

[0061] 2.4 Clinical sample testing

[0062] The established cdPCR and RT-PCR methods were used to test nucleic acid samples from 20 dead sheep tissues stored in our laboratory. The results showed that the cdPCR results were consistent with the RT-PCR results, with a concordance rate of 100%, with 3 positive samples and 27 negative samples in each case.

[0063] Table 3cdPCR clinical test results

[0064]

Claims

1. A cdPCR primer-probe combination for detecting ENTV-2, characterized in that: The primer-probe combination consists of an upstream primer, a downstream primer and a probe; wherein, The sequence of the upstream primer was: 5′-ACCTCTGATTCTCCTGTGACACAT-3′; The sequence of the downstream primer was: 5′-TTCCTGCACCAGCTGTTGTG-3′; The sequence of the probe is: 5'-fluorescent reporter group-CGATCAGTGGCCCCTAACACAAGAGA-fluorescent quencher group-3'.

2. The cdPCR primer-probe combination for detecting ENTV-2 according to claim 1, characterized in that: The fluorescent reporter group includes a FAM group.

3. The cdPCR primer-probe combination for detecting ENTV-2 according to claim 1, characterized in that: The fluorescence quenching group includes a BHQ1 group.

4. A kit for detecting ENTV-2, characterized in that The kit comprises the primer-probe combination according to any one of claims 1 to 3.

5. Use of the primer-probe combination according to any one of claims 1 to 3 or the kit according to claim 4 in detecting ENTV-2 for non-disease diagnosis purposes.

6. A method for detecting ENTV-2 for non-disease diagnosis purposes, characterized in that: The method comprises using the primer-probe combination according to claims 1 to 3 to conduct a reaction by constructing a cdPCR reaction system.

7. The method according to claim 6, characterized in that The cdPCR reaction system was as follows: 2.0 μL of 10×dPCR Mix, 1.0 μL of upstream primer and downstream primer each at a concentration of 10 μmol / L, 0.5 μL of probe primer at a concentration of 10 μmol / L, and 2.0 μL of template cDNA, which was made up to 20.0 μL with DEPC water.

8. The method according to claim 6 or 7, characterized in that The reaction procedure of the reaction was: 95°C for 10 min, 95°C for 30 s, 60°C for 45 s, for a total of 40 cycles.

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