Dual real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for simultaneously detecting porcine hemagglutination encephalomyelitis virus and porcine teschovirus

Abstract

The invention discloses a dual real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for simultaneously detecting porcine hemagglutinated encephalomyelitis virus (PHEV) and porcine teschovirus (PTV), and belongs to the technical field of animal virus molecular biological detection. Aiming at the problems of similar clinical symptoms, high fatality rate and lack of synchronous detection and identification means after the suckling piglets are infected by PHEV and PTV, a specific primer pair and a probe are designed and screened through sequence comparison based on conserved regions of genomes of the two viruses. The detection method provided by the invention can realize qualitative and quantitative analysis of the two viruses, has the advantages of high sensitivity, high specificity and simplicity and convenience in operation, can quickly identify single infection or mixed infection of the two viruses, and provides an effective technical means for accurate detection in laboratories and epidemiological investigation.

Description

Technical Field

[0001] This invention belongs to the field of animal virus molecular biological detection technology, and relates to a dual real-time quantitative PCR detection method for simultaneously detecting porcine hemagglutinating encephalomyelitis virus and porcine cerebrospinal virus. Background Technology

[0002] Porcine hemagglutinating encephalomyelitis virus (PHEV) was first isolated in Canada in 1962, but research on PHEV remains limited and there is no commercially available vaccine. PHEV belongs to the genus *β-coronavirus* of the family Coronaviridae and is an enveloped, single-stranded, positive-sense RNA virus. The PHEV genome is approximately 30 kb in length, consisting of a 5'-UTR, a large open reading frame (ORF), and a 3'-UTR. It includes the hemagglutinin-esterase (HE) protein, spike protein (S), small membrane (E) protein, membrane (M) protein, and nucleocapsid (N) protein. PHEV can infect birds and many other mammals, but pigs are the only naturally susceptible animal. PHEV can infect pigs of all ages, but typically only piglets under 4 weeks of age show obvious clinical symptoms after infection; adult pigs often remain asymptomatic. PHEV is currently the only known neurotropic coronavirus affecting pigs. In piglets under 4 weeks of age, clinical symptoms typically include vomiting, depression, encephalomyelitis, tremors, ataxia, and other neurological symptoms, with a mortality rate approaching 100%. Current molecular biological detection methods for PHEV include RT-PCR, real-time fluorescent RT-PCR, and loop-mediated isothermal amplification (LAMP), while serological detection methods include hemagglutination inhibition assays, indirect ELISA, and colloidal gold immunochromatography (GICA).

[0003] Porcine Teschovirus (PTV) was first discovered in the Tescho region of Teloskov, Czechoslovakia in 1929. In 2003, the Harbin Veterinary Research Institute in my country first isolated PTV in Inner Mongolia Autonomous Region. PTV belongs to the genus Teschovirus of the family Picornaviridae. It is a non-enveloped, single-stranded, positive-sense RNA virus. The PTV genome is approximately 7.1 kb in length and contains only one large open reading frame (ORF). Its genome structure is 5'-UTR-L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3'-UTR. Existing research indicates that pigs are the most common susceptible animals for PTV, with piglets exhibiting the most severe clinical symptoms after infection. Clinical symptoms in infected piglets typically include fever, lethargy, diarrhea, convulsions, opisthotonus, and neurological symptoms such as poliomyelitis. The mortality rate in suckling piglets is close to 100%.

[0004] Both PHEV and PTV are RNA viruses. Infected piglets exhibit similar clinical symptoms and high mortality rates, causing severe losses to pig farming. Research on whether PHEV and PTV can co-infect is lacking, thus necessitating differential diagnosis between them. Given the current lack of a method for simultaneously detecting, identifying, and quantifying both viruses, a rapid, specific, sensitive method capable of simultaneously detecting, identifying, and quantifying PHEV and PTV is needed for clinical monitoring. Real-time quantitative PCR (qPCR) offers advantages such as high sensitivity, high specificity, and ease of operation. It also eliminates the need for further processing of PCR products required in traditional PCR methods and is now widely used for virus detection and differentiation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention provides the following technical solution:

[0006] In a first aspect, this invention uses the gene sequences of PHEV (OQ798818.1) and PTV (DQ355222.1) in GenBank as references, performs sequence alignment using Oligo 7 software, screens highly conserved regions, and designs specific primer-probe combinations. These specific primer-probe combinations can simultaneously detect porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV). The nucleotide sequences of the forward primer PHEV-F, reverse primer PHEV-R, and probe PHEV-Taq for PHEV are shown in SEQ ID NO:1-3, respectively. The nucleotide sequences of the forward primer PTV-F, reverse primer PTV-R, and probe PTV-Taq for PTV are shown in SEQ ID NO:4-6, respectively.

[0007] Furthermore, the amplification fragment length of the forward primer PHEV-F and the reverse primer PHEV-R is 158 bp; the amplification fragment length of the forward primer PTV-F and the reverse primer PTV-R is 111 bp.

[0008] Furthermore, the PHEV-Taq probe is labeled with the fluorescent reporter group FAM at its 5' end and BHQ1 at its 3' end; the PTV-Taq probe is labeled with the fluorescent reporter group VIC at its 5' end and BHQ2 at its 3' end.

[0009] A second aspect of the present invention provides a product comprising the above-described primer-probe combination for the simultaneous detection of porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV).

[0010] The third aspect of this invention provides the application of the above-mentioned primer-probe combination in the preparation of products that simultaneously detect porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV).

[0011] The fourth aspect of this invention provides a method for simultaneously detecting porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV) without the purpose of disease diagnosis and treatment, characterized by comprising the following steps:

[0012] (1) Sample processing: RNA was extracted from the samples to be tested using a nucleic acid extraction kit;

[0013] (2) Reverse transcription: The RNA of the sample to be tested is reverse transcribed to obtain cDNA;

[0014] (3) Real-time quantitative PCR: Using cDNA products as templates, amplification reaction solutions were prepared using various primers and probes. Real-time quantitative PCR was performed using primer and probe combinations that could simultaneously detect porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV).

[0015] (4) Result detection: The result is determined based on the Ct value corresponding to the amplification curve drawn by the software of the real-time PCR instrument.

[0016] Furthermore, the reaction system for the real-time PCR reaction is as follows: 10 μL of qPCR SuperMix (2×), 2 μL of cDNA, 2.8 μL of primer mixture, 0.8 μL of probe mixture, and ddH2O to bring the total to 20 μL.

[0017] Furthermore, the amplification program for the fluorescence quantitative PCR reaction is as follows: 94℃ pre-denaturation for 30s, 94℃ denaturation for 5s, 60℃ annealing for 30s, for 45 cycles.

[0018] Furthermore, the determination of the detection results is as follows: when the Ct value of the FAM detection channel is ≤35 and a specific amplification curve appears, the result is determined to be positive for porcine hemagglutinating encephalomyelitis virus (PHEV); when the Ct value of the VIC detection channel is ≤35 and a specific amplification curve appears, the result is determined to be positive for porcine cerebrospinal virus (PTV); when the Ct value of the FAM detection channel is >35 and no specific amplification curve appears, the result is determined to be negative for porcine hemagglutinating encephalomyelitis virus (PHEV); when the Ct value of the VIC detection channel is >35 and no specific amplification curve appears, the result is determined to be negative for porcine cerebrospinal virus (PTV). The results are as follows: Porcine hemagglutinating encephalomyelitis virus (PHEV) is negative when the Ct value of the FAM detection channel is >35 and a specific amplification curve appears. If the experiment is repeated and a specific amplification curve still appears, the result is considered positive. If no further specific amplification curve appears, the result is considered negative. Similarly, if the Ct value of the VIC detection channel is >35 and a specific amplification curve appears, the experiment is repeated. If a specific amplification curve still appears, the result is considered positive. If no further specific amplification curve appears, the result is considered negative.

[0019] The beneficial effects of this invention are:

[0020] (1) Achieving precise simultaneous detection and identification: This invention designs specific primers based on highly conserved regions of the genomes of porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV), and combines them with specific probes bearing different fluorescent labels (FAM, VIC), enabling simultaneous detection of both viruses in a single reaction system. Through fluorescence signal difference and Ct value analysis, it is possible to accurately determine whether a sample is infected with PHEV or PTV, distinguish between single or mixed infections, and effectively solve the problem of similar clinical symptoms and difficulty in differential diagnosis after piglets are infected with the two viruses.

[0021] (2) Excellent technical performance:

[0022] High sensitivity: This dual real-time quantitative PCR detection method has a detection limit of 4 gene copies for PHEV and 5 gene copies for PTV, and can detect extremely low concentrations of the virus, making it suitable for the detection of early or latent infections.

[0023] High specificity: It shows no cross-reaction with multiple related viruses such as porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV), and the detection results are accurate and reliable;

[0024] Simple and efficient operation: Based on real-time quantitative PCR technology, there is no need for subsequent processing of PCR products. The reaction procedure is standardized (45 cycles, completed in about 1.5-2 hours). The operation steps are simple and the test results can be obtained quickly. Attached Figure Description

[0025] Figure 1 : Optimal reaction amplification results, amplification curves, and standard curves for PHEV real-time quantitative PCR;

[0026] Figure 2 : Optimal reaction amplification results, amplification curves, and standard curves for PTV real-time quantitative PCR;

[0027] Figure 3 : Optimal reaction detection results, amplification curves, standard curves, and sensitivity detection results for PHEV and PTV dual real-time quantitative PCR;

[0028] Figure 4 Specific detection results of PHEV and PTV in dual real-time quantitative PCR;

[0029] Figure 5 Results of intra- and inter-group repeatability of PHEV and PTV in dual real-time quantitative PCR. Detailed Implementation

[0030] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0031] Example 1 Primer Design and Synthesis

[0032] Based on the partially known sequences of PHEV (GenBank No. OQ798818.1) and PTV (GenBank No. DQ355222.1) published in GenBank, specific primer-probe combinations were designed (Table 1).

[0033] Table 1 Primer and probe nucleotide sequences

[0034]

[0035] Example 2: Establishment of the dual real-time quantitative PCR amplification reaction system and optimization of primer and probe concentrations.

[0036] Includes the following steps:

[0037] (1) Sample processing: Viral RNA was extracted using a nucleic acid extraction kit;

[0038] (2) Reverse transcription: The viral RNA is reverse transcribed to obtain cDNA;

[0039] (3) Real-time quantitative PCR: Using cDNA products as templates, amplification reaction solutions were prepared using various primers and probes, and real-time quantitative PCR was performed; the reaction system of the real-time quantitative PCR reaction was: 10 μL of qPCR SuperMix (2×), 2 μL of cDNA, 2.8 μL of primer mixture, 0.8 μL of probe mixture, and ddH2O to bring the total to 20 μL; the amplification program of the real-time quantitative PCR reaction was: 94℃ pre-denaturation for 30 s, 94℃ denaturation for 5 s, 60℃ annealing for 30 s, for 45 cycles;

[0040] (4) Result detection: The result is determined based on the Ct value corresponding to the amplification curve drawn by the software of the real-time PCR instrument.

[0041] Primer pairs for the two viruses were amplified at different concentrations, with the primer concentration for each virus decreasing sequentially from 0.8 μmol / L, 0.7 μmol / L, 0.6 μmol / L, 0.5 μmol / L, 0.4 μmol / L, 0.3 μmol / L, and 0.20 μmol / L, to obtain the optimal reaction concentration for each primer (Table 2). Similarly, different probe concentrations were selected for amplification reactions, decreasing sequentially from 0.4 μmol / L, 0.3 μmol / L, and 0.2 μmol / L, to obtain the optimal concentration for each probe (Table 3).

[0042] Table 2 Optimal final concentrations of primers for dual real-time quantitative PCR detection method

[0043]

[0044] Table 3 Optimal final concentrations of each probe in the dual real-time quantitative PCR detection method.

[0045]

[0046] Example 3 Sensitivity Detection

[0047] Primers for amplifying porcine hemagglutinating encephalospinal virus (PHEV) and porcine cerebrospinal virus (PCV) were used to amplify corresponding fragments. The amplified fragments from PHEV and PTV were cloned into the pUC57 vector. Positive plasmids were screened through transformation, plasmid extraction, enzyme digestion, and PCR identification. After purification and recovery, the concentration and copy number were calculated. The two constructed positive plasmids were serially diluted 10-fold to obtain the following concentrations: PHEV: 4 × 10⁻⁶ 6 copies / μL, 4×105 copies / μL, 4×10 4 copies / μL, 4×10 3 copies / μL, 4×10 2 copies / μL, 4×10 1 copies / μL, 4copies / μL; PTV: 5×10 6 copies / μL, 5×10 5 copies / μL, 5×10 4 copies / μL, 5×10 3 copies / μL, 5×10 2 copies / μL, 5×10 1 The detection methods described in this application were used for single-template amplification and double-template amplification. The results showed that the sensitivity for detecting PHEV could detect 4 gene copies, and the sensitivity for detecting PTV could detect 5 gene copies. Figures 1-2 The dual-template sensitivity can detect 4 gene copies and 5 gene copies, respectively. Figure 3 The results show that the sensitivity of this detection method is highly consistent and extremely high in both single pathogen and mixed pathogen detection.

[0048] Example 4 Specificity Verification

[0049] PHEV, PTV, and positive plasmid standards were used as positive controls, and sterile distilled water was used as a negative control. The detection method constructed in this application was applied to real-time quantitative PCR reactions of samples containing porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine cytomegalovirus (PCMV), classical swine fever virus (CSFV), and porcine pseudorabies virus (PRV). The results showed that only PHEV and PTV samples exhibited positive amplification curves. Figure 4 The positive result of the positive result indicates that the detection method of the present invention does not cross-react with other viruses and has strong specificity.

[0050] Example 5 Repeatability Verification

[0051] The dual real-time quantitative PCR detection method constructed in this application was used to detect mixed samples of PHEV and PTV. Intra-group repeatability was achieved by performing three replicate assays on the same sample, and inter-group repeatability was achieved by performing three assays at different times. Figure 5 The results showed that the dual real-time quantitative PCR method established in this application has good reproducibility.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the invention should fall within the protection scope defined by the claims.

Claims

1. A primer-probe combination for simultaneous detection of porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV), characterized in that, The nucleotide sequences of the forward primer PHEV-F, reverse primer PHEV-R, and probe PHEV-Taq of the porcine hemagglutinating encephalomyelitis virus (PHEV) are shown in SEQ ID NO:1-3, respectively, and the nucleotide sequences of the forward primer PTV-F, reverse primer PTV-R, and probe PTV-Taq of the porcine cephalovirus (PTV) are shown in SEQ ID NO:4-6, respectively.

2. The primer-probe combination according to claim 1, characterized in that, The amplified fragment length of the forward primer PHEV-F and the reverse primer PHEV-R is 158 bp; the amplified fragment length of the forward primer PTV-F and the reverse primer PTV-R is 111 bp.

3. The primer-probe combination according to claim 1, characterized in that, The PHEV-Taq probe is labeled with the fluorescent reporter group FAM at its 5' end and BHQ1 at its 3' end; the PTV-Taq probe is labeled with the fluorescent reporter group VIC at its 5' end and BHQ2 at its 3' end.

4. A product for simultaneously detecting porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV), characterized in that, Includes the primer-probe combination as described in any one of claims 1-3.

5. The application of the primer-probe combination according to any one of claims 1-3, characterized in that, Prepare a product for the simultaneous detection of porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV).

6. A method for simultaneously detecting porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV) without the purpose of disease diagnosis and treatment, characterized in that, The product described in claim 4 was used for testing.

7. The detection method according to claim 6, characterized in that, Includes the following steps: (1) Sample processing: RNA was extracted from the samples to be tested using a nucleic acid extraction kit; (2) Reverse transcription: The RNA of the sample to be tested is reverse transcribed to obtain cDNA; (3) Real-time quantitative PCR: Using cDNA products as templates, amplification reaction solutions were prepared using various primers and probes. Real-time quantitative PCR was performed using primer and probe combinations that could simultaneously detect porcine hemagglutinating encephalomyelitis virus (PHEV) and porcine cerebrospinal virus (PTV). (4) Result detection: The result is determined based on the Ct value corresponding to the amplification curve drawn by the software of the real-time PCR instrument.

8. The detection method according to claim 7, characterized in that, The reaction system for the quantitative real-time PCR reaction is as follows: 10 μL of qPCR SuperMix (2×), 2 μL of cDNA, 2.8 μL of primer mixture, 0.8 μL of probe mixture, and ddH2O to bring the total to 20 μL.

9. The detection method according to claim 7, characterized in that, The amplification program for the quantitative PCR reaction was as follows: 94℃ pre-denaturation for 30s, 94℃ denaturation for 5s, 60℃ annealing for 30s, for 45 cycles.

10. The detection method according to claim 7, characterized in that, The specific determination of the detection results is as follows: when the Ct value of the FAM detection channel is ≤35 and a specific amplification curve appears, the result is determined to be positive for porcine hemagglutinating encephalomyelitis virus (PHEV); when the Ct value of the VIC detection channel is ≤35 and a specific amplification curve appears, the result is determined to be positive for porcine cerebrospinal virus (PTV); when the Ct value of the FAM detection channel is >35 and no specific amplification curve appears, the result is determined to be negative for porcine hemagglutinating encephalomyelitis virus (PHEV); when the Ct value of the VIC detection channel is >35 and no specific amplification curve appears, the result is determined to be porcine cerebrospinal virus (PTV). The results are as follows: Porcine hemagglutinating encephalomyelitis virus (PHEV) is negative when the Ct value of the FAM detection channel is >35 and a specific amplification curve appears. If the experiment is repeated and a specific amplification curve still appears, the result is considered positive. If no further specific amplification curve appears, the result is considered negative. Similarly, if the Ct value of the VIC detection channel is >35 and a specific amplification curve appears, the experiment is repeated. If a specific amplification curve still appears, the result is considered positive for porcine hemagglutinating encephalomyelitis virus (PHEV). If no further specific amplification curve appears, the result is considered negative.

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