Primer probe combination, kit and method for synchronously detecting PCV2 and Mhp

Abstract

The invention provides a primer probe combination, a kit and a method for synchronously detecting PCV2 and Mhp, and belongs to the technical field of animal epidemic disease molecular diagnosis. According to the technical scheme provided by the invention, the porcine circovirus type 2 and the mycoplasma hyopneumoniae can be simultaneously, rapidly and accurately detected in one reaction system, the operation steps are remarkably simplified, the detection efficiency is improved, and the detection cost is reduced. The method is high in sensitivity, and infection can be found early; the specificity is strong, the cross reaction is effectively avoided, and the result is reliable. The matched kit is convenient for standardized production and clinical popularization, provides an efficient and convenient technical tool for mixed infection monitoring of the two important pathogens in a pig farm and early diagnosis and precise prevention and control of epidemic situations, and has good application prospects and market values.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostic technology for animal diseases, and in particular to a primer-probe combination, kit, and method for simultaneous detection of PCV2 and Mhp. Background Technology

[0002] Porcine circovirus type 2 (PCV2) is the main pathogen causing porcine circovirus-associated disease (PCVAD), which can lead to various clinical manifestations such as multisystemic wasting syndrome in weaned piglets and porcine dermatitis-nephropathy syndrome, causing growth retardation, immunosuppression, and even death, resulting in significant economic losses to the global pig industry. Mycoplasma hyopneumoniae (Mhp) is the main pathogen of swine endemic pneumonia (SEP), characterized by chronic dry cough, decreased growth performance, and susceptibility to secondary bacterial or viral infections.

[0003] In large-scale pig farming practices, co-infection of PCV2 and Mhp is very common. Both can attack the respiratory system of pigs, and their synergistic effect exacerbates the severity of porcine respiratory disease syndrome (PRDC), leading to more complex clinical symptoms, higher mortality rates, and more difficult-to-control outbreaks, becoming one of the key factors restricting the health of pig herds and production efficiency.

[0004] Existing detection methods include pathogen isolation, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR). Among these, nucleic acid-based PCR technology is widely used due to its high sensitivity and specificity. However, traditional detection methods typically target a single pathogen. To determine whether there is a mixed infection, two independent testing procedures are required for the same sample. This approach not only increases sample and reagent consumption and prolongs testing time but also increases operational complexity and labor costs, proving inefficient for large-scale sample screening or rapid disease diagnosis. Therefore, the industry urgently needs to develop detection technologies that can simultaneously and efficiently identify the infection status of PCV2 and Mhp to meet the pressing needs of modern aquaculture for rapid disease diagnosis and precise prevention and control. Summary of the Invention

[0005] The purpose of this invention is to provide a primer and probe combination, kit, and method for simultaneous detection of PCV2 and Mhp, which solves the technical problems of existing technologies that require separate detection of porcine circovirus type 2 (PCV2) and porcine mycoplasma pneumoniae (Mhp), which are cumbersome, inefficient, and difficult to effectively deal with common clinical mixed infections.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for simultaneous detection of porcine circovirus type 2 and Mycoplasma hyopneumoniae. In the same reaction system, a primer and probe set targeting the ORF1 gene of porcine circovirus type 2 and a primer and probe set targeting the LepA gene of Mycoplasma hyopneumoniae are used to perform real-time PCR detection on the sample to be tested.

[0008] The primer and probe set targeting the porcine circovirus type 2 ORF1 gene includes an upstream primer with nucleotide sequences as shown in SEQ ID NO. 1, a downstream primer as shown in SEQ ID NO. 2, and a probe as shown in SEQ ID NO. 3;

[0009] The primer and probe set targeting the LepA gene of Mycoplasma hyopneumoniae includes an upstream primer with nucleotide sequences as shown in SEQ ID NO.4, a downstream primer with nucleotide sequences as shown in SEQ ID NO.5, and a probe with SEQ ID NO.6.

[0010] Preferably, the probe shown in SEQ ID NO.3 is labeled with a FAM fluorescent reporter group.

[0011] Preferably, the probe shown in SEQ ID NO.6 is labeled with a HEX fluorescent reporter group.

[0012] Preferably, the reaction procedure for the real-time PCR detection includes a pre-denaturation step and an amplification step of 35 to 50 cycles;

[0013] The pre-denaturation step is performed at 90 to 98 degrees Celsius for 30 to 90 seconds.

[0014] Each amplification cycle includes a denaturation step of 5 to 20 seconds at 90 to 98 degrees Celsius, and an annealing extension step of 30 to 60 seconds at 55 to 62 degrees Celsius.

[0015] Preferably, the method for simultaneous detection of porcine circovirus type 2 and porcine mycoplasma pneumoniae further includes a result determination step. When the Ct value of the fluorescence channel corresponding to the detection of porcine circovirus type 2 is less than or equal to 40, the sample is determined to be positive for porcine circovirus type 2. When the Ct value of the fluorescence channel corresponding to the detection of porcine mycoplasma pneumoniae is less than or equal to 40, the sample is determined to be positive for porcine mycoplasma pneumoniae.

[0016] The present invention also provides an application of a primer-probe combination in the simultaneous detection of porcine circovirus type 2 and Mycoplasma hyopneumoniae, wherein the primer-probe combination comprises a first primer pair and a first probe that specifically amplifies the target sequence of the ORF1 gene of porcine circovirus type 2, and a second primer pair and a second probe that specifically amplifies the target sequence of the LepA gene of Mycoplasma hyopneumoniae.

[0017] The nucleotide sequences of the first primer pair and the first probe are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and the nucleotide sequences of the second primer pair and the second probe are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0018] The present invention also provides an application of a kit for the simultaneous detection of porcine circovirus type 2 and porcine mycoplasma pneumoniae, comprising the above-mentioned primer-probe combination.

[0019] Preferably, the kit further comprises DNA polymerase, deoxyribonucleoside triphosphate, and reaction buffer.

[0020] Preferably, the kit further includes a positive control containing a porcine circovirus type 2 ORF1 gene fragment and a porcine mycoplasma pneumoniae LepA gene fragment.

[0021] The present invention also provides the application of a primer-probe combination in the preparation of a detection reagent for diagnosing or screening mixed infection of porcine circovirus type 2 and Mycoplasma hyopneumoniae, wherein the primer-probe combination comprises a first primer pair and a first probe that specifically amplifies the target sequence of the ORF1 gene of porcine circovirus type 2, and a second primer pair and a second probe that specifically amplifies the target sequence of the LepA gene of Mycoplasma hyopneumoniae.

[0022] The nucleotide sequences of the first primer pair and the first probe are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and the nucleotide sequences of the second primer pair and the second probe are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0023] The beneficial effects of this invention are:

[0024] The technical solution provided by this invention enables the simultaneous, rapid, and accurate detection of porcine circovirus type 2 and Mycoplasma hyopneumoniae in a single reaction system, significantly simplifying the operation steps, improving detection efficiency, and reducing detection costs. This method boasts high sensitivity, enabling early detection of infection; it also exhibits strong specificity, effectively avoiding cross-reactions and ensuring reliable results. Its accompanying reagent kit facilitates standardized production and clinical application, providing an efficient and convenient technical tool for monitoring mixed infections of these two important pathogens in pig farms, as well as for early diagnosis and precise control of outbreaks. It has promising application prospects and market value. Attached Figure Description

[0025] Figure 1 Construct a graph of the results for the standard curve;

[0026] Figure 2 This is a graph showing the results of specific detection.

[0027] Figure 3 The graphs show the qPCR amplification curves for PCV2 and Mhp, where: Channel 1 - PCV2 and Channel 2 - Mhp, 1-8:10. 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 9 copies / μL; negative control group. Detailed Implementation

[0028] This invention provides a method for the simultaneous detection of Porcine Circovirus Type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp). The core of this method lies in the use of a carefully designed primer and probe system to simultaneously amplify and detect the specific nucleic acid sequences of both pathogens within the same quantitative PCR (qPCR) reaction system, thereby enabling the identification of mixed infections in a single step. This "dual" or "multiplex" qPCR strategy is a commonly used technical approach in modern molecular diagnostics to improve throughput, save samples and reagents, and enhance detection efficiency.

[0029] Specifically, the method involves using a primer and probe set targeting the porcine circovirus type 2 (ORF1) gene and a primer and probe set targeting the porcine mycoplasma pneumoniae (LepA) gene simultaneously in the same reaction system to perform quantitative real-time PCR detection and signal acquisition on the nucleic acid extract (usually a DNA template) of the sample to be tested. In real-time quantitative PCR, the reaction system typically includes thermostable DNA polymerase, dNTPs, primers, probes, buffer, and template. The term "same reaction system" means that all primers, probes, and other necessary reaction components targeting both targets are pre-mixed or simultaneously added to a separate reaction unit (such as a PCR tube or plate well), and then the same thermal cycling program is run on the same quantitative PCR instrument, collecting signals from different fluorescence channels at specific stages of each cycle.

[0030] The primer-probe set targeting the ORF1 gene of porcine circovirus type 2 (PCV2) comprises a set of oligonucleotides capable of specifically recognizing and binding to a specific sequence in the ORF1 gene region of the PCV2 genome. Specifically, this set includes: an upstream primer (or forward primer) with the nucleotide sequence shown in SEQ ID NO. 1, a downstream primer (or reverse primer) with the nucleotide sequence shown in SEQ ID NO. 2, and a probe with the nucleotide sequence shown in SEQ ID NO. 3. The upstream and downstream primers define the target region of the ORF1 gene to be amplified and enable exponential replication of this fragment under the action of DNA polymerase. The probe is used to generate a detectable fluorescent signal during amplification. The principle typically employs probe hydrolysis technology such as TaqMan. The probe is labeled with a fluorescent reporter group and a quencher group at both ends. When the probe binds to the amplification product and is hydrolyzed by the 5'→3' exonuclease activity of DNA polymerase, the reporter group and quencher group separate, thereby generating a fluorescent signal. The design of primers and probes follows conventional principles of molecular biology, ensuring specificity (no significant homology with non-target sequences), appropriate Tm values ​​(melting temperature), and avoiding the formation of primer dimers or secondary structures. The sequences shown in SEQ ID NO.1 to NO.3 were obtained through optimized screening and can efficiently and specifically target the PCV2 ORF1 gene.

[0031] Similarly, the primer-probe set targeting the LepA gene of Mycoplasma hyopneumoniae comprises a set of oligonucleotides capable of specifically recognizing and binding to a specific sequence in the LepA gene region (a gene encoding a ribosome-associated protein, often used as a detection target) of the Mycoplasma hyopneumoniae genome. Specifically, this set includes: an upstream primer with the nucleotide sequence shown in SEQ ID NO.4, a downstream primer with the nucleotide sequence shown in SEQ ID NO.5, and a probe with the nucleotide sequence shown in SEQ ID NO.6. Its mechanism of action is similar to that of the PCV2 primer-probe set described above, used to specifically amplify the LepA gene fragment and generate a corresponding detection signal. The sequences shown in SEQ ID NO.4 to NO.6 have been optimized and screened to efficiently and specifically target the Mycoplasma hyopneumoniae LepA gene.

[0032] In a preferred embodiment, to distinguish signals from two pathogens in the same reaction, two probes need to be labeled with fluorescent reporter groups with different spectral characteristics. This is a standard technique for multiplex qPCR detection. Specifically, the probe shown in SEQ ID NO.3 (for PCV2) is preferably labeled with a FAM fluorescent reporter group. FAM (6-carboxyfluorescein) is a commonly used green fluorescent dye with a maximum excitation wavelength of approximately 494 nm and a maximum emission wavelength of approximately 518 nm. The probe shown in SEQ ID NO.6 (for Mhp) is preferably labeled with a HEX fluorescent reporter group. HEX (hexachloro-6-carboxyfluorescein) is another commonly used fluorescent dye with a fluorescence spectrum that differs somewhat from FAM, typically having a maximum emission wavelength of around 556 nm, facilitating differentiation from the FAM channel on a quantitative PCR instrument equipped with appropriate filters. Both probes are typically labeled with suitable quenching groups, such as BHQ1 (black hole quencher 1), to effectively quench the fluorescence of the reporter group when the probes are intact.

[0033] The reaction procedure for quantitative real-time PCR detection is a crucial step driving nucleic acid amplification and signal acquisition, typically consisting of a series of precisely temperature-controlled cycles. In the method of this invention, the reaction procedure mainly includes a pre-denaturation step followed by 35 to 50 cycles of amplification. The purpose of the pre-denaturation step is to completely unwind the double-stranded DNA template and potentially activate a hot-start DNA polymerase. This step is typically performed at a relatively high temperature, for example, in the range of 90 to 98 degrees Celsius, for 30 to 90 seconds. Each subsequent amplification cycle typically includes two main phases: a denaturation step and an annealing / extension step. The denaturation step aims to unwind the newly formed double-stranded PCR product and template DNA back into single strands, preparing for the next round of primer binding. This step is typically performed at a temperature of 90 to 98 degrees Celsius for 5 to 20 seconds (this time can be shorter as the instrument's heating and cooling rates increase). In the annealing and extension step, the temperature is lowered to a level where the primers can specifically bind to the template target sequence (annealing). Simultaneously, DNA polymerase uses the primer-bound single strand as a template to catalyze the synthesis of a new complementary strand (extension). For many standard qPCR processes using Taq polymerase, annealing and extension can be performed at the same temperature, typically between 55°C and 62°C for 30 to 60 seconds. The number of cycles (35 to 50 cycles) should be set to strike a balance between ensuring sufficient detection sensitivity and avoiding excessive accumulation of nonspecific background signals.

[0034] The synchronous detection method also includes a step of determining the results of quantitative real-time PCR (qPCR). The determination is primarily based on the Ct value (Threshold Cycle) of each fluorescence channel. The Ct value refers to the number of cycles required for the fluorescence signal to reach a set threshold; it is inversely proportional to the logarithm of the initial template amount and is the basis for qPCR quantification. In this invention, the result determination step is as follows: when the Ct value of the fluorescence channel corresponding to porcine circovirus type 2 detection (i.e., the channel detecting the FAM signal) is less than or equal to 40, the sample is determined to be positive for porcine circovirus type 2 nucleic acid, suggesting possible PCV2 infection; when the Ct value of the fluorescence channel corresponding to porcine mycoplasma pneumoniae detection (i.e., the channel detecting the HEX signal) is less than or equal to 40, the sample is determined to be positive for porcine mycoplasma pneumoniae nucleic acid, suggesting possible Mhp infection. If the Ct value is greater than 40 or there is no typical amplification curve (i.e., no Ct value), it is usually determined to be negative for the corresponding pathogen. This threshold (Ct≤40) is a commonly used preliminary positive cut-off value in the field of molecular diagnostics. In practical applications, it can be fine-tuned according to laboratory validation and should be combined with the results of negative and positive controls for comprehensive judgment.

[0035] This invention also provides the application of a specific primer-probe combination in the simultaneous detection of porcine circovirus type 2 and Mycoplasma hyopneumoniae. The term "primer-probe combination" is a collective concept, referring to all primers and probes containing the aforementioned specific sequences. Specifically, the combination includes a first primer pair and a first probe for specifically amplifying the ORF1 gene target sequence of porcine circovirus type 2, and a second primer pair and a second probe for specifically amplifying the LepA gene target sequence of Mycoplasma hyopneumoniae. The first primer pair consists of the nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the first probe is the nucleotide sequence shown in SEQ ID NO. 3; the second primer pair consists of the nucleotide sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5, and the second probe is the nucleotide sequence shown in SEQ ID NO. 6. The application scenario for this composition is to prepare detection reagents and construct detection systems to perform the aforementioned simultaneous detection method.

[0036] This invention further provides an application of a kit for the simultaneous detection of porcine circovirus type 2 and Mycoplasma hyopneumoniae. The kit contains the aforementioned primer-probe combination. In practical product form, this kit is a user-friendly package, typically pre-aliquoting and combining the core or all components required for the detection. In addition to the core primer-probe combination, the preferred kit also contains other necessary biochemical reagents to complete a full quantitative PCR assay. For example, the kit preferably also contains DNA polymerase (such as a thermostable Taq DNA polymerase, possibly a hot-start type to improve specificity), deoxyribonucleoside triphosphates (dNTPs, including dATP, dTTP, dCTP, and dGTP, which are the raw materials for synthesizing new DNA strands), and reaction buffer (to provide a suitable pH, ionic strength, and other environment for the enzymatic reaction). These are conventionally essential components for PCR amplification.

[0037] To ensure quality control and accurate result interpretation during the testing process, the preferred kit also includes a positive control. The positive control is a sample known to contain the target nucleic acid sequence, used to verify that the entire testing system is functioning correctly. In this invention, the positive control preferably contains a fragment of the porcine circovirus type 2 ORF1 gene and a fragment of the Mycoplasma hyopneumoniae LepA gene. These fragments can be specific DNA fragments cloned into a plasmid vector, in vitro synthesized oligonucleotides, or validated pathogen genomic DNA, etc. During the test, the positive control should produce the expected positive signal (Ct value within a specific range); otherwise, it indicates a potential problem with the test.

[0038] Furthermore, this invention also provides the application of the primer-probe combination in the preparation of a detection reagent for diagnosing or screening mixed infections of porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae. Here, "detection reagent" is a broader concept, referring to any chemical or biological product containing the primer-probe combination for performing a detection purpose. Its preparation includes, but is not limited to: aliquoting the primers and probes into dry powder or solution form, combining them with a PCR reaction mix (containing enzymes, dNTPs, buffers, etc.) to form a single reagent or premixed reagent, or integrating them into more complex detection devices (such as microfluidic chips). The "diagnostic" or "screening" application clarifies that the ultimate purpose of this technical solution is directed towards clinical or surveillance practices for animal diseases, aiming to help veterinarians or breeders determine whether pigs have single or mixed infections of PCV2 and Mycoplasma hyopneumoniae, providing laboratory evidence for disease control decisions.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] This embodiment provides a method for detecting Mycoplasma hyopneumoniae, the steps of which are as follows:

[0042] 1. Materials and Methods

[0043] 1.1 Main Instruments and Materials

[0044] Viral genomic DNA / RNA extraction kit was purchased from Aisjin Biotechnology (Hangzhou) Co., Ltd.; Taq DNA enzyme, TB Green Premix Ex Taq enzyme, ROX fluorescent dye, RNase-free water, PUC57 vector, gel extraction kit, E. coli DH5α competent cells, and other reagents were purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; column-based plasmid DNA mini-extraction kit was purchased from Tiangen Biotech Co., Ltd.; DL 2000 DNA Marker was purchased from Beijing Qingke Biotechnology Co., Ltd.; agarose was purchased from BioFROXX GmbH, Germany; and the Applied Biosystems QuantStudio 5 real-time quantitative PCR instrument was purchased from Thermo Fisher Scientific.

[0045] 1.2 Primer Design

[0046] Highly conserved regions were screened based on the PCV2 whole genome sequences already registered in GenBank (GenBank accession numbers: KT719404, KX814348, HM038017, MT543004, HQ378160, EF565346, KX828222, KP231116, MH920569, MT814850). A set of probe primers targeting the ORF1 gene was designed, with a fragment size of 104 bp. The genome sequences of Mycoplasma hyopneumoniae (GenBank accession numbers: MG813457.1, MG813446.1, AE017332.1, CP007229.1, CP079200.1) were also used. Highly conserved sequences (GU644440.1) were screened, and a set of 120bp probes and primers targeting the LepA gene was designed and sent to Shanghai Sangon Biotech for synthesis. The primers and probes were stored at -20℃. See Table 1 below:

[0047] Table 1 Primer and probe sequences

[0048]

[0049] 1.3 Establishment of Standards

[0050] Positive plasmids were constructed based on the PCV2 CAP and Mhp LepA sequences from NCBI. These plasmids were synthesized by Beijing Qingke Biotechnology Co., Ltd. The purified PCR products were ligated into the PUC57 vector to construct recombinant plasmids. The copy number of the PCV2 recombinant plasmid was 2.04 × 10⁻⁶. 9 The copy number of the Mhp recombinant plasmid was 1.86 × 10^10 copies / μL. 9 After calculation, the copy number of the PCV2 recombinant plasmid was first diluted to 1 × 10⁻⁶ copies / μL. 9 copies / μL, Mhp recombinant plasmid copy number diluted to 1×10 9 copies / μL; then the recombinant plasmids diluted with PCV2 and Mhp were diluted 10-fold to obtain copy numbers of 1×10⁻⁶. 9 ~1×10 0 Recombinant plasmids of PCV2 and Mhp at 10 dilutions (copies / μL, etc.) were used as standard templates.

[0051] 1.4 Preparation of mixed template DNA of PCV2 and Mhp:

[0052] Take 10 μL of PCV2 recombinant plasmid with a copy number of 1×10⁹ copies / μL, and 10 μL of plasmid with a copy number of 1×10⁹ copies / μL. 9 Mixing Mhp recombinant plasmids in copies / μL yields 1×10 9 copies / μL of mixed template DNA; 1×10 copies / μL of this mixture were then used to prepare 1×10 copies / μL of the mixed template DNA. 8 copies / μL ~1×10 0 Mixed template DNA copies / μL;

[0053] 1.5 Optimization of reaction conditions

[0054] The recombinant plasmids PUC57-PCV2-ORF1 and PUC57-Mhp-LepA were used as templates. Takara Premix Ex Taq™ (Probe qPCR) reagents were used: 12.5 μL TaqMan Fast Advanced Master Mix, 0.4 μL ROX Reference Dye II, and PCV2 and Mhp were optimized by varying the annealing temperatures (56℃, 57℃, 58℃, 59℃, 60℃, 61℃) and primer and probe concentrations at ratios of 1:1:2, 1:1:1.5, 1:1:1, 1:1:0.5, and 1:1:0.25. RNase-free ddH2O was added to bring the total volume to 25 μL. The reaction conditions were: 95℃ pre-denaturation for 60 s; 95℃ denaturation for 15 s, 45 cycles, with fluorescence signals collected simultaneously. Amplification was performed using a real-time quantitative PCR instrument.

[0055] Example 2

[0056] Establishment of standard curve

[0057] The PCR amplification reaction system was prepared using the specific primer combination screened in Example 1, with a copy number of 1×10⁻⁶ in Example 1. 7 copies / μL ~1×10 0 A mixed template of PCV2 and Mhp (copies / μL) and a blank control (using sterile RNase-free water as the test sample) were used as template DNA for TaqMan multiplex real-time quantitative PCR, and standard curves were plotted (the results are shown in the figure, where the horizontal axis represents the copy number logarithm, and the vertical axis represents the Ct value; red indicates the standard curve for PCV2; blue indicates the standard curve for Mhp). The specific steps are as follows:

[0058] The reaction system for Taqman multiplex real-time fluorescent PCR amplification is as follows: 12.5 μL of premix Ex Taq, 5 μL of template DNA, 0.4 μL of 10 μM primer PCV2-F, 0.4 μL of 10 μM primer PCV2-R, 0.4 μL of 10 μM primer Mhp-F, 0.4 μL of 10 μM primer Mhp-R, 0.8 μL of 10 μM primer PCV2-Probe, 0.8 μL of 10 μM primer Mhp-Probe, and RNase-free water to a final volume of 25 μL.

[0059] The reaction conditions for Taqman multiplex real-time fluorescence PCR amplification are: 95℃ pre-denaturation for 60s, 95℃ denaturation for 15s, 59℃ annealing for 34s, for 45 cycles.

[0060] As shown in the figure, the Ct values ​​obtained from each sample exhibit a good linear relationship with the logarithm of the plasmid standard copy number. The PCV2 standard curve equation is y = -3.63x + 42.92, with a correlation coefficient R² of 0.997; the PPV standard curve equation is y = -3.17x + 38.75, with a correlation coefficient R² of 0.998.

[0061] Example 3

[0062] Establishment of sensitivity curves

[0063] 1) Establishment of PCV2 sensitivity curve

[0064] With a diluted concentration of 1×10 8 copies / μL ~1×10 0 PCV2 recombinant plasmid (copies / μL) was used as template DNA for Taqman multiplex real-time quantitative PCR and conventional PCR. An RNase-free water blank control was also performed. Each PCR reaction and blank control was repeated in triplicate. A sensitivity curve for PCV2 detection was constructed. The Taqman multiplex real-time quantitative PCR results are shown below. Figure 3 ( Figure 3 In the diagram, 1 through 8 represent a copy number of 1 × 10⁻⁸. 1 ~1×10 8 (Curve of PCV2 recombinant plasmid amplification with Copies / μL).

[0065] Primer and probe information is as shown in Example 1;

[0066] The reaction system for Taqman multiplex real-time fluorescent PCR amplification includes: 12.5 μL of premix Ex Taq, 5 μL of template DNA, 0.4 μL of 10 μM primer PCV2-F, 0.4 μL of 10 μM primer PCV2-R, 0.8 μL of 10 μM primer PCV2-Probe, and RNase-free water to a final volume of 25 μL.

[0067] The Taqman multiplex real-time fluorescence PCR amplification reaction program is as follows: 95℃ pre-denaturation for 60s, 95℃ denaturation for 15s, 59℃ annealing for 34s, for 45 cycles.

[0068] Depend on Figure 3 It can be seen that the limit of detection for PCV2 using TaqMan for singlet real-time quantitative PCR is 1×10⁻⁶. 1 Copies / μL. Therefore, the method provided by this invention has higher detection sensitivity.

[0069] 2) Establishment of Mhp sensitivity curve

[0070] With a diluted concentration of 1×10 8 copies / μL ~1×10 0 Using Mhp recombinant plasmid (copies / μL) as template DNA, Taqman multiplex real-time quantitative PCR and conventional PCR were performed. A blank control was set up using RNase-free water. Each PCR reaction and blank control was performed in triplicate. A sensitivity curve for detecting Mhp was constructed. The Taqman multiplex real-time quantitative PCR amplification results are shown below. Figure 3 ( Figure 3 In the diagram, 1 through 8 represent a copy number of 1 × 10⁻⁸. 1 ~1×10 8 (Curve after amplification of Mhp recombinant plasmid with Copies / μL).

[0071] Primer and probe information is as shown in Example 1;

[0072] The reaction system for Taqman multiplex real-time fluorescent PCR amplification includes: 12.5 μL of premix Ex Taq, 5 μL of template DNA, 0.4 μL of 10 μM primer Mhp-F, 0.4 μL of 10 μM primer Mhp-R, 0.8 μL of 10 μM primer Mhp-Probe, and RNase-free water to a final volume of 25 μL.

[0073] The Taqman multiplex real-time fluorescence PCR amplification reaction program is as follows: 95℃ pre-denaturation for 60s, 95℃ denaturation for 15s, 59℃ annealing for 34s, for 45 cycles.

[0074] Depend on Figure 3 It can be seen that when using TaqMan for singlet real-time quantitative PCR, the limit of detection for Mhp is 1×10⁻⁶. 0 Copies / μL. Therefore, the method provided by this invention has higher detection sensitivity.

[0075] Example 4

[0076] Specific experiments

[0077] A Taqman multiplex real-time quantitative PCR reaction system was prepared using the primers and probes shown in Example 1. Porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus DNA, TGEV, PEDV, PRV, PPV, PCV3, and rotavirus (RV) were used as template DNA. A positive result was set up (the positive result consisted of a mixed template DNA of PVC2 and Mhp prepared in Example 1, 1×10⁻⁶). 6 Specificity analysis was performed using copies / μL and a negative control.

[0078] The system and conditions for Taqman multiplex real-time quantitative PCR amplification are the same as those in step 4) of Example 3, which establishes sensitivity curves for simultaneous detection of PVC2 and Mhp. Judgment criteria: When the Ct value of the quantitative PCR amplification curve for porcine circovirus PCV2 primers and probes is <40, the sample contains PCV3; when the Ct value of the quantitative PCR amplification curve for porcine mycoplasma pneumoniae Mhp primers and probes is <40, the sample contains Mhp; when there is no quantitative PCR amplification curve or the Ct value of the quantitative PCR amplification curve is ≥40, the sample is considered negative. Results are shown in [link to results]. Figure 2 .

[0079] Depend on Figure 2 As can be seen, the positive control showed a typical amplification curve, while the negative control showed no amplification signal, confirming the validity of the experiment. In this experiment, no amplification signals were observed for PRRSV, TGEV, PEDV, PRV, PPV, PCV3, and rotavirus (RV). Therefore, the method provided by this invention exhibits good specificity and no cross-reactivity when simultaneously detecting PVC3, PVC2, and PPV.

[0080] Example 5

[0081] Repeatable experiments

[0082] A Taqman multiplex real-time quantitative PCR reaction system was prepared using the primers and probes shown in Example 1. The copy number of 1×10⁻⁶ was selected from the samples in Example 1. 6 copies / μL ~1×10 2 The mixed template DNA of PVC2 and Mhp was sampled in copies / μL and subjected to three repeated Taqman multiplex real-time quantitative PCR tests (the system and conditions for Taqman multiplex real-time quantitative PCR amplification are the same as step 4 in Example 3). The sensitivity curves for simultaneous detection of PVC2 and Mhp were established in the same way, and the coefficient of variation between groups was calculated. The same Taqman multiplex real-time quantitative PCR test was repeated three times, and the coefficient of variation within groups was calculated. The results are shown in Table 2.

[0083] Table 2. Repeatability of qPCR

[0084]

[0085] As can be seen from the table, the detection method provided by this invention has an intra-group coefficient of variation of 0.01% to 0.04% and an inter-group coefficient of variation of 0.01% to 0.02%, which is less than 0.05%. Therefore, the repeatability of this method is good.

[0086] Example 6

[0087] Preliminary application of Taqman multiplex real-time fluorescence PCR method for simultaneous detection of PCV2 and Mhp:

[0088] Samples from the same batch of pigs in large-scale pig farms in Hubei Province exhibiting clinical symptoms such as coughing, wheezing, emaciation, paleness, and skin inflammation were collected, including 140 blood samples, 400 oral and nasal swabs, and 60 lung tissue samples. A total of 600 samples were analyzed to assess their clinical infection status. The results were compared with established single-fluorescence quantitative methods for PCV2 and Mhp (Table).

[0089] The results of the established dual TaqMan qPCR method for PCV2 and Mhp showed that the overall positive rate for simultaneous detection of PCV2 was 51.67%, and the overall positive rate for Mhp was 21%. Consistent with the results of single TaqMan qPCR, the positive rates in mixed infections were 12.86% for serum samples, 10.75% for nasal and oral swabs, 40% for lung tissue, and 14.17% overall. The concordance rate between the two methods was 100%.

[0090] Table 3 Clinical Sample Testing

[0091]

[0092] As demonstrated by the above embodiments, this invention provides a dual-fluorescent quantitative PCR method for the simultaneous detection of porcine circovirus type 2 and Mycoplasma hyopneumoniae, exhibiting high sensitivity, good specificity, and excellent repeatability. This method successfully established a standard curve, enabling accurate quantification of both targets over a wide concentration range. Specificity experiments confirmed no cross-reactivity with other common porcine pathogens. Repeatability tests showed low intra- and inter-group coefficients of variation, resulting in stable and reliable results. Preliminary clinical sample application results indicate that this method is applicable to various sample types, including blood, swabs, and tissues, with detection results completely consistent with single-detection methods, validating its practicality and accuracy in field testing and providing an effective tool for the rapid diagnosis of mixed infections in clinical settings.

[0093] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneous detection of porcine circovirus type 2 and Mycoplasma hyopneumoniae, characterized in that, In the same reaction system, the sample to be tested is detected by fluorescence quantitative PCR using a primer probe set for the porcine circovirus type 2 ORF1 gene and a primer probe set for the Mycoplasma hyopneumoniae LepA gene; The primer probe set for the porcine circovirus type 2 ORF1 gene comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 2, and a probe with a nucleotide sequence as shown in SEQ ID NO. 3; The primer probe set for the Mycoplasma hyopneumoniae LepA gene comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 4, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 5, and a probe with a nucleotide sequence as shown in SEQ ID NO.

6.

2. The method of claim 1, wherein, The probe as shown in SEQ ID NO. 3 is labeled with a FAM fluorescent reporter group.

3. The method of claim 1, wherein, The probe as shown in SEQ ID NO. 6 is labeled with a HEX fluorescent reporter group.

4. The method of claim 1, wherein, The reaction procedure of the fluorescence quantitative PCR detection comprises a pre-denaturation step and 35 to 50 cycles of amplification steps; The pre-denaturation step is performed at 90 to 98 degrees Celsius for 30 to 90 seconds; Each amplification cycle comprises a denaturation step at 90 to 98 degrees Celsius for 5 to 20 seconds, and an annealing and extension step at 55 to 62 degrees Celsius for 30 to 60 seconds.

5. The method of claim 1, wherein, The method for simultaneously detecting porcine circovirus type 2 and Mycoplasma hyopneumoniae further comprises a result determination step, when the fluorescence channel Ct value corresponding to the detection of porcine circovirus type 2 is less than or equal to 40, the sample is determined to be positive for porcine circovirus type 2, and when the fluorescence channel Ct value corresponding to the detection of Mycoplasma hyopneumoniae is less than or equal to 40, the sample is determined to be positive for Mycoplasma hyopneumoniae.

6. The application of a primer probe combination in simultaneous detection of porcine circovirus type 2 and Mycoplasma hyopneumoniae, characterized in that, The primer probe combination comprises a first primer pair and a first probe that specifically amplify a target sequence of the porcine circovirus type 2 ORF1 gene, and a second primer pair and a second probe that specifically amplify a target sequence of the Mycoplasma hyopneumoniae LepA gene; The nucleotide sequences of the first primer pair and the first probe are shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively, and the nucleotide sequences of the second primer pair and the second probe are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

7. The use of a kit in the simultaneous detection of porcine circovirus type 2 and Mycoplasma hyopneumoniae, characterized in that, The kit comprises the primer probe combination of claim 6.

8. Use according to claim 7, characterized in that, The kit further comprises a DNA polymerase, deoxyribonucleotide triphosphates, and a reaction buffer.

9. Use according to claim 7, characterized in that, The kit further comprises a positive quality control containing a porcine circovirus type 2 ORF1 gene fragment and a Mycoplasma hyopneumoniae LepA gene fragment.

10. A primer probe combination for use in the preparation of a diagnostic or screening test for the diagnosis or screening of mixed infection of porcine circovirus type 2 and Mycoplasma hyopneumoniae, characterized in that, The primer probe combination comprises a first primer pair and a first probe that specifically amplify a target sequence of the porcine circovirus type 2 ORF1 gene, and a second primer pair and a second probe that specifically amplify a target sequence of the Mycoplasma hyopneumoniae LepA gene; The kit comprises the primer probe combination of claim 6. The nucleotide sequences of the first primer pair and the first probe are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and the nucleotide sequences of the second primer pair and the second probe are shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively. The nucleotide sequences of the first primer pair and the first probe are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and the nucleotide sequences of the second primer pair and the second probe are shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

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