Multiplex fluorescent quantitative PCR primer group for simultaneously detecting PPV, PCV2 and JEV, kit and application thereof

Abstract

The invention discloses a multiplex fluorescent quantitative PCR primer group and kit for simultaneously detecting PPV, PCV2 and JEV and application of the multiplex fluorescent quantitative PCR primer group and kit, and belongs to the technical field of porcine virus detection. According to the invention, the exclusive multiplex fluorescent quantitative detection of three high-frequency co-infected sow reproductive disorder viruses, namely PCV2, PPV and JEV, is realized for the first time, the sensitivity is superior to that of the prior art, the lowest detection limit of PCV2 reaches 1 copy / mu L, both PPV and JEV are 10 copy / mu L, the detection period is only 1.5-2 hours, subsequent product treatment is not needed, a result can be directly judged through a fluorescent signal, and the method is suitable for rapid screening of large-scale samples. In practical application, the detection flux can be greatly improved, time and economic cost are remarkably saved, a powerful tool is provided for rapid diagnosis and large-scale epidemiological monitoring of porcine reproductive disorder diseases, and the technical blank in exclusive and efficient detection of PCV2, PPV and JEV in the prior art is filled.

Description

Technical Field

[0001] This invention relates to the field of swine virus detection technology, and in particular to a multiplex real-time PCR primer set, kit, and application for simultaneous detection of PPV, PCV2, and JEV. Background Technology

[0002] Porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine circovirus type 2 (PCV2) are the main pathogens causing reproductive disorders in sows. Mixed infections are common clinically, with similar symptoms that are difficult to distinguish based on clinical manifestations. Existing detection methods cannot simultaneously detect all three pathogens, resulting in low efficiency, long detection cycles, and high costs, failing to meet the needs of farms for rapid, simultaneous monitoring of multiple pathogens.

[0003] Although multiplex quantitative PCR technology has been applied to the detection of other porcine viruses, no multiplex quantitative PCR methods have been reported for the detection of PPV, JEV, and PCV2, three viruses that cause sow reproductive disorders. Chinese invention patent CN202311141562.3 (A primer and probe combination, kit, and method for simultaneous detection of PCV3, PCV2, and PPV) only detects PCV3, PCV2, and PPV, lacking JEV. The current detection methods are insufficient to address clinical scenarios involving co-infection of JEV with PCV2 and PPV. Chinese invention patent CN202310247430.2 (RT-qPCR kit and method for simultaneous detection of PHEV, PRV, CSFV, and JEV) only detects PHEV, PRV, CSFV, and JEV, omitting PCV2 and PPV, thus failing to accommodate the simultaneous detection of these two core viruses causing porcine reproductive disorders. Chinese invention patent CN201510180486.6 (a multiplex RT-PCR detection kit for simultaneous detection or diagnosis of multiple porcine viral infections and its application), while including PCV2, PPV, and JEV, is designed for mixed detection of six viruses (PRV, PPV, PCV2, PRRSV, CSFV, and JEV), not specifically optimized for PCV2, PPV, and JEV. Furthermore, it employs conventional RT-PCR technology, requiring post-processing after agarose gel electrophoresis (a process taking 4-5 hours), resulting in a detection limit of only approximately 10 for PPV, PCV2, and JEV. 3 -10 4 Copy / μL. Therefore, there is an urgent need in the field for a multiplex fluorescence quantitative detection method to simultaneously detect PPV, JEV, and PCV2. Summary of the Invention

[0004] The purpose of this invention is to provide a multiplex quantitative PCR primer set, kit, and application for the simultaneous detection of PPV, PCV2, and JEV, in order to solve the problems existing in the prior art. This invention establishes a multiplex quantitative PCR primer set, kit, and detection method that can simultaneously, rapidly, and accurately distinguish and detect PPV, JEV, and PCV2 in a single reaction tube, realizing accurate diagnosis and efficient monitoring of these three major pathogens causing reproductive disorders in sows.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a set of primers for simultaneous detection of PPV, PCV2 and JEV, including a PPV primer set, a PCV2 primer set and a JEV primer set;

[0007] The PPV primer set includes PPV-F with nucleotide sequence as shown in SEQ ID NO.1, PPV-R with nucleotide sequence as shown in SEQ ID NO.2, and PPV-Probe with nucleotide sequence as shown in SEQ ID NO.3;

[0008] The PCV2 primer set includes PCV2-F with nucleotide sequence as shown in SEQ ID NO.4, PCV2-R with nucleotide sequence as shown in SEQ ID NO.5, and PCV2-Probe with nucleotide sequence as shown in SEQ ID NO.6;

[0009] The JEV primer set includes JEV-F with nucleotide sequence as shown in SEQ ID NO.7, JEV-R with nucleotide sequence as shown in SEQ ID NO.8, and JEV-Probe with nucleotide sequence as shown in SEQ ID NO.9.

[0010] Optionally, the PPV-Probe is characterized by having a 6-FAM group modified at its 5' end and a BHQ1 group modified at its 3' end.

[0011] The PCV2-Probe is modified with a ROX group at its 5' end and a BHQ2 group at its 3' end.

[0012] The JEV-Probe has a 5' end modified with a HEX group and a 3' end modified with a BHQ1 group.

[0013] The present invention also provides the application of the aforementioned multiplex quantitative PCR primer set in the preparation of a multiplex quantitative PCR kit for simultaneous detection of PPV, PCV2 and JEV.

[0014] The present invention also provides a multiplex quantitative PCR kit for simultaneous detection of PPV, PCV2 and JEV, comprising the aforementioned multiplex quantitative PCR primer set.

[0015] Optionally, the steps for simultaneously detecting PPV, PCV2, and JEV using the kit are as follows:

[0016] Using the DNA of the sample to be tested as a template, a PCR reaction was performed using the aforementioned multiplex quantitative PCR primer set, and the fluorescence signal was collected to obtain the Ct value of the sample to be tested.

[0017] Optionally, if the FAM channel shows a typical amplification curve and the Ct value is ≤35, then the sample to be tested is determined to contain PPV;

[0018] If the ROX channel shows a typical amplification curve and the Ct value is ≤35, then the sample to be tested is determined to contain PCV2.

[0019] If a typical amplification curve appears in the HEX channel and the Ct value is ≤35, then the sample to be tested is determined to contain JEV.

[0020] Optionally, the PCR reaction system consists of 10 μL of 2X Pro Taq HS Probe Premix III, 0.4 μL of PPV-F, 0.4 μL of PPV-R, 0.4 μL of PPV-Probe, 0.4 μL of PCV2-F, 0.4 μL of PCV2-R, 0.4 μL of PCV2-Probe, 0.4 μL of JEV-F, 0.4 μL of JEV-R, 0.4 μL of JEV-Probe, 3 μL of DNA template, and 3.4 μL of DEPC water.

[0021] Optionally, the concentrations of PPV-F, PPV-R, PPV-Probe, PCV2-F, PCV2-R, PCV2-Probe, JEV-F, JEV-R, and JEV-Probe are all 10 μM.

[0022] Optionally, the PCR reaction conditions are 95℃ for 30s for 1 cycle; or 95℃ for 5s and 56℃ for 30s for 45 cycles.

[0023] The present invention discloses the following technical effects:

[0024] This invention is the first to achieve specific multiplex quantitative PCR detection of three high-frequency co-infecting sow reproductive disorder viruses: PCV2, PPV, and JEV. It utilizes primer and probe combinations and amplification fragments designed for the conserved ORF2 region of PCV2, the conserved VP2 region of PPV, and the conserved E gene region of JEV. These are compared with the primers and probes for PCV2 / PPV (targeting the PCV2 CAP sequence and PPV VP2 sequence, without JEV design) in CN202311141562.3, the primers and probes for JEV (targeting the JEV E gene, without PCV2 / PPV design) in CN202310247430.2, and the primers and probes for PCV2 / PPV / JEV (targeting PCV2 ORF2, PPV NS1, and JEV) in CN201510180486.6. The E gene (different in sequence and amplified fragment length) shows no homology; and its sensitivity is superior to the three patents mentioned above. The detection limit for PCV2 is as low as 1 copy / μL, while for PPV and JEV it is 10 copies / μL (1-2 orders of magnitude higher than CN201510180486.6, and 100 times higher than the PCV2 detection sensitivity in CN202311141562.3). The detection cycle is only 1.5-2 hours, no subsequent product processing is required, and the results can be directly judged by fluorescence signal. It is suitable for rapid screening of large-scale samples and fills the technological gap of the three patents in the dedicated high-efficiency detection of PCV2, PPV, and JEV.

[0025] Compared to existing technologies, the most prominent advantage of this invention lies in its successful integration of the detection of three key pathogens—PPV, JEV, and PCV2—into a single reaction system, achieving a technology that moves from "multiple single tests" to "one triple test." This results in significant advantages of high efficiency, low cost, and high accuracy. Through carefully designed specific primers and multiple fluorescently labeled probes, the method ensures high sensitivity and strong specificity, effectively identifying mixed infections. The optimized and stable system guarantees the reproducibility of results. In practical applications, this method can greatly increase detection throughput, significantly save time and economic costs, and provide a powerful tool for the rapid diagnosis and large-scale epidemiological monitoring of porcine reproductive disorders. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The results show the optimized annealing temperatures of 60℃ (A), 58℃ (B), and 56℃ (C) in the multiplex quantitative PCR reaction system.

[0028] Figure 2 Positive amplification curves and negative controls for PPV (A), PCV2 (B), and JEV (C);

[0029] Figure 3 The specific amplification curves for triple real-time PCR of PPV, PCV2, JEV, and blank control are shown.

[0030] Figure 4 Sensitivity assays for quantitative real-time PCR of PPV (A), PCV2 (B), and JEV (C);

[0031] Figure 5 Standard curves for quantitative real-time PCR of PPV (A), PCV2 (B), and JEV (C);

[0032] Figure 6 This refers to the triple test results for the positive sample;

[0033] Figure 7 This is the result of a single test on a positive sample. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Example 1

[0040] 1. Design a primer set for simultaneous detection of PPV, PCV2, and JEV in multiplex quantitative PCR.

[0041] The primer set for simultaneous detection of PPV, PCV2, and JEV in this invention is mainly used to amplify the VP2 gene fragment of PPV (MZ706996.2), the Cap gene fragment of PCV2 (PV087256.1), and the E gene fragment of JEV (KY083679.1). The 5' end of the probe is attached to different fluorescent groups, namely 6-FAM, ROX, or HEX; the 3' end is attached to corresponding quenching groups, namely BHQ1 or BHQ2. The primer and probe sequences included in this embodiment are shown in Table 1.

[0042] Table 1. Primer sequences for Taqman multiplex real-time quantitative PCR

[0043]

[0044] 2. Construction of Multiplex Real-Time PCR Reaction System

[0045] DNA was extracted from the test samples using conventional techniques and stored at -20℃ for later use. The conditions of the multiplex quantitative PCR reaction system were optimized. The optimized scheme was as follows: in a 20 µL reaction system, the amount of 10 µM primer added was 0.4, 0.5, 0.6, 0.7, and 0.8 μL, respectively; the amount of 10 µM probe added was 0.4, 0.6, and 0.8 μL, respectively. Specifically, the primer addition amounts corresponding to 0.4 and 0.6 μL of 10 µM probe were 0.4, 0.5, 0.6, 0.7, and 0.8 μL, respectively; and the primer addition amounts corresponding to 0.8 μL of 10 µM probe were 0.4, 0.5, 0.6, and 0.7 μL, respectively. Three annealing temperature gradients were set at 56℃, 58℃, and 60℃. Quantitative PCR experiments were conducted under these different reaction conditions. After obtaining the results, the optimal reaction system and reaction conditions were selected by comprehensively analyzing factors such as Ct value, fluorescence intensity, and reaction cost.

[0046] Amplification curves as follows Figure 1 As shown in Tables 2-4, the final optimized total reaction volume (20 μL) was determined based on the results as follows: Reaction solution A (PCR amplification reagent 2X Pro Taq HS Probe Premix III) 10 μL, Reaction solution B (PPV-F (10 μM) 0.4 μL, PPV-R (10 μM) 0.4 μL, PPV-Probe (10 μM) 0.4 μL, PCV2-F (10 μM) 0.4 μL, PCV2-R (10 μM) 0.4 μL, PCV2-Probe (10 μM) 0.4 μL, JEV-F (10 μM) 0.4 μL, JEV-R (10 μM) 0.4 μL, JEV-Probe (10 μM) 0.4 μL (i.e., the concentration of primers and probes for the three viruses is 10 μM, and the amount used is 0.4 μL, for a total of 3.6 μL)), Sample DNA template 3 μL of reaction solution C (DEPC water) and 3.4 μL of reaction solution C. The reaction conditions were 95℃ for 30s, 1 cycle; 95℃ for 5s, 56℃ for 30s, 45 cycles.

[0047] Result interpretation: If the FAM channel shows a typical amplification curve and the Ct value is ≤35, the sample to be tested is determined to contain PPV; if the ROX channel shows a typical amplification curve and the Ct value is ≤35, the sample to be tested is determined to contain PCV2; if the HEX channel shows a typical amplification curve and the Ct value is ≤35, the sample to be tested is determined to contain JEV.

[0048] If the sample to be tested does not have an amplification curve in a certain gene channel, or the Ct value is ≥38, then it is determined that the sample to be tested does not contain the virus.

[0049] If a gene channel in the sample to be tested has an amplification curve, but 35 < Ct < 38, it is judged as an uncertain sample and needs to be retested.

[0050] Table 2 Results at annealing temperature of 56℃

[0051]

[0052] Table 3 Results at annealing temperature of 58℃

[0053]

[0054] Table 4 Results at annealing temperature of 60℃

[0055]

[0056] 3. Specificity verification

[0057] 3.1 Specificity Validation of Multiplex Quantitative PCR Detection System

[0058] To verify the single specificity of the multiplex quantitative PCR detection system constructed in this invention, genomic DNA / RNA of PPV, PCV2, and JEV was extracted and reverse transcribed as PCR templates. A blank control group and a negative control group were also set up. The blank control was DEPC water, and the negative control was sterilized diethyl pyrocarbonate-treated water. The specificity verification results of the multiplex quantitative PCR are as follows: Figure 2 As shown, PPV, PCV2, and JEV only exhibit amplification curves in their respective fluorescence channels; the blank control and negative control showed no amplification curves in any of the three channels. This result demonstrates that the multiplex quantitative PCR detection system of this invention possesses high single-channel specificity.

[0059] 3.2 Validation of the overall specificity of the multiplex quantitative PCR detection system

[0060] Genomes of five common porcine viruses—pseudorabies virus (PRV), porcine epidemic diarrhea virus (PEDV), porcine gettavirus (GETV), porcine rotavirus (RV), and porcine reproductive and respiratory syndrome virus (PRRSV)—were routinely extracted. A mixture of plasmids constructed from the VP2 gene fragment of PPV, the Cap gene fragment of PCV2, and the E gene fragment of JEV (in a 1:1:1 mass ratio) served as a positive control, while DEPC water was used as a negative control. Real-time quantitative PCR (qPCR) amplification and detection were performed to determine the specificity of the established multiplex qPCR detection method. Results showed that the established multiplex qPCR detection method exhibited good specificity. Figure 3 ).

[0061] 4. Sensitivity validation of the multiplex quantitative PCR detection system

[0062] like Figure 4 As shown, positive plasmids at different dilutions (A: PPV, 10) 9 ~10 0 copies / μL; B: PCV2, 10 9 ~10 0 copies / μL; C: JEV, 10 9 ~10 0 Using copies / μL as templates, sensitivity tests were performed on three pairs of primers and probes. The results showed that the lowest detection value for PPV and JEV was 10 copies / μL, and the lowest detection value for PCV2 was 1 copy / μL.

[0063] 5. Establishment of standard curve for multiplex quantitative PCR detection

[0064] With 1.0×109 Copies / μL ~ 1.0 × 10⁻⁶ 3 Recombinant plasmid standards at a mass ratio of 1:1:1 were mixed and used as templates for multiplex quantitative PCR amplification. A standard curve was constructed based on the average Ct value, as shown below. Figure 5 As shown, the correlation coefficient R 2 All values ​​are greater than 0.99, indicating a good linear relationship.

[0065] 6. Repeatability assay of multiplex quantitative PCR

[0066] With 1.0×10 8 Copy / μL, 1.0×10 6 Copy / μL, 1.0×10 4 Three recombinant plasmid standards (copies / μL) were mixed at a 1:1:1 ratio and used as templates for multiplex quantitative PCR. Triple replicates were performed at 24h, 48h, and 72h to obtain within-group and inter-group results. The coefficient of variation (CV) was calculated using the formula. The results showed that the CV for both within-group and inter-group repeatability tests was less than 3%, indicating that the method established in this study has good reproducibility. (See Table 5).

[0067] Table 5 Repeatability assays for multiplex quantitative PCR

[0068]

[0069] 7. Validation of the validity of actual detection results from multiplex quantitative PCR

[0070] Multiplex quantitative PCR was used to detect clinically positive cases, and the results were successfully confirmed upon retesting. Figures 6-7 ).

[0071] Example 2: Establishment of a multiplex real-time PCR detection kit for simultaneous detection of PPV, PCV2, and JEV

[0072] A multiplex quantitative PCR kit for simultaneous detection of PPV, PCV2, and JEV includes reaction solution A, reaction solution B, reaction solution C, a positive control, a negative control, instructions, and a kit body. Reaction solution A is PCR amplification reagent 2X Pro Taq HSProbe Premix III. Preferably, the PCR amplification reagent described in this invention includes DNA polymerase, dNTPs, and Mg. 2+This invention does not specifically limit the source or amount of the DNA polymerase, dNTPs, and buffer reagents; commercially available products in the field can be used. In a specific embodiment of this invention, the PCR amplification reagent 2X Pro Taq HS Probe Premix III was purchased from AG Ericsson. Reaction solution B is a mixture of primers and probes for PPV, PCV2, and JEV, with the concentration of primers and probes for all three viruses being 10 μM, and the amount used being 0.4 μL each, totaling 3.6 μL; reaction solution C is DEPC water. The positive control is a mixture of plasmids constructed based on the VP2 gene fragment of PPV, the Cap gene fragment of PCV2, and the E gene fragment of JEV, in a mass ratio of 1:1:1. The negative control is DEPC water.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A multiplex fluorescent quantitative PCR primer set for simultaneously detecting PPV, PCV2 and JEV, characterized in that, The PPV primer group, the PCV2 primer group and the JEV primer group; The PPV primer group comprises a PPV-F with a nucleotide sequence as shown in SEQ ID NO. 1, a PPV-R with a nucleotide sequence as shown in SEQ ID NO. 2 and a PPV-Probe with a nucleotide sequence as shown in SEQ ID NO. 3; The PCV2 primer group comprises a PCV2-F with a nucleotide sequence as shown in SEQ ID NO. 4, a PCV2-R with a nucleotide sequence as shown in SEQ ID NO. 5 and a PCV2-Probe with a nucleotide sequence as shown in SEQ ID NO. 6; The JEV primer group comprises a JEV-F with a nucleotide sequence as shown in SEQ ID NO. 7, a JEV-R with a nucleotide sequence as shown in SEQ ID NO. 8 and a JEV-Probe with a nucleotide sequence as shown in SEQ ID NO.

9.

2. The multiplex fluorescent quantitative PCR primer set according to claim 1, wherein The 5' end of the PPV-Probe is modified with a 6-FAM group, and the 3' end is modified with a BHQ1 group; The 5' end of the PCV2-Probe is modified with a ROX group, and the 3' end is modified with a BHQ2 group; The 5' end of the JEV-Probe is modified with a HEX group, and the 3' end is modified with a BHQ1 group.

3. Use of the multiplex fluorescent quantitative PCR primer group according to claim 1 or 2 in the preparation of a multiplex fluorescent quantitative PCR kit for simultaneously detecting PPV, PCV2 and JEV.

4. A multiplex fluorescent quantitative PCR kit for simultaneously detecting PPV, PCV2 and JEV, characterized in that, The multiplex fluorescent quantitative PCR primer group according to claim 1 or 2.

5. The kit of claim 4, wherein The kit simultaneously detects PPV, PCV2 and JEV according to the following steps: DNA of a sample to be tested is used as a template to perform PCR reaction by using the multiplex fluorescent quantitative PCR primer group, fluorescence signals are collected, and Ct values of the sample to be tested are obtained.

6. The kit of claim 5, wherein If a typical amplification curve appears in the FAM channel and the Ct value is ≤ 35, it is determined that the sample to be tested contains PPV; If a typical amplification curve appears in the ROX channel and the Ct value is ≤ 35, it is determined that the sample to be tested contains PCV2; If a typical amplification curve appears in the HEX channel and the Ct value is ≤ 35, it is determined that the sample to be tested contains JEV.

7. The kit of claim 5, wherein The reaction system of the PCR reaction is 2X Pro Taq HSProbe Premix III 10 μL, PPV-F 0.4 μL, PPV-R 0.4 μL, PPV-Probe 0.4 μL, PCV2-F 0.4 μL, PCV2-R 0.4 μL, PCV2-Probe 0.4 μL, JEV-F 0.4 μL, JEV-R 0.4 μL, JEV-Probe 0.4 μL, DNA template 3 μL and DEPC water 3.4 μL.

8. The kit of claim 7, wherein The concentrations of the PPV-F, the PPV-R, the PPV-Probe, the PCV2-F, the PCV2-R, the PCV2-Probe, the JEV-F, the JEV-R and the JEV-Probe are all 10 μM.

9. The kit of claim 5, wherein The reaction condition of the PCR reaction is 95℃ 30s, 1 cycle; 95℃ 5s, 56℃ 30s, 45 cycles. The reaction condition of the PCR reaction is 95℃ 30s, 1 cycle; 95℃ 5s, 56℃ 30s, 45 cycles.

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