Primers and probe for detecting porcine epidemic diarrhea virus subtype g2c by real-time fluorescence quantitative PCR and application thereof

Abstract

The application discloses primers and a probe for real-time fluorescent quantitative PCR detection of a porcine epidemic diarrhea virus G2c subtype and application thereof and belongs to the field of animal virus molecular biology testing methods. The application compares S genes of different gene subtype strains of the porcine epidemic diarrhea virus, designs a pair of primers and a Taqman-MGB probe on the basis of a mutation site specific to the G2c subtype strain, and proves that the primers and the probe can effectively distinguish the G2c and other PEDV gene subtype strains through virus liquids and clinical samples of different gene subtype strains of the PEDV, meanwhile, the primers and the probe have no cross reaction to other viruses of the porcine enteric diarrhea syndrome, and the detection sensitivity of the kit can reach 5 copies / muL. The application can realize specific and sensitive detection of the G2c subtype, and has a positive significance for diagnosis and epidemiological investigation of the porcine epidemic diarrhea virus.

Description

Technical Field

[0001] This invention relates to the field of molecular biological testing methods for animal viruses, and in particular to primers, probes and their applications for real-time quantitative PCR detection of porcine epidemic diarrhea virus G2c subtype. Background Technology

[0002] Porcine epidemic diarrhea (PED) is caused by porcine epidemic diarrhea virus (PEDV), which belongs to the family Coronaviridae, genus Alphacoronavirus. Its genome is a single-stranded positive-sense RNA, prone to mutation. Based on genetic variations in the S gene, PEDV is classified into G1, G2, and S-INDEL through sequencing and evolutionary analysis. The G1 genotype is further divided into G1a and G1b, and the G2 genotype into G2a, G2b, and G2c. Conventional genotype analysis and identification are time-consuming, involving sample nucleic acid amplification, amplified fragment sequencing, sequence alignment, and phylogenetic tree analysis, generally requiring more than 3 days. The proportion of clinically isolated G2c strains is significantly higher than other genotypes. Therefore, it is necessary to establish a simplified PEDV G2c detection technology. This is essential for rapidly identifying whether the causative strain belongs to the currently prevalent PEDV genotype G2c and for assessing the effectiveness of existing vaccinations in pig farms.

[0003] Real-time quantitative PCR (qPCR) is a mature technology for diagnosing animal diseases. Compared to traditional PCR, it offers advantages such as single-tube sealing, high automation, and strong specificity. It also allows for real-time monitoring of template amplification, effectively overcoming the limitations of traditional PCR endpoint determination. Furthermore, qPCR can establish standard curves for absolute quantification of samples. Probe-based qPCR offers higher specificity than dye-based methods. The Taqman-MGB probe binds a minor groove binder (MGB) to its 3' end. MGB conforms to the DNA helical structure, significantly increasing the probe's Tm value compared to ordinary Taqman probes. Therefore, MGB probes allow for shorter probe sequences, improving binding specificity, accuracy, and sensitivity, which is widely used in the detection of single nucleotide polymorphisms. Currently, there are no patents or literature on the application of qPCR based on Taqman-MGB probes for specific genotypes of PEDV G2c. Summary of the Invention

[0004] The purpose of this invention is to provide primers, probes, and their applications for real-time quantitative PCR detection of porcine epidemic diarrhea virus G2c subtype, in order to solve the problems existing in the prior art. The detection primers achieve specific and sensitive detection of G2c subtype, which is of positive significance for the diagnosis and epidemiological investigation of porcine epidemic diarrhea virus.

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

[0006] This invention provides a real-time fluorescence quantitative PCR detection primer and probe for porcine epidemic diarrhea virus G2c subtype Taqman-MGB probe. The detection primer and probe include an upstream primer G2c-F, a downstream primer G2c-R, and a probe G2c-P, with nucleotide sequences as shown in SEQ ID NO.1-3, respectively.

[0007] Preferably, the probe G2c-P has a fluorescent reporter group labeled at its 5' end and a non-fluorescent quencher group labeled at its 3' end.

[0008] The present invention also provides the application of the detection primers and probes described herein in the preparation of a kit for detecting the genotype of porcine epidemic diarrhea virus G2c subtype.

[0009] The present invention also provides the application of the detection primers and probes described herein in the preparation of a kit for distinguishing porcine epidemic diarrhea virus G2c subtype from other porcine epidemic diarrhea virus genotype strains.

[0010] The present invention also provides a real-time fluorescence quantitative PCR detection kit for porcine epidemic diarrhea virus G2c subtype Taqman-MGB probe, the detection kit comprising an amplification reaction solution, the amplification reaction solution comprising the detection primers and probes described above.

[0011] Preferably, the amplification reaction solution comprises 0.4 μL of upstream primer G2c-F, 0.4 μL of downstream primer G2c-R, 0.2 μL of probe G2c-P, 10 μL of amplification buffer, 0.4 μL of ROX Passive Reference DyeII, and 7.6 μL of sterile deionized water.

[0012] Preferably, when amplifying the genome of the sample to be tested using the amplification reaction solution, the amplification conditions are as follows: pre-denaturation at 95°C for 5 min; 95°C for 10 s; 60°C for 35 s for 40 cycles; and fluorescence signal acquisition at 60°C for 35 s.

[0013] Preferably, the genotype of porcine epidemic diarrhea virus G2c subtype is determined based on the collected fluorescence signal. The determination method is as follows: if the Ct value of the test sample is ≤38 and a standard S-shaped amplification curve appears, it is judged as positive; if the test sample has no Ct value, or the Ct value is >38 and no standard amplification curve appears, it is judged as negative.

[0014] Preferably, the test kit also includes a positive control and a negative control.

[0015] Preferably, the positive control is a recombinant plasmid of the S gene of the porcine epidemic diarrhea virus G2c subtype strain, and the negative control is Vero cell nucleic acid.

[0016] The present invention discloses the following technical effects:

[0017] This invention compares and analyzes the S gene sequences of multiple PEDV subtypes, selects characteristic base sites that distinguish most G2c subtypes from other subtypes, and designs these sites into the middle of a TaqMan-MGB probe, enabling the probe to specifically recognize the G2c subtype sequence. Based on the mutation sites specific to the G2c subtype strain, a pair of primers and a TaqMan-MGB probe (upstream primer G2c-F, downstream primer G2c-R, and probe G2c-P) were designed. When used for PEDV detection, these probes exhibit the following characteristics: ① High specificity: The synthesized TaqMan-MGB probe precisely identifies the characteristic base sites of the G2c subtype, enabling detection of specific PEDV G2c strains; ② Rapid and efficient amplification: The overall detection time is 50 minutes; ③ High sensitivity: The detection limit for the G2c subtype reaches 5 copies / μL; ④ Simple result interpretation: Results can be quickly determined from the amplification curve without requiring further analytical steps.

[0018] Furthermore, this invention has demonstrated through viral fluids and clinical samples from different PEDV genotype subtypes that the one pair of primers and one Taqman-MGB probe designed in this invention can effectively distinguish the G2c subtype strain from other PEDV genotype subtype strains, while showing no cross-reactivity with other viruses causing swine enteric diarrhea syndrome, exhibiting high specificity and sensitivity. Attached Figure Description

[0019] 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.

[0020] Figure 1 The results of sequence alignment of the S gene of different PEDV subtypes and the S gene sequence of PEDV-positive samples;

[0021] Figure 2 The results of the specificity assay for the porcine epidemic diarrhea virus G2c Taqman-MGB probe by real-time fluorescence quantitative PCR are as follows;

[0022] Figure 3 The results of the sensitivity test of the real-time fluorescence quantitative PCR for the porcine epidemic diarrhea virus G2c Taqman-MGB probe;

[0023] Figure 4 The results of a repeatability test for real-time quantitative PCR using the Taqman-MGB probe for porcine epidemic diarrhea virus G2c.

[0024] Figure 5 The standard curve for real-time quantitative PCR of porcine epidemic diarrhea virus G2c Taqman-MGB probe;

[0025] Figure 6 This study describes the real-time quantitative PCR detection of porcine epidemic diarrhea virus G2c Taqman-MGB probe in clinical samples. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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 obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] 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.

[0031] Example 1: Primer and Taqman-MGB probe design and synthesis

[0032] Primers and probes for real-time quantitative PCR amplification of porcine epidemic diarrhea virus (PEDV) G2c subtype were used. S gene sequences of different PEDV genotypes were downloaded from the NCBI database. A phylogenetic tree was constructed using MEGA11 software, and alignment analysis was performed using SnapGene software. The characteristic bases distinguishing the G2c genotype from other genotypes were identified (see [link to relevant documentation]). Figure 1 One primer pair and one Taqman-MGB probe were designed using Primer Express 3.0 software and synthesized by Sangon Biotech (Shanghai) Co., Ltd. An ABI 7500 real-time fluorescence PCR instrument was used to monitor parameters such as the baseline position and morphology of the amplification curve, the onset time and slope of the exponential growth of the amplification curve, and the onset time of the plateau phase during the amplification process, optimizing reaction conditions. The primer pair and probe combination were determined to be the G2c upstream primer (G2c-F), the G2c downstream primer (G2c-R), and the probe (G2c-P). PCR primers for the PEDV-S positive recombinant plasmid were designed using Primer Premier 5 software, including PEDV-SF and the downstream primer PEDV-SR. The sequences of the primers and probes are shown in Table 1, and the concentration of each was 10 μmol / L.

[0033] Table 1 Primer and probe sequences

[0034]

[0035] In Table 1 above, the 5' end of the probe G2c-P is labeled with a FAM reporter group, and the 3' end is labeled with an MGB SQ non-fluorescent quencher group.

[0036] Example 2: Preparation of positive control

[0037] Preparation of positive plasmids for porcine epidemic diarrhea virus S gene: RNA genome was extracted from porcine epidemic diarrhea virus HK2021 (G2c) strain using a rapid RNA extraction kit (Novizan Group, catalog number RC113-01). cDNA was obtained by reverse transcription using a reagent (Kangwei Century, catalog number CW2020M). PCR was performed using upstream primer PEDV-SF and downstream primer PEDV-SR, and the amplified bands were recovered using a gel recovery kit.

[0038] The amplification reaction system consisted of: 18 μL of amplification reaction solution (1 μL of 10 μmol / L PEDV-SF, 1 μL of 10 μmol / L PEDV-S-R, 10 μL of 2 × Phanta Max Master Mix (Novizan Group, catalog number P525-01), and 6 μL of ddH2O); 2 μL of cDNA template or a positive or negative control; the total volume of the reaction system was 20 μL. The reaction program was as follows: pre-denaturation at 95℃ for 3 min; 95℃ for 15 s; 56℃ for 15 s; 72℃ for 30 s for 35 cycles; and extension at 72℃ for 5 min.

[0039] The recovered product was incubated with pMD19-T vector (Takara, catalog number 3271) at 16°C for 2 h, followed by Trans5α competent cell transformation. After antibiotic selection and PCR identification, plasmids were extracted and sequenced for identification. The concentration of positive plasmids and their copy number were determined using Nanodrop 2000 (Thermo).

[0040] Example 3: Preparation of negative control

[0041] The whole genome was extracted from Vero cells using a rapid RNA extraction kit (Novizan Group, catalog number RC113-01). cDNA was obtained by reverse transcription using a reagent (Kangwei Century, catalog number CW2020M). The cDNA was identified by PCR and used as a negative control. The identification method is the same as in Example 2.

[0042] Example 4: Real-time quantitative PCR detection method for porcine epidemic diarrhea virus G2c subtype using Taqman-MGB probe, comprising the following steps:

[0043] 1) Extract RNA from the sample to be tested: Use a commercially available RNA extraction kit to extract genomic RNA from the sample to be tested.

[0044] 2) Obtaining cDNA template: Using commercially available reverse transcription reagent, the RNA of the sample to be tested is reverse transcribed into cDNA to obtain the cDNA template.

[0045] 3) The amplification reaction system consisted of: 19 μL amplification reaction solution (0.4 μL of 10 μmol / L G2c-F, 0.4 μL of 10 μmol / L G2c-R, 0.2 μL of 10 μmol / L G2c-P, 10 μL of 2 × AceQ qPCR Probe Master Mix buffer, 0.4 μL of ROX Passive Reference Dye II, and 7.6 μL of sterile deionized water); 1 μL of cDNA template or positive or negative control; the total volume of the reaction system was 20 μL.

[0046] The positive control tube contained a recombinant plasmid of the S gene of the porcine epidemic diarrhea virus G2c subtype strain, with a volume of 20 μL.

[0047] The negative control tube contained Vero cell nucleic acid, with a volume of 20 μL.

[0048] 4) Real-time quantitative PCR amplification of porcine epidemic diarrhea virus G2c subtype Taqman-MGB probe: The amplification reaction system prepared in step 3) was subjected to PCR amplification. The reaction conditions were: pre-denaturation at 95℃ for 5 min; 95℃ for 10 s; 60℃ for 35 s for 40 cycles; fluorescence signal was collected at 60℃ for 35 s.

[0049] 5) Result determination:

[0050] The conditions for the experiment to be valid are: the positive control amplification curve is obvious and the Ct value is <30; at the same time, the negative control amplification curve has no logarithmic growth phase.

[0051] Judgment: A sample with a Ct value ≤ 38 and a standard S-shaped amplification curve is judged as positive; a sample with no Ct value, or a Ct value > 38 and no standard amplification curve, is judged as negative.

[0052] Example 5: Specificity assay of porcine epidemic diarrhea virus G2c subtype Taqman-MGB probe by real-time fluorescence quantitative PCR

[0053] Specificity tests were conducted using the reaction system and conditions described in Example 4. The templates used were porcine epidemic diarrhea virus HK2021 (G2c subtype), porcine epidemic diarrhea virus JS2008 (G1b subtype), porcine epidemic diarrhea virus AH2012 / 12 (G2b subtype), porcine epidemic diarrhea virus NJ-32 (S-INDEL type), porcine rotavirus (PRV), porcine deltavirus (PDCoV), porcine transmissible gastroenteritis virus (TGEV), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and a negative control.

[0054] like Figure 2 As shown, the results indicated that only the porcine epidemic diarrhea G2c subtype was positive, while all other samples were negative.

[0055] Example 6: Sensitivity test of real-time quantitative PCR using Taqman-MGB probe for porcine epidemic diarrhea virus G2c subtype.

[0056] The constructed PEDV-S positive recombinant plasmid was diluted to 5 copies / μL, and steps 3)-5) in Example 4 were performed 10 times in each case. The results showed that the amplification curve exhibited dispersion as the template copy number decreased; however, all 10 repeated tests using this method on the 5 copy / μL diluted plasmid were positive. Figure 3 .

[0057] Example 7: Repeatability test of real-time quantitative PCR using Taqman-MGB probe for porcine epidemic diarrhea virus G2c subtype

[0058] The constructed PEDV-S positive recombinant plasmid was diluted to 2×10⁻⁶. 6 copies / μL and 2×10 3 The detection was performed three times, with copies / μL, following steps 3)-5) in Example 4. The coefficients of variation of the Ct values ​​for high and low copy detections were 0.32% and 0.56%, respectively. (See attached table). Figure 4 .

[0059] Example 8: Establishment of a standard curve for real-time quantitative PCR using the Taqman-MGB probe for porcine epidemic diarrhea virus G2c subtype.

[0060] The constructed PEDV-S positive recombinant plasmid was diluted to obtain 10 7 ~10 0 Plasmids at concentrations of 1 copy / μL were analyzed according to steps 3)-5) in Example 4, and a standard curve was plotted. The standard curve showed R... 2 The amplification efficiency was 103% (0.997); the amplification equation was Y = -3.243x + 38.932. Therefore, this method exhibits good amplification efficiency, a good correlation between its fluorescence curve and the detected target gene concentration, and high accuracy. (See attached image.) Figure 5 .

[0061] Example 9: Real-time quantitative PCR detection of porcine epidemic diarrhea virus G2c subtype in clinical samples using Taqman-MGB probe.

[0062] Five clinically collected pig fecal samples (samples 1-5) were used to extract RNA genomes using a rapid RNA extraction kit (Novizan Group, catalog number RC113-01). cDNA was then obtained using a reverse transcription reagent (Kangwei Century, catalog number CW2020M) and used as the template for testing. HK2021 (G2c) cDNA was used as a positive control, and N as a negative control. The reaction system and conditions of Example 4 were used for detection.

[0063] like Figure 6As shown in the results, amplification curves were detected in samples 1-4 of G2c, while no amplification was detected in sample 5 of G2b. This indicates that the method has a good distinguishing effect on clinical samples. Figure 1 Sequencing results further confirmed that the sites corresponding to samples 1-4 are characteristic G2c bases.

[0064] 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 primer and probe for detecting porcine epidemic diarrhea virus G2c subtype by real-time fluorescence quantitative PCR with Taqman-MGB probe, characterized in that, The detection primer and probe comprise an upstream primer G2c-F, a downstream primer G2c-R, and a probe G2c-P, and the nucleotide sequences are shown in SEQ ID NO. 1-3, respectively. The probe G2c-P is labeled with a fluorescent reporter group at the 5' end and a non-fluorescent quencher group at the 3' end.

2. The use of the detection primer and probe according to claim 1 in the preparation of a kit for detecting the genotype of the porcine epidemic diarrhea virus G2c subtype.

3. The use of the detection primer and probe according to claim 1 in the preparation of a kit for distinguishing the porcine epidemic diarrhea virus G2c subtype from other porcine epidemic diarrhea virus genetic subtype strains.

4. A real-time fluorescent quantitative PCR detection kit for porcine epidemic diarrhea virus G2c subtype Taqman-MGB probe, characterized in that, The detection kit comprises an amplification reaction solution, and the amplification reaction solution comprises the detection primer and probe according to claim 1 or 2.

5. The test kit of claim 4, wherein The amplification reaction solution comprises 0.4 μL of the upstream primer G2c-F, 0.4 μL of the downstream primer G2c-R, 0.2 μL of the probe G2c-P, 10 μL of an amplification buffer, 0.4 μL of ROX Passive Reference Dye II, and 7.6 μL of sterilized deionized water.

6. The test kit of claim 4, wherein When the amplification reaction solution is used to amplify the genome of a sample to be tested, the amplification conditions are as follows: pre-denaturation at 95℃ for 5 min, 95℃ for 10 s, 60℃ for 35 s for 40 cycles, and fluorescence signal collection at 60℃ for 35 s.

7. The test kit of claim 6, wherein The genotype of the porcine epidemic diarrhea virus G2c subtype is determined according to the collected fluorescence signal, and the determination method is as follows: if the Ct value of the sample to be tested is ≤38 and a standard S-shaped amplification curve appears, the sample to be tested is determined to be positive; if the sample to be tested has no Ct value or the Ct value is >38 and no standard amplification curve appears, the sample to be tested is determined to be negative.

8. The test kit of claim 4, wherein The detection kit further comprises a positive control and a negative control.

9. The test kit of claim 8, wherein The positive control is a porcine epidemic diarrhea virus G2c subtype strain S gene recombinant plasmid, and the negative control is a Vero cell nucleic acid.

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