AU2019100992A4

AU2019100992A4 - Primer set, kit, and method for detecting porcine epidemic diarrhea virus

Info

Publication number: AU2019100992A4
Application number: AU2019100992A
Authority: AU
Inventors: Xinsheng Liu
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2019-09-02
Filing date: 2019-09-02
Publication date: 2019-10-10
Anticipated expiration: 2027-09-02

Abstract

The present invention discloses a primer set, a kit, and a method for detecting porcine epidemic diarrhea virus (PEDV), and relates to the field of viral molecular biological detection. The primer set of the present invention includes an upstream primer, a downstream primer, and a probe. The sequence of the upstream primer is depicted in SEQ ID NO. 1, the sequence of the downstream primer is depicted in SEQ ID NO. 2, and the sequence of the probe is depicted in SEQ ID NO. 3. Using the primer set of the present invention, the lower limit of detection (LLD) of the PDEV is 102 copies/pL, featuring strong specificity, high sensitivity, and good between-run and within-run reproducibilities. One hundred and fifty clinical porcine fecal samples were assayed by the present invention, and the result showed a PEDV positive rate of 74.6%.

Description

PRIMER SET, KIT, AND METHOD FOR DETECTING PORCINE EPIDEMIC DIARRHEA VIRUS

TECHNICAL FIELD

The present invention relates to the field of viral molecular biological detection, and in particular to a primer set, a kit, and a method for detecting porcine epidemic diarrhea virus (PEDV).

BACKGROUND

Porcine epidemic diarrhea virus (PEDV) is a common virus that causes porcine acute intestinal infectious diseases, featuring susceptibility, very high morbidity, certain seasonality, and similar clinical symptoms to porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine kobuvirus (PKV), porcine reproductive and respiratory syndrome virus (PRRSV), and foot and mouth disease virus (FMDV).

There have been many methods for detecting PEDV in China and overseas, including ELISA, RT-PCR, and fluorogenic quantitative PCR assays, but there is a difference in sensitivity among different methods. Among the foregoing methods, TaqMan probe fluorescent quantitative RT-PCR assay has higher sensitivity and specificity compared with other methods. So far, little research has been done on detecting PEDV by TaqMan probe fluorescent quantitative RT-PCR assay, and the existing detection methods cannot deal well with the diagnosis of clinical PEDV samples and epidemiological investigation due to their poor sensitivity, low positive rate, and bad reproducibility.

SUMMARY

In view of this, the objective of the present invention is to provide a primer set, a kit, and a method for detecting porcine epidemic diarrhea virus (PEDV). The positive rate of the virus is high, and the specificity and sensitivity of the method satisfy the requirements of diagnosis of a clinical PEDV sample and epidemiological investigation.

In order to achieve the foregoing invention objective, the present invention provides the following technical solutions:

The present invention provides a primer set for detecting PEDV The primer set includes an upstream primer, a downstream primer, and a probe, where the sequence of the upstream primer is depicted in SEQ ID NO. 1, the sequence of the downstream primer is depicted in SEQ ID NO. 2, and the sequence of the probe is depicted in SEQ ID NO. 3.

The present invention further provides a kit for detecting PEDV, including the upstream primer, the downstream primer, and the probe according to claim 1.

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Preferably, concentrations of both the upstream and downstream primers are 10 μΜ, and the concentration of the probe is 5 puM.

The present invention further provides a method for detecting PEDV, including the following steps: (1) extracting analyte RNAto obtain a template RNA;

(2) extracting PEDV RNA to obtain a positive standard; and (3) using the primer set or the kit to conduct the template RNA and the positive standard on TaqMan fluorogenic quantitative PCR;

there is no chronological relationship between step (1) and step (2).

Preferably, in the system of the TaqMan fluorogenic quantitative PCR of step (3), 20 pL each of the reaction system includes: 10 pL of 2x RT-PCR buffer, 0.4 pL of ExTaq HS, 0.4 pL of reverse transcriptase mixture, 0.4 pL each of upstream and downstream primers, 0.6 pL of probe, 5.8 pL of RNase-free water, and 2 pL of template RNA or positive standard;

the reaction procedure of the TaqMan fluorogenic quantitative PCR is: preheating at 42°C for 5 min, predenaturation at 95°C for 10 s, denaturation at 95°C for 5 s, and annealing and extension at 57°C for 20 s; the procedure of the denaturation and annealing extends for a total of 40 cycles.

The present invention provides a primer set, a kit, and a method for detecting PEDV. The primer set includes an upstream primer, a downstream primer, and a probe, where the sequence of the upstream primer is depicted in SEQ ID NO. 1, the sequence of the downstream primer is depicted in SEQ ID NO. 2, and the sequence of the probe is depicted in SEQ ID NO. 3. In an embodiment of the present invention, the primer set could specifically detect PEDV, but the test was negative for porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine kobuvirus (PKV), porcine reproductive and respiratory syndrome virus (PRRSV), and foot and mouth disease virus (FMDV). The primer set had a good linear relationship within the range from 10⁸ to 10² copies/pL, the detection limit of sensitivity was 10²copies/pL, its sensitivity was high, and coefficients of variation (CVs) of both between-run and within-run reproducibilities were below 1%, with good reproducibility. One hundred and fifty clinical porcine fecal samples collected between 2017 and 2018 were assayed by the present invention, and their positive rate was 74.6%; however, conventional RT-PCR assay was used for controlled trial, and the positive rate was merely 50.6%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of a PCR amplified product of PEDV N gene provided by an embodiment of the present invention;

FIG. 2 shows a standard curve plot of TaqMan real-time fluorogenic quantitative PCR of PEDV provided by an embodiment of the present invention;

FIG. 3 shows a graph of kinetic curves of TaqMan real-time fluorogenic quantitative PCR of

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PEDV provided by an embodiment of the present invention;

FIG. 4 shows a graph of sensitivity test results of TaqMan real-time fluorogenic quantitative PCR of PEDV provided by an embodiment of the present invention;

FIG. 5 shows a graph of specificity test results of TaqMan real-time fluorogenic quantitative PCR of PDCoV provided by an embodiment of the present invention.

DETAILED DESCRIPTION

The present invention provides a primer set for detecting porcine epidemic diarrhea virus (PEDV). The primer set includes an upstream primer, a downstream primer, and a probe, where the sequence of the upstream primer is depicted in SEQ ID NO. 1, the sequence of the downstream primer is depicted in SEQ ID NO. 2, and the sequence of the probe is depicted in SEQ ID NO. 3.

For the primers of the present invention, results are preferably compared according to a DNA Star software sequence of PEDV N gene, and a pair of primers and a probe are designed by means of Primer Express5.0, a probe and primer design software, as shown in Table 1:

Table 1 Primers and TaqMan probe sequences of PEDV

	Name of the primer	Primer sequence	SEQ ID NO.
Upstream	PEDV-F	CTATAAGGTTGCTACTGGCGT
primer
Downstream	PEDV-R	GAACTCGGATTACTCACAGCT	-
primer
Probe	PEDV-Probe	FAM-ACAGTCGCCAAGGCCACTACAACAAT-BHQ1

In the kit of the present invention, concentrations of both the upstream and downstream primers are 10 μΜ, and the concentration of the probe is 5 puM. The kit of the present invention further includes: 2x RT-PCR buffer, ExTaq HS, reverse transcriptase mixture, and RNase-free water.

(2) extracting PEDV RNA to obtain a positive standard; and (3) using the primer set or the kit to conduct the template RNA and the positive standard on

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TaqMan fluorogenic quantitative PCR;

there is no chronological relationship between step (1) and step (2).

When the present invention detects PEDV, the analyte RNA is extracted and reversely transcribed to obtain a template cDNA. The analyte of the present invention preferably includes pig feces. The extraction method of the RNA is not specifically limited in the present invention, and conventional methods in the field may be used.

The present invention extracts PEDV RNA to obtain a positive standard. Extraction of the RNA of the present invention is preferably conducted in accordance with the operating instructions of RNeasy Mini kit(50)QIAGen, specifically including the steps of: pipetting 350 pL of viral suspension into a 1.5 mL centrifuge tube, adding isovolumetric RLT and 3.5 pL of β-mercaptoethanol, standing on ice for 5-10 min, then adding 700 pL of absolute alcohol rapidly, slowly mixing well, taking out a minicolumn, adding 700 pL of mixture, centrifuging for 15 s at 12000 rpm/min, discarding the fluid from a collection tube, and repeating once; then adding 700 pL of RW1, standing on ice for 1 min, centrifuging for 15 s at 12000 rpm/min, changing a new collection tube, adding 500 pL of RPE, centrifuging for 15 s at 12000 rpm/min, discarding the fluid from the collection tube, adding 500 pL of RPE, centrifuging for 2 min at 12000 rpm/min, placing the minicolumn into an RNase-free 1.5 mL centrifuge tube, adding 30 pL of RNase-free water, standing for 1 min, centrifuging for 1 min at 12000 rpm/min, taking out the minicolumn, and storing the extracted RNA in a freezer at -20°C for use.

The preparation method of the positive standard of the present invention includes: using cDNA obtained by reverse transcription of RNA as a template, conducting specific primers containing fluorescent quantitative target fragments on RT-PCR amplification, and setting up a negative control (without template cDNA). The sequences of the specific primers containing fluorescent quantitative target fragments are preferably:

EDVFLTGCATTCCAGTGCTTGGAGCA (SEQ ID NO.4);

PEDV RLAATGAAGCACTTTCTCACTATCT (SEQ ID NO.5).

In the present invention, the amplified and purified product is ~630 bp, and the PCR amplified product of PEDV N gene is depicted in FIG. 1. The DNA of the amplified product is assayed by 1.5% agarose gel electrophoresis and result was observed. Amplification result is in consistent with the designed target fragment. In the present invention, positive PCR product is purified, ligated to a PMD-20T carrier, and transformed into DH5a competent cells; white transformants are screened out by means of blue-white selection, culture is expanded, and then plasmids are extracted, which are re-sequenced by Beijing Genomics Institute Group after restriction enzyme digestion. With successfully sequenced positive plasmid as in vitro transcription standard and using an RNA transcript of T7 in vitro Transcription Kit, an RNA

2019100992 02 Sep 2019 standard is purified and obtained; after purification and measurement with Nanodrop 2000 Spectrophotometer, the concentration of the extracted RNA standard is 839.3 ng/pL, and OD260/OD280 is 1.96, storing at -70°C for use.

After the template RNA and the positive standard are obtained, the present invention uses the primer set or the kit to conduct the template RNA and the positive standard on TaqMan fluorogenic quantitative PCR.

In the system of the TaqMan fluorogenic quantitative PCR of the present invention, 20 pL each of the reaction system preferably includes: 10 pL of 2x RT-PCR buffer, 0.4 pL of ExTaq HS, 0.4 pL of reverse transcriptase mixture, 0.4 pL each of upstream and downstream primers, 0.6 pL of probe, 5.8 pL of RNase-free water, and 2 pL of template RNA or positive standard. The reaction procedure of the TaqMan fluorogenic quantitative PCR of the present invention is preferably: preheating at 42°C for 5 min, predenaturation at 95°C for 10 s, denaturation at 95°C for 5 s, and annealing and extension at 57°C for 20 s; the procedure of the denaturation and annealing extends for a total of 40 cycles.

In the present invention, the criteria for judgment of positivity or negativity of the analyte are preferably that it will be:

(1) positive: if the analyte has a Ct value of <35.0, and shows a curve with an obvious exponential growth phase;

(2) suspicious: if the analyte has a Ct value of >35.0 and there is a sample with a typical amplification curve, then the test will be repeated; and (3) negative: if the Ct value of the analyte is undetectable, and there is no obvious amplification curve.

The primer set, kit, and method for detecting PEDV will be described in detail below in ligation with the embodiment of the present invention and should not be construed as limiting the scope of the invention.

Embodiment 1

1. Extraction of PEDV RNA

Extraction of PEDV RNA was conducted in accordance with the operating instructions of RNeasy Mini kit(50)QIAGen, specifically including the steps of: pipetting 350 pL of viral suspension into a 1.5 mL centrifuge tube, adding isovolumetric RLT and 3.5 pL of β-mercaptoethanol, standing on ice for 5-10 min, then adding 700 pL of absolute alcohol rapidly, slowly mixing well, taking out a minicolumn, adding 700 pL of mixture, centrifuging for 15 s at 12000 rpm/min, discarding the fluid from a collection tube, and repeating once; then adding 700 pL of RW1, standing on ice for 1 min, centrifuging for 15 s at 12000 rpm/min, changing a new collection tube, adding 500 pL of RPE, centrifuging for 15 s at 12000 rpm/min, discarding the

2019100992 02 Sep 2019 fluid from the collection tube, adding 500 pL of RPE, centrifuging for 2 min at 12000 rpm/min, placing the minicolumn into an RNase-free 1.5 mL centrifuge tube, adding 30 pL of RNase-free water, standing for 1 min, centrifuging for 1 min at 12000 rpm/min, taking out the minicolumn, and storing the extracted RNA in a freezer at -20°C for use.

2. Preparation of RNA standard

Specific primers containing fluorescent quantitative target fragments were used for RT-PCR amplification. A product of 630 bp was obtained after PCR amplification and purification. PCR amplified product of PEDV N gene is depicted in FIG. 1. The DNA of the amplified product was assayed by 1.5% agarose gel electrophoresis and result was observed. Amplification result was in consistent with the designed target fragment. Positive PCR product was purified, ligated to a PMD-20T carrier, and transformed into DH5a competent cells; white transformants were screened out by means of blue-white selection, culture was expanded, and then plasmids were extracted, which were re-sequenced by Beijing Genomics Institute Group after restriction enzyme digestion. With successfully sequenced positive plasmid as in vitro transcription standard and using an RNA transcript of T7 in vitro Transcription Kit, an RNA standard was purified and obtained; after purification and measurement with Nanodrop 2000 Spectrophotometer, the concentration of the extracted RNA standard was 839.3 ng/pL, and OD260/OD280 was 1.96, storing at -70°C for use.

3. Fluorogenic quantitative PCR system and optimization of reaction procedure

Primers: PEDV-F: CTATAAGGTTGCTACTGGCGT;

PEDV-R: GAACTCGGATTACTCACAGCT;

PEDV-Probe: FAM-ACAGTCGCCAAGGCCACTACAACAAT-BHQ1;

Concentrations of primers and probe were 0.2 mol/L and 0.15 mol/L, respectively.

Amplified reaction system: 2* RT-PCR buffer, E*Taq HS, reverse transcriptase mixture, forward and reverse primers, probe, and RNase-free water;

Comparative experiment was conducted on the amplified reaction system,

The amount of each substance is depicted in Table 2:

Table 2 The amount of each substance in the optimization experiment of the amplified reaction system (pL)

2xbpffer

ExTaq HS

RT-Enzy me

Primer F

Primer R

Probe

Water

RNA

Final probe concentr ation

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10	0.4	0.4	0.4	0.4	0.2	6.2	2	0.05
10	0.4	0.4	0.4	0.4	0.3	6.1	2	0.075
10	0.4	0.4	0.4	0.4	0.4	6.0	2	0.1
10	0.4	0.4	0.4	0.4	0.5	5.9	2	0.125
10	0.4	0.4	0.4	0.4	0.6	5.8	2	0.15
10	0.4	0.4	0.4	0.4	0.7	5.7	2	0.175
10	0.4	0.4	0.4	0.4	0.8	5.6	2	0.2

By comparison of the gradient of probe concentrations added in the reaction system, the optimal probe concentration was determined as 0.15 mol/ L; for primer concentration, empirical 0.2 mol/L was the optimal concentration. From the Table 2, the optimal amplified reaction system was: 10 pL of 2^χ RT- PCR buffer, 0.4 pL of ExTaq HS, 0.4 pL of reverse transcriptase mixture, 0.4 pL of forward primer, 0.4 pL of reverse primer, 0.6 pL of probe, and 5.8 pL of RNase-free water;

pL each of RNA and negative control was added to a total volume of 20 pL of amplified reaction system, a well-prepared PCR tube was preheated at 42°C for 5 min, predenatured at 95°C for 10 s, denatured at 95°C for 5 s, and annealed and extended at 57°C for 20 s; the procedure of the denaturation and annealing extended for a total of 40 cycles.

(4) Result determination: The corresponding Ct values were read by means of fluorogenic quantitative PCR system built-in software in order to determine the positivity or negativity of an analyte.

4. Establishment of fluorogenic quantitative standard curve

The concentration of the purified RNA transcript was determined. Using a calculation formula of copy number, i.e., copy concentration = [6.023 ^χ 10^{23 χ} (concentration ng/pL ^χ10'⁹)]/base number ^χ 660, the copy number was calculated as 2.4^χ10¹² copies/pL; after 10-fold serial dilution, fluorogenic quantitative PCR was conducted with 2.4 ^χ 10⁹, 2.4 ^χ 10⁸, 2.4 ^χ 10⁷, 2.4 ^x 10⁶, 2.4 ^x 10⁵, 2.4 ^χ 10⁴ copies/pL standards as templates, and an amplification kinetic curve for detecting PEDV was obtained, as shown in FIG. 3 (in FIG. 3, 1-6 indicate that RNA template concentrations are 2.4 ^χ 10⁹, 2.4 ^χ 10⁸, 2.4 ^χ 10⁷, 2.4 ^χ 10⁶, 2.4 ^χ 10⁵, and 2.4 ^χ 10⁴copies/pL, respectively), and the corresponding standard curve was calculated; combined with the contents depicted in FIG. 2, there was a good linearity when the concentration of fluorogenic quantitative PCR standard of PEDV ranged from 2.4 ^χ 10⁸ to 2.4 ^χ 10² copies/pL, with Y = -3.365 x lg(x) + 49.36, an amplification efficiency of 98.2% and a correlation coefficient (R²) of 1.000.

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5. Sensitivity test

After 10-fold serial dilution of the RNA transcript, fluorogenic quantitative PCR was conducted with 2.4 χ 10⁸, 2.4 χ 10⁷, 2.4 χ 10⁶, 2.4 χ 10⁵, 2.4 χ 10⁴, 2.4 χ 10³, 2.4 χ 10², 2.4 χ 10¹copies/pL standards as templates in order to determine the sensitivity of the method. If the analyte has a Ct value of <35.0, and shows a curve with an obvious exponential growth phase, it will be positive; if the analyte has a Ct value of >35.0 and there is a sample with a typical amplification curve, then the test will be repeated; if the Ct value of the analyte is undetectable, and there is no obvious amplification curve, it will be negative. With a Ct value of <35.0, the analyte was positive. Test results are depicted in FIG. 4 (in FIG. 4, 1-8 indicate that PEDV RNA standard concentrations are 2.4 χ 10⁸, 2.4 χ 10⁷, 2.4 χ 10⁶, 2.4 χ 10⁵, 2.4 χ 10⁴, 2.4 χ 10³, 2.4 χ 10², 2.4 χ 10¹ copies/pL, respectively). Combined with FIG. 2, it could be seen that when the copy number of the standard was 2.4 χ 10² copies/pL, it remained detectable, but the blank control was not amplified, suggesting that the lower limit of detection (LLD) of the detection method could be up to 240 copies/pL.

7. Specificity test

RNA extraction kit was used to extract RNAs of porcine reproductive and respiratory syndrome virus (PRRSV), foot and mouth disease virus (FMDV), porcine kobuvirus (PKV), porcine transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) and reversely transcribe them into cDNAs, which were amplified by PCR with their respective primers to obtain the corresponding specific fragments; the five viruses were verified as positive strains, and the reversely transcribed cDNAs could be amplified normally. Fluorogenic quantitative PCR amplification was carried out with cDNAs of PRRSV, FMDV, PKV, TGEV, and PDCoV as templates, and negative and positive controls were set up. Test results are shown in FIG. 5 (where all numbers separately indicate: 1 as PEDV, 2 as PRRSV, 3 as FMDV, 4 as PKV, 5 as TGEV, and 6 as PDCoV). It could be seen that, for PRRSV, FMDV, PKV, TGEV, PDCoV, and negative control, fluorogenic quantitative PCR showed no amplification curve, but only PEDV showed a good amplification curve. It was indicated that the TaqMan real-time fluorogenic quantitative PCR assay established by the experiment had good specificity.

8. Reproducibility test (1) Between-run reproducibility test: Taking three different concentrations (2.4 χ 10⁷, 2.4 χ 10⁶, and 2.4 χ 10⁵ copies/pL) of RNA standard samples, reproducibility test was carried out in octuplicate under the same conditions; test results are shown in Table 3:

Table 3 Results of the between-run reproducibility test

Sample Between-run reproducibility Mean cycle CV%

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2.4xl0⁷	24.46	24.36	24.21	24.52	24.12	24.33	24.10	24.23	24.29	0.60%
2.4xl0⁶	27.49	27.42	27.35	27.32	27.23	27.03	27.92	27.66	27.42	0.98%
2.4xl0⁵	31.43	31.40	31.33	31.47	31.43	31.39	31.44	31.41	31.41	0.13%

From Table 3, coefficients of variation (CVs) of 2.4 x 10⁷, 2.4 x 10⁶, and 2.4 x 10⁵ copies/pL RNA standards were 0.60%, 0.98%, and 0.13%, respectively, all of which were below 1%, indicating that the TaqMan real-time fluorogenic quantitative PCR assay for PEDV established by the experiment had good between-run reproducibility.

(2) Within-run reproducibility test: Taking three different concentrations (2.4 x 10⁷, 2.4 x 10⁶, and 2.4 x 10⁵ copies/pL) of RNA standard samples, reproducibility test was carried out in octuplicate under the same conditions, and test results were tested and analyzed. Their CVs were calculated: CV (β) = standard deviation (SD)/mean (x). Results are depicted in Table 4:

Table 4 Within-run reproducibility of real-time fluorogenic quantitative PCR assay for

PEDV

Sample Within-run reproducibility Mean cycle CV% threshold

2.4xl0⁷	25.19	25.28	25.39	25.42	25.50	25.39	25.42	25.32	25.36	0.38%
2.4xl0⁶	28.05	28.13	28.30	28.29	28.15	28.95	28.26	28.45	28.32	0.99%
2.4xl0⁵	32.23	32.41	32.32	32.28	32.32	32.29	32.31	32.36	32.31	0.16%

From Table 4, CVs of 2.4 x 10⁷, 2.4 x 10⁶, and 2.4 x 10⁵ copies/pL RNA standards were 0.38%, 0.99%, and 0.16%, respectively, all of which were below 1%, indicating that the TaqMan real-time fluorogenic quantitative PCR assay for PEDV established by the experiment had good within-run reproducibility.

8. Detection of clinical sample

One hundred and fifty porcine fecal samples were collected from Gansu, Xinjiang, Henan, Shaanxi, and Hunan between 2017 and 2018. The porcine fecal samples collected were assayed by means of the method established by the present invention. Meanwhile, samples were also assayed by conventional RT-PCR assay, and negative and positive controls were set up. Detection results were compared with those by conventional RT-PCR assay. Conventional RT-PCR assay was done by means of specific primers. For detailed method, refer to operating instructions for

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RT-PCR kit. Detection results are shown in Table 5:

Table 5 Detection results of clinical samples by real-time fluorogenic quantitative PCR assay for PEDV

Amount of positive sample and positive rate

Sample collection----Sample size Conventional Positive rate of Positive rate of site qPCR

PCR conventional PCR qPCR

Gansu	36	16	44.4%	32	88.8%
Xinjiang	28	12	42.8%	19	67.8%
Henan	50	23	46.0%	28	56.0%
Shaanxi	16	12	75.0%	16	100.0%
Hunan	20	13	65.0%	17	85.0%

From Table 5, 76 positive samples could be detected by conventional RT-PCR assay, with a positive rate of 50.6%, while 112 positive samples could be detected by the TaqMan real-time fluorogenic quantitative PCR assay of the present invention, with a positive rate of 74.6%, indicating that the TaqMan real-time fluorogenic quantitative PCR assay established by the present invention was more sensitive.

The present invention provides a primer set, a kit, and a method for detecting PEDV, featuring strong specificity, high sensitivity, and good between-run and within-run reproducibilities with regard to detection of PEDV.

The foregoing descriptions are only preferred implementation manners of the present invention. It should be noted that for a person of ordinary skill in the art, several improvements and modifications may further be made without departing from the principle of the present invention. These improvements and modifications should also be deemed as falling within the protection scope of the present invention.

Claims

1. A primer set for detecting porcine epidemic diarrhea virus (PEDV), wherein the primer set comprises an upstream primer, a downstream primer, and a probe, the sequence of the upstream primer is depicted in SEQ ID NO. 1, the sequence of the downstream primer is depicted in SEQ ID NO. 2, and the sequence of the probe is depicted in SEQ ID NO. 3.

(2) extracting PEDV RNA to obtain a positive standard; and (3) using the primer set according to claim 1 or the kit according to claim 2 or 3 to conduct the template RNA and the positive standard on TaqMan fluorogenic quantitative PCR; wherein there is no chronological relationship between step (1) and step (2).

2. A kit for detecting porcine epidemic diarrhea virus (PEDV), comprising the upstream primer, the downstream primer, and the probe according to claim 1.

3. The kit according to claim 2, wherein concentrations of both the upstream and downstream primers are 10 μΜ, and the concentration of the probe is 5 puM.

4. A method for detecting porcine epidemic diarrhea virus (PEDV), comprising the following steps: (1) extracting analyte RNAto obtain a template RNA;

5. The method according to claim 4, wherein, in the system of the TaqMan fluorogenic quantitative PCR of step (3), 20 pL each of the reaction system includes: 10 pL of 2x RT-PCR buffer, 0.4 pL of ExTaq HS, 0.4 pL of reverse transcriptase mixture, 0.4 pL each of upstream and downstream primers, 0.6 pL of probe, 5.8 pL of RNase-free water, and 2 pL of template RNA or positive standard;