Micro-droplet type digital PCR (Polymerase Chain Reaction) primer group and probe for detecting porcine epidemic diarrhea virus

Abstract

The invention aims to provide the microdroplet type digital PCR primer group and the probe for detecting the porcine epidemic diarrhea virus, so that the porcine epidemic diarrhea virus can be more sensitively and efficiently detected. The primer group is used for amplifying all or part of fragments with the sequence of SEQ ID NO: 1, the nucleotide sequence of an upstream primer of the primer group is SEQ ID NO: 2, the nucleotide sequence of a downstream primer of the primer group is SEQ ID NO: 3, and the sequence of a probe is SEQ ID NO: 4. Through optimized screening of the primers and the probes, a primer and probe combination with the best effect is obtained, and a reaction system and reaction conditions are optimized, so that the detection sensitivity of the method established by the invention reaches 2.13 copies / reaction on the basis of ensuring specificity and repeatability, and the requirement of rapid virus detection can be met.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus molecular biology detection, and specifically relates to a primer set and a probe for detecting porcine epidemic diarrhea virus based on droplet digital PCR. Background Art

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious enteric disease of pigs caused by porcine epidemic diarrhea virus (PEDV). It is characterized by vomiting, acute watery diarrhea, and dehydration in newborn piglets. Pigs of all ages are susceptible to infection, with the most serious disease in newborn piglets, and the mortality rate can be as high as 100%. Currently, my country's Ministry of Agriculture and Rural Affairs has classified it as a Category II animal epidemic disease.

[0003] Porcine epidemic diarrhea virus (PEDV) belongs to the order Nidovirales, family Coronaviridae, genus Alphacoronavirus. The PEDV genome is about 28k long and is a single-stranded positive-strand RNA virus, containing a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR) and seven open reading frames (ORFs): two encoding polyproteins (pp1a and pp1b) and the remaining five ORFs encoding five important proteins: spike (S), ORF3, envelope (E), membrane (M) and nucleocapsid (N). Genetic evolution analysis based on the full-length genome sequence of PEDV showed that PEDV is divided into two major categories in genetic evolution: the classical PEDV strain (G1) that began in the 1870s and the emerging PEDV strain (G2) that appeared in late 2010. Among them, variant strains include G1 type, G2 type or non-S gene insertion or deletion strains. After the emergence of a mutant strain of porcine epidemic diarrhea virus in 2010, the disease quickly spread around the world, often in an explosive manner, causing huge economic losses to the global pig industry. At present, porcine epidemic diarrhea caused by the new mutant strain is still a major problem that plagues the healthy development of the pig industry in major pig producing areas in my country and even the world.

[0004] Although coronavirus is the RNA virus with the largest known genome, the 3'-5' exoribonuclease activity of its non-structural protein nsp14 can remove mismatches and embedded nucleic acid analogs, so coronavirus has maintained its genome stability and high fidelity during evolution. Among them, the nucleoprotein (N protein) encoded by the N gene is the structural protein with the highest content in PEDV, providing a structural basis for the helical nucleocapsid and playing a major role in the viral replication cycle. The N gene consists of 1326 nucleotides, encodes 441 amino acids, and has a molecular mass of approximately 57ku. The N protein of PEDV is a multifunctional protein that participates in biological processes related to the survival of PEDV. The N protein has a great connection with the genome and binds to the viral particle RNA to provide a structural basis for the helical nucleocapsid. In the early stage of PEDV infection, a large amount of N protein is detected in the infected cells, inducing the production of a large amount of antibodies. These characteristics of the N protein make it an excellent target molecule in the diagnosis of PEDV, and some PEDV antigen detection methods are based on the N gene.

[0005] At present, the main diagnostic methods for PEDV are traditional methods, such as virus isolation and identification, electron microscopy diagnosis; immunological detection technology, including enzyme-linked immunosorbent assay, immunochromatographic test strip detection technology; molecular biology technology, including RT-PCR, real-time fluorescence quantitative RT-PCR, loop-mediated isothermal amplification technology, in situ hybridization technology and other PCR-based pathogen nucleic acid detection technologies. It solves the problems of time-consuming and labor-intensive traditional etiological diagnosis and window period of immunoserology, and has become one of the most commonly used methods for animal disease pathogen detection in veterinary laboratories. However, its sensitivity still needs to be improved, especially when the virus content is extremely low, false negative or suspicious results often occur, so it is necessary to establish a more sensitive and accurate method. Summary of the invention

[0006] The purpose of the present invention is to provide a droplet digital PCR primer set and probe for detecting porcine epidemic diarrhea virus, which can detect porcine epidemic diarrhea virus more sensitively and efficiently, thereby making up for the shortcomings of the prior art.

[0007] The present invention first provides primers and probes for detecting porcine epidemic diarrhea virus based on a droplet digital PCR method. The primers and probes are used to detect a nucleic acid fragment of a conservative sequence of porcine epidemic diarrhea virus whose sequence is SEQ ID NO: 1.

[0008] Furthermore, the specific sequences of the primers and probes are as follows:

[0009] Upstream primer PEDV-F3: 5′-CCGTGGTGAGCGAATTGAA-3′ (SEQ ID NO: 2);

[0010] Downstream primer PEDV-R3: 5′-GGTCCTGTTCCGAGGTAGTAGAAA-3′ (SEQ ID NO: 3);

[0011] Probe PEDV-P3: 5′-FAM-AACCTTCCAATTGGC-MGB-3′ (SEQ ID NO: 4).

[0012] The primer set and probe provided by the present invention are used to prepare a PCR detection kit for detecting porcine epidemic diarrhea virus;

[0013] The PCR detection kit is a droplet digital PCR detection kit.

[0014] In one aspect, the present invention also provides a method for detecting porcine epidemic diarrhea virus for non-disease treatment purposes, which uses the above-mentioned primer set and probe for detection.

[0015] The method for detecting porcine epidemic diarrhea virus based on droplet digital PCR method comprises the following steps:

[0016] 1) Extract nucleic acid from the sample tissue to be tested according to the instructions of the DNA / RNA extraction kit and store it at -20°C for future use; if the sample is a swab, milk sample, or bacterial culture, extract nucleic acid using lysis, magnetic bead enrichment, washing, elution, and other steps;

[0017] 2) Connect the microdroplet preparation instrument (Sinafo SQD-X4) to the power supply for preheating.

[0018] 3) Add 2 μL of reverse transcriptase, 4 μL of water, 1.5 μL of upstream and downstream primers at a concentration of 10 μM and 0.5 μL of 10 μM probe to 10 μL of reaction buffer (dPCR Mix), and then add 2 μL of the nucleic acid sample obtained in step 1), mix well, and obtain an aqueous phase reaction system;

[0019] 4) The eight rows of the prepared 20 μL aqueous phase reaction system are placed in the instrument, and the oil tank is filled with microdroplet generation oil.

[0020] 5) Install the droplet generation pipette tip.

[0021] 6) Close the chamber door and set the reaction program: 50℃ for 20min; 95 for 3min; 94℃ for 15s, annealing temperature at 58℃ for 45s, 40 cycles; heating and cooling rate of 2.0℃ / s, and start the digital PCR experiment.

[0022] Furthermore, during the detection, the probe concentration was 250 nmol / L, the upstream and downstream concentrations were both 750 nmol / L, and the annealing temperature of the reaction system was 58°C.

[0023] The reaction system is 20 μL: 2×One-step ddPCR supermix 10 μL, Reversetranscriptase (enzyme) 2.0 μL, upstream and downstream primers 0.75 μL each, both with a final concentration of 750 nmol / L, probe 0.5 μL, both with a final concentration of 250 nmol / L, template 2 μL, and water 4 μL.

[0024] The ddPCR reaction program was: 50°C for 20 min; 95°C for 3 min; 94°C for 15 s, annealing temperature of 58°C for 45 s, 40 cycles, and a heating and cooling rate of 2.0°C / S.

[0025] The present invention obtains the best primer-probe combination through optimized screening of primers and probes, and optimizes the reaction system and reaction conditions, so that the method established by the present invention can achieve a detection sensitivity of 2.13 copies / reaction on the basis of ensuring specificity and repeatability, which can meet the requirements of rapid virus detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : The result diagram of the primer-probe combination screening of the present invention;

[0027] Figure 2 : The result diagram of the optimal concentration of primers and probes of the present invention;

[0028] Figure 3 : The result diagram of the optimal annealing temperature of the primer probe of the present invention;

[0029] Figure 4 : The sensitivity test results of the present invention, from left to right are 2.13×10 4 copies / μL~2.13×10 0 copies / μL;

[0030] Figure 5 : Specificity experimental result diagram of the present invention;

[0031] Figure 6 : Clinical sample test result diagram of the present invention. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings and attached tables in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034]

[0035] The highly conserved sequences obtained through screening are used as the target gene fragments for detection, and primers and probes are designed, screened and tested.

[0036] Based on the conservative sequence of the N gene of porcine epidemic diarrhea virus BJ-2011-C, Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize a DNA plasmid, and the vector plasmid was pMD-18T.

[0037] 1) Primer design

[0038] Based on the conservative sequence of the N gene of porcine epidemic diarrhea virus, primers for detection by droplet digital PCR method were designed with a length of 20 to 24 bp. The sequences are shown in Table 1 below.

[0039] Table 1: Sequence information of designed primers

[0040]

[0041] Three pairs of primers and probes were tested and verified. The results showed that with PEDV virus nucleic acid as the template, the third pair of primers and probes separated the amplified droplets of the same template better under the experimental conditions, and the third pair of primers and probes had a higher concentration, and the separation effect could meet the experimental expectations. In order to meet the needs of later experiments, the third pair of primers and probes was still selected as the primer and probe combination for later experiments, specifically:

[0042] PEDV-F3: 5'-GGTGTTGATCTTGGGTGACT-3';

[0043] PEDV-R3: 5'-GGTCCTGTTCCGAGGTAGTAGAAA-3';

[0044] PEDV-P3: 5'-FAM-AACCTTCCAATTGGC-MGB-3'.

[0045] 2) Optimal concentrations of primers and probes

[0046] To determine the optimal concentrations of primers and probes, three sets of primer and probe concentrations were set according to the gradient for droplet digital PCR, as shown in Table 2.

[0047] Table 2: Primer probe concentration screening table

[0048] 1 2 3 Primers 600nmol / L 700nmol / L 900nmol / L Probe 150nmol / L 250nmol / L 350nmol / L

[0049] like Figure 2As shown in the results, when the final concentration of the upstream and downstream primers was 750 nmol / L and the probe concentration was 250 nmol / L, at the same concentration of the plasmid containing the PEDV N gene, the copy value was the highest and the value was the most stable. Therefore, the final concentration of the upstream and downstream primers of 750 nmol / L and the probe concentration of 250 nmol / L were selected as the preferred concentrations of the droplet number.

[0050] 3) Optimal annealing temperature for the reaction

[0051] In order to determine the optimal annealing temperature of primers and probes, four groups of temperatures were set according to the gradient: 55°C, 58°C, 60°C, and 65°C.

[0052] like Figure 3 The results show that the copy number is higher when the annealing temperature is 55°C, 58°C, and 60°C, and the droplets are more scattered when the annealing temperature is 65°C. In order to ensure the specificity of the probe, the temperature of 58°C is selected as the preferred annealing temperature for droplet digital PCR.

[0053] 4) Primers, probes and plasmids were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0054] 5) The reaction system for detecting porcine epidemic diarrhea virus based on droplet digital PCR is 20 μL: One-step ddPCR supermix 10 μL, reverse transcriptase (enzyme) 2 μL, upstream and downstream primers 0.75 μL each, the final concentration is 750 nmol / L, probe 0.5 μL, the final concentration is 250 nmol / L, template 2 μL, ddH 2 O 4μL. The ddPCR reaction program was: 50℃20min; 95 3min; 94℃15s, annealing temperature 58℃45s, 40 cycles; heating and cooling speed 2.0℃ / s.

[0055] Example 2

[0056] The sensitivity, repeatability and specificity of the primers of the present invention to the probe are tested below.

[0057] 1. Sensitivity test

[0058] 1) Primer probe

[0059] Upstream primer PEDV-F3: 5′-CCGTGGTGAGCGAATTGAA-3′;

[0060] Downstream primer PEDV-R3: 5′-GGTCCTGTTCCGAGGTAGTAGAAA-3′;

[0061] Probe PEDV-P3: 5′-FAM-AACCTTCCAATTGGC-MGB-3′.

[0062] The probe was modified with a fluorescent reporter group (FAM) and a fluorescent quencher group (MGB).

[0063] 2) Prepare plasmid positive standards, which are:

[0064] Positive standard 1, containing 2.13×10 4 copies / μl porcine epidemic diarrhea virus plasmid non-infectious RNA fragment;

[0065] Positive standard 2, containing 2.13×10 3 copies / μl porcine epidemic diarrhea virus plasmid non-infectious RNA fragment;

[0066] Positive standard 3, containing 2.13×10 2 copies / μl porcine epidemic diarrhea virus plasmid non-infectious RNA fragment;

[0067] Positive standard 4, containing 2.13×10 1 copies / μl porcine epidemic diarrhea virus plasmid non-infectious RNA fragment;

[0068] Positive standard 5, containing 2.13×10 0 copies / μl porcine epidemic diarrhea virus plasmid non-infectious RNA fragment;

[0069] 3) Perform detection according to the above-mentioned droplet digital PCR method for detecting porcine epidemic diarrhea virus.

[0070] 4) Sensitivity test results

[0071] The results showed that the PEDV ddPCR detection method established in the present invention can detect a minimum of 2.13 copies / μL ( Figure 4 ).

[0072] 2. Specificity experiment

[0073] 1) Primers, probes and negative control products are the same as those in Example 1.

[0074] 2) Sample nucleic acids of porcine rotavirus, porcine viral gastroenteritis virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, and porcine circovirus used in specific experiments are preserved by this laboratory.

[0075] 3) Sample extraction method:

[0076] The samples were pretreated first, and then the nucleic acid was extracted using the Tianlong Nucleic Acid Extractor's automatic DNA / RNA extraction method and stored at -20°C for later use.

[0077] 4) Perform detection according to the above-mentioned droplet digital PCR method.

[0078] 4) Specificity test results:

[0079] Test results such as Figure 5 As shown: The results showed that only porcine epidemic diarrhea virus was significantly amplified, while porcine rotavirus, porcine viral gastroenteritis virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, and porcine circovirus nucleic acid had no obvious amplification, showing good specificity.

[0080] 3. Repeatability experiment

[0081] 1) Primers, probes and negative control products are the same as those in Example 1.

[0082] 2) Take two tubes of the national standard secondary standard material porcine epidemic diarrhea virus (GHN / HB01 strain) inactivated virus standard material GBE(E)091302, and verify the repeatability of each tube of standard material three times.

[0083] 3) Perform detection according to the above-mentioned droplet digital PCR method for detecting porcine epidemic diarrhea virus.

[0084] 4) The results of the repeatability test are shown in Table 3. The calculated coefficient of variation is 2.8%, indicating that the digital PCR method has good repeatability and the detection results are stable and reliable.

[0085] Table 3 Repeatability test

[0086]

[0087]

[0088] Example 3 Clinical sample detection

[0089] 1) The primers and probes are the same as those in Example 1.

[0090] 2) Test the 9 inactivated samples collected and stored in our laboratory and verified by sequencing;

[0091] 3) The samples were pre-treated (homogenized) and then nucleic acid was extracted using the Tianlong Nucleic Acid Automatic Extractor method and stored at -20°C for later use.

[0092] 4) The digital PCR method established by the present invention is used for detection, and positive droplets are determined as positive, and the detection results of the method for clinical samples are analyzed.

[0093] 5) The results are as follows Figure 6 The results showed that 4 samples were positive and 5 were negative, indicating that this method was consistent with the clinical test results of the national standard GB / T34757-2017 RT-PCR method for porcine epidemic diarrhea virus.

[0094] In summary, the present invention obtains the best primer-probe combination through optimized screening of primers and probes, and optimizes the reaction system and reaction conditions, so that the method established by the present invention can meet the requirements for rapid detection of porcine epidemic diarrhea virus while ensuring specificity and repeatability.

Claims

1. A primer and probe for detecting porcine epidemic diarrhea virus based on a droplet digital PCR method, characterized in that: The primers and probes are used to detect the nucleic acid fragment of porcine epidemic diarrhea virus, the sequence of which is SEQ ID NO:

1.

2. The amplification primer and probe according to claim 1, wherein: The upstream primer sequence of the primers is SEQ ID NO: 2, the downstream primer sequence is SEQ ID NO: 3, and the probe primer sequence is SEQ ID NO:

4.

3. Use of the amplification primers and probes according to claim 1 in the preparation of a PCR detection kit for detecting porcine epidemic diarrhea virus.

4. The use according to claim 3, characterized in that The PCR detection kit is a droplet digital PCR detection kit.

5. A droplet digital PCR detection kit, characterized in that: The kit comprises the amplification primers and probes according to claim 1 or 2.

6. A method for detecting porcine epidemic diarrhea virus for non-disease treatment purposes, characterized in that: The method is to use the kit described in claim 5 for detection.

7. The method according to claim 6, characterized in that The probe concentration of the method described is 250 nmol / L, the upstream and downstream concentrations are both 750 nmol / L, and the annealing temperature of the reaction system is 58°C.

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