CN111440902A - Porcine epidemic diarrhea virus detection primer, kit and application thereof - Google Patents

Abstract

The invention provides a porcine epidemic diarrhea virus detection primer, a kit and its application. In the present invention, the DPO primer is designed according to the highly conserved specific sequence of porcine epidemic diarrhea virus (PEDV) by introducing the dual priming oligonucleotide primer (Dual Priming Oligonucleotide, DPO) design technology, and the reaction system is optimized to establish a DPO primer. DPO‑RT‑PCR detection methods for PEDV and corresponding detection kits. The kit has the characteristics of rapidity, simplicity, high specificity, high sensitivity, and good reliability. It can realize the accurate detection of PEDV and help the prevention, control, diagnosis and treatment of PEDV, and has a good application prospect.

Description

A porcine epidemic diarrhea virus detection primer, kit and application thereof

technical field

The invention belongs to the technical field of animal disease detection, and in particular relates to a porcine epidemic diarrhea virus detection primer, a kit and applications thereof.

Background technique

Porcine Epidemic Diarrhea (PED) first appeared in the United Kingdom in 1971, and there were successive reports of the disease in my country in the early 1980s. Porcine epidemic diarrhea, commonly known as winter diarrhea, often occurs in outbreaks in non-immunized weaned piglets (type I) or pigs of all ages (type II), and is caused by porcine epidemic diarrhea virus (PEDV) in pigs An acute intestinal infectious disease that causes watery diarrhea, vomiting and dehydration in pigs.

PEDV belongs to the family Coronavirinae and belongs to the genus Coronavirus. Its genome is an infectious single-stranded positive-stranded RNA virus with a total length of about 28kb, mainly including 6 ORFs, which are encoded and replicated in order from 5' to 3'. Genes for the enzymes polyprotein 1ab (pp1ab), spike protein (S), ORF protein, small membrane protein (E), membrane glycoprotein (M) and nucleocapsid protein (N). So far, no different serotypes of PEDV have been found. PEDV is sensitive to ether and chloroform. The clinical changes and symptoms of porcine epidemic diarrhea are very similar to those of porcine infectious gastroenteritis, and there is no significant difference in epidemiology and clinical symptoms from porcine infectious gastroenteritis, so it is difficult to diagnose, and laboratory diagnosis is required.

At present, domestic laboratory diagnostic methods include pathological examination, serological detection and RT-PCR detection, etc. However, the existing detection methods have limitations and are easily limited by factors such as detection conditions, cycle, and accuracy, and it is difficult to achieve efficient and rapid pathogen diagnosis. , such as virus isolation and serological detection methods are time-consuming and labor-intensive, have low sensitivity and are prone to false positives, while conventional RT-PCR methods have complex primer design processes, and primer parameters need to be optimized repeatedly. Avoid the appearance of non-specific amplification. DPO dual priming oligonucleotide primer (Dual Priming Oligonucleotide, DPO) is a novel primer design method. The specificity of the primer is stronger than that of the conventional primer. The DPO primer and the template DNA have more than 3 base mismatches, and the amplification efficiency will be reduced. The reaction is greatly reduced or terminated, and the effective annealing temperature range of DPO primers is wide, and the design process is simple, which greatly simplifies the tedious steps of conventional primer design.

In view of this, the present invention expects to establish a DPO-RT-PCR method for detecting PEDV by introducing dual priming oligonucleotide primer (Dual Priming Oligonucleotide, DPO) design technology, so as to improve the sensitivity and specificity of detection, and realize the detection of PEDV. Accurate detection of PEDV.

SUMMARY OF THE INVENTION

The present invention provides a porcine epidemic diarrhea virus detection primer, a kit and application thereof. By introducing the dual priming oligonucleotide primer (Dual Priming Oligonucleotide, DPO) design technology, the gene sequence of PEDV is compared, and its high specificity is selected. The DPO primers were designed by using the sex sequence, and a highly sensitive and high-specific detection method for PEDV was established, so as to realize the accurate detection of PEDV and help the prevention, control, diagnosis and treatment of PEDV.

To this end, a first aspect of the present invention provides a porcine epidemic diarrhea virus detection primer, the primer includes:

Upstream primer: 5'-GGTGAGCGAATTGAACAACCTTCIIIIIGGCATTTC-3' (SEQ ID NO. 1),

Downstream primer: 5'-GACCCTGGTTATTTCCACGATTCTIIIIITTACCAC-3' (SEQ ID NO. 2),

Wherein, I represents inosine.

The second aspect of the present invention provides a porcine epidemic diarrhea virus detection kit, which includes the upstream and downstream primers as described in SEQ ID NO.1 and SEQ ID NO.2, and also includes a positive quality control product and a negative quality control product.

In an embodiment of the present invention, in the porcine epidemic diarrhea virus detection kit, the final reaction concentration of the upstream and downstream primers in the reaction system is 0.025-0.6 μM.

In a preferred embodiment of the present invention, in the porcine epidemic diarrhea virus detection kit, the final reaction concentration of the upstream and downstream primers in the reaction system is 0.2-0.6 μM.

In an embodiment of the present invention, in the porcine epidemic diarrhea virus detection kit, the reaction conditions include:

In an embodiment of the present invention, in the porcine epidemic diarrhea virus detection kit, the reaction conditions include:

In an embodiment of the present invention, the porcine epidemic diarrhea virus detection kit has a detection sensitivity of up to 6.84×10 ³ copies/μL.

In an embodiment of the present invention, the porcine epidemic diarrhea virus detection kit can specifically identify porcine epidemic diarrhea virus in a sample, wherein the sample contains porcine infectious gastroenteritis virus, porcine acute diarrhea virus One or more of syndrome coronavirus, porcine deltacoronavirus, porcine rotavirus, porcine respiratory coronavirus disease, and porcine Seneca Valley virus.

A third aspect of the present invention provides a porcine epidemic diarrhea virus detection method, comprising the following steps:

1) Extract nucleic acid from the sample to be tested;

2) using the nucleic acid obtained in step 1) as an amplification template, using the primers described in the first aspect of the present invention, performing RT-PCR amplification to obtain an amplification product;

3) The amplified product is subjected to agarose gel electrophoresis to obtain the detection result.

In an embodiment of the present invention, the agarose gel in step 3) is 1% agarose gel.

The fourth aspect of the present invention provides the porcine epidemic diarrhea virus detection primer according to the first aspect of the present invention, the porcine epidemic diarrhea virus detection kit according to the second aspect of the present invention, or the porcine epidemic diarrhea virus detection kit according to the third aspect of the present invention Application of porcine epidemic diarrhea virus detection method in the detection of porcine epidemic diarrhea virus.

The kit provided by the invention has the advantages of quickness and convenience, strong specificity, high sensitivity, good reliability, low detection cost, low requirements on the level of detection instruments and operators, and high detection sensitivity and specificity at the same time. It can accurately differentiate and diagnose trace latent infection of porcine epidemic diarrhea virus. Therefore, it is suitable for monitoring and early warning of porcine epidemic diarrhea virus trace latent infection period. application and promotion.

Description of drawings

Fig. 1 is the primer annealing temperature optimization result of the porcine epidemic diarrhea virus DPO-RT-PCR method provided in the embodiment of the present invention, wherein M is DNAMarker1000, 1-6 are respectively: 42 ℃, 46 ℃, 50 ℃, 54 ℃, 58°C and 62°C;

Figure 2 shows the results of primer concentration optimization of the porcine epidemic diarrhea virus DPO-RT-PCR method provided in the embodiment of the present invention, wherein M is DNAMarker1000, and 1 to 8 are: 0.025 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM;

Fig. 3 is the specificity test result of the porcine epidemic diarrhea virus DPO-RT-PCR method provided in the embodiment of the present invention, wherein M is DNAMarker1000, 1 is a positive control, and 2-7 are respectively: porcine infectious gastroenteritis virus ( TGEV), porcine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), porcine rotavirus (PoRV), porcine respiratory coronavirus disease (PRCV) and porcine Seneca Valley virus (SVV) ) sample, 8 is a negative control;

Fig. 4 is the sensitivity test result of the porcine epidemic diarrhea virus DPO-RT-PCR method provided in the embodiment of the present invention, wherein M is DNAMarker1000, and 1 to 8 are respectively 10-fold gradient dilution samples, and the dilutions are in sequence: ^10-1 , ^10-2 , ^10-3 , ^10-4 , ^10-5 , ^10-6 , ^10-7 , ^10-8 ;

Fig. 5 is the detection result of the porcine epidemic diarrhea virus DPO-RT-PCR method on clinical samples provided in the embodiment of the present invention, wherein M is DNAMarker1000, 1 is a negative control, 2 is a positive control, and 3-24 are clinical samples.

Detailed ways

The following description is the preferred embodiment of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made, and these improvements and modifications are also regarded as It is the protection scope of the present invention. The experimental methods that do not specify specific conditions in the examples are usually in accordance with conventional conditions. Unless otherwise specified in the examples of the present invention, the reagents and consumables used are all commercially available commodities.

Example 1 Construction of RT-PCR rapid detection method

1. Primer design:

According to the gene sequence of porcine epidemic diarrhea virus (PEDV) registered in GenBank, use Oligo6.0 primer design software to select N gene as the primer design region. The primer sequence is as follows:

Upstream primer PEDV-DPO-F:

5'-GGTGAGCGAATTGAACAACCTTCIIIIIGGCATTTC-3' (SEQ ID NO: 1)

Downstream primer PEDV-DPO-R:

5'-GACCCTGGTTATTTCCACGATTCTIIIIITTACCAC-3' (SEQ ID NO: 2)

2. Extraction of sample RNA:

Use Corning Life Sciences (Wujiang) Co., Ltd.'s body fluid viral DNA/RNA preparation kit (Cat. No.: PID0320425) to extract template RNA according to the instructions, and store at -70°C for later use.

3. RT-PCR reaction:

Use the one-step RT-PCR kit (RR057A) of Baori Doctor Biotechnology (Beijing) Co., Ltd., and perform RT-PCR reaction with the sample RNA extracted in step 2 as template RNA according to the instruction manual. The 25 μl reaction system is as follows:

The reaction procedure is as follows:

4. Judgment of test results:

The RT-PCR product was identified by 1% agarose gel electrophoresis. If a single target band with a size of 361 bp could be amplified, the sample was determined to be positive for porcine epidemic diarrhea virus.

5. Gene cloning and construction of plasmid containing the target gene

The PCR product was recovered with an agarose gel DNA recovery kit, and the PCR product was ligated with the PMD-19T vector overnight and transformed into E. coli DH5α. The colony plasmids grown after transformation were extracted with plasmid extraction kit.

6. Sequencing identification

The obtained plasmid was identified by PCR and double-enzyme digestion, and the plasmid containing the target gene was sent to Guangdong Wenshi Dahuanong Biotechnology Co., Ltd. for sequencing. After comparison and confirmation, a positive plasmid was obtained.

7. Optimization of DPO-RT-PCR conditions

For different annealing temperatures (42°C, 46°C, 50°C, 54°C, 58°C and 62°C) and primer concentrations (0.025μM, 0.05μM, 0.1μM, 0.2μM, 0.3μM, 0.4μM, 0.5μM and 0.6μM ) PCR amplification was carried out on a gradient PCR machine, and the optimal reaction conditions for PCR were explored. The results of annealing temperature optimization are shown in Figure 1. The results show that all the annealing temperatures used can amplify the target bands, indicating that the effective annealing temperature range of DPO primers is wide. The results of primer concentration optimization are shown in Figure 2. The effective concentration of DPO primers is 0.025-0.6 μM. When the DPO primer concentration is 0.2-0.6 μM, the PCR amplification effect is better.

8. Specificity of DPO-RT-PCR method

According to the RT-PCR reaction system and reaction procedure described in step 3, the sample template RNA was replaced with porcine transmissible gastroenteritis virus (TGEV), porcine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus, respectively. Virus (PDCoV), Porcine Rotavirus (PoRV), Porcine Respiratory Coronavirus Disease (PRCV) and Porcine Seneca Valley Virus (SVV) samples, porcine epidemic diarrhea virus detected by our laboratory and identified by sequencing The positive sample is a positive control, and RT-PCR reaction is performed. The RT-PCR product is identified by 1% agarose gel electrophoresis. The electrophoresis result is shown in Figure 3. The result shows that only a single band appears at 361bp of the positive control lane, indicating the method. Good specificity.

9. Sensitivity of DPO-RT-PCR method

According to the RT-PCR reaction system and reaction procedure described in step 3, using the positive sample RNA (6.84×10 ⁹ copies/μL) in step 8 as the template, press 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^{- 4} , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 were subjected to 10-fold gradient dilution, and deionized water was used as a negative control to carry out RT-PCR reaction, and RT-PCR products were identified by 1% agarose gel electrophoresis , the electrophoresis results are shown in Figure 4, the results show that the dilution is ^10-5 band is still obvious, the dilution is ^10-6 band is still inconspicuous but visible to the naked eye, indicating that ^10-6 is the lowest detection PEDV single-item DPO-RT-PCR detected the lowest 6.84×10 ³ copies/μL.

10. Clinical sample testing

According to the RT-PCR reaction system and reaction procedure described in step 3, with the positive control RNA in step 8 as the positive control and deionized water as the negative control, 22 clinical samples suspected of porcine epidemic diarrhea virus were detected , the extraction of template RNA and RT-PCR reaction refer to steps 2 and 3, the RT-PCR product was identified by 1% agarose gel electrophoresis, and the electrophoresis result is shown in FIG. 5 . The results showed that the negative and positive controls were established, and 7 samples were PEDV positive.

To sum up, the kit for detecting porcine epidemic diarrhea virus provided by the present invention has high detection sensitivity and specificity, and at the same time, the detection kit provided by the present invention has low requirements on equipment and personnel skills, and is suitable for the detection of pigs. Monitoring and early warning during the period of micro-recessive infection of epidemic diarrhea virus is conducive to its application and promotion in breeding enterprises, which can greatly increase the pathogen detection rate in the case of micro-infection of porcine epidemic diarrhea virus, and can accurately differentiate and diagnose pigs The etiology of infection is conducive to the implementation of the correct treatment plan, and has a good application prospect.

The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be Modifications or equivalent substitutions are made without departing from the spirit and scope of the technical solutions of the present invention.

sequence listing

<110> South China Agricultural University

<120> A porcine epidemic diarrhea virus detection primer, kit and its application

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<213> Artificial Sequence

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gaccctggtt atttccacga ttctttacca c 31

Claims (8)

1. The primers for detecting the porcine epidemic diarrhea virus are characterized by comprising the following components:

an upstream primer: 5'-GGTGAGCGAATTGAACAACCTTCIIIIIGGCATTTC-3' (SEQ ID NO.1),

a downstream primer: 5'-GACCCTGGTTATTTCCACGATTCTIIIIITTACCAC-3' (SEQ ID NO.2),

wherein I represents inosine.

2. A porcine epidemic diarrhea virus detection kit is characterized by comprising upstream and downstream primers shown in SEQ ID No.1 and SEQ ID No.2, and further comprising a positive quality control substance and a negative quality control substance.

3. The porcine epidemic diarrhea virus detection kit of claim 2, wherein the final concentration of the upstream and downstream primer reactions in the reaction system of the kit is 0.025-0.6 μ M.

4. The porcine epidemic diarrhea virus detection kit of claim 2, wherein the kit reaction conditions comprise:

5. the porcine epidemic diarrhea virus detection kit of claim 2, wherein the kit has a detection sensitivity of up to 6.84 × 10³copies/μL。

6. The porcine epidemic diarrhea virus detection kit of claim 2, wherein the kit specifically identifies the porcine epidemic diarrhea virus in a sample comprising one or more of porcine transmissible gastroenteritis virus, porcine acute diarrhea syndrome coronavirus, porcine delta coronavirus, porcine rotavirus, porcine respiratory coronavirus disease, and porcine sendai valley virus.

7. A method for detecting porcine epidemic diarrhea virus comprises the following steps:

1) extracting nucleic acid from a sample to be detected;

2) performing RT-PCR amplification by using the nucleic acid obtained in the step 1) as an amplification template and using the primer of claim 1 to obtain an amplification product;

3) and carrying out agarose gel electrophoresis on the amplification product to obtain a detection result.

8. The use of the primer for detecting porcine epidemic diarrhea virus according to claim 1, the kit for detecting porcine epidemic diarrhea virus according to claim 2, or the method for detecting porcine epidemic diarrhea virus according to claim 7 for detecting porcine epidemic diarrhea virus.

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