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Nucleic acid, real-time fluorescent RT-RPA kit and method for detecting porcine epidemic diarrhea virus

A porcine epidemic diarrhea, RT-RPA technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of unsuitable for on-site detection, long-term detection, complicated operation, etc., and achieve easy operation. , good detection effect, simple operation effect

Inactive Publication Date: 2017-10-27
河北省检验检疫科学技术研究院
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Problems solved by technology

[0003] Early, rapid and effective detection of PEDV is one of the important means to prevent and control the spread of PED. At present, a variety of PEDV detection methods have been established, mainly including virus isolation and identification, indirect immunofluorescence method, immunoelectron microscopy and ELISA, etc. Traditional methods, however, these traditional methods are time-consuming, laborious and less sensitive. With the development of molecular biology, a series of PCR methods have also been established, such as RT-PCR, nanoparticle RT-PCR, real-time fluorescent RT-PCR, etc. However, the RT-PCR method requires a high-precision, complicated and expensive PCR instrument, as well as a good laboratory and skilled technicians. Therefore, the RT-PCR series method is not suitable for poorly equipped experiments in poor and backward areas. It is not suitable for on-site detection. At present, a variety of isothermal detection methods for PEDV detection have been established, mainly including insulating isothermal PCR (iiPCR), RT-LAMP and cross-primed amplification method (RT-CPA). The method generally takes 45-60min to complete the detection, and the RT-LAMP method requires 6 primers, while the RT-CPA method requires 3 primers and 2 probes
These all make these methods difficult to design and take a long time to complete the detection

Method used

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  • Nucleic acid, real-time fluorescent RT-RPA kit and method for detecting porcine epidemic diarrhea virus
  • Nucleic acid, real-time fluorescent RT-RPA kit and method for detecting porcine epidemic diarrhea virus
  • Nucleic acid, real-time fluorescent RT-RPA kit and method for detecting porcine epidemic diarrhea virus

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Experimental program
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Effect test

Embodiment 1

[0052] Viral DNA / RNA Extraction

[0053] PEDV (JSCZ1601 strain, mutant strain), transmissible gastroenteritis virus (TGEV, attenuated H strain), porcine rotavirus (PoRV, HB-BD / 2016 strain), Lawsonia intracellularis (Lintracellularis, LX5 strain) ) are kept in our laboratory. PEDV (CV777 strain, classic strain), classical swine fever virus (CSFV, AV1412 strain), porcine parvovirus (PPV, BJ-2 strain) were all obtained from commercial attenuated vaccines; porcine Delta coronavirus (PDCoV) RNA was obtained from real-time fluorescent RT -Pig small intestine samples that were negative for PEDV and positive for PDCoV by PCR.

[0054] 76 pig small intestine samples were obtained from 7- to 14-day-old piglets with watery diarrhea and dehydration symptoms from 11 pig farms in Hebei Province between April 2016 and February 2017. Pig small intestine samples were homogenized to make a 10% (w / v) homogenate, centrifuged at 3000 g for 10 min at 4°C, and 200 µL was used for viral RNA extract...

Embodiment 2

[0057] In vitro transcription of PEDV RNA

[0058] Using N-F and N-R as primers, as shown in Table 3, RT-PCR was carried out using the PEDV CV777 strain viral genome RNA as a template to obtain the PEDV nucleocapsid gene (N gene), with a full length of 1326 bp. The RT-PCR reaction system was 50 μL, including 2×one-step Buffer25 μL, one-step enzyme mixture (Takara, Dalian, China) 2 μL, N-F (20 μmol / L) 0.5 μL, N-R (20 μmol / L) 0.5 μL, PEDV RNA 5 μL, ddH2O 17 μL, RT-PCR reaction conditions are: 50°C, 30min, 1 cycle; 94°C, 2min, 1 cycle; 94°C, 30s, 55°C, 30s, 72°C, 60s, 32 cycles.

[0059] The RT-PCR product was purified using the Tiangen DNA Purification Kit, then connected to the pGEM-T Easy vector (Promega, Madison, USA) and transformed into Escherichia coli competent cells DH5α, the transformed positive clones were picked, and the plasmid was extracted And sequenced, confirmed the transformed positive clone by sequencing method, obtained the recombinant plasmid pGEM-T-PEDV-N, ...

Embodiment 3

[0063] Design of upstream and downstream primers and exo probe for detection of porcine epidemic diarrhea virus

[0064] The PEDV N gene is highly conserved and can replicate during virus replication, so the PEDV N gene is used as the target gene of real-time fluorescent RT-RPA. According to different reference sequences of PEDV in GenBank (accession numbers: JN173295; KX168410; JQ743654; KF840555; KJ646621; KT323979; KT800000), design upstream and downstream primers and exo probes according to the PEDV N gene. See Table 1 for specific information. and exo probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

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Abstract

The invention relates to the technical field of biological monitoring and specifically discloses a nucleic acid, a real-time fluorescent RT-RPA kit and a method for detecting porcine epidemic diarrhea virus. The real-time fluorescent RT-RPA method comprises the following steps: extracting RNA from a sample; performing isothermal amplification on the obtained RNA; adding a reaction system into a reaction tube filled with a lyophozyme preparation; adding a magnesium acetate solution, and reacting for 20min at a constant temperature of 40 DEG C; when an obvious amplification curve appears in the to-be-detected sample, determining that the result is positive. The real-time fluorescent RT-RPA method provided by the invention has the advantages of easiness in operation, strong specificity and high sensitivity; quick detection of a PEDV positive sample can be realized within 4-15min without using complicated instrument.

Description

technical field [0001] The invention relates to the technical field of biological monitoring, in particular to a nucleic acid, real-time fluorescent RT-RPA kit and method for detecting porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is caused by porcine epidemic diarrhea virus (PEDV). Piglets can occur, and the morbidity and death of suckling piglets are the most serious. PEDV belongs to the Alphacoronavirus genus of the Coronaviridae family. It is a single-stranded positive-sense RNA virus with an envelope. It was first reported, and then spread rapidly to many countries in Europe and Asia. In 1973, PED first occurred in my country, and it existed sporadically in pigs in my country before 2010. Since the winter of 2010, a highly pathogenic PEDV It has become popular in my country's pig herds and has caused serious economic losses. After the pig herds are infected with highly pathogenic PEDV, the inciden...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2522/101C12Q2521/507C12Q2537/1376
Inventor 王建昌刘立兵王金凤石蕊寒南汇珠
Owner 河北省检验检疫科学技术研究院
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