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Nested RT-PCR detection method for differentiating variant strains and classic strains of PEDV

A technology of RT-PCR and detection method, which is applied in the field of rapid detection of PEDV virus, can solve the problems of declaration and authorization without the identification and detection technology of PEDV classic strain and variant strain.

Inactive Publication Date: 2016-08-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Searched by SOOPAT, there is currently no application and authorization for the design of PEDVS gene primers for the identification and detection of PEDV classic strains and variant strains

Method used

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  • Nested RT-PCR detection method for differentiating variant strains and classic strains of PEDV
  • Nested RT-PCR detection method for differentiating variant strains and classic strains of PEDV
  • Nested RT-PCR detection method for differentiating variant strains and classic strains of PEDV

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The extraction of RNA in the sample of embodiment 1

[0033] 1 Experimental materials

[0034] Samples containing PEDV strains were named MX-2014, MX-2015, HA-2015, NC-2014, NC-2015 according to the sample collection time and place; samples containing TGEV strains were named QW; samples containing PDCoV strains The samples containing the RV strain were designated as BC; and the samples containing the RV strain were designated as JC.

[0035] 1.1 Main reagents

[0036] Trizol total RNA extraction reagent; TaqTM enzyme, reverse transcriptase M-MLV, RNase inhibitor, 5×M-MLVbuffer, 10×PCR buffer, agarose, DL2000 DNAMaker, dNTPs, 6×Loadingbuffer were all purchased from Takara Company.

[0037] 2 test method

[0038] 2.1 Trizol method to extract total RNA

[0039] 2.1.1 Take samples MX-2014, MX-2015, HA-2015, NC-2014, NC-2015, QW, BC and JC grinding solution 250μL into 2mlEP tube, add 500μL pre-cooled Trizol.

[0040] 2.1.2 The samples were mixed vigorously and left at r...

Embodiment 2

[0045] Embodiment 2: Nested RT-PCR amplification reaction

[0046] 1 Design of primers

[0047] Two pairs of primers were designed and screened by Primer Premier 5.0 software. The upstream PEDV / F-1 of the outer primer: 5-AACACTTAGCCTACCACA—3', and the downstream PEDV / F-2 of the outer primer: 5'—GTGGAATCATTGGACAA—3'.

[0048] 2 The first nested RT-PCR amplification reaction

[0049] After total RNA was extracted by Trizol method, reverse transcription reaction was performed: 2 μL of RNA, 1 μL of PEDV / F-2 downstream primer and 3 μL of ddH2O were mixed, reacted at 70°C for 10 min, and then cooled on ice for two minutes.

[0050] 2.1 Add 2 μL 5×MLV buffer, 0.5 μL dNTPMixture, 0.25 μL RNase Inhibitor, 0.25 μL M-MLV and 1 μL ddH to this reaction mixture 2 O.

[0051] 2.2 After being incubated at 42°C for 1 hour, and then incubated at 70°C for 15 minutes, the reaction was terminated by cooling on ice to obtain cDNA.

[0052] 2.3 Take 2μL cDNA as a template for the first PCR ampli...

Embodiment 3

[0054] Embodiment 3: the second nested RT-PCR amplification reaction

[0055] 1 Design of primers for the second PCR

[0056] Two pairs of primers were designed and screened by Primer Premier 5.0 software. The upstream PEDV / R-1 of the internal primer: 5'-GAAAACCAGGGTGTCAA-3', and the downstream PEDV / R-2 of the internal primer: 5'-GAAATACCATCCTCCACCAG-3'.

[0057] 2 Carry out the second nested RT-PCR amplification reaction

[0058] Take 2 μL of the first PCR product as a template for the second PCR amplification, and configure a 25 μL reaction system: 10×PCR buffer 2.5 μL, dNTP Mixture 2 μL, external primers R1 and R2 1 μL each, r-Taq enzyme 0.5 μL, ddH 2 O16μL mix well.

[0059] 2.1 Amplify according to the following conditions: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 5 minutes, renaturation at 51°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; final extension at 72°C for 10 minutes to terminate the reaction. Obtain the second PCR pro...

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Abstract

The invention discloses a nested RT-PCR detection method for differentiating variant strains and classic strains of PEDV, belonging to the technical field of rapid detection of PEDV virus. According to the nested RT-PCR detection method, the disadvantages that the steps of isolation, identification and gene sequencing of viruses are tedious, the cost is high, and the consumed time is long are overcome, and the problems of low specificity and poor sensitivity of the traditional RT-PCR are solved. The nested RT-PCR detection method has the beneficial effects that the operation is simple and convenient, the use is simple, the accuracy is high, the specificity is strong, and the sensitivity is high; the method is capable of detecting whether PEDV on infected animal engine bodies is the variant strain and qualitatively detecting whether PEDV exists in epidemic materials, so that the detection technique for differentiating whether PEDV is the variant strain in the production is greatly facilitated, and the PEDV research contents in the laboratory are enriched.

Description

technical field [0001] The invention belongs to the technical field of rapid detection of PEDV virus, in particular to a nested RT-PCR detection method for distinguishing PEDV mutant strains and classic strains. Background technique [0002] Epidemic diarrhea (porcineepidemicdiarrhea, PED) is a highly contagious disease of pigs caused by porcine epidemic diarrhea virus. Its main features are vomiting, watery diarrhea and death from dehydration; pigs of all ages are susceptible, Especially for 1-2 week-old suckling piglets, the harm is the most serious, and the mortality rate can be as high as 90%-100%. SunRQ et al. reported that since October 2010, PEDV has broken out in more than ten provinces in southern China, resulting in more than 1 million pigs. The death of piglets has brought great economic losses to the pig industry. Early detection and early diagnosis are of great significance to the prevention and treatment of the disease. To achieve early diagnosis, it is neces...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6848C12Q2531/113C12Q2549/119C12Q2565/125
Inventor 贺东生焦臣鹏王飞陈小芬苏丹萍罗华林
Owner SOUTH CHINA AGRI UNIV
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