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Nested RT-PCR(reverse transcription-polymerase chain reaction)kit and method for detecting porcine Kobuvirus

A technology of RT-PCR and reagent kit, which is applied in the field of genetic engineering to achieve the effects of good specificity, high sensitivity and simple operation

Inactive Publication Date: 2013-04-24
武汉康湃特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is in order to overcome the defect that the detection of porcine Kobu virus basically all adopts common RT-PCR method at present, and provide a kind of porcine Kobu virus nested RT-PCR detection kit and detection method thereof, this method specificity It has strong sex and high sensitivity, and has a good detection effect on samples containing a small amount of porcine Kobu virus genome, and can be used for laboratory detection and molecular epidemiological investigation of porcine Kobu virus

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  • Nested RT-PCR(reverse transcription-polymerase chain reaction)kit and method for detecting porcine Kobuvirus
  • Nested RT-PCR(reverse transcription-polymerase chain reaction)kit and method for detecting porcine Kobuvirus
  • Nested RT-PCR(reverse transcription-polymerase chain reaction)kit and method for detecting porcine Kobuvirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The design of embodiment 1 specific primer

[0055] Specific primers were designed for the conserved sequence of porcine Kobu virus S-1-HUN strain gene (GenBank accession number EU787450), as shown in the table below, and specific nested PCR primers were synthesized according to the following sequence. Dilute the primers with DEPC-H2O to 20pmol / L, and store them at -20°C for later use.

[0056]

[0057] The establishment and optimization of embodiment 2 amplification system

[0058] 1. Overcoat PCR amplification reaction system and amplification program optimization

[0059] Using the cDNA obtained at RT as a template, optimize the 50 μL coat PCR amplification reaction system. The reaction system consists of 10×PCR Buffer (Mg 2+ plus) 5μL, 2.5mM dNTP Mixture4μL, 5U / μL Taq enzyme 1μL, 20pmol / μL outward primer 1μL each, ddH 2 O33~35μL, cDNA3~5μL composition. The amplification conditions are: ①94~95°C, 3~5min, ②93~94°C, 35~45s, ③58~60°C, 30~40s, ④70~73°C, 35~45s, 3...

Embodiment 3

[0062] Embodiment 3 specificity and sensitivity test

[0063] Using the optimized system and amplification conditions described in Example 2, get porcine Kobu virus (PKV) positive samples, swine influenza virus (SIV), porcine Jieshen virus (PTV), porcine Japanese encephalitis virus (JEV), RNA was extracted from 6 samples including porcine pseudorabies virus (PRV) and negative control (N), and after reverse transcription, nested PCR was used to test the specificity of the kit. Among them, RNA extraction reagents include Trizol, chloroform, isopropanol, 75% DEPC-ethanol, DEPC-H 2 O; The conditions of nested PCR are as follows: the amplification conditions of MIXⅠPCR are: ①95°C, 3min, ②94°C, 40s, ③59°C, 30s, ④72°C, 40s, steps ② to ④, a total of 35 cycles, ⑤72°C, 8min; The conditions for MIXⅡPCR amplification are: ① 95°C, 3 min, ② 94°C, 30s, ③ 59°C, 30s, ④ 72°C, 35s, steps ② to ④ for a total of 30 cycles, ⑤ 72°C, 8min.

[0064]Nested PCR results showed that only porcine Kobu vir...

Embodiment 4

[0066] The assembly of embodiment 4 kits

[0067] The preparation of kit components is completed according to the three parts of total RNA extraction, RT and nested PCR. The data and reagent composition provided below are the reagents required for a single nested RT-PCR detection:

[0068] (1) Total RNA extraction (stored at 4°C)

[0069] Sample lysate tube: Trizol750μL;

[0070] Chloroform tube: 200 μL of chloroform;

[0071] 75% ethanol tube: 800 μL of 75% DEPC-ethanol;

[0072] Isopropanol tube: 600 μL of isopropanol;

[0073] DEPC-H 2 O tube: 11 μL;

[0074] (2) Reverse transcription (RT) (stored at -20°C)

[0075] RT master mix tube: 2 μL of 10× reverse transcription buffer, 25 mM MgCl 2 4 μL, 10 mM dNTP 2 μL, 40 U / μL RNasin 0.5 μL, 0.5 μg / μL random primer 2 μL, 25 U / μL MV reverse transcriptase 1.5 μL, DEPC-H 2 O 3 μL.

[0076] (3) Nested PCR (stored at -20°C)

[0077] Overcoat PCR reaction solution tube (MIXⅠ): 10×PCR Buffer (Mg 2+ plus) 5 μL, 2.5 mM dNTP Mixt...

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Abstract

The invention discloses a nested RT-PCR(reverse transcription-polymerase chain reaction)primer, a kit containing the primer and a detection method, which are used for detecting porcine Kobuvirus. The nested RT-PCR primer comprises (1) outward primers P1 and P2, the sequences of which are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2, and (2) inward primers P3 and P4, the sequences of which are respectively as shown in SEQ ID NO.3 and SEQ ID NO.4. The detection method comprises the steps of: (1) extracting the total RNA (Ribose Nucleic Acid) of a sample; (2) obtaining cDNA (complementary deoxyribonucleic acid) by taking the total RNA as a template through RT (Reverse Transcription); and (3) detecting by taking the cDNA as a template after carrying out nested PCR (Polymerase Chain Reaction). The detection method has the advantages of good specificity, high sensitivity and simplicity in operation; and the kit has the advantages of low cost, fast detection speed, difficulty in pollution, easiness in determination of results and suitability for diagnosis of porcine Kobuvirus infection and very good application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a detection kit for porcine Kobu virus and a detection method thereof. Background technique [0002] Porcine kobuvirus (porcine kobuvirus, PKV) belongs to the Picornaviridae family and the genus Rigivirus. It was first detected by Reuter G et al. in February 2007 from 60 fecal samples collected from a healthy pig herd in a pig farm in Hungary. Previous studies have shown that in clinically asymptomatic pigs without diarrhea, the infection rate of PKV is very high; PKV is not limited to intestinal infection, but can also cause viremia. Recent studies have shown that PKV is associated with porcine diarrhea, and PKV infection is prevalent in pigs exhibiting diarrhea symptoms in Korea. [0003] In 2009, Khamrin P et al. collected 293 fecal samples from healthy pigs in 28 pig farms in Japan for RT-PCR detection. The positive rate of PKV was 45.4%. Positive samples were f...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 杨德全刘佩红周锦萍葛菲菲鞠厚斌刘健王建张维谊邓波葛杰
Owner 武汉康湃特生物科技有限公司
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