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Quadruple fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting porcine epidemic diarrhea virus and porcine rotavirus and application of quadruple fluorescent quantitative PCR detection kit

A technology of porcine epidemic diarrhea and porcine rotavirus, applied in the biological field, can solve the problems of high harm, high infectivity and pathogenicity of diarrhea pathogens, and achieve the best specificity and repeatability, excellent sensitivity, and reduce workload Effect

Active Publication Date: 2022-07-29
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These viral diarrhea pathogens have high infectivity and pathogenicity, and are relatively harmful, but it is difficult to effectively distinguish them only by clinical symptoms, and the complexity of their etiology has brought great challenges to related detection, diagnosis and prevention.

Method used

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  • Quadruple fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting porcine epidemic diarrhea virus and porcine rotavirus and application of quadruple fluorescent quantitative PCR detection kit
  • Quadruple fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting porcine epidemic diarrhea virus and porcine rotavirus and application of quadruple fluorescent quantitative PCR detection kit
  • Quadruple fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting porcine epidemic diarrhea virus and porcine rotavirus and application of quadruple fluorescent quantitative PCR detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0045] A quadruple fluorescence quantitative PCR detection kit for the detection of porcine epidemic diarrhea virus and porcine rotavirus, including hot-start Taq DNA polymerase, enzyme-free water, PCR reaction solution, and probe-based fluorescence quantitative PCR matching Buffer, And four pairs of specific primers and corresponding TaqMan probes and controls for the detection of G1 genotype porcine epidemic diarrhea virus, G2 genotype porcine epidemic diarrhea virus, porcine group A rotavirus, and porcine group C rotavirus ; wherein, the PCR reaction solution contains Mg 2+ ions, PCR buffer, dNTPs mix.

[0046] The control substance includes positive control substance and negative control substance: the positive control substance is the S gene fragment of G1 genotype porcine epidemic diarrhea virus and G2 genotype porcine epidemic diarrhea virus, porcine group A rotavirus and porcine epidemic diarrhea virus respectively. Four target genes of VP6 gene fragment of porcine gr...

Embodiment 2 4

[0078] Example 2 Establishment of standard curve of quadruple TaqMan qPCR detection method

[0079] 1. All clinical samples are stored in -80℃ low temperature refrigerator. The clinical samples were freeze-thawed and placed in a 1.5 mL centrifuge tube, PBS buffer was added and ground sufficiently, and then centrifuged at 12,000 rpm for 10-15 min to collect the sample supernatant. The viral nucleic acid was extracted from the supernatant according to the instructions of the nucleic acid extraction kit Viral DNA / RNA Kit.

[0080] 2. Using the extracted viral nucleic acid as a template for reverse transcription, use the RevertAid First StrandcDNA Synthesis Kit to reverse-transcribe the viral nucleic acid into cDNA. The total reaction volume is 20 μL, including: 1 μL RandomHexamer Primer, 1 μL Oligo(dT) 18Primer, 5μL nuclease-free Water, 5μL total RNA, incubate at 65°C for 5min after mixing, ice bath for 3min, continue to add: 4μL 5×Reaction Buffer, 2μL 10×dNTP master mix, 1μLRev...

Embodiment 3 4

[0088] Example 3 The quadruple TaqMan qPCR detection method is most suitable for the exploration of the reaction system

[0089] 1. Use a concentration of 1×10 4 copies / μL of PEDV-G1, PEDV-G2, RVA and RVC plasmid standards were placed into the qPCR reaction system, each system used primers of different concentrations, PCR amplification was performed, and fluorescent signals were collected.

[0090] 2. The optimal primer concentration qPCR reaction system is: 10 μL Probe Master Mix (containing Mg 2+ ions, dNTPs mixture, hot-start Taq DNA polymerase, etc.), 0.1 μL each of PEDV-G1-Probe, PEDV-G2-Probe, RVA-Probe and RVC-Probe, 2 μL cDNA template, nuclease-free water was added to the total volume of the system 20 μL, PEDV-G1(F), PEDV-G1(R), PEDV-G2(F), PEDV-G2(R), RVA(F), RVA(R), RVC(F) and RVC(R) 0.1 μL, 0.2 μL, 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, 0.7 μL, 0.8 μL were added, respectively.

[0091] 3. The optimal probe concentration qPCR reaction system is: 10 μL Probe Master Mix (c...

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Abstract

The invention discloses a quadruple fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting porcine epidemic diarrhea virus and porcine rotavirus and application of the quadruple fluorescent quantitative PCR detection kit. The kit comprises a hot start Taq DNA polymerase, enzyme-free water, a PCR reaction solution and a probe method fluorescent quantitative PCR matched Buffer, the four pairs of specific primers, the corresponding TaqMan probes and the reference substances are used for detecting the G1 genotype porcine epidemic diarrhea virus, the G2 genotype porcine epidemic diarrhea virus, the porcine group A rotavirus and the porcine group C rotavirus. The kit provided by the invention realizes simultaneous detection of four viruses capable of causing porcine diarrhea in one PCR reaction tube, has excellent specificity and repeatability, and can be directly applied to conventional laboratory diagnosis of porcine clinical diarrhea samples; and a rapid and accurate detection method is provided for epidemiological research of the porcine epidemic diarrhea virus and the porcine rotavirus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a quadruple fluorescence quantitative PCR detection kit for detecting porcine epidemic diarrhea virus and porcine rotavirus and its application. Background technique [0002] Porcine viral diarrhea is a major dilemma faced by the pig industry in terms of production and reproduction. Currently, viral diarrhea caused by Porcine Epidemic Diarrhea Virus (PEDV) infection is the most common clinical disease, especially the G2 gene. Type PEDV became widespread after 2010, and was often accompanied by mixed infection of multiple diarrheal pathogens, such as porcine group A rotavirus (Rotavirus A, RVA) or porcine group C rotavirus (Rotavirus C, RVC). These viral diarrhea pathogens are highly contagious and pathogenic, and cause great harm, but it is difficult to distinguish them effectively by clinical symptoms alone. Therefore, establishing a simple, rapid, efficient and economi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/107C12Q2545/114C12Q2537/143Y02A50/30
Inventor 粟硕张乐天姜智文周子彤孙久萌盛守志
Owner NANJING AGRICULTURAL UNIVERSITY
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