Fluorescence quantitative PCR kit used for detecting PRV, and application thereof

A detection kit and fluorescence quantitative technology, applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the inaccuracy, specificity, sensitivity and reproducibility of real-time quantitative PCR detection technology Meet the problems that have not yet been developed, and achieve the effect of fast detection, high accuracy, and improved signal-to-noise ratio

Active Publication Date: 2014-01-15
WUHAN CHOPPER BIOLOGY
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  • Abstract
  • Description
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Problems solved by technology

However, when using real-time fluorescent quantitative PCR technology, laboratory personnel usually need to purchase fluorescent dyes and enzyme systems separately, and spend a lot of time on designing suitable primers and probes and optimizing reaction systems. In scientific research practice, a kit product that can quickly detect PRV through simple operation has not been developed, and the accuracy, specificity, sensitivity and reproducibility of real-time quantitative PCR detection technology cannot meet the actual needs. The problem

Method used

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  • Fluorescence quantitative PCR kit used for detecting PRV, and application thereof
  • Fluorescence quantitative PCR kit used for detecting PRV, and application thereof
  • Fluorescence quantitative PCR kit used for detecting PRV, and application thereof

Examples

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Effect test

Embodiment 1

[0040] Example 1 Screening of Primers and Probes for Fluorescent Quantitative PCR Detection of Porcine Pseudorabies Virus (PRV)

[0041] 1. Design of primers and probes

[0042] Referring to the gene sequence of PRV in my country published in Genebank, combined with the design characteristics of fluorescent PCR primers and probes, Primer Express and DNAStar software were used to design primers and probes for PRV conserved region fragments, and the designed multiple pairs of primers and probes (see Table 1) Screening tests were carried out, and the literature (Zhao Li, Cui Baoan, Chen Hongying, etc., the establishment and preliminary application of TaqMan real-time fluorescent quantitative PCR method for detecting porcine pseudorabies virus [J]. Chinese Journal of Preventive Veterinary Medicine, 2009, 31 (2) : 137-140.) reported in the detection of PRV primers and probe sequences (that is, the sixth group in Table 1) as a control for comparative research, from which the primers...

Embodiment 2

[0049] Example 2 Composition and optimization of fluorescent quantitative PCR detection kit for porcine pseudorabies virus

[0050] Optimization of the concentration of primers and probes: When other components in the reaction system remain unchanged, the concentrations of the specific primer pairs are respectively selected as 0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, and 0.5 μmol / L, and the concentration of specific probes was respectively selected as 0.05 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, and 0.4 μmol / L for the PCR pre-experiment, and the preferred specific primer pair was 0.2 μmol / L L, the specific probe concentration is 0.1 μmol / L.

[0051] Optimization of the concentration of the dNTP mixture: When other components in the reaction system remain unchanged, the concentrations of the dNTP mixture are respectively selected as 0.1mmol / L, 0.2mmol / L, 0.3mmol / L, 0.4mmol / L, 0.5mmol / L, 0.6mmol / L, for PCR, the preferred dNTP mixture concentration is 0.5mmol / L.

...

Embodiment 3

[0061] Example 3 Application of Fluorescent Quantitative PCR Rapid Detection Kit for Porcine Pseudorabies Virus

[0062] 1 Extraction of viral DNA

[0063] 1.1 Extraction of viral DNA in the sample to be tested (tissue type): Take 50-100 mg of the sample to be tested, put it in a sterile 1.5ml centrifuge tube, and add it according to the ratio of tissue sample: PBS weight volume ratio of 1:5 PBS solution, fully homogenized with a homogenizer, centrifuged at 4000rpm for 10min, took 200μL of the supernatant, and put it into a new sterile 1.5mL centrifuge tube free of DNase contamination, numbered for future use.

[0064] Take a 1.5mL centrifuge tube that is equivalent to the number of samples to be tested without DNase contamination and mark it. Add 600 μL of DNA extraction solution, and then add 200 μL of the corresponding supernatant of the sample to be tested and 200 μL of the negative quality control product, shake for 30 sec and mix, then place at room temperature for 10...

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Abstract

The invention relates to a fluorescence quantitative PCR rapid detection kit used for specifically detecting pig pseudorabies virus (PRV) infection, and an application thereof. The kit comprises a specific primer pair used for amplifying PRV gE gene conserved region, and a specific TaqMan fluorescent-labeled probe. An upstream primer sequence of the specific primer pair is 5'-GAGGACGACGGGCTGTAC-3', and a downstream primer sequence of the specific primer pair is 5'-GGACATCAACAGGCGGTTG-3'. The sequence of the TaqMan probe is 5'-TGGGTCCATTCGTCACTTCCG-3'. The 5' end of the TaqMan probe is labeled with a fluorescent reporter group FAM. The 3' end of the TaqMan probe is labeled with a fluorescence quenching group BHQ1 which is not intrinsically fluorescent. The kit can be used for rapid qualitative and quantitative detections of PRV infection, and can be used in identification detections of PRV gE gene-deleted vaccine strains and/or PRV wild strains.

Description

technical field [0001] The invention belongs to the field of virus nucleic acid detection, in particular to a fluorescent quantitative PCR rapid detection kit for specifically detecting porcine pseudorabies virus (PRV) infection and an application thereof. Background technique [0002] Pseudorabies (PR), also known as Aujeszky's disease, is an acute infectious disease caused by pseudorabies virus (Pseudorabies Virus, PRV) of the herpesviridae α-herpesvirus subfamily. Breeding domestic animals and wild animals are the main infected objects of the virus, and they show infection symptoms such as fever, itching, and encephalomyelitis. Pseudorabies was first reported in Hungary in 1902, and has since spread widely around the world. Since the first report of the disease in my country in 1947, so far, the disease has been prevalent in more than 20 provinces (Li Sulian. Thoughts on the study of pseudorabies gene deletion vaccine strains [J]. Sichuan Animal Husbandry and Veterinary ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
CPCC12Q1/686C12Q1/70C12Q2561/101C12Q2531/113
Inventor 薛霜陈其兵朱薇李晶梅漆世华温文生谢红玲
Owner WUHAN CHOPPER BIOLOGY
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