Porcine Delta coronavirus detection kit based on constant temperature isolation type fluorescent polymerase chain reaction (PCR) platform, and application of porcine Delta coronavirus detection kit

A detection kit and coronavirus technology, applied in the biological field, can solve the problems of long reaction time and complicated operation, and achieve the effects of short detection time, high sensitivity and easy transportation

Inactive Publication Date: 2017-08-08
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the common fluorescent quantitative PCR method restricts its application in rapid on-site detection due to factors such as large-scale equipment, complicated operation, and long reaction time. Therefore, it is imminent to establish a fast, sensitive, specific, and on-site detection method for PDCoV.

Method used

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  • Porcine Delta coronavirus detection kit based on constant temperature isolation type fluorescent polymerase chain reaction (PCR) platform, and application of porcine Delta coronavirus detection kit
  • Porcine Delta coronavirus detection kit based on constant temperature isolation type fluorescent polymerase chain reaction (PCR) platform, and application of porcine Delta coronavirus detection kit

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Effect test

Embodiment 1

[0031] Example 1 Porcine Delta coronavirus detection kit based on constant temperature isolation fluorescent PCR platform

[0032] A pig Delta coronavirus detection kit based on a constant temperature isolation fluorescent PCR platform, comprising the following components:

[0033] (1) Reaction buffer: 3ml, composed of 500mM KCl, 100mM Tris-HCl (pH 8.3), 15mM MgCl 2 and ddH 2 O (balance) composition.

[0034] (2) Freeze-dried tubes of fluorescent quantitative PCR reaction solution (50 tubes):

[0035] Fluorescent quantitative PCR reaction solution including Taq enzyme 1 μL / tube (5U·μL -1 ), reverse transcriptase 1.25μL / tube (20U·μL -1), each 3.5 μL / tube of upstream and downstream primers for detection (10 μmol·μL -1 ) and probe 0.25μL / tube (10μmol·μL -1 ), dNTPs 4μL / tube (2.5mM); add the above mixture into a 0.5ml PCR tube to drop to -50°C, then freeze-dry at 10pa pressure for 24h, form a dry powder and close the tube to prepare a fluorescent quantitative PCR reaction so...

Embodiment 2

[0039] Example 2 The method of using the porcine Delta coronavirus detection kit based on the constant temperature isolation fluorescent PCR platform

[0040] The method includes the following steps:

[0041] 1) RNA template: Take 200 μL of the supernatant of the stool sample, and refer to the PetNAD kit extraction manual of Jinrui Hongjie (Xiamen) Biotechnology Co., Ltd. to extract the total RNA from the supernatant of the tested sample to prepare the RNA template;

[0042] 2) Preparation of the fluorescent PCR reaction system: the preparation of the fluorescent PCR reaction system was performed on ice;

[0043] Take 100 μL of reaction buffer and add it to the freeze-dried tube of the positive control substance and mix it to prepare the positive control substance, then take 50 μL of the reaction buffer solution and 5 μL of the positive control substance and add it to the freeze-dried tube of the fluorescent quantitative PCR reaction solution to prepare the positive control re...

Embodiment 3

[0046] Embodiment 3 standard substance preparation and the mensuration of sensitivity

[0047] Take 200 μL of the supernatant of the feces sample, refer to the PetNAD kit extraction manual of Jinrui Hongjie (Xiamen) Biotechnology Co., Ltd. Total RNA was reverse transcribed into cDNA.

[0048] The cDNA prepared above was amplified by PCR with PDCoV primers respectively, and the corresponding pathogenic cDNA was used as a positive control, and no template was used as a negative control. The reaction system is: 2.0 μL of cDNA, 12.5 μL of 2×Taq PCR Master Mix, 1.0 μL of upstream and downstream primers, 8.5 μL of ddH2O, and a total volume of 25 μL. The PCR amplification conditions were: 95°C for 5min, 95°C for 30s, 55°C for 30s, 72°C for 40s, 30 cycles, 72°C for 10min. The PCR product was identified by 3% agarose gel electrophoresis, and the target fragment was recovered with a gel recovery kit, and cloned into the pMD19-T vector (Bao Biological Engineering Co., Ltd.), and transf...

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Abstract

The invention discloses a porcine Delta coronavirus detection kit based on a constant temperature isolation type fluorescent polymerase chain reaction (PCR) platform, and application of the porcine Delta coronavirus detection kit. The porcine Delta coronavirus detection kit consists of reaction buffer, a fluorescent quantitative PCR reaction liquid freeze-drying tube, a positive reference substance freeze-drying tube and a negative reference substance freeze-drying tube, wherein the fluorescent quantitative PCR reaction liquid freeze-drying tube is formed by mixing and freeze-drying Taq enzyme, reverse transcriptase, a detection primer, a probe and deoxynucleoside triphosphate (dNTP). The kit provided by the invention is short in detection time (the reaction time is only 42min), high in specificity and sensitivity and convenient to store (at 4 DEG C); when being matched with a constant temperature isolation type PCR instrument for use, the kit is very suitable for the rapid field detection of the porcine Delta coronavirus and can be widely popularized at the grassroots level.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a porcine Delta coronavirus detection kit based on a constant temperature isolation type fluorescent PCR platform and its application. Background technique [0002] Porcine Deltacoronavirus (PDCoV) was first identified in molecular surveillance studies in Hong Kong in 2009, but its clinical significance was not investigated. The disease first appeared in Ohio in February 2014 and quickly spread to other states in the United States. PDCoV was also found in pigs in South Korea in April 2014. Recently, the disease has also appeared in mainland China and Thailand. Studies have shown that PDCoV can cause gastric alveolar epithelial necrosis, small intestinal villi atrophy, and mild interstitial lung. Animal experiments have confirmed that PDCoV can cause severe diarrhea, vomiting, and enteritis. The disease has a high incidence rate and a mortality rate of 30%-40%, which ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107C12Q2521/107
Inventor 岳华汤承沈思思
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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