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EHP and SHIV dual real-time fluorescent quantitative PCR detection primer and probe combination and kit

A real-time fluorescent quantitative, primer-probe technology, applied in recombinant DNA technology, microbial assay/inspection, microorganisms, etc., can solve the problem of long time-consuming detection, and achieve the effect of improving sensitivity and accuracy and reducing time-consuming.

Inactive Publication Date: 2019-08-13
珠海科艺普检测科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The established conventional PCR technology mostly detects one pathogen at a time, which takes a long time to detect, and the detection sensitivity and accuracy need to be further improved

Method used

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  • EHP and SHIV dual real-time fluorescent quantitative PCR detection primer and probe combination and kit
  • EHP and SHIV dual real-time fluorescent quantitative PCR detection primer and probe combination and kit
  • EHP and SHIV dual real-time fluorescent quantitative PCR detection primer and probe combination and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Viral nucleic acid extraction

[0035] Get diseased shrimp tissue (hepatopancreas, gills, intestinal tract, muscle), carry out homogenization process, obtain tissue homogenate, specific method is as follows:

[0036] 1) First add about 3 steel balls into the 2mL homogenate tube, 1 large steel ball (4.5mm in diameter) and 2 small steel balls (2.5mm in diameter);

[0037] 2) Take about (100 mg) diseased shrimp tissue (hepatopancreas, gills, intestines, muscles) into the tube, and add 500 μL PBS;

[0038] 3) Place the homogenization tube in a tissue homogenizer for homogenization for 1 min;

[0039] 4) Freeze the tissue homogenate at -40°C for at least 30 minutes, then return it to room temperature to dissolve, repeat freezing and thawing 2 to 3 times, and put it in a tissue homogenizer for homogenization once each time;

[0040] 5) Centrifuge at 10,000×g for 5 minutes, transfer about 200 μL of the supernatant to a 1.5 mL centrifuge tube, numbered for future use.

[...

Embodiment 2

[0056] specificity test

[0057] Using the extracted nucleic acids of SHIV, EHP, infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) as templates, PCR was carried out according to the method established by the standard curve. Amplify to verify its specificity.

[0058] Using the genomes of SHIV, EHP, IHHNV and WSSV as templates for real-time fluorescent quantitative PCR amplification, the results showed that only the corresponding fluorescent signals of SHIV and EHP were positive, and the detection of other viruses were negative, indicating that the constructed The dual real-time fluorescent quantitative PCR method has strong specificity ( image 3 ).

Embodiment 3

[0060] Sensitivity test The template with the determined DNA concentration was serially diluted 10 times, and the sensitivity was determined by the established dual real-time fluorescent quantitative PCR method.

[0061] Measure the concentration of DNA standard products of SHIV and EHP with a nucleic acid protein detector, calculate their copy number, and then carry out 10-fold serial dilution to obtain 1×10 7 copies / μL~1×10 1 copies / μL, using SHIV and EHP dual real-time fluorescence quantitative amplification established in this study, and obtained the kinetic amplification curve of SHIV and EHP probe method real-time fluorescent quantitative PCR ( Figure 4 ), the minimum amount of DNA that can be detected in the range of Ct Figure 5 , 6 ).

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Abstract

The invention provides an EHP and SHIV dual real-time fluorescent quantitative PCR detection primer and probe combination and kit, and belongs to the technical field of aquatic animal pathogen detection. The primer and probe combination adopts a dual real-time fluorescent quantitative PCR detection method, and amplification of two kinds of pathogenic DNA can be conducted simultaneously at a time by means of specialized detection primers and probes of EHP and SHIV. According to the detection kit, convenience, rapidness and low detection limit are achieved, the specificity is high, the sensitivity is high, the accuracy is high, shrimp enterocytozoon hepatopenaei and shrimp hemocyte iridovirus can be detected simultaneously through dual real-time fluorescent quantitative PCR detection, and the combination and kit are suitable for early monitoring of cover infection of shrimps.

Description

technical field [0001] The invention belongs to the technical field of aquatic animal pathogen detection, and in particular relates to a combination of primers and probes for EHP and SHIV double real-time fluorescent quantitative PCR detection and a kit. Background technique [0002] Shrimp Hemocyte Iridovirus (SHIV) was first discovered by Lightner and Redman in 1993 in Protrachypene precipua Burkenroad in a shrimp farm near Ecuador. The symptoms of the diseased shrimp were poor vitality and obvious hepatopancreas Atrophy, whitish muscles, reddened and broken intestines, empty stomach in the jejunum, blackened gills, walking legs and swimming legs. In 2017, researchers from the Yellow Sea Research Institute of the Chinese Academy of Sciences discovered a new virus on the severely dead Penaeus vannamei in Zhejiang. It does not belong to the five genera established under the Iridoviridae family. , named Shrimp Hematocytic Iridescent Virus (SHIV). Shrimp Enterocytozoon hepat...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6893C12Q1/6851C12Q1/06C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/6893C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 侯月娥曾俊霞陈斯娜蓝间媛许枣珠万绍莉
Owner 珠海科艺普检测科技有限公司
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