Kit and method for detecting aeromonas veronii

A technology of Aeromonas viridis and a kit, which is applied in the field of detection of Aeromonas viridis, can solve the problems of low sensitivity, low detection limit, and specificity needs further research, and achieves high sensitivity and improved economical efficiency. Benefit, detect fast effect

Active Publication Date: 2016-04-20
TONGWEI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Bian Yu et al., "Establishment and Preliminary Application of RCR Rapid Detection Method for Aeromonas vickerii", Chinese Journal of Veterinary Medicine, Issue 01, 2013, disclosed a method for detecting Aeromonas vickerii, but its sensitivity was low. The minimum detection limit is 0.158pg / μL
Wang Hui, Bian Yu, etc., "Establishment of a double-gene PCR detection method for Aeromonas verkisii" discloses a method for detecting Aeromonas veroris by simultaneously detecting two genes, but its specificity needs to be further studied. The sensitivity is also low, and the minimum detection limit is still 0.158pg / μL

Method used

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  • Kit and method for detecting aeromonas veronii
  • Kit and method for detecting aeromonas veronii
  • Kit and method for detecting aeromonas veronii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Primer Design

[0032] 1. Experimental method

[0033] 1. PCR primer design and synthesis

[0034] A pair of primers were designed according to the sequence of the aerolysin gene of Aeromonas verkirea (Table 1). Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0035] Table 1PCR primers

[0036]

[0037] 2. Template preparation

[0038] Take 5 strains of Aeromonas verkirea from different sources (identified as Aeromonas verkirea by 16SrDNA sequence), and use the Bacterial Genomic DNA Extraction Kit produced by Tiangen Biochemical Technology Co., Ltd. (to extract genomic DNA as a template.

[0039] 3. PCR amplification and detection

[0040] The PCR amplification system is shown in Table 2.

[0041] Table 2 PCR amplification system

[0042] Reactant

volume

2×PCR Mix

12.5μl

10 μM Weishi1F / Weishi1R

0.25μl each

template

2μl

wxya 2 o

10μl

[0043] P...

Embodiment 2

[0048] Embodiment 2 primer specificity test

[0049] 1. Test method

[0050] 1. PCR primers

[0051] With embodiment 1.

[0052] 2. Template preparation:

[0053] Aeromonas hydrophila, Edwardsiella tarda, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus iniae, Vibrio cholerae, Vibrio parahaemolyticus, Aeromonas temperatus, Pseudomonas fluorescens Bacteria, Salmonella enterica, Vibrio alginolyticus, Vibrio vulnificus and Aeromonas virtienii, using the bacterial genomic DNA extraction kit produced by Tiangen Biochemical Technology Co., Ltd. to extract genomic DNA as a template.

[0054] 3. PCR amplification and detection

[0055] With embodiment 1.

[0056] 2. Results

[0057] The result is as figure 2As shown, except that the positive sample (Aeromonas victoria) can amplify the target band with a size of 309bp, 12 strains of other common aquatic pathogens including Aeromonas bacteria, namely Aeromonas hydrophila, Edwardsiella tarda, Stenotrophomona...

Embodiment 4

[0059] Example 4 Primer Sensitivity Test

[0060] 1. Test method

[0061] 1. PCR primers

[0062] With embodiment 1.

[0063] 2. Template preparation:

[0064] Bacteria sensitivity: Plate count the overnight culture of Aeromonas victoria, and follow the original concentration of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 The bacterial genomic DNA extraction kit produced by Tiangen Biochemical Technology Co., Ltd. was used to extract genomic DNA.

[0065] DNA sensitivity: Take Aeromonas verkisii, use the bacterial genome DNA extraction kit produced by Tiangen Biochemical Technology Co., Ltd. to extract genomic DNA, and measure the concentration of the extracted Aeromonas -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 Doubling serial dilutions.

[0066] 3. PCR amplification and detection

[0067] With embodiment 1.

[0068] 2. Results

[0069] Cell sensitivity test electrophoresis results are as follows: image 3 As shown, for each dilutio...

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Abstract

The invention discloses a method and a kit for detecting aeromonas veronii and relates to a detection technology of aeromonas veronii. The kit for detecting fish pathogenic bacteria, disclosed by the invention, comprises primer pairs of which the sequence is shown as SEQ ID NO: 1-2, and genes from aeromonas veronii are amplified. The detection kit and detection method provided by the invention can accurately and effectively detect the aeromonas veronii, are high in sensitivity, high in specificity, low in time consumption and fast in detection and can be used for rapidly detecting and predicting fish diseases, preventing fish diseases and improving economic benefits.

Description

technical field [0001] The invention relates to a detection method and a detection kit for Aeromonas vernerii. Background technique [0002] Aeromonas verdeii is a facultative anaerobic and motile Gram-negative bacillus, which is an important zoonotic pathogenic microorganism. In aquatic products, it mainly causes local infections such as septicemia or skin ulcers in aquaculture animals, such as rot skin disease, perforation disease, hemorrhage disease of Chinese soft-shelled turtle, ascites diseases such as carp and channel catfish, rotten tail disease of Chinese eel, skin ulcer disease of loach, Eriocheir sinensis and other septicemia. The mortality rate of sick aquatic animals is as high as 60% to 100%, causing serious economic losses. At present, the clinical diagnosis of Aeromonas verdeii mostly relies on general bacteriological tests such as the observation of the appearance and pathology, the isolation and identification of bacteria, and the pathological phenomenon ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 敬小兵刘天强黄冠军阳涛刘衍鹏
Owner TONGWEI
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