Kit and detection method for simultaneously detecting vibrio bacteria and edwardsiella ictaluri

The technology of a Vibrio genus and a kit, applied in the field of bacterial detection, can solve the problems of simultaneous detection of Vibrio genus bacteria and Edwardsiella spp., unfavorable mixed infection detection, heavy workload, etc., and achieve good application prospects, Inexpensive and time-consuming results

Inactive Publication Date: 2015-12-09
TONGWEI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection of Vibrio and Edwardsiella is performed separately, and only one pathogen can be detected at a time, which is not conducive to the detection of mixed infections, ...

Method used

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  • Kit and detection method for simultaneously detecting vibrio bacteria and edwardsiella ictaluri
  • Kit and detection method for simultaneously detecting vibrio bacteria and edwardsiella ictaluri
  • Kit and detection method for simultaneously detecting vibrio bacteria and edwardsiella ictaluri

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Primer Design

[0028] 1. Experimental method

[0029] 1. PCR primer design and synthesis

[0030] Two pairs of primers were designed according to the common rpoA gene sequence of Vibrio bacteria and the phosphoserine aminotransferase gene sequence of Edwardsiella spp. (Table 1). Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0031] Table 1PCR primer information

[0032]

[0033] 2. Template preparation

[0034] Activated Edwardsiella and Vibrio parahaemolyticus were extracted using the Bacterial Genomic DNA Extraction Kit produced by Tiangen Biochemical Technology Co., Ltd.

[0035] 3. PCR amplification

[0036] Use the DNA extracted in step 2 as a template, and the template for the negative control is ddH 2 O, get two pairs of primers in step 1 and amplify respectively according to the following system and program:

[0037] PCR reaction system (20μl):

[0038]

[0039] PCR reaction program:

[0040] A...

Embodiment 2

[0050] Embodiment 2 primer specificity test

[0051] 1. Test method

[0052] 1. PCR primers

[0053] Take two pairs of primers shown in Table 1 in Example 1.

[0054] 2. Template preparation:

[0055] Streptococcus agalactiae, Streptococcus iniae, Pseudomonas fluorescens, Aeromonas hydrophila, Flavobacterium columnar, Aeromonas caviae standard, Flavobacterium columnar, Salmonella, Bacillus, Aeromonas temperatus Genomic DNA was used as a template and extracted using a bacterial genomic DNA extraction kit produced by Tiangen Biochemical Technology Co., Ltd.

[0056] 3. PCR amplification

[0057] Take the genomic DNA obtained in step 2 as a template, and the template for the negative control is ddH 2 O, using a mixture of 2.5 μl of Edwardsiella DNA and 2.5 μl of Vibrio parahaemolyticus DNA as a positive template, amplify according to the amplification procedure in Example 1.

[0058] 4. Result detection

[0059] 1.5% agarose gel was used to perform electrophoresis at a const...

Embodiment 3

[0063] Embodiment 3 primer sensitivity test

[0064] 1. Test method

[0065] 1. PCR primers

[0066] Take two pairs of primers shown in Table 1 in Example 1.

[0067] 2. Template preparation:

[0068] Take the positive sample DNA (a mixture of 2.5 μl Edwardsiella DNA and 2.5 μl Vibrio parahaemolyticus DNA) for concentration determination, and follow the original concentration of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7、 10 -8 、10 -9 Doubling dilutions were performed.

[0069] 3. PCR amplification

[0070] Take the genomic DNA obtained in step 2 as a template, and the template for the negative control is ddH 2 O, amplify according to the amplification procedure in Example 1.

[0071] 4. Result detection

[0072] 1.5% agarose gel was used to perform electrophoresis at a constant voltage of 100V for 60 min, and the results of PCR amplification were observed under a gel imaging system.

[0073] 2. Results

[0074] Such as image 3 As shown, according to th...

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Abstract

The invention discloses primer pairs shown in SEQ ID NO.1-2 and SEQ ID NO.3-4 and an application thereof, and further, discloses a kit and detection method for detecting vibrio bacteria and edwardsiella ictaluri. The kit and the method can amplify vibrio bacteria and edwardsiella ictaluri gene fragments respectively and simultaneously, achieve one-step detection of two pathogenic bacteria in a same reaction system, have the advantages of strong specificity, high sensitivity, short consuming time, simple steps, small workload, and low cost, are used for rapid detection of culture water bodies, can timely and effectively prevent happening of fish diseases, and have good application prospects.

Description

technical field [0001] The invention relates to a detection technology of bacteria, in particular to a kit and a detection method for simultaneous detection of bacteria belonging to the genus Vibrio and Edwardsiella spp. Background technique [0002] Yellow catfish belongs to the order Siluriformes, and belongs to the genus Catfish. It is also known as yellow catfish. Its tender meat, low fat content and high protein content are deeply loved by consumers. However, with the expansion of the breeding scale and the increase of the breeding density, the breeding environment continues to deteriorate, and diseases continue to break out in the breeding process, which brings huge economic losses to the farmers. Among them, Vibrio and Edwardsiella have developed into the main pathogens of yellow catfish. [0003] Edwardsiella catfish, belonging to the Enterobacteriaceae Edwardsia genus bacteria, can cause the "splitting head syndrome" of yellow catfish; Vibrio bacteria, such as Vibr...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/04C12R1/01
Inventor 刘衍鹏刘天强黄冠军敬小兵王浩丞
Owner TONGWEI
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