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Detection kit for aeromonas veronii and application method thereof

A technology of Aeromonas viridis and detection kit, which is applied in the field of genetic engineering, can solve the problems of unreasonable detection of target genes, low accuracy, complicated operation, etc., to avoid repeated cultivation, high specificity and strong sensitivity Effect

Inactive Publication Date: 2017-02-15
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the technical defects of the prior art, and provides a detection kit for Aeromonas versicolor and its use method, so as to solve the problem of accurate detection method of Aeromonas veroris based on PCR technology in the prior art. less technical issues
[0005] Another technical problem to be solved by the present invention is the technical problem that the accuracy of the detection method of Aeromonas victorii based on PCR technology in the prior art is low due to the unreasonable selection of the detection target gene.
[0006] Another technical problem to be solved by the present invention is that some detection methods for Aeromonas verkisii in the prior art are cumbersome to operate and take a long time

Method used

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  • Detection kit for aeromonas veronii and application method thereof
  • Detection kit for aeromonas veronii and application method thereof
  • Detection kit for aeromonas veronii and application method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Design of primers

[0037] According to the gyrB gene sequence (KX058388) and Aha gene sequence (accession number: KU877437) of Aeromonas verkisii in GenBank, two pairs of specific fragment primers were designed and screened. The primer sequences and expected lengths of amplified fragments are shown in Table 1:

[0038] Table 1 The specific primer sequences and expected amplification results of gyrB gene and Aha gene

[0039]

[0040] 2. Double PCR reaction system and reaction conditions

[0041] Reaction system: 1 μL of sample solution to be tested, 2.5 μL of 10×PCR buffer, 0.25 μL of 5 U / μL rTaq DNA polymerase solution, 250 mmol / L of MgCl 2 Solution 1.5 μL, 20 μmol / L gyrB gene upstream primer 0.5 μL, 20 μmol / L gyrB gene downstream primer 0.5 μL, 20 μmol / L Aha gene upstream primer 0.6 μL, 20 μmol / L Aha gene downstream primer 0.6 μL, 2.5 mmol 0.5 μL / L dNTP solution, ddH 2 O to make up to 25 μL.

[0042] Reaction conditions: pre-denaturation at 94°C for 4 min;...

Embodiment 2

[0055] The invention discloses a detection kit for Aeromonas vernerii, which comprises a pair of primers for the gyrB gene of the Aeromonas vernerii and a pair of primers for the Aha gene of the Aeromonas veroris.

[0056] On the basis of the above technical solutions, the following conditions are met:

[0057] The pair of primers for the gyrB gene of Aeromonas vernerii is an upstream primer whose sequence is shown in SEQ ID NO.1 and a downstream primer whose sequence is shown in SEQ ID NO.2.

[0058] The pair of primers for the Aha gene of Aeromonas vernerii is an upstream primer whose sequence is shown in SEQ ID NO.3 and a downstream primer whose sequence is shown in SEQ ID NO.4.

[0059] The kit also includes 10×PCR buffer, dNTP, MgCl 2 , rTaq DNA polymerase, positive quality control, negative quality control.

[0060] The test kit also includes Aeromonas verkis bacteria liquid as a positive quality control and ddH as a negative quality control 2 O.

[0061] A method fo...

Embodiment 3

[0069] The invention discloses a detection kit for Aeromonas vernerii, which comprises a pair of primers for the gyrB gene of the Aeromonas vernerii and a pair of primers for the Aha gene of the Aeromonas veroris.

[0070] On the basis of the above technical solutions, the following conditions are met:

[0071] The pair of primers for the gyrB gene of Aeromonas vernerii is an upstream primer whose sequence is shown in SEQ ID NO.1 and a downstream primer whose sequence is shown in SEQ ID NO.2.

[0072] The pair of primers for the Aha gene of Aeromonas vernerii is an upstream primer whose sequence is shown in SEQ ID NO.3 and a downstream primer whose sequence is shown in SEQ ID NO.4.

[0073] A method for applying the above-mentioned kit to detect Aeromonas victorii, comprising the following steps:

[0074] 1) Use the sample solution to be tested as the PCR template, use the DNA fragment with the sequence shown in SEQ ID NO. For the downstream primer of the Aeromonas vervetii ...

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Abstract

The invention provides a detection kit for aeromonas veronii and an application method thereof. In the technical scheme, gyrB gene is found to be a single-copy house-keeping gene through experiments and has more remarkable advantages than conventional 16S rRNA in the distinguishing and identifying of aeromonas veronii and sibling species thereof; and meanwhile, Aha gene is found to be stably exist in the aeromonas veronii and related to the toxicity thereof. Based on the beneficial discoveries, the gyrB gene and Aha gene are adopted as target genes to develop a duplex PCR detection method for aeromonas veronii; in the method, specific primers are designed for the two target genes, and a PCR reaction system and reaction conditions are determined. In the invention, the sensitivity and specificity of aeromonas veronii detection are high, the pelteobagrus fulvidraco-source pathogenic aeromonas veronii can be quickly and accurately detected, and the wrong detection and missed detection are effectively avoided. Meanwhile, repeated culture and redundant series of biochemical reactions are prevented, and the time, labor and cost are saved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and further relates to a biological detection method based on genetic engineering technology, in particular to a detection kit for Aeromonas verkirea and a use method thereof. Background technique [0002] Yellow catfish (Pelteobagrus fulvidraco), commonly known as Langsi, Huanglatin, Jiangqian, belongs to Siluriformes (Siluriformes), family (Bagridae), Pelteobagrus (Pelteobagrus), is a kind of bottom dwelling small economic fish common in rivers and lakes in my country Because of its tender meat and delicious taste, it is favored by consumers. However, as a disease-prone fish species, yellow catfish often suffers from diseases during the breeding process. The common diseases reported mainly include bacterial diseases, fungal diseases, parasites, and nutritional diseases. Among them, bacterial diseases are the most serious hazards, which can easily lead to large-scale death of yellow ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143Y02A50/30
Inventor 朱成科刘桂嘉周朝伟郑宗林张争世朱龙李艳苹杨成年
Owner SOUTHWEST UNIVERSITY
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