Multiple PCR primer set and probes and detecting method for simultaneously detecting three kinds of aeromonas

An Aeromonas and detection method technology, applied in the field of biological detection, can solve the problems of high gene homology and difficult to distinguish, and achieve the effects of good repeatability, improved specificity and high sensitivity

Inactive Publication Date: 2016-03-23
于辉
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high gene homology between members of the Aeromonas genus, common PCR detection methods are difficult to distinguish

Method used

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  • Multiple PCR primer set and probes and detecting method for simultaneously detecting three kinds of aeromonas
  • Multiple PCR primer set and probes and detecting method for simultaneously detecting three kinds of aeromonas
  • Multiple PCR primer set and probes and detecting method for simultaneously detecting three kinds of aeromonas

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Design and synthesis of specific primers and molecular beacon probes

[0065] The Aer gene sequence of Aeromonas hydrophila (accession number: M16495), the Aer gene sequence of Aeromonas victoria (accession number: EF034117) and the gyrB gene sequence of Aeromonas schubertius published on Genbank (accession number: JQ319030.1) carry out homology comparison, determine the selection range of each strain-specific primer and probe, apply Primer5.0 software to find all possible primer and probe sequence combinations, and screen qualified primer and probe sequences, The specificity was analyzed by BLAST software, and three pairs of specific primers and corresponding TaqMan probe target sequences were obtained. 5'-labeled FAM fluorophore for Aeromonas hydrophila probe, 5'-labeled HEX fluorophore for Aeromonas victoria probe, 5'-labeled CY5 fluorophore for Aeromonas schubertii probe group.

[0066] Primers and probes are as follows:

[0067] Aeromonas hydrophila u...

Embodiment 2

[0077] Sensitivity Analysis of Embodiment 2 Aeromonas hydrophila, Aeromonas victorii and Aeromonas schubert multiplex PCR detection method

[0078] 1. Experimental method

[0079] (1) Preparation of samples: construction method of standard plasmids: use the above-mentioned three kinds of Aeromonas upstream and downstream primers (SEQ ID NO: 1-2; SEQ ID NO: 4-5; SEQ ID NO: 7-8) respectively to corresponding bacterial DNA Amplified by conventional PCR method to obtain figure 2 The calculation fragments of corresponding sizes are shown, and the nucleic acid sequences are shown in SEQ ID NO: 10-12. After the amplified fragments were purified, they were cloned into the pMD19-T vector by TA, identified by sequencing, and the recombinant vectors with correct sequencing results were transformed into DH5α competent cells, and the plasmids were extracted after amplification to obtain the plasmid DNAs corresponding to the three bacteria . The plasmids of the three bacteria were then ...

Embodiment 3

[0091] Example 3 Aquatic Animal Infection Aeromonas Detection and Specificity Analysis

[0092] 1. Experimental method

[0093] (1) Preparation of samples: samples were taken from diseased fish positive for Aeromonas hydrophila, Aeromonas vieterii and Aeromonas schubert, and the tissues of the liver, spleen and kidney of the fish were taken The samples were mixed a little, and the diseased fish tissues positive for Aeromonas temperatus, Escherichia coli, Flavobacterium columnar and Pseudomonas fluorescens were mixed as the control. DNA from the above tissues was extracted using a microbial DNA extraction kit to obtain a DNA template.

[0094] (2) Reaction solution preparation:

[0095] Prepare the reaction solution according to the following table:

[0096] Element

Volume (μl)

2×SG probe Master Mix

10

DNA template

1

Upstream primer (10μM)×3

0.5×3

Downstream primer (10μM)×3

0.5×3

Probe (10μM)×3

0.5×3

d...

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Abstract

The invention discloses a multiple PCR primer set and probes and a detecting method for simultaneously detecting three kinds of aeromonas and discloses a detecting method for simultaneously and fast detecting aeromonas hydrophila (AH), aeromonas veronii (AV) and aeromonas schubertii (AS) and a primer set and probes used in the detecting method. The detecting method is characterized in that three pairs of characteristic primers and three nucleic acid probes are used and can distinguish the three aeromonas high in homology, and the three kinds of aeromonas are simultaneously detected and distinguished in a same pipe reaction system. By means of the primers and the probes and the detecting method, the three kinds of zoonosis pathogenic bacteria can be fast detected, and the advantages of easy and convenient operation, high sensitivity, good specificity and the like are achieved; suspected cases can be found and determined in time, and the detection level of the kind of pathogens can be improved.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a multiple PCR primer set, a probe and a detection method for simultaneously detecting three types of Aeromonas. Background technique [0002] my country is a big aquaculture country in the world, however, the disease loss seriously restricts the healthy and sustainable development of aquaculture industry. Aeromonas Hydrophila (AH for short), Aeromonas Veronii (AV for short) and Aeromonas Schubertii (AS for short) are pathogenic Aeromonas prevalent in my country. The diseases caused by these three pathogenic bacteria not only bring serious economic losses to the aquaculture industry, but also infect humans and animals through aquatic animals and aquatic products, leading to diarrhea and food poisoning in humans and animals. Establishing accurate and reliable detection technology for the main pathogenic strains is of great significance for the prevention and control o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2600/16
Inventor 杨映李华于辉
Owner 于辉
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