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Specific primer pair, probe and detection reagent kit for detecting aeromonas hydrophila

A technology of Aeromonas hydrophila and detection kits, applied in the field of probes, kits, and primers, which can solve the problems of limiting the use of LAMP methods and false positive results

Active Publication Date: 2020-01-31
江苏省渔业技术推广中心 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although LAMP increases the amplification efficiency, non-specific pairing between primers easily leads to false positive results, which limits the use of LAMP methods

Method used

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  • Specific primer pair, probe and detection reagent kit for detecting aeromonas hydrophila
  • Specific primer pair, probe and detection reagent kit for detecting aeromonas hydrophila
  • Specific primer pair, probe and detection reagent kit for detecting aeromonas hydrophila

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] 1. Acquisition of the positive plasmid of Aeromonas hydrophila: through the NCBI search literature, the common Aeromonas hydrophila genes are listed, and the Vector NTI software is used to compare and find out its conserved region, and select the partial region of the aerA gene as The target amplified segment was constructed into the vector PUC57 to prepare a positive plasmid for Aeromonas hydrophila. The plasmid was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The partial sequence of the selected aerA gene is as follows:

[0065] GGGTATCGTTGTGGCGAGAAGACGGCCATCAAGGTCAGCAACTTTGCATACAACCTGGACCCTGACAGTTTCAAACATGGTGACGTGACCCAGTCTGATCGCCAGCTGGTCAAGACGGTGGTGGGCTGGGCGATCAACGACAGCGACACCCCGCAATCCGGTTATGATGTCACCCTACGTTACGATACTGCCACCAACTGGTCCAAGACCAATACCTATGGCCTGAGCGAGAAGGTGACCACCAAGAACAAGTTCAAGTGGCCACTGGTAGGAGAAACCGAACTCTCCATCGAGATTGCGGCCAACCAGTCCTGGGCATCCCAGAACGGGGGCTCTACCACCACCTCCCTGTCGCAATCCGTGCGGCCAACTGTGCCGGCCCGCTCCAAGATCCCGGTGAAGATCGAGCTCTACAAGGCTGACATCTCCTATCCCT...

Embodiment 2

[0092] Select the following primers and probe sequences:

[0093] The upstream primer sequence is: 5'-CGGTTATGATGTCACCCCTACGTTACGATAC-3' (SEQ ID NO: 3);

[0094] The downstream primer sequence is: 5'-CACGGATTGCGACAGGGAGGTGGTGGTAGA-3' (SEQ ID NO: 4);

[0095] The probe sequence is: 5'-TCACCCTACGTTACGATACTGCCACCAAC(FAM-dT)G(THF)(BHQ1-dT)CCAAGACCAATACCTA(C3-SPACER)-3'(SEQ ID NO: 9)

[0096] By synthesizing a plasmid containing the aerA gene sequence of Aeromonas hydrophila as the detection target, the method version amplification test of recombinase polymerase amplification (combined with exonuclease III) was carried out, and a 50 μl amplification reaction system was constructed as follows:

[0097] 60mM tris-acetic acid buffer pH8.0

[0098] 100mM potassium acetate

[0099] 14mM magnesium acetate

[0100] 3mM Dithiothreitol

[0101] 5% polyethylene glycol (20000)

[0102] 2mM ATP

[0103] 20mM creatine phosphate

[0104] 100ng / μl creatine kinase

[0105] 600ng / μl E. col...

Embodiment 3

[0120] Select the primer pair and probe sequence designed in Example 2, utilize the recombinase polymerase amplification (in conjunction with endonuclease IV) method amplification reaction system to amplify, construct 50 μ l amplification reaction system as follows:

[0121] 60mM tris-acetic acid buffer pH8.0

[0122] 100mM potassium acetate

[0123] 14mM magnesium acetate

[0124] 3mM Dithiothreitol

[0125] 5% polyethylene glycol (molecular weight 20000)

[0126] 2mM ATP

[0127] 20mM creatine phosphate

[0128] 100ng / μl creatine kinase

[0129] 400ng / μl E. coli recA protein

[0130] 200ng / μl E. coli SSB protein

[0131] 60ng / μl E. coli recO protein

[0132] 40ng / μl E. coli recR protein

[0133] 60ng / μl E. coli recF protein

[0134] 8Units Bacillus subtilis DNA polymerase I

[0135] 50ng / μl Endonuclease IV

[0136] 450 μM dNTPs

[0137] 420nM per upstream primer

[0138] 420nM each downstream primer

[0139] 120nM fluorescent probe

[0140] The template is a ...

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Abstract

The invention discloses a specific primer pair, probe and detection reagent kit for detecting aeromonas hydrophila, and further discloses a detection reagent kit. An aerA gene in the aeromonas hydrophila is used as a detection target point, and through a constant-temperature amplification technique, the specific primer and the probe are combined, so that the detection convenience and the specificity of the aeromonas hydrophila are improved, and besides, the detection time is greatly shortened. Compared with a PCR detection method, the method disclosed by the invention omits an electrophoresisverification process of products, can avoid the appearance of false positive results, and can improve the detection precision. Compared with qPCR, the method is simpler and easier to operate, complexinstrument and equipment do not need to be operated, the cost is saved, the detection efficiency is improved, and besides, promotion and application in a large range can be convenient. Compared with other constant-temperature amplification methods, the detection method disclosed by the invention is shorter in required time, and the detection accuracy rate is higher.

Description

technical field [0001] The invention relates to primers, probes and kits, in particular to specific primer pairs, probes and detection kits for detecting Aeromonas hydrophila. Background technique [0002] Aeromonas hydrophila (Aeromonas hydrophila) is a common pathogenic bacteria that widely exists in the water environment. It can not only cause infectious diseases of various aquatic animals, but also cause reptiles, amphibians, birds and mammals. A variety of animals such as systemic sepsis or local infection, and often lethal animals. Since the 1990s, this pathogen has often caused fulminant sepsis in freshwater cultured fish in my country, causing significant economic losses, and has become one of the main bacterial diseases of aquaculture animals. In recent years, a large number of studies have proved that Aeromonas hydrophila can also be infected alone or with other pathogenic bacteria, causing food poisoning, diarrhea, sepsis and local wound infections in humans, aff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2537/1376C12Q2563/107C12Q2531/119C12Q2522/101
Inventor 吴亚锋刘肖汉王楠楠王晶晶刘训猛陈静袁锐于继彬
Owner 江苏省渔业技术推广中心
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