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CN-121975964-A - Fluorescent PCR primer, probe and kit for detecting aeromonas hydrophila of silver carp and bighead carp as well as detection method and application thereof

CN121975964ACN 121975964 ACN121975964 ACN 121975964ACN-121975964-A

Abstract

The invention relates to the technical field of biological detection, and discloses a primer, a probe and a kit for detecting aeromonas hydrophila based on fluorescence PCR and application thereof. The primer and fluorescent probe combination is obtained through design and screening of a specific gene conservation region of aeromonas hydrophila. The specific amplification of the aeromonas hydrophila gene based on the fluorescent PCR technology can be successfully realized. The method improves the detection accuracy and sensitivity, can detect aeromonas hydrophila in the early stage of the silver carp bacterial sepsis, is convenient for the prevention and control of the silver carp bacterial sepsis, and can meet the basic requirements of the on-site detection of a farm.

Inventors

  • FENG DEXIANG
  • YUAN GAOLIANG
  • ZHANG YANWEI
  • LIN XIN
  • LIU KAIFANG
  • WANG RUOXUAN
  • LIU HUIYU

Assignees

  • 信阳农林学院

Dates

Publication Date
20260505
Application Date
20260311

Claims (9)

  1. 1. The fluorescent quantitative PCR primer for detecting aeromonas hydrophila of silver carp and bighead carp is characterized in that the upstream primer F is selected from any one of SEQ ID NO.1-SEQ ID NO. 5; the downstream primer R is selected from any one of SEQ ID NO.6-SEQ ID NO. 10; the sequence of the primer is from left to right, the left side is a starting end 5', and the right side is an extension end 3'.
  2. 2. The fluorescent quantitative probe for detecting aeromonas hydrophila of silver carp and bighead carp is characterized in that the probe P is selected from any one of SEQ ID NO.11-SEQ ID NO. 15; The sequence of the gene of the probe is from left to right, the left side is a starting end 5', the right side is an extension end 3', the starting end 5 'is marked with a fluorescent group, and the extension end 3' is marked with a quenching group.
  3. 3. The probe of claim 2, wherein the fluorophore is FAM and the quencher is BHQ.
  4. 4. A fluorescent quantitative kit for detecting aeromonas hydrophila of silver carp and bighead carp, which is characterized by comprising the primer and the probe according to claim 1 and 2.
  5. 5. The kit of claim 4, further comprising a plasmid standard.
  6. 6. The kit according to claim 5, wherein the plasmid standard is constructed by inserting a specific gene gyrA of aeromonas hydrophila into a pcDNA3.1 plasmid serving as a vector through a genetic engineering method, and the nucleotide sequence of the specific gene gyrA is shown as SEQ ID No. 16.
  7. 7. A fluorescent quantitative detection method of aeromonas hydrophila of silver carp and bighead carp is characterized by comprising the following steps of (1) taking a sample to be detected and extracting DNA; (2) Performing fluorescent PCR detection using the extracted DNA as a template and the upstream primer F and the downstream primer R as defined in claim 1 and the fluorescent probe P as defined in claim 2; (3) The fluorescence signal appears, the sample is positive, otherwise, the sample is negative.
  8. 8. The method according to claim 7, wherein in the step (2), the reaction system of the fluorescent PCR is 2X TAQ PCR MASTER Mix 10. Mu.L, each of the fluorescent probe, the upstream primer and the downstream primer is 0.5. Mu.L, 1. Mu.L of the DNA template to be detected or the plasmid standard according to claim 5 is supplemented to 20. Mu.L with sterilized ddH 2 O; the reaction procedure was 95℃pre-denaturation 30s, 95℃denaturation 5s,60℃annealing/extension 34s,40 cycles.
  9. 9. Use of the kit according to claim 4, characterized in that the kit is used for the following applications: (1) Qualitative detection and/or analysis of aeromonas hydrophila of silver carps and bighead carps; (2) Monitoring the bacterial load of the culture water body and the bait, and early warning the explosion risk of the silver carp and bighead carp bacterial septicemia in advance; (3) Quantitative determination of the bacterial load of the diseased fish body, and auxiliary grading, disease course evaluation and prognosis judgment of the disease; (4) The effects of the sterilizing preparation and the antibacterial drug are evaluated, the accurate medication is guided, and the abuse of antibiotics is reduced; (5) Micro-ecological safety evaluation of the cultivation environment, and auxiliary optimization of a water quality management and control and cultivation management mode; (6) Compliance screening of pathogenic bacteria before aquatic products are marketed, so that edible safety and market circulation are ensured; (7) Epidemiological investigation of aeromonas hydrophila, tracking propagation paths and regional epidemic rules; (8) Quantitatively detecting virulence genes of the bacterial strains, and evaluating pathogenicity and outbreak risk of pathogenic bacteria; (9) And (3) researching disease resistance breeding of silver carp and bighead carp, and assisting in screening high-quality germplasm resources resistant to aeromonas hydrophila.

Description

Fluorescent PCR primer, probe and kit for detecting aeromonas hydrophila of silver carp and bighead carp as well as detection method and application thereof Technical Field The invention belongs to the technical field of biological detection, and particularly relates to a fluorescent PCR primer, a probe, a kit for detecting aeromonas hydrophila of silver carp and bighead carp, and a detection method and application thereof. Background The silver carp and bighead carp are filter-feeding fishes with important ecological and economic values in freshwater aquaculture in China, and the silver carp (Hypophthalmichthys molitrix) and bighead carp (ARISTICHTHYS NOBILIS) can effectively regulate the eutrophication state of the aquaculture water body by feeding plankton in the water body, so that the silver carp and bighead carp are key varieties in a pond polyculture mode. The method has the advantages of fast growth and strong adaptability, and has an important share in the yield of large freshwater fish in China. In recent years, with the increase of culture density, bacterial septicemia and frequent outbreaks of enteritis caused by aeromonas hydrophila (Aeromonas hydrophila) occur, and the body surface bleeding and visceral organ swelling of diseased fish are accompanied by ascites, so that the death rate is high, and serious economic loss is caused. And aeromonas hydrophila is a typical zoonotic primordium of human-animal-fish zoonotic, and the public health safety significance of the aeromonas hydrophila is not ignored. The bacteria not only harm aquatic animals, but also can infect human beings by contacting with polluted water sources or eating uncooked infected aquatic products and the like, and are important food-borne and opportunistic pathogenic bacteria. The human infection can cause epidermic wound infection, gastroenteritis and diarrhea, and even necrotizing fasciitis or septicemia can be caused in severe cases, so that the injury to people with low immune functions is particularly remarkable. Therefore, the establishment of a rapid and accurate aeromonas hydrophila detection method is not only the need of guaranteeing the healthy development of the aquaculture industry, but also an important link of preventing and controlling the food safety risk and maintaining the public health safety. The detection of aeromonas hydrophila at present mainly depends on bacterial isolation culture and biochemical identification, the operation is tedious and takes longer time, in addition, the ELISA detection method (Ren Yan, pan Zihao, liu Chengping, yao Huochun, wu Shuqin. Dot-ELISA method is used for detecting pathogenic aeromonas hydrophila [ J ]. Animal husbandry and veterinary school report, 2011, 42 (10): 1409-1415) established by Ren Yan et al is quick, but has limited sensitivity and is easily influenced by cross reaction, and the conventional PCR and isothermal amplification technology (such as RPA and LAMP) disclosed in the CN103305613B, CN117089638A, CN111793702A, CN110734994B, CN119506444A patent improves the detection sensitivity, but still has the problems of complex primer design, unstable amplification specificity or easy generation of false positive and the like, and is difficult to meet the requirements of quick and accurate diagnosis on site of a fishing ground. Therefore, a simpler and more convenient aeromonas hydrophila detection method is developed, and the high-sensitivity, high-specificity and rapid quantitative detection of aeromonas hydrophila of silver carps is realized by designing specific primers and probes, so that a reliable technical means is provided for early diagnosis, prevention and control of a cultivation site. Disclosure of Invention In order to effectively overcome the defects and limitations in the technology, the invention establishes a detection method based on fluorescence PCR (qPCR), wherein the used primer F is selected from any one of SEQ ID NO.1-SEQ ID NO.5, and the downstream primer R is selected from any one of SEQ ID NO.6-SEQ ID NO. 10. Further, the kit also comprises a probe P, wherein the probe P is selected from any one of SEQ ID NO.11-SEQ ID NO. 15. Furthermore, the sequence of the primer group gene is from left to right, the left side is a starting end 5', and the right side is an extension end 3'. Furthermore, the sequence of the gene of the probe is from left to right, the left side is a starting end 5', the right side is an extension end 3', the starting end 5 'is marked with a fluorescent gene, and the extension end 3' is marked with a quenching gene. Further, the fluorescent group is FAM, and the quenching group is BHQ. BHQ is a quencher available from LGC company, and is of the type BHQ1-3, and the invention can use a quencher of the type 1-3. The invention also provides a fluorescent quantitative kit for detecting aeromonas hydrophila of silver carp and bighead carp, which comprises the primer group and the probe. Further, the kit also comprises a plasmid