Search

CN-122012760-A - Primer pair, composition, kit and method for detecting pseudomonad for killing yellow croaker visceral ichthyophthiriasis based on RPA-CRISPR/Cas12a technology

CN122012760ACN 122012760 ACN122012760 ACN 122012760ACN-122012760-A

Abstract

The invention relates to a primer pair, a composition, a kit and a method for detecting pseudomonad for killing yellow croaker visceral ichthyophthiriasis of large yellow croaker based on an RPA-CRISPR/Cas12a technology, wherein the nucleotide sequence of an upstream primer RPA-F is respectively shown as SEQ ID NO. 1-4, the nucleotide sequence of a downstream primer RPA-R corresponding to the upstream primer RPA-F is respectively shown as SEQ ID NO. 5-8, and the composition comprises a primer pair, crRNA, cas12a protein and a fluorescent report probe. Compared with the prior art, the P. plecoglossicida detection method is simple and convenient to operate, short in detection time, good in specificity and high in sensitivity, only needs common constant-temperature equipment, and is suitable for on-site rapid detection of basic laboratories and large yellow croaker farms.

Inventors

  • ZHANG YIBEI
  • WANG QIYAO
  • LI YIMING
  • MA YUE

Assignees

  • 华东理工大学

Dates

Publication Date
20260512
Application Date
20260213

Claims (10)

  1. 1. The primer pair for detecting the pseudomonad for killing the yellow croaker visceral ichthyophthiriasis based on the RPA-CRISPR/Cas12a technology is characterized by comprising one of the following a) to d) for amplifying the pseudomonad for killing the pseudomonad DVB73_RS07365 gene: a) An upstream primer RPA-F1 with a nucleotide sequence shown as SEQ ID NO.1 and an upstream primer RPA-R1 with a nucleotide sequence shown as SEQ ID NO. 5; b) An upstream primer RPA-F2 with a nucleotide sequence shown as SEQ ID NO.2 and an upstream primer RPA-R2 with a nucleotide sequence shown as SEQ ID NO. 6; c) An upstream primer RPA-F3 with a nucleotide sequence shown as SEQ ID NO.3 and an upstream primer RPA-R3 with a nucleotide sequence shown as SEQ ID NO. 7; d) The nucleotide sequence is shown as an upstream primer RPA-F4 shown as SEQ ID NO.4 and an upstream primer RPA-R4 shown as SEQ ID NO. 8.
  2. 2. A composition for detecting pseudomonad for killing yellow croaker visceral ichthyophthiriasis based on the RPA-CRISPR/Cas12a technology, which is characterized by comprising the primer pair as set forth in claim 1 and crRNA, wherein the nucleotide sequence of the crRNA is shown as SEQ ID NO. 9.
  3. 3. The composition of claim 2, further comprising a Cas12a protein and a fluorescent reporter probe having a nucleotide sequence of TTATT.
  4. 4. A kit for detecting pseudomonad aromaticalis responsible for visceral ichthyophthiriasis in large yellow croaker based on RPA-CRISPR/Cas12a technology, comprising the primer pair of claim 1, and/or comprising the composition of claim 2.
  5. 5. Use of a primer pair according to claim 1, a composition according to any one of claims 2 or 3 and/or a kit according to claim 4 in any one of the following: The application in preparing a product for detecting pseudomonad for killing the yellow croaker visceral ichthyophthiriasis; use in the manufacture of a product for the diagnosis or co-diagnosis of a disease caused by pseudomonas infection of a aromaticillium fish; The application of the method in preparing products for screening diseases caused by pseudomonas infection of the fragrant fishes.
  6. 6. The method for detecting the pseudomonad for killing the yellow croaker visceral ichthyophthiriasis based on the RPA-CRISPR/Cas12a technology is characterized by comprising the following steps of: s1, extracting DNA of a sample to be detected; S2, carrying out RPA amplification by using the DNA of the sample to be detected obtained in the step S1 as a template and using the primer pair as defined in claim 1 to obtain an RPA amplification product; S3, detecting the RPA amplification product obtained in the step S2 by using a CRISPR/Cas12a cutting reaction system; S4, judging the result according to the fluorescence intensity returned under ultraviolet irradiation.
  7. 7. The method of claim 6, wherein the reaction system for RPA amplification in step S2 is: 29.4 mu L of A buffer,2 mu L of 10 mu M upstream primer, 2 mu L of 10 mu M downstream primer, 2 mu L of DNA template, 2.5 mu L of B buffer and dd H 2 O are added into a freeze-dried powder reagent reaction tube to complement the system to 50 mu L, Wherein the freeze-dried powder reagent reaction tube, the buffer A and the buffer B are derived from a DNA amplification kit; The concentration of the DNA template when added is 10 fg/μl or more.
  8. 8. The method of claim 6, wherein the reaction conditions for the amplification of RPA in step S2 are 37℃incubation (15-20) min.
  9. 9. The method of claim 6, wherein in step S3, the CRISPR/Cas12a cleavage reaction system is 2. Mu.L 10x Cas12a reaction solution, 2. Mu.L Cas12a protein, 5. Mu.L ddH 2 O, 4. Mu.L 1. Mu.L crRNA, 2. Mu.L fluorescent reporter probe, wherein the final content of Cas12a protein is 100 ng and the final concentration of fluorescent reporter probe is 2. Mu.M.
  10. 10. The method of claim 6, wherein the reaction conditions of the CRISPR/Cas12a cleavage reaction system in step S3 are 37 ℃ incubation (15-60) min.

Description

Primer pair, composition, kit and method for detecting pseudomonad for killing yellow croaker visceral ichthyophthiriasis based on RPA-CRISPR/Cas12a technology Technical Field The invention belongs to the technical field of biological detection, and relates to a primer pair, a composition, a kit and a method for detecting pseudomonad for killing visceral ichthyophthiriasis of large yellow croaker based on an RPA-CRISPR/Cas12a technology. Background It was first discovered in 2000 that visceral ichthyophthiriasis in large yellow croakers is caused by Pseudomonas fragi (Pseudomonas plecoglossicida). Pseudomonad of the pseudosciaena crocea shows a series of remarkable clinical symptoms after being infected by the pseudomonad of the pseudosciaena crocea, and main symptoms comprise white spots of viscera, wherein the white spots are caused by accumulation of thalli and metabolites thereof, so that viscera tissues show mildew-like white spots. In addition, fish infected with Pseudomonas fragi can have increased mucous secretion on the body surface, congestion and bleeding on the gill, reduced fish body movement and decreased appetite, and seriously affects the growth and survival rate. Research shows that pseudomonas fish is easy to burst under the conditions of water temperature change and over-high culture density, and researchers are working on developing effective diagnosis and control measures to reduce the influence of the disease on fishery production. Early measures for preventing visceral ichthyophthiriasis of large yellow croakers include strengthening water quality management, periodically cleaning the culture environment, controlling the culture density and performing preventive treatment by using antibacterial agents. In addition, enhancing quarantine of introduced fish is also one of the key measures. Diagnostic techniques commonly used today include PCR, LAMP, ELISA, etc. The diagnosis method for the pseudomonas fragi mainly comprises the traditional method (such as blood smear method, histopathological analysis and the like), the immunological method (such as enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody detection technology (IFAT) and the like), and the molecular biology technology (including conventional PCR, nested PCR, real-time fluorescent quantitative PCR, loop-mediated isothermal amplification technology (LAMP) and the like). The diagnosis techniques have advantages and application ranges, but have higher requirements on detection conditions and operators, and are not beneficial to large-area popularization and application. Therefore, it is very necessary to develop a diagnosis technique which is specific, sensitive, rapid, convenient and easy to popularize. The RPA-CRISPR/Cas12a is a novel technology which combines a recombinase polymerase isothermal amplification technology and a CRISPR/Cas12a technology, and can realize exponential amplification of a target sequence in an RPA reaction system within 10-20 min by using a pair of upstream and downstream primers with a gap of generally not more than 500 bp, and then the designed crRNA contains a complementary region of a target fragment, guides and activates ssDNA around a template of the Cas12a to cut, and a fluorescent reporter molecule is cut at the moment to release FAM groups, so that macroscopic fluorescence can be observed under ultraviolet after 30 min. The combination of RPA and CRISPR/Cas12a has the advantages of simple operation, low equipment requirement, low cost, high speed and the like, and is suitable for detection on site or production line. Disclosure of Invention The invention aims to provide a primer pair, a composition, a kit and a method for detecting pseudomonad for killing yellow croaker visceral ichthyophthiriasis of large yellow croaker based on an RPA-CRISPR/Cas12a technology, so as to solve the problems of the prior art, and the method is suitable for rapidly detecting the visceral ichthyophthiriasis of large yellow croaker in a basic laboratory and a large yellow croaker farm site, reduces the rapid spreading and spreading risk of the new pathogenic agent, improves the prevention and control effect on the visceral ichthyophthiriasis of large yellow croaker, and provides technical support for improving the growth and survival rate of large yellow croaker. The aim of the invention can be achieved by the following technical scheme: One of the technical schemes of the invention is to provide a primer pair for detecting pseudomonad for killing the viscera ichthyophthiriasis of large yellow croaker based on an RPA-CRISPR/Cas12a technology, which is an RPA primer pair for amplifying the gene of the pseudomonad for killing the DVB73_RS07365 and comprises one of the following a) to d): a) An upstream primer RPA-F1 with a nucleotide sequence shown as SEQ ID NO.1 and an upstream primer RPA-R1 with a nucleotide sequence shown as SEQ ID NO. 5; b) An upstream primer RPA-F2 with a nucleotide sequen