CN-122012819-A - Multiplex MIRA primer group for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi and application thereof

Abstract

The invention discloses a multiplex MIRA primer group for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi and application thereof, belonging to the technical field of molecular biology detection. The invention designs four pairs of specific MIRA primers, respectively targets specific genes of four pathogens of nervous necrosis virus, iridovirus, streptococcus iniae and vibrio harveyi, and provides a kit containing the primer group. The invention can be used for establishing a multiple MIRA detection method, the method can be completed after the reaction is carried out for 30 minutes under the constant temperature condition of 41 ℃, a complex thermal cycler is not needed, and one-tube synchronous detection of DNA and RNA pathogens can be realized without a separate reverse transcription step. The method has the advantages of rapidness, simplicity and convenience in operation, strong specificity and high sensitivity, and is particularly suitable for on-site rapid screening, monitoring and early diagnosis of important diseases in the scenes such as aquiculture farms, ports and the like.

Inventors

• ZHENG NAN
• WANG ZHUOYU
• QI JIE
• HE YAN

Assignees

• 中国海洋大学三亚海洋研究院
• 中国海洋大学

Dates

Publication Date

20260512

Application Date

20260410

Claims (8)

1. 1. A multiplex MIRA primer group for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi is characterized in that the sequences of the primer groups are shown as SEQ ID NO. 1-8.
2. 2. A multiplex MIRA detection kit for simultaneous detection of NNV, RSIV, s, iniae and v, harveyi, comprising the primer set of claim 1.
3. 3. The kit according to claim 2, further comprising a buffer solution, an enzyme premix, dNTPs and enzyme-free water required for the MIRA isothermal amplification reaction.
4. 4. A method for simultaneously detecting NNV, RSIV, s, iniae, and v, harveyi using the primer set of claim 1 or the kit of claim 3, said method being for the purpose of non-disease treatment or prevention, comprising the steps of: (1) Extracting total nucleic acid of a sample to be detected; (2) Establishing a multiple MIRA reaction system, wherein the total volume of the reaction system is 50 mu L, and the reaction system comprises 4 mu L of a nucleic acid template to be detected, 29.4 mu L of a conventional MIRA reaction buffer solution A, 8 mu L of a mixture of the primer group according to claim 1, 2.5 mu L of a conventional MIRA reaction buffer solution B and the balance of enzyme-free water; (3) Isothermal amplification reaction, namely reacting the reaction system in the step (2) at 39-43 ℃ for 25-30 min; (4) Analysis of results agarose gel electrophoresis analysis of the amplified product of step (3), if specific bands appear at positions 303 bp, 357, bp, 239 bp and 141 bp, then the presence of nervous necrosis virus, red sea bream iridovirus, streptococcus iniae and Vibrio harveyi is indicated, respectively.
5. 5. The method according to claim 4, wherein in the step (3), the isothermal amplification reaction is performed at a temperature of 41℃and a reaction time of 30 min.
6. 6. The method according to claim 4, wherein in the step (2), the final concentration of each primer in the mixture of primer sets is 0.2. Mu.M.
7. 7. The method of claim 4, wherein in step (1), the sample to be tested comprises a fish tissue, a body of water, or a sample of a farming environment.
8. 8. Use of a multiplex MIRA primer set according to claim 1 for the preparation of a product for simultaneous detection or assisted diagnosis of infection with nervous necrosis virus, red sea bream iridovirus, streptococcus iniae and vibrio harveyi.

Description

Multiplex MIRA primer group for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi and application thereof Technical Field The invention belongs to the field of molecular biology, and particularly discloses a multiplex MIRA primer group for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi and application thereof. Background The nervous necrosis virus (Nervous necrosis virus, NNV) belongs to a fish viral pathogen belonging to the family nodaviridae (Nodaviridae) and genus beta nodavirus (Betanodavirus) and can cause viral nervous necrosis. The genome of the virus consists of two single-stranded sense RNAs, RNA1, encoding an RNA polymerase and RNA2, encoding a species-specific capsid protein. NNVs can be classified into five genotypes of Epinephelus akaara nervous necrosis virus (RGNNV), turbot Nervous Necrosis Virus (TNNV), fugu rubripes nervous necrosis virus (TPNNV), scirpus pseudolaricis nervous necrosis virus (SJNNV) and Verasper nervous necrosis virus (BFNNV) according to the homology of RNA2 sequences. Because of the specificity of RNA2 sequences in different virus species, designing primers for RNA2 single-stranded sequences is a major method for early detection of NNV. The red sea bream iridovirus (Red sea bream iridovirus, RSIV) is an important pathogen for the aquaculture industry. RSIV mainly causes splenomegaly and liver blushing, has serious influence on the fish culture survival rate, and RSIV has the characteristics of latency and high mortality rate, and is difficult to control clinically. Streptococcus ragmitis (Streptococcus iniae, S. iniae) belongs to the order of Lactobacillus, the family Streptococcaceae, the genus Streptococcus, gram positive bacteria is one of important aquatic animal pathogenic bacteria, has wide host adaptability, and the host range comprises tens of freshwater fishes such as tilapia, rainbow trout, turbot, paralichthys olivaceus and the like, and has become one of important pathogenic bacteria of bacterial infectious diseases of various farmed fishes in the global scope. While the current control of s. iniae is still dependent on a number of antibiotics for the therapeutic approach of s. iniae. However, the use of antibiotics in large amounts is not only liable to cause the occurrence of pathogenic bacterial resistance but also may destroy the ecological balance of the aquaculture water, and thus it is of great importance to strengthen the early prevention of s iniae. Vibrio harveyi (Vibrio harveyi, V. harveyi) is a main pathogenic vibrio of various aquaculture animals and is widely distributed in aquaculture water environments and body surfaces and bodies of various aquaculture animals. In recent years, the development of the aquaculture industry in China is seriously influenced by frequent outbreaks of vibriosis caused by V. harveyi. Research shows that V. harveyi can infect various important aquatic economic animals such as sea bream, penaeus vannamei boone, weever and variegated abalone, and the host range covers fish, shrimp and shellfish. Traditional methods for identifying bacterial and viral pathogens are cumbersome in steps, complex in operation, time-consuming and labor-consuming. In recent years, immunology and molecular biology technologies are continuously advanced, such as a practical fluorescence quantitative PCR method, an LAMP method, an enzyme-linked immunosorbent assay method and the like, and the technologies have certain limitations on detection time and sensitivity, and a recombinase-mediated isothermal amplification (Multienzyme Isothermal Rapid Amplification ‌, MIRA) technology is a novel nucleic acid detection technology, the reaction is carried out at a constant temperature of 25-42 ℃, and can be completed by 5-30 min without complex temperature cycling equipment, so that the method is suitable for on-site rapid detection. However, there is no report on a multiplex MIRA method capable of simultaneously detecting the four important aquatic pathogens described above. Disclosure of Invention The invention aims to provide a primer group capable of simultaneously detecting NNV, RSIV, S. iniae and V. harveyi in a short time and application of a detection method, so as to establish a simple and effective synchronous detection method for various fish pathogens. The invention aims to solve the technical problem of providing a multiplex MIRA system for simultaneously detecting nervous necrosis virus, red sea bream iridovirus, streptococcus iniae and vibrio harveyi, and the amplification products can be amplified and detected by utilizing agarose gel electrophoresis technology. The invention is realized by the following technical scheme: one of the purposes of the present invention is to provide a multiplex MIRA primer set for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi, wherein the sequences of the primer sets are shown as SEQ ID NO. 1-8. Specifically, the primer group consists of the following primers: NNV-RNA2-F（