CN-122012813-A - Primer group for identifying novel goose astrovirus gene type 1 and type 2, application thereof and kit

CN122012813ACN 122012813 ACN122012813 ACN 122012813ACN-122012813-A

Abstract

The application belongs to the field of biology, and discloses a primer set for identifying novel goose astrovirus genes type 1 and type 2, wherein the primer set is a primer set for a nicking endonuclease mediated isothermal amplification system, the upstream and downstream primer sequences in the primer set for identifying the novel goose astrovirus genes type 1 are shown as SEQ ID NO.1 and SEQ ID NO.2, and the upstream and downstream primer sequences in the primer set for identifying the novel goose astrovirus genes type 2 are shown as SEQ ID NO.3 and SEQ ID NO. 4. The primer group is developed aiming at a nicking endonuclease mediated isothermal amplification system, expensive detection equipment and reagents are not needed, the primer design is relatively short, pain points of other technologies are solved, and meanwhile, the problems of serious side reaction and severe requirements on a reaction system and a detection method are overcome. Meanwhile, the application also provides a kit based on the primer group and application thereof.

Inventors

JI JUN
XU XIN
WANG HAOYANG
Man Yuanzhuo
DONG YUZHU
YAO LUNGUANG

Assignees

南阳师范学院

Dates

Publication Date: 20260512
Application Date: 20260131

Claims (5)

1. The primer group for identifying the novel goose astrovirus gene type 1 and type 2 is characterized by being used for a nicking endonuclease mediated isothermal amplification system; the upstream and downstream primer sequences in the primer group for identifying the novel goose astrovirus gene 1 are shown as SEQ ID NO.1 and SEQ ID NO. 2; The upstream and downstream primer sequences in the primer group for identifying the novel goose astrovirus gene type 2 are shown as SEQ ID NO.3 and SEQ ID NO. 4.
2. The use of the primer group as set forth in claim 1 for preparing a detection kit for identifying novel goose astrovirus genes type 1 and type 2, wherein the kit is based on a nicking endonuclease-mediated isothermal amplification technology.
3. A kit based on a nicking endonuclease-mediated isothermal amplification technology, which is characterized by comprising the primer set according to claim 1.
4. The kit according to claim 3, wherein the kit comprises a buffer solution, and the buffer solution is a composite phosphate buffer solution with a pH of 7.4.
5. The kit according to claim 3, wherein the kit comprises a polymerase and a nicking enzyme, wherein the polymerase is Bst DNA3.0 polymerase, and wherein the nicking enzyme is WARMSTART Nt.BstNBI nicking enzyme.

Description

Primer group for identifying novel goose astrovirus gene type 1 and type 2, application thereof and kit Technical Field The invention relates to the field of biology, in particular to a primer group for identifying novel goose astrovirus genes type 1 and type 2, application thereof and a kit. Background The novel astrovirus (novel goose astrovirus, N-GoAstV) of goose mainly induces the deposition of uric acid salts on joints and viscera of young geese of 7-20 days old, death occurs 2-3 days after infection, the death peak can be reached within 5-7 days, the resistant gosling becomes stiff goose, the growth and development are seriously hindered, and researches show that the virus can also cause vertical transmission through genital tracts to pollute goose strain. In recent years, different students in China sequentially find that the virus can infect duckling and young muscovy ducks and find infection in chicken flocks, and the economic loss caused by the virus is difficult to measure. Meanwhile, with the spread and variation of viruses, different genotypes of N-GoAstV have been found in the industry (N-GoAstV-1 and N-GoAstV-2). N-GoAstV-1 (represented by AHDY strain, FLX strain, etc.) was reported earliest, but epidemic early epidemic strains were mainly N-GoAstV-2 genotype strains (represented by HLJ01 strain, AHAU strain, etc.). With epidemiological evolution, research reports from 2022 years indicate that the "dominant positive duty" of the N-GoAstV-2 genotype strain is gradually replaced by the N-GoAstV-1 genotype strain, and that many positive farms are suffering from mixed infection of both genotypes N-GoAstV. In addition, mixed infection of N-GoAstV with goose parvovirus (goose parvovirus, GPV), goose reovirus (Goose reovirus, GREOV) and Riemerella epidemic (RIEMERELLA ANATIPESTIFER, RA) further increases the difficulty of clinical diagnosis. Up to now, no effective medicine or biological agent exists in clinic to prevent and treat the disease, and establishing a diagnosis method suitable for clinic is crucial to prevent and control the disease. Thus, it is urgent to establish a simple, accurate and rapid dual diagnosis method of N-GoAstV-1/2. Since the common symptom of N-GoAstV infection is urate deposition (gout syndrome) of multiple organs and joints, the gout syndrome caused by non-pathogenic is difficult to distinguish, and is difficult to distinguish only from clinical and sectional inspection. The laboratory diagnosis method for N-GoAstV-1/2 is mainly divided into culture identification, serological diagnosis and molecular biology. Wherein, the separation culture takes a lot of time, which is unfavorable for the control of illness and epidemic. The established serologic diagnosis method aiming at GoAstV mainly comprises enzyme-linked immunosorbent (enzyme linked immunosorbent assay, ELISA), immunochromatography (immunochromatographic STRIP ASSAY, ICS), photoelectrochemical immunosensor (photoelectrochemical immunosensor, PEC-IS) and other methods, wherein ELISA IS a sensitive and accurate serologic immunology diagnosis technology, the operation IS relatively complex, the detection flow IS time-consuming, the ICS and the PEC-IS are short in flow and easy to operate, and the clinical application of the ICS and the PEC-IS IS limited due to low sensitivity. In addition, the antibodies selected for serodiagnostic methods may be incompatible or cross-reactive to varying degrees with N-GoAstV-1 and N-GoAstV-2, affecting the accuracy of such methods and presenting a greater challenge in differentiating between the two genotypes N-GoAstV. In recent years, with the development of related technologies in molecular biology, nucleic acid amplification has become the most central and common method in gene diagnosis technology. PCR and PCR derived techniques have been increasingly used and developed for N-GoAstV diagnostics. However, these methods have the disadvantages of (1) requiring expensive PCR apparatus and relatively complicated operation, increasing the cost of basic layer detection, (2) having to use a nucleic acid extraction kit to obtain a "high quality" nucleic acid template due to sensitivity of polymerase to various body fluid components, (3) requiring a precise temperature cycle process, otherwise affecting nucleic acid amplification, (4) having a long amplification reaction time, requiring the addition of a reverse transcription process in advance, adding complicated cycle processes such as denaturation, annealing and extension, and generally requiring several hours, adding electrophoresis and imaging processes, further prolonging detection time, and being difficult to popularize and apply in basic layers. Regarding the identification technology of the type 1 and type 2 of the goose astrovirus, publication number CN116103441A is visible, the subject is a patent application of a multiplex fluorescence PCR primer probe set, a method and application for detecting the type 1 and type 2 goose a