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CN-122012816-A - Fluorescent quantitative PCR primer group for detecting hyena exendin virus type 2, detection kit and application thereof

CN122012816ACN 122012816 ACN122012816 ACN 122012816ACN-122012816-A

Abstract

The invention discloses a fluorescent quantitative PCR primer group for detecting the type 2 of the herpes simplex virus, a detection kit and application thereof. The invention designs a pair of specific primers IghV-F and IghV-R aiming at a conserved region of a hyena herpesvirus type 2 (Iguanid herpesvirus, ighV2) DNA dependent DNA polymerase (pol) gene, and establishes a SYBR Green dye method fluorescence quantitative PCR detection method, and has the advantages of high sensitivity, high specificity, wide linear range, rapidness, simplicity, good repeatability and the like. The invention provides important technical support for herpesvirus infection monitoring, quarantining and protecting work of rare or endangered lizard species, fills the blank of the detection method in the field, and is suitable for non-clinical medical fields such as scientific research, epidemiological investigation, quarantine monitoring, protecting management and the like.

Inventors

  • LIU HAO
  • ZHANG JIELING
  • Wu Qinhuang
  • MA XINXIN
  • LI LIYANG
  • ZHOU WENSI
  • WANG JIALIN

Assignees

  • 佛山大学

Dates

Publication Date
20260512
Application Date
20260313

Claims (8)

  1. 1. The primer set for detecting the type 2 of the hyena herpesvirus is characterized by comprising an upstream primer IghV-F and a downstream primer IghV-R, wherein the nucleotide sequence of the upstream primer IghV-F is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer IghV2R is shown as SEQ ID NO. 2.
  2. 2. A fluorescent quantitative PCR kit for detecting the type 2 of the herpes hyena virus, which is characterized by comprising the primer set as claimed in claim 1 and a SYBR Green dye method fluorescent quantitative PCR reagent.
  3. 3. The kit of claim 2, further comprising a positive control and a negative control, wherein the positive control is a recombinant plasmid containing the pol gene in the genome of the herpes simplex virus type 2, and the negative control is ddH 2 O.
  4. 4. The kit according to claim 3, wherein the concentration of the recombinant plasmid containing the pol gene in the genome of the type 2 herpes simplex virus is 10 7 copies/. Mu.L or more.
  5. 5. The kit according to claim 3, wherein the GenBank accession number of the pol gene in the genome of the type 2 herpes simplex virus is nc_043063.1.
  6. 6. A fluorescent quantitative PCR method for detecting the type 2 of the herpes simplex virus for non-disease diagnosis purposes, comprising the steps of: S1, extracting DNA of a sample to be detected; S2, using the fluorescent quantitative PCR kit, and carrying out fluorescent quantitative PCR amplification by taking the DNA obtained in the step S1 as a template; And S3, judging whether the sample to be detected contains the type 2 of the herpes simplex virus according to the amplification curve condition after the reaction is finished, wherein the judging method comprises the steps that the Ct value is less than or equal to 35, the amplification curve is in a typical S shape, the sample to be detected contains the type 2 of the herpes simplex virus, the Ct values are all more than 35 or no Ct value, and the sample to be detected does not contain the type 2 of the herpes simplex virus.
  7. 7. The method according to claim 6, wherein the reaction system for fluorescent quantitative PCR amplification is 10. Mu.L of 2 XSYBR Green fluorescent quantitative PCR reaction premix, 0.6. Mu.L of the upstream primer IghV-F having a nucleotide sequence of SEQ ID NO.1 and 0.6. Mu.L of the downstream primer IghV R having a nucleotide sequence of 10. Mu.M as shown in SEQ ID NO.2, and 2. Mu.L of DNA of the sample to be detected, and ddH 2 O is added to 20. Mu.L.
  8. 8. The method of claim 6, wherein the fluorescent quantitative PCR amplification is performed by a reaction sequence of about 30 s at 95℃and 40 cycles of about 5 s at 95℃and 30 s at 60℃annealing extension, collecting fluorescent signals during the 60℃annealing extension step, and performing a melting curve analysis after the amplification, wherein the temperature is increased from about 60℃to about 95℃and the temperature is increased by about 0.5℃/5 s.

Description

Fluorescent quantitative PCR primer group for detecting hyena exendin virus type 2, detection kit and application thereof Technical Field The invention belongs to the technical field of wild animal pathogen detection and molecular diagnosis, and particularly relates to a fluorescent quantitative PCR primer set for detecting the type 2 of the herpes simplex virus, a detection kit and application thereof. Background Herpesviridae (Herpesviridae) is a class of double stranded DNA viruses widely distributed in vertebrates, with the property of establishing latent infections and periodically reactivating, and constitutes a constant threat to host health and aquaculture. The herpesviridae are divided into three subfamilies, α, β, γ, depending on host range and genomic characteristics. In the veterinary and farming fields, various herpesviruses have been shown to cause serious diseases, namely porcine herpesvirus type 1 (PRV-1, belonging to the subfamily alpha herpesvirus) causing pseudorabies manifested by neurological symptoms, dyspnea and sow reproductive disorders, causing significant economic losses to the pig industry, bovine herpesvirus type 1 (BoHV-1) causing infectious rhinotracheitis, leading to respiratory tract infections, abortion and milk production decline, equine herpesvirus type 1 and type 4 (EHV-1/4) being closely related to respiratory diseases, neurological diseases and abortion in horses, and in aquatic animals, cyprinid herpesvirus type 3 (CyHV-3) leading to massive death of fancy carp and carp manifested clinically as gill necrosis, kidney hemorrhage and dyspnea. Herpes viruses are also detected in lizards. The type 2 of the hepatitis b virus (Iguanid herpesvirus, ighv 2) was detected in a piece of color-changing speckle of diffuse liver necrosis, and Iguanid HV 2 was successfully isolated (Wellehan et al, 2003). Iguanid HV 1 (Clark and Karzon, 1972) was isolated from cell cultures of the spleen, kidney and heart of green hyena (Iguana iguana) which could cause cytopathic effects. VARANID HV 1 (Literak et al, 2010) was detected in the oral mucosa and brain of chloral giant exendin (Varanus prasinus) with proliferative stomatitis. New herpes viruses Iguanid Alphaherpesvirus 4 (IgHV 4) were detected in endangered Galagos land Pink (Conolophus marthae) (Ainoa Nieto-Claud i n et al, 2024). DNA polymerase (pol/UL 30 homology) is a core conserved gene of herpes virus, suitable as a molecular detection target. The pol gene is essential gene for viral DNA replication, bears strong evolution selection pressure, has sequence conservation obviously higher than that of envelope glycoprotein and other easily-changed genes, has higher amino acid homology of the pol gene of the same subfamily, and can simultaneously meet the requirements of broad-spectrum detection and type identification. The reference sequence of the herpesvirus pol gene in the NCBI and other databases has clear annotation, complete coverage and clear conserved region, and is convenient for designing high-specificity and high-sensitivity primers. In molecular epidemiological researches of amphibious reptile herpesviruses (such as mossback herpesviruses and tortoise herpesviruses), pol and gB (UL 27) are the most commonly used PCR targets, and the methods such as broad-spectrum PCR, semi-nested PCR and the like based on pol conservation regions are widely verified in human and animal herpesvirus detection, and the method is mature and reliable. Currently, the detection of IghV2 mainly depends on the traditional method, and has obvious technical limitations: (1) Virus isolation culture, which is a gold standard for etiology diagnosis, requires a specific cell line (e.g., a reptile-derived cell line) and stringent culture conditions, takes a long time (usually 2-4 weeks), requires a high technical requirement, and many herpesviruses are difficult to culture in vitro, resulting in a low detection rate; (2) Conventional PCR (polymerase chain reaction) can detect viral nucleic acid, but has limited sensitivity (the detection lower limit is usually 10 3-104 copies/. Mu.L), can only perform qualitative analysis, cannot accurately evaluate viral load, and requires subsequent operations such as agarose gel electrophoresis, so that pollution risk and detection time are increased; (3) Serological methods are limited by the lack of specific antibody reagents and the latent infection characteristics of herpes viruses make antibody detection difficult to reflect the current infection status; (4) Histopathological examination, although characteristic pathological changes (such as nuclear inclusion bodies) can be observed, requires biopsy or autopsy samples, is highly invasive in operation, is not suitable for in vivo monitoring of rare endangered species, and cannot be diagnosed in early or latent stages of infection. The limitations of the above method severely restrict the early diagnosis, epidemiological monitoring and disease prevention and control