Primer for detecting goat endemic intranasal tumor virus and application of primer in HRM (High Resolution Melting) detection reagent

A tumor virus, endemic technology, applied in the field of molecular biology, to achieve fast cost, good reproducibility, and strong specificity

Pending Publication Date: 2021-05-25
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
View PDF4 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is mostly used to detect gene mutations, and has the advantages of high sensitivity, good specificity, low cost, and closed-tube operation. At present, there is no application of HRM method for goat endemic intranasal tumors at home and abroad.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer for detecting goat endemic intranasal tumor virus and application of primer in HRM (High Resolution Melting) detection reagent
  • Primer for detecting goat endemic intranasal tumor virus and application of primer in HRM (High Resolution Melting) detection reagent
  • Primer for detecting goat endemic intranasal tumor virus and application of primer in HRM (High Resolution Melting) detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: the design of primer

[0046] The present invention compares the complete genome sequence of ENTV-2 published on NCBI and viruses with higher homology with ENTV-2, such as ENTV-1, endogenous viruses (ERVs), and sheep lung adenoma virus (JSRV), A pair of primers were designed, verified by NCBI BLAST, and synthesized by Bosun Biotechnology (Shanghai) Co., Ltd. The nucleotide sequence is as follows:

[0047] S1-enHRM1223 (SEQ ID NO. 1): 5'-TCATACTGTGGATTCCCTGTC-3' and R2-enHRM1223 (SEQ ID NO. 1): 5'-CTAACTACATCATATAAAGCTGATAGTC-3'.

Embodiment 2

[0048] Embodiment 2: the application of primer in HRM detection reagent

[0049] The reagent containing the above primers is applied to the HRM detection of goat endemic intranasal tumor virus, and the HRM detection method comprises the following steps:

[0050] (1) RNA extraction and reverse transcription: Extract ENTV-2 RNA according to the instructions of the EasyPure Viral DNA / RNA Kit, store at -70°C, and remove residual genomic DNA after treatment with deoxyribonuclease I (DNaseI). Reverse Transcriptase Instructions Reverse transcribe RNA into cDNA and store at -20°C.

[0051] (2) Construction of standard products: using the cDNA obtained in step (1) as a template, use the above primers to perform PCR amplification of ENTV-2, and primers S1-enHRM1223 and R2-enHRM1223 amplify the specific target fragment of ENTV-2 , the length is 108bp.

[0052] Use 20 μL of PCR amplification system: 1 μL of template, 1 μL of upstream and downstream primers (final concentration: 0.5 μmo...

Embodiment 3

[0056] Embodiment 3: the specificity test of HRM

[0057] DNA was extracted from goat skin cells, sheep lung cells and sheep kidney cells as negative samples of endogenous sheep intranasal tumor virus (enENTV), oral ulcer virus (ORFV), bovine coronavirus (BCV), Mycoplasma pneumoniae ( M0), mycoplasma mycoplasma goat subspecies (Mmc), mycoplasma capricoides goat pneumonia subspecies (Mccp) and the sheep intranasal tumor virus (ENTV-2) cDNA that obtains in embodiment 2 steps (1) are template, implement step by step The HRM reaction was carried out under the optimized reaction conditions in step (3) of Example 2.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a primer for detecting a goat endemic intranasal tumor virus and the application of the primer in an HRM (High Resolution Melting) detection reagent, and relates to the technical field of molecular biology. The primer provided by the invention is designed by comparison according to an ENTV-2 whole genome sequence and gene sequences of viruses such as ENTV-1, JSRV, ERVs and the like with high homology with the ENTV-2, the length of a corresponding PCR amplification target fragment is 108bp, the primer is applied to an HRM detection reagent, and by analyzing a dissolution curve of a PCR product and according to a specific dissolution peak and a TM value, the HRM detection reagent with high homology with the ENTV-2 can be used for detecting the ENTV-2 in the HRM detection reagent. According to the present invention, whether the amplification product is the target fragment is recognized, the specific probe and the sequencing are not required, the whole process is closed, the experiment accuracy is improved, the advantages of strong specificity, good repeatability, rapidness and low cost are provided, the method is suitable for the border inspection and quarantine of the sheep intranasal tumor, the blank of the research in the field at home and abroad is filled, and the important economic significance is provided.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer for detecting goat endemic intranasal tumor virus and its application in HRM detection reagents. Background technique [0002] Goat endemic intranasal tumor (Enzootic nasal tumor, ENT) is a chronic, progressive, contagious infectious disease characterized by malignant transformation of the ethmoid epithelial cells of the turbinates of affected sheep. The main clinical symptom is loss of appetite in affected sheep. , continuous runny nose, dyspnea, later developed into a large number of rhinorrhea, extreme weight loss, some protruding eyes and skull deformation, autopsy showed cauliflower tumors on one or both sides of the nasal cavity; the incidence of the disease is not high, It is about 0.5-15%, but the mortality rate can reach 100%, and the incubation period is as long as several months or even a year, causing great losses and potential threats to the sheep ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686
Inventor张靖鹏胡奇林江锦秀林欲胜游伟
OwnerINST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI