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Primer for detecting type 1 and type 3 duck hepatitis A virus and duck tembusu virus and application

A technology for duck tembusu virus and duck hepatitis A, which is applied to the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problems of cumbersome operation, error-prone, time-consuming and labor-intensive, etc. Achieve the effect of overcoming mutual interference, high sensitivity and good repeatability

Inactive Publication Date: 2021-09-17
GUANGXI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] DTMUV can infect poultry and other birds. Virus-infected ducklings show some clinical symptoms and pathological changes similar to DVH. When the above-mentioned two or more viruses are mixed-infected, it is difficult to make an accurate judgment in clinical diagnosis. Routine Diagnostic methods (such as pathogen isolation and identification and serological tests, etc.) are complicated, time-consuming, and difficult to carry out differential diagnosis. Single molecular detection is cumbersome and error-prone when the number of samples is large; mutual interference

Method used

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  • Primer for detecting type 1 and type 3 duck hepatitis A virus and duck tembusu virus and application
  • Primer for detecting type 1 and type 3 duck hepatitis A virus and duck tembusu virus and application
  • Primer for detecting type 1 and type 3 duck hepatitis A virus and duck tembusu virus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Nucleic acid extraction and reverse transcription of RNA

[0038] Extract type 1 duck hepatitis A virus (DHAV-1, GX050028), type 3 duck hepatitis A virus (DHAV-3, GXNN201201) and duck Tembusu according to the operation instructions of the RNA extraction kit [purchased from Beijing Tiangen Biochemical Co., Ltd.] Virus (DTMUV, GXD0915), duck paramyxovirus (NDV), avian influenza virus (AIV), avian reovirus (ARV) RNA and according to ReverseTransciptase M-ML V (RNaseH-) [purchased from Bao Biological Engineering ( Dalian) Co., Ltd.] Reverse transcription manual Reverse transcription to obtain the cDNA of the above virus, the volume should be 25 μL, the reaction system is: 5×Buffer 5 μL, dNTP (10mM) 2 μL, random primer (Randon Primer) 1 μL, reverse transcriptase (M-MLV, 200U / μL) 1 μL, inhibitor (Rnase Inhibitor) 0.5 μL, RNA extract 15.5 μL. The above system was placed in a 42°C water bath for 70 minutes to obtain cDNA, which was stored at -20°C until use. According to the ...

Embodiment 2

[0040] 1. Establishment of triple RT-PCR amplification system

[0041] 1. Optimization of the reaction system

[0042]Set concentration gradients for the primer concentration, rTaq DNA polymerase concentration, and dNTPs concentration in the reaction system (as shown in Table 1), and use the PCR instrument program gradient to optimize and explore the best ratio.

[0043] Table 1

[0044]

[0045] Finally, the triple RT-PCR reaction system was determined to be 50 μL: 10×PCR buffer 4 μL, 10 mM dNTPs 3.5 μL, rTaq DNA polymerase 0.8 μL (200 U / μL), DHAV-1 upstream and downstream primers 1 μL each (both primer concentrations were 50 μM / mL) , the final concentration in the reaction system is 1 μM / mL), 1 μL of DHAV-3 upstream and downstream primers (both primer concentrations are 50 μM / mL, and the final concentration in the reaction system is 1 μM / mL), DTMUV upstream and downstream primers 1 μL each (primer concentration is 50 μM / mL, and the final concentration in the reaction sy...

Embodiment 4

[0058] Triple RT-PCR detection of virus-infected samples

[0059] 4 samples of artificial virus infection, namely: type 3 duck hepatitis A virus + duck Tembusu virus, type 1 duck hepatitis A virus + duck Tembusu virus, type 3 duck hepatitis A virus + type 1 duck hepatitis A virus, type 1 duck hepatitis A Virus + Type 3 Duck Hepatitis A Virus + Duck Tembusu Virus. The virus samples obtained from the disease materials were processed separately, the total RNA of each virus was extracted and reverse-transcribed into a cDNA template, and the kit was used for amplification operation. The amplification results were as follows: Figure 7 As shown, the expected amplified bands could be detected.

[0060] Each embodiment in this specification is described in a progressive manner, each embodiment focuses on the difference from other embodiments, and the same and similar parts of each embodiment can be referred to each other.

[0061] The above description of the disclosed embodiments i...

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Abstract

The invention discloses a primer for detecting a type 1 duck hepatitis A virus (DHAV-1), a type 3 duck hepatitis A virus (DHAV-3) and a duck tembusu virus (DTMUV). A detection method is established, and a detection kit is prepared. The kit is simple in method, accurate in result and high in sensitivity, the detection limit of DHAV-3 nucleic acid is 10 pg, the detection limit of DTMUV nucleic acid is 10 pg, the detection limit of DHAV-1 nucleic acid is 100 pg, the problem of mutual interference of multiple primers is solved, and the detection result is stable in clinical detection.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, in particular to a primer for detecting type 1 and type 3 duck hepatitis A virus and duck Tembusu virus and its application. Background technique [0002] Duck viral hepatitis (DVH) is an acute, highly contagious and fatal infectious disease of ducklings within 3 weeks of age caused by duck hepatitis virus (DHV). At present, the pathogens that have been identified to cause duck hepatitis mainly include duck hepatitis A virus (DHAV) and duck astrovirus (DAstV). DHAV is divided into three types, namely DHAV type 1 (DHAV-1), DHAV type 2 (DHAV-2) and DHAV type 3 (DHAV-3). Among them, DHAV-1 and DHAV-3 are prevalent in mainland China and can infect ducklings alone or in combination. [0003] Duck tembusu virus (DTMUV), also known as duck yellow virus, has been outbreaks since April 2010 in Fujian, Shandong, Zhejiang, Shanghai, Jiangsu, Guangxi, etc., the main breeding duck and laying duc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/706C12Q1/701C12Q1/686C12Q2600/166C12Q2545/113C12Q2565/125Y02A50/30
Inventor 韦天超兰奉瑜秦桂清唐华丽韦平磨美兰黄腾杨飞燕刘颖张廉
Owner GUANGXI UNIV