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Primer set for detecting duck hepatitis A virus type 3, kit and applications

A technology of duck hepatitis A virus and detection kit, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of low sensitivity, difficulty in standardization, and long time consumption, and achieve high sensitivity Simple operation and fast effect

Active Publication Date: 2020-09-04
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these tests have shortcomings such as time-consuming, low sensitivity, and difficult standardization, which have certain limitations in practical applications.
[0004] At present, although the PCR detection method and the fluorescent quantitative PCR detection method of type 3 duck hepatitis A virus have been established, the detection sensitivity is relatively high, but the conventional PCR detection method Need to use PCR instrument and gel electrophoresis, fluorescent quantitative PCR method needs to use more expensive fluorescent quantitative instrument and reagents, etc., so it is difficult to meet the needs of grassroots on-site detection

Method used

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  • Primer set for detecting duck hepatitis A virus type 3, kit and applications
  • Primer set for detecting duck hepatitis A virus type 3, kit and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A primer set for detecting type 3 duck hepatitis A virus, comprising inner primers 1 and 2, outer primers 3 and 4, and the primer sequences are as follows:

[0025] Internal primer FIP: 5'-CACCACTGAATCTTGCCTTCA-ACACTCGTAAGATAGTTGGAG-3', SEQ ID No.1;

[0026] Internal primer BIP: 5'-CAAAGGCCCAGAAGCATTCAAA-GTAGTTGTAATATTCAGGACCAT-3', SEQ ID No.2;

[0027] Outer primer F3: 5'-AACACCTGCATTTCTGCC-3', SEQ ID No.3;

[0028] Outer primer B3: 5'-GAGCTTTCAACTTGGAACAA-3', SEQ ID No.4.

[0029] A kit containing the above primers, wherein the reaction solution: contains 10×BstBuffer, Bst DNA polymerase 8U / μL, 10mM dNTPs, 100mM magnesium sulfate, 20μM inner primer 1, 20μM inner primer 2, 20μM outer primer 3, 20μM outer primer 4 , 10M betaine, 625μM calcein, 12.5mM manganese chloride and double distilled water.

[0030] Specifically, the reaction solution is 24 μL per tube, and its composition is: 2.5 μL 10×BstBuffer, 1 μL Bst DNA polymerase 8U / μL, 3.5 μL 10 mM dNTPs, 2 μL 100 mM m...

Embodiment 2

[0032] The method for detecting type 3 duck hepatitis A virus by using the type 3 duck hepatitis A virus LAMP detection kit in Example 1 of the present invention.

[0033] 1. Extraction of RNA in tissue samples (total RNA extraction commercial kit)

[0034] A. Add 250 μL virus allantoic fluid and 750 μL lysate RZ to the centrifuge tube, shake and mix well, add 200 μL chloroform, shake vigorously for 30 seconds and place at room temperature for 3 minutes.

[0035] B. Centrifuge at 12000r / min at 4°C for 10min. The sample is divided into three layers: yellow organic layer, middle layer and supernatant colorless water phase. The extract mainly exists in the water phase. Transfer the water to a new centrifuge tube (about 600μL ).

[0036] C. Slowly add 0.5 times the volume (about 300 μL) of absolute ethanol, mix well and transfer the mixture into the adsorption column CR3. Centrifuge at 12000r / min at 4°C for 1min. (If the entire solution and mixture cannot be added to the adsorp...

Embodiment 3

[0047] specificity test

[0048] DHAV-3 and DHAV-1, duck Tambusu virus (DTMUV), Newcastle disease virus (NDV), avian influenza virus (AIV), avian reovirus (ARV), duck plague virus (DEV), large intestine Bacillus, Pasteurella, Salmonella, Riemerella anatipestifer, Listeria, and Staphylococcus aureus were used as templates, and nucleic acids extracted from healthy poultry tissues and reverse-transcribed were used as negative controls. Viral LAMP should be reacted according to the system and conditions.

[0049] see results figure 1 , the detection of duck hepatitis A virus type 3 was positive (shown in green), while DHAV-1, DTMUV, NDV, AIV, ARV, DEV, Escherichia coli, Pasteurella, Salmonella, Riemerella anatipestifer, Listeria The virus tests of Tetrabacterium and Staphylococcus aureus were all negative (shown in orange). As a result of electrophoresis, only DHAV-3 had positive bands.

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Abstract

The invention discloses a primer set for detecting duck hepatitis A virus type 3, a kit and applications, and belongs to the technical field of virus detection. Two specific internal primers and two specific external primers, the kit based on the detection primers and applications for non-diagnostic purposes are included. The primer set, the kit and the applications are good in specificity, high in sensitivity, rapid, low in cost, more convenient in operation method and very suitable for primary laboratories to detect the duck hepatitis A virus type 3; and therefore, the defects in the prior art of long detection time, complex operation and expensive instruments can be solved.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a primer set, kit and application for detecting type 3 duck hepatitis A virus. Background technique [0002] Type 3 duck hepatitis A virus (Duckhepatitis A virus type 3, DHAV-3) is an acute, highly contagious and fatal infectious disease pathogen that causes ducklings within 4 weeks of age. The lethal rate to ducklings can be as high as 95%, and it is one of the main diseases that endanger the healthy development of the duck industry. [0003] The traditional detection methods of duck hepatitis A virus type 3 include serum neutralization test, agar diffusion test and ELISA. However, these tests have disadvantages such as time-consuming, low sensitivity, and difficult standardization, which have certain limitations in practical application. [0004] At present, although the PCR detection method and the fluorescent quantitative PCR detection method of type 3 duck hepatiti...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/706C12Q1/6844C12Q2521/107C12Q2531/119C12Q2563/103Y02A50/30
Inventor 韦天超兰奉瑜韦平唐华丽秦桂清磨美兰黄腾
Owner GUANGXI UNIV
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