Primer group and kit for detecting fowl adenovirus serotype 4 (FAdV-4) as well as detection method and application of primer group

A technology of poultry adenovirus and detection method, which is applied in the field of detection of poultry adenovirus serotype 4, can solve problems such as aerosol pollution, fuzziness, and reduced reaction sensitivity, and achieve the effect of good specificity and easy operation

Inactive Publication Date: 2019-07-23
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the formation of white precipitates by visual observation is sometimes very vague and not enough to judge; when using AGE or SYBR Green for detection, it is necessary to open the cover step, which sometimes cau

Method used

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  • Primer group and kit for detecting fowl adenovirus serotype 4 (FAdV-4) as well as detection method and application of primer group
  • Primer group and kit for detecting fowl adenovirus serotype 4 (FAdV-4) as well as detection method and application of primer group
  • Primer group and kit for detecting fowl adenovirus serotype 4 (FAdV-4) as well as detection method and application of primer group

Examples

Experimental program
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Effect test

Embodiment 1

[0025] The purpose of this example is to provide two pairs of specific primers for detecting avian adenovirus serotype 4.

[0026] According to the genome sequence of avian adenovirus serotype 4, two pairs of primers were designed according to the principles of primer design and LAMP amplification technology, including outer primers and inner primers, wherein the outer primers were F3 and B3, and the inner primers were FIP and BIP. The nucleotide sequence of F3 is shown in SEQ ID NO.1, the nucleotide sequence of B3 is shown in SEQ ID NO.2; the nucleotide sequence of FIP is shown in SEQ ID NO.3, the The nucleotide sequence of the BIP is shown in SEQ ID NO.4, the FIP is a sequence labeled with biotin at the 5-terminal, and the BIP is the sequence labeled with fluorescein isothiocyanate at the 5-terminal. The sequence of F3 / B3 / FIP / BIP is shown in Table 1:

[0027] Table 1

[0028]

Embodiment 2

[0030] This embodiment provides a LAMP-LFD kit for detecting poultry adenovirus serotype 4, including amplification reagents, positive control substances, negative control substances, lateral flow test strips and the primer set in Example 1, the positive The control substance is avian adenovirus SC-Neijiang, and the negative control substance is double distilled water.

[0031]Wherein, the amplification reagents include Isothermal Amplification Buffer with a concentration of 10×, Bst 2.0 WarmStart DNA polymerase with a concentration of 8000U / mL, dNTPs MIX with a concentration of 10mmol / L, and MgSO with a concentration of 100mmol / L. 4 .

[0032] Wherein, the Isothermal Amplification Buffer with a concentration of 10× contains Tris-HCl with a concentration of 200mmol / L, (NH 4 ) 2 SO 4 , KCl with a concentration of 500mmol / L, and MgSO with a concentration of 20mmol / L 4 and 0.1% Tween 20 by volume, the pH of the Isothermal AmplificationBuffer is 8.8.

Embodiment 3

[0034] This embodiment provides a method for detecting poultry adenovirus serotype 4 with the LAMP-LFD kit in Implementation 2, comprising the following steps:

[0035] S1, extract the DNA of the sample to be tested;

[0036] S2, preparing the LAMP-LFD reaction system;

[0037] S3. Prepare the reaction system in step S2 in a PCR tube, and set a positive control group and a negative control group at the same time;

[0038] S4. LAMP-LFD amplification reaction: Mix the PCR tube containing the reaction system and centrifuge to perform the amplification reaction to obtain the reaction solution;

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Abstract

The invention discloses a primer group and a kit for detecting a fowl adenovirus serotype 4 (FAdV-4) as well as a detection method and an application of the primer group. The primer group comprises outer primers and inner primers, wherein the outer primers include F3 and B3, the inner primers include FIP and BIP, the nucleotide sequence of the F3 is shown as SEQ ID NO.1, and the nucleotide sequence of the B3 is shown as SEQ ID NO.2; and the nucleotide sequence of the FIP is shown as SEQ ID NO.3, the nucleotide sequence of the BIP is shown as SEQ ID NO.4, the FIP is a 5-end biotin labeled sequence, and the BIP is a 5-end fluorescein isothiocyanate labeled sequence. By combination of a loop-mediated isothermal amplification (LAMP) technology with a lateral flow dipstick (LFD) technology, a set of primers are designed for an important factor 52 k gene of the FAdV-4, and an LAMP-LFD rapid detection technology of the FAdV-4 is established. The LAMP-LFD kit provided by the invention is convenient to operate, safe, reliable, sensitive, efficient, good in specificity and particularly suitable for field rapid visual detection.

Description

technical field [0001] The invention belongs to the technical field of detecting poultry adenovirus serotype 4, and in particular relates to a primer set, a kit, a detection method and an application for detecting poultry adenovirus serotype 4. Background technique [0002] Avian adenovirus is a member of Adenoviridae Adenovirus genus. According to restriction endonuclease test, avian adenovirus is divided into five subspecies (A-E), and according to serum cross-neutralization test, it is divided into 12 serotypes ( 1-8a, 8b-11). Avian adenovirus is one of the pathogens that harm poultry. It mainly causes in farms: adenoviral gizzard erosion (AGE), inclusion body hepatitis (IBH), pericardium syndrome (hydropericardium syndrome, HPS) etc. Among these diseases, pericardial effusion syndrome, mainly caused by avian adenovirus serotype 4 (FAdV-4), has a mortality rate ranging from 10% to 100%, which is susceptible to 3-5 week-old broilers and occurs in the heart Pericardial f...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2565/625
Inventor 王红宁翟熙雯雷昌伟梅雪然吴暄
Owner SICHUAN UNIV
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