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
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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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