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A PCR detection primer and kit for Riemerella anatipestifer virulent phage

A technology of Riemerella anatipestifer and detection kit, which is applied in the fields of biotechnology and diagnostic testing to achieve the effect of filling gaps in the research field

Active Publication Date: 2022-08-09
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it has been found that the virulent phages of R. anatipestifer have the potential of clinical application in the prevention and control of R. anatipestifer. The existing technology has also isolated and identified some virulent phages from the sewage and duck manure of duck farms. The existing duck The identification method of Riemeria anatipestifer phage mainly includes pathogen isolation and observation, and electron microscope observation. At present, there is no PCR detection method for specific detection of Riemeria anatipestifer virulent phage at home and abroad. The establishment of the present invention can fill the gap in related fields at home and abroad.

Method used

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  • A PCR detection primer and kit for Riemerella anatipestifer virulent phage
  • A PCR detection primer and kit for Riemerella anatipestifer virulent phage
  • A PCR detection primer and kit for Riemerella anatipestifer virulent phage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Primer design and screening

[0047] 1.1. Primer design

[0048] According to the specific conserved region (p36874~p41656) of the gene sequence of Riemerella anatipestifer virulent phage RAP44 strain (HQ396194.1) in the National Center of Biotechnology Information (NCBI) database, Lasergene DNAStar was used for nuclear analysis. The glucoside sequence was analyzed and aligned, and 4 sets of primers were designed using the primer design software Oligo7.0, and the sequences were as follows:

[0049]

[0050] The above primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0051] 1.2. Primer screening

[0052] 1.2.1. Extraction of genomic DNA

[0053] Corresponding genomic DNA was extracted from Riemerella anatipestifer virulent phage RAP44 strain according to the method of EasyPure Viral DNA / RNA Kit, and frozen at -80°C for future use.

[0054] Control strains Riemerella anatipestifer type 1 (FQ, MH, ZZ and NP strains), Riemerella anati...

Embodiment 2

[0058] Example 2 Establishment of PCR method

[0059] 1.1. Extraction of genomic DNA

[0060] The corresponding genomic DNAs of RAP44 and control strains DEV, MDPV, GPV, DuCV, and DAdV-3 were extracted according to the method of EasyPure ViralDNA / RNA Kit, and frozen at -80°C for future use.

[0061] The control strains FQ, RAf32, and R267 were all extracted according to the method of EasyPure Bacteria Genomic DNA Kit, and the corresponding genomic DNA was extracted and stored at -80°C for future use.

[0062] 1.2. Configuration of reaction solution and optimization of annealing temperature

[0063] Follow 2 x TransTaq -T PCR SuperMix kit recommended 20μL system for amplification, in which 10μL of 2×PCR Master Mix amplification reaction solution, 1.0μL of primer SEQ ID NO:5 / SEQ ID NO:6 (each 10μM), extracted nucleic acid template 2.0 μL, supplemented with sterile deionized water to a final volume of 20 μL, and PCR amplification was performed after mixing. Amplification con...

Embodiment 3

[0066] Example 3 Specificity test

[0067] The optimized PCR system and amplification conditions were used for phage RAP44 strain, Riemerella anatipestifer FQ strain, RAf32 strain and R267 strain, DEV, MDPV, GPV, DuCV, DAdV-3 and sterilized deionized water control. Amplification. As a result, only the phage RAP44 strain (lane 1) was positive, and the rest were negative, indicating that the established method has strong specificity and no cross-reaction to common waterfowl pathogens ( Figure 5 ).

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Abstract

The invention belongs to the technical field of biotechnology and diagnostic detection, and discloses a PCR detection primer for Riemerella anatipestifer virulent phage and a kit thereof, wherein the primers are shown in SEQ ID NO: 5 and SEQ ID NO: 6, The Riemerella anatipestifer virulent phage can be quickly identified simply by using a pair of specific primers to amplify the sample to be tested by PCR and then electrophoresis. At present, there is no report on the identification of Riemerella anatipestifer virulent phage based on molecular biology methods. This study can fill the gap in related research fields.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and diagnosis and detection, and more particularly relates to a PCR detection primer of Riemerella anatipestifer virulent phage and a kit thereof. Background technique [0002] Riemerella anatipestifer (Riemerella anatipestifer) is an important pathogen that is currently endangering the duck industry. It has few sensitive drugs and is prone to drug resistance. It is not uncommon for multiple drugs to be used in combination with overdose or over-course of treatment in clinical practice. On the one hand, the consequences lead to residues of veterinary drugs in the duck carcass, which indirectly threatens human health; . Therefore, it is urgent to develop an efficient, specific and non-polluting R. anatipestifer biological control preparation. [0003] Bacteriophage is a class of viruses that lyse bacteria (or spirochetes, fungi). Because of its strict host tropism, fast proliferation, low pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/92
CPCC12Q1/701C12Q1/686C12Q2565/125Y02A50/30
Inventor 刘荣昌程龙飞陈红梅黄瑜傅光华施少华万春和傅秋玲
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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