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Muscovy duckling parvovirus LAMP (loop-mediated isothermal amplification) detection reagent kit

A Muscovy duck parvovirus and kit technology, which is applied in recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems such as the establishment of MDPVLAMP visual detection kits and methods, and achieve high specificity High reliability, good application prospects, and high sensitivity

Active Publication Date: 2013-01-16
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are no relevant reports on the establishment of MDPV LAMP visual detection kits and methods at home and abroad

Method used

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  • Muscovy duckling parvovirus LAMP (loop-mediated isothermal amplification) detection reagent kit
  • Muscovy duckling parvovirus LAMP (loop-mediated isothermal amplification) detection reagent kit
  • Muscovy duckling parvovirus LAMP (loop-mediated isothermal amplification) detection reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the design of primer

[0037] According to the conserved gene sequence of Muscovy duck parvovirus MDPV in GenBank, LAMP primers were designed using the online software Primer Explorer V4 (http: / / primerexplorer.jp / e / v4-manual / index.html). Primers were synthesized by Guangzhou Invitrogen Company. The sequences of the 6 primers are listed in Table 1.

[0038] Table 1 is the LAMP primer sequence

[0039]

[0040] The addition of LF and LB primers can also improve the sensitivity of detection.

Embodiment 2

[0041] Example 2, the application of primers in the detection of Muscovy duck parvovirus by LAMP

[0042] 1. Extraction of nucleic acid

[0043] Refer to the instructions of the EasyPure viral DNA / RNA kit extraction kit to extract the RNA of duck type I hepatitis virus AV2111 strain, the DNA of muscovy duck parvovirus MDPV (AV238), the DNA of muscovy duck parvovirus (A17F4), and the DNA of duck circovirus , DNA of goose plague virus, RNA of H9 subtype avian influenza virus (AIV H9), RNA of Newcastle disease virus, DNA of duck plague virus. The above RNAs were reverse transcribed to obtain cDNAs.

[0044] 2. Optimizing the LAMP reaction system and reaction conditions

[0045] 25μL LAMP reaction system: 1-4μL dNTPs (10mmol / L, final concentration 0.4mmol / L-1.6mmol / L), 2.5μL 10×Bst buffer, 1ul Bst DNA polymerase 8U (final concentration 320U / L), 4 -7μL Betaine (5mmol / L, the final concentration is 0.8mmol / L-1.4mmol / L), 2-9μL MgSO 4 (25mmol / L, the final concentration is 2mmol / L-9...

Embodiment 3

[0062] Embodiment 3, the detection of clinical sample

[0063] Viruses were isolated and identified, and duck disease materials were extracted for LAMP detection.

[0064] The samples to be tested are 50 duck disease samples (numbered 1-50) collected from duck farms in Guangxi in 2012, and the DNA of duck disease materials (liver of sick duck, spleen of sick duck or lung of sick duck) was extracted respectively.

[0065] The DNA of each sample numbered 1-50 was used as a template, and the LAMP reaction was carried out according to the optimal reaction system and optimal reaction conditions in the above two of Example 2.

[0066] The result is as Figure 4 As shown, picture A is the result of electrophoresis detection, picture B is the result of visual observation, where M is 100bp DNAladder, 1 is the positive control Muscovy duck parvovirus MDPV (AV238), 2-5 are the samples numbered 1-4 respectively, and 4 is the Negative control (water); it can be seen that samples numbered...

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Abstract

The invention discloses a muscovy duckling parvovirus LAMP (loop-mediated isothermal amplification) detection reagent kit. A loop-mediated isothermal amplification primer group for detecting muscovy duckling parvoviruses is provided and comprises a primer 1, a primer 2, a primer 3 and a primer 4, and nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table sequentially. Experiments prove that six primers aiming at eight specific areas are designed by the aid of conserved sequence design of MDPV (muscovy duckling parvoviruses), the muscovy duckling parvovirus LAMP detection reagent kit is more specific and sensitive than a conventional detection method when used for LAMP detection, only one temperature-controllable water bath kettle needs to be used, and results can be judged by naked eyes without apparatuses. Therefore, the muscovy duckling parvovirus LAMP detection reagent kit is suitable for rapid detection of grass-root veterinary stations and livestock farms and has good application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection kit for Muscovy duck parvovirus LAMP. Background technique [0002] Muscovy duckling parvovirus (MDPV) is an important pathogen that endangers muscovy duck breeding. It can cause disease in young muscovy ducks aged 1-3 weeks, and the mortality rate can reach 50%-80%. This virus is the most susceptible virus to ducklings and has a very high lethality rate. [0003] Traditional detection methods require the use of expensive instruments and reagents. Loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) is a new nucleic acid amplification technology established by Notomi et al. in 2000. This technology has the advantages of simple operation, fast response, low cost and visualized results. At present, it is widely used in the detection of some pathogenic microorganisms. [0004] At present, there are no related reports on the establishment o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 谢芝勋谢丽基刘加波庞耀珊邓显文谢志勤范晴
Owner GUANGXI VETERINARY RES INST
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