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Primers and method for fast detecting koi herpes viruses (KHV)

A koi herpes virus, fast technology, applied in the field of animal pathogen detection, can solve expensive problems, achieve the effect of avoiding pollution and optimizing reaction conditions

Pending Publication Date: 2019-06-21
广州动佰生物科技有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, despite its advantages, PCR technology requires expensive instrumentation, cyclic denaturation, annealing steps during target sequence amplification, and often takes several hours to produce results

Method used

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  • Primers and method for fast detecting koi herpes viruses (KHV)
  • Primers and method for fast detecting koi herpes viruses (KHV)
  • Primers and method for fast detecting koi herpes viruses (KHV)

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Embodiment 1

[0033] 1. Virus Extraction

[0034] DNA was extracted from fish tissues infected with koi herpesvirus according to the instructions of TIANamp Genomic DNAKit kit from TIANGEN Company.

[0035] 2. Primer Design

[0036] According to the TK gene sequence published by KHV in GenBank, PCR primers were designed, among which: KHV-TK-F: 5`GGGTTACCTGTACGAG`3, KHV-TK-R: 5`CACCCAGTAGAT TATGC`3, using this pair of primers to amplify the gene, the reaction The system is: rTaq enzyme 20 μL, upstream primer (SEQ ID NO: 1) 2 μL, downstream primer (SEQ ID NO: 2) 2 μL, template DNA (DNA extracted in step 1) 2 μL, dH 2 O 14 μL to make up 40 μL. The amplification program is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 10 min; storage at 12°C, see figure 1 . The amplified PCR product was electrophoresed with agarose gel, and after the electrophoresis was completed, t...

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Abstract

The invention discloses primers and method for fast detecting koi herpesviruses (KHV) and belongs to the field of animal pathogen detection. The primers and method are high in specificity and sensitivity and good in repeatability. According to the technical scheme, the nucleotide sequence of the primers is as shown in SEQ ID No. 1 and SEQ ID No. 2, and the nucleotide sequence of a probe is as shown in SEQ ID No. 3. The detecting method includes the steps of firstly, extracting virus genome DNA from a clinical sample; secondly, using the extracted sample DNA as the template, using the primer group in claim 1 and the probe in claim 2 to prepare an RPA reaction system, and using a TwistAmp exo kit to perform RPA amplification reaction; thirdly, placing the reaction tube after the RPA amplification into a fluorescence detector, monitoring fluorescent signals in real time, determining that the sample contains KHV if an evident fluorescent signal curve exists, and determining that the sampledoes not contain KHV if no fluorescent signal curves exist.

Description

technical field [0001] The invention belongs to the field of animal pathogen detection, and in particular relates to a primer for rapid detection of koi herpes virus and a method thereof. Background technique [0002] Koi herpes virus disease (KHVD) is a highly contagious viral disease caused by Koi herpes virus disease (KHV), which threatens the reproduction of carp and koi. Koi herpes virus, also known as cyprinidherpesvirus 3 (CyHV-3), belongs to the family Herpesviridae. The virus has an envelope, and the nucleocapsid is icosahedral, with a diameter of 170-230nm. The virus consists of 31 species The viral polypeptide is composed of viral nucleic acid as double-stranded DNA, and the genome size is about 227kb. All carp and koi age groups are susceptible to KHV infection and exhibit high morbidity and mortality. [0003] Koi herpes virus occurs mostly in spring and autumn, and the incubation period is about 2 weeks. Death will occur within 24-48 hours after the onset of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
Inventor 王华南胡明明董建国丛潇杨忠艳孙杰
Owner 广州动佰生物科技有限公司
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