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RT-PCR (reverse transcription polymerase chain reaction) Detection kit and method of koi herpesvirus

A koi herpes virus and RT-PCR technology, which is applied in the field of RT-PCR detection kits for koi herpes virus, can solve the problems of great harm to the aquaculture industry and achieve the effect of strong sensitivity

Inactive Publication Date: 2018-08-10
张朝明 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Because the mortality rate of the disease koi and carp is as high as 75% to 95%, it is extremely harmful to the aquaculture industry, and there is currently no effective drug and commercial vaccine for the treatment of koi herpes virus disease, so a koi herpes virus disease is established. The reverse transcription PCR diagnostic method and special kit for carp herpes virus can facilitate the rapid and accurate diagnosis of the disease in the early stage of the disease, and provide an effective reference for the prevention and control of the disease

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1, the construction of differential diagnosis method

[0022] 1. Primer design

[0023] Two specific primers were designed as follows:

[0024] S1:5'-AAGGCTTAGTAGCTTACCAT-3' (SEQ ID NO. 1)

[0025] S2: 5'-CAAGGTAGCTGAGTCGACCTATG-3' (SEQ ID NO. 2)

[0026] S3: 5'-ATAAGGGCTGAGTCCTGAT-3' (SEQ ID NO. 3)

[0027] Using S1, S2 and S3 for PCR amplification, the expected amplified fragment size of the vaccine strain is 836 bp, and the expected amplified fragment size of the current isolate is 578 bp.

[0028] 2. Extraction of viral RNA

[0029] The virus strain cell culture medium was taken, freeze-thawed 3 times, centrifuged at 5000 g for 15 min, and the supernatant was taken for use.

[0030] (1) Take 0.2 ml of chloroform, add an equal volume of supernatant, shake vigorously for 15 s, place at room temperature for 3 min, and then centrifuge at 12,000 g at 4°C for 5 min. After centrifugation, it is divided into 3 layers, the bottom red is phenol-chloroform pha...

Embodiment 2

[0049] Example 2. Sensitivity experiment

[0050] Dilute the virus solution of the local isolate by 10-fold gradient to make the virus concentration 5.4×10 4 -5.4×10 - 4 copies / μL, and detected according to the method of Example 1, the result shows that the concentration in the 8th swimming lane is 5.4×10 - 3 Copies / μL can amplify the expected fragments, and the minimum nucleic acid detection amount of the primers and detection method of the present invention is 5.4×10 - 3 copies / μL, but lane 9 is 5.4×10 -4 copies / μL of fragments with no expected size, the sensitivity is 5.4×10 - 3 copies / μL.

[0051] Dilute the virus solution of the vaccine strain by 10-fold gradient to make the virus concentration 1.5×10 4 -1.0×10 -4 copies / μL, and detected according to the method of Example 1, the results show that the concentration in the 8th lane is 1.5×10 -3Copies / μL can amplify the expected fragments, and the minimum nucleic acid detection amount of the primers and detection ...

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Abstract

The invention discloses an RT-PCR (reverse transcription polymerase chain reaction) detection kit and method of koi herpesvirus. A primer group includes primers S1 and S2, wherein S1 is 5'-AAGGCTTAGTAGCTTACCAT-3', S2 is 5'-CAAGGTAGCTGAGTCGACCTATG-3', and S3 is 5'-ATAAGGGCTGAGTCCTGAT-3'. The specific primers S1, S2 and S3 are designed and capable of carrying out specific amplification on koi herpesvirus. The detection kit as a special kit has high sensitivity, reaches 100% in coincidence rate as compared to virus separation, IFA (indirect immunofluorescent assay) and other methods, and is widely applicable to identification diagnosis of koi herpesvirus.

Description

technical field [0001] The invention relates to the technical field of veterinary biological virus detection, in particular to an RT-PCR detection kit for koi herpes virus and a detection method thereof. Background technique [0002] Koi herpesvirus (Koi herpesvirus), referred to as KHV. Because it is mainly caused by DNA herpes virus infection, also known as carp nephritis and gill gangrene virus, it was also reported in Asia in 2004 that it was infected with koi herpes virus. my country lists it as a Category II animal disease, and it is a disease that must be declared by the World Organization for Animal Health (OIE). Also known as carp herpes virus type 3 (CyHV-3), it belongs to the family of herpesviruses. The disease mostly occurs in spring and autumn, with an incubation period of 14 days. Death begins 24 to 48 hours after the onset of symptoms, and the mortality rate is rapid within 2 to 4 days, reaching 80% to 100%. [0003] Since the mortality rate of koi and car...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/705C12Q2521/107C12Q2565/125
Inventor 张朝明严裕舟张胜辉李文得
Owner 张朝明
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