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Koi herpesvirus RT-LAMP detection primer group, kit and detection method thereof

A technology of RT-LAMP and koi herpes virus, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of expensive and long detection cycle, and achieve easy operation and specificity High, Low Probability Effects

Inactive Publication Date: 2015-06-03
INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, it generally takes more than 15 days to obtain the results of koi herpes virus by cell culture, the detection period is too long, and the PCR detection method requires expensive equipment and professional operation level, so the establishment of a fast, accurate, high-throughput and Detection kits and detection methods that do not require high instruments have become an urgent problem to be solved at present

Method used

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  • Koi herpesvirus RT-LAMP detection primer group, kit and detection method thereof
  • Koi herpesvirus RT-LAMP detection primer group, kit and detection method thereof
  • Koi herpesvirus RT-LAMP detection primer group, kit and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Establishment of Koi Herpes Virus RT-LAMP Detection Kit

[0043] RT-LAMP (Real-Time Loop-mediated Isothermal Amplification, constant temperature real-time fluorescence amplification reaction) detection kit for koi herpes virus, including RT-LAMP primer set, RT-LAMP reaction solution, Bst DNA polymerase, positive and negative controls.

[0044] (1) RT-LAMP primer design: the main envelope protein gene of koi herpesvirus (KHV) ( env Gene, GenBank accession number is AB195965.1) as the target gene, KHV-specific RT-LAMP primers were designed online. The primer sequences are listed in Table 1.

[0045] Table 1. KHV-specific RT-LAMP primer sequence list

[0046]

[0047] (2) RT-LAMP reaction solution: 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 Aqueous solution, 10×SYTO-9 solution.

[0048] (3) The positive control contains the main envelope protein gene of koi herpes virus (KHV) envThe plasmid DNA of gene fragment, its preparation method is: tak...

Embodiment 2

[0050] Example 2 RT-LAMP detection method for koi herpes virus

[0051] Use the above kit to detect koi herpes virus (KHV) in the following way:

[0052] (1) Extraction of DNA from samples to be tested: DIO’s Aquatic Animal Virus Genomic DNA Extraction Kit extracts DNA from samples to be tested;

[0053] (2) Real-Time Loop-mediated Isothermal Amplification (RT-LAMP):

[0054] The 25μl reaction system contains: KHV-F3 0.2μM, KHV-B3 0.2μM, KHV-FIP 1.6μM, KHV-BIP 1.6μM, KHV-LF 0.8μM, KHV-LB 0.8μM, RT-LAMP reaction solution 13.0μl, BstDNA 8U of polymerase, 1~100ng of DNA to be tested, made up to 25μl with DEPC water; set positive control and negative control; mix the prepared PCR tubes, centrifuge, and react at 65°C for 60min.

[0055] (3) Judgment of results: Place the above-mentioned reaction tube in the constant temperature fluorescence detector 308C of Diao Bio, set the reaction program, and judge the amplification result according to the fluorescence signal read by the ins...

Embodiment 3

[0056] Embodiment 3 specificity experiment

[0057] Carp herpes virus (KHV) DNA, carp pox virus (CyHV-1) DNA, goldfish hematopoietic necrosis virus (CyHV-2) DNA, forktail catfish virus (CVV) DNA were separately purified by the method of Example 2. detection.

[0058] The identification results showed that: with koi herpes virus (KHV) RT-LAMP primers as primers, KHV DNA showed a normal amplification curve, and the negative water control and CyHV-1, CyHV-2, CVV, etc. had no amplification curves (see figure 1 ), showing that the detection primer and method of the present invention have good specificity.

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Abstract

The invention discloses a koi herpesvirus RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection primer group, a kit and a detection method thereof. The detection primer group comprises a pair of outer primers, a pair of loop primers and a pair of inner primers; the detection kit comprises a primer group, an RT-LAMP reaction liquid, Bst DNA (deoxyribonucleic acid) polymerase and negative / positive control. The detection method of the detection kit comprises the following steps: extracting DNA of a to-be-detected virus, amplifying the sample DNA template at 63-65 DEG C with six specific primers and the Bst DNA polymerase with strand displacement activity, observing whether an S amplification curve exists through a convenient constant temperature fluorescence detector, and determining whether the to-be-detected sample contains koi herpesvirus (KHV) DNA. The kit has the advantages of high specificity, high sensitivity, rapidity and high efficiency, simplicity and convenience in operation, easily readable result, and so on, and is suitable for being generalized and applied to detection units of different levels and aquaculture users.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method for aquaculture, in particular to a RT-LAMP (Real-Time Loop-mediated Isothermal Amplification, constant temperature real-time fluorescent amplification reaction) detection of koi herpes virus (KHV) Primer set, detection kit and detection method. Background technique [0002] Koi herpes virus disease is a highly pathogenic disease of koi (Cyprinus carpio koi) and carp (Cyprinus carpio carpio) caused by Koi herpesvirus (KHV). The disease mostly occurs in spring and autumn when the water temperature is 18-28°C, and is highly contagious, with a morbidity and mortality rate as high as 80%-100%. Due to the lack of relevant quarantine system, the international trade of koi and carp has promoted the outbreak of the disease worldwide, which has had a devastating impact on the koi and carp aquaculture industry. The disease was first reported in Germany, and the...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/705C12Q1/6844
Inventor 刘助红邱德义张璜陈洵石磊
Owner INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE
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