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LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method for letalurus punetaus herpesviruses

A channel catherpes virus and detection kit technology is applied in the field of LAMP detection primer sets of channel catfish herpes virus, which can solve the problems of high harm, expensive equipment and serious problems, and achieve the effects of simple operation, high sensitivity and rapid detection.

Active Publication Date: 2020-05-05
HOHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the continuous expansion of the channel catfish breeding scale, the increasing degree of intensification, and the deterioration of the breeding environment, channel catfish diseases are becoming more and more serious, especially channel catfish virus disease (CCVD) caused by channel catfish virus (CCV). The largest, causing serious economic losses to the channel catfish farming industry
[0003] At present, the traditional detection method of CCVD mainly adopts cell culture technology to diagnose CCVD, and then combines serum neutralization test, fluorescent antibody technology, ELISA or PCR identification, the above Although the method is accurate and reliable, it requires expensive instruments and equipment, high testing costs and high technical requirements for testing personnel, making it unsuitable for the popularization and use of channel catfish in the field

Method used

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  • LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method for letalurus punetaus herpesviruses
  • LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method for letalurus punetaus herpesviruses
  • LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method for letalurus punetaus herpesviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 : Establishment of a kit for detecting herpes virus in channel catfish

[0039] A kit for detecting herpes virus in channel catfish based on LAMP technology, including LAMP primer set, Bst DNA polymerase, LAMP reaction solution, positive control and negative control, and chromogenic reagent SYTO-9.

[0040] (1) LAMP primer design: LAMP primers were designed with the protein OFR77 gene carried by channel catfish herpes virus as the target. The primer sequences are listed in Table 1.

[0041] Table 1 Primer sequence list

[0042]

[0043] (2) LAMP reaction solution: containing 10mM dNTP, 10×ThermoPol reaction buffer, and 150mM MgSO4 aqueous solution, the volume ratio of the three is 8:5:2.

[0044] (3) The positive control is a plasmid DNA containing channel catfish herpes virus ORF77 gene fragment, and its preparation method is: extract catfish herpes virus DNA, and use the external primers (SEQ ID NO: 1 and SEQ ID NO: 2) in Table 1 to pair Amplify, the...

Embodiment 2

[0046] Example 2 : Using a real-time fluorescent quantitative PCR instrument to establish a method for detecting herpes virus in channel catfish:

[0047] The method utilizing the kit of embodiment 1 to detect channel catfish herpes virus comprises the steps:

[0048] (1) Preparation of catfish herpes virus DNA: cut the tissue material from the channel catfish tissue to be tested, place it in a sterilized mortar, add liquid nitrogen to grind it, transfer it to a centrifuge tube, add 200 μl DNA extraction buffer (10 mmol / L Tris-cl, 0.1mol / L EDTA, 05% SDS), 5μl proteinase K (100μg / ml), mixed evenly, heated in a water bath at 50-60℃ for 2h, cooled to room temperature, and extracted with an equal volume of phenol Once, the aqueous phase was collected by centrifugation at 2500rpm / min, extracted once with an equal volume (phenol-chloroform-isopropanol) mixture, and the aqueous phase was collected by centrifugation at 2500rpm / min. Add an equal volume of isopropanol for precipitat...

Embodiment 3

[0051] Example 3 : detection specificity experiment:

[0052] With the method of embodiment 1 respectively to catfish herpes virus positive DNA and white spot syndrome virus, infectious subcutaneous and hematopoietic tissue necrosis virus, koi herpes virus, red sea bream iridescent virus, infectious spleen and kidney necrosis virus, grouper iridescent virus DNA testing.

[0053] Identification results such as figure 1 Shown: the channel catfish herpes virus LAMP primer set was used for amplification reaction, catfish herpes virus positive DNA amplified normally, negative water control and koi herpes virus, frog iridescent virus, mandarin fish infectious spleen and kidney necrosis virus did not amplify, showed good specificity.

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) detection primer group, a kit and a detection method for letalurus punetaus herpesviruses and belongs to the field of disease prevention and control in aquaculture. The kit consists of an LAMP primer group, DNA (deoxyribonucleic acid) polymerase, an LAMP reaction liquid, positive control, negative control and a developing agent, wherein the LAMP primer group comprises a pair of outer primers, a pair of inner primers and a pair of loop primers. The detection kit comprises the following application steps in detection on theletalurus punetaus herpesviruses: preparing DNA of the letalurus punetaus herpesviruses, performing a constant-temperature gene amplification reaction and performing result judgment by using a real-time fluorescence quantitative PCR (polymerase chain reaction) instrument. The kit disclosed by the invention has the advantages of being simple to operate, rapid in detection, good in specificity, high in sensitivity, reliable in result, and the like, can play roles in early-stage rapid diagnosis and real-time monitoring on channel catfish virus diseases, and is applicable to rapid detection on pathogens on a letalurus punetaus breeding site.

Description

technical field [0001] The invention belongs to the field of aquaculture disease prevention and control, and relates to a molecular detection method for aquaculture animal pathogens, in particular to a LAMP detection primer set, kit and detection method for channel catfish herpes virus. Background technique [0002] Channel catfish (Letalurus Punetaus), native to North America, is a warm-water freshwater farmed fish. After it was introduced to China in 1984, its aquaculture area and total output have continued to increase. Currently, it has been cultured in more than 20 provinces in my country. With an output of 285,000 tons, my country has become one of the most important channel catfish breeding countries in the world. Channel Catfish Virus (CCV) is an enveloped double-stranded DNA virus, also known as Ictalurid herpesvirus 1, which can cause fatal infection to channel catfish fry and fingerlings. With the continuous expansion of the channel catfish breeding scale, the inc...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/705C12Q1/6844C12Q2600/166C12Q2531/119C12Q2563/107C12Q2537/1376C12Q2545/113
Inventor 赵哲郝凯史燕喻飞
Owner HOHAI UNIV
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