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LAMP detection primer group for Siniperca chuatsi-perch rhabdovirus, application of LAMP detection primer group and detection kit

A detection kit and detection primer technology, applied in recombinant DNA technology, microbial determination/inspection, microorganisms, etc., can solve the problems of expensive instruments and equipment, high technical requirements, high detection costs and detection personnel, etc., and achieve high sensitivity, Simple to use effects

Inactive Publication Date: 2020-12-11
HOHAI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection method of mandarin fish and California perch rhabdovirus mainly relies on the traditional PCR method. Although this method can provide accurate and reliable detection results, it requires expensive equipment, high detection costs and high labor costs for the detection personnel. The technical requirements make it unsuitable for large-scale screening use and promotion in mandarin fish and sea bass farming sites

Method used

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  • LAMP detection primer group for Siniperca chuatsi-perch rhabdovirus, application of LAMP detection primer group and detection kit
  • LAMP detection primer group for Siniperca chuatsi-perch rhabdovirus, application of LAMP detection primer group and detection kit
  • LAMP detection primer group for Siniperca chuatsi-perch rhabdovirus, application of LAMP detection primer group and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 is used to detect the kit of mandarin perch rhabdovirus to establish

[0026] A kit for detecting Rhabdovirus in mandarin perch based on LAMP technology, including LAMP primer set, AMV reverse transcriptase, Bst DNA polymerase, LAMP reaction solution, positive control and negative control, and chromogenic agent SYTO-9.

[0027] (1) Design of LAMP primers: The RNA-dependent RNA polymerase gene (L gene) of mandarin perch rhabdovirus was used as the target to design LAMP primers. The primer sequences are listed in Table 1. The molar ratio of outer primer, inner primer and loop primer is 1:8:4.

[0028] Table 1 Primer sequence list

[0029]

[0030] (2) LAMP reaction solution: containing 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 Aqueous solution, the volume ratio of the three is 8:5:2.

[0031] (3) The positive control is a plasmid DNA containing the rhabdovirus L gene fragment, and its preparation method is: extract the genomic RNA of the ma...

Embodiment 2

[0033] Example 2 Using a constant temperature fluorometer to establish a detection method for rhabdovirus in mandarin perch

[0034] The method utilizing the kit of embodiment 1 to detect California perch rhabdovirus may further comprise the steps:

[0035] (1) Preparation of California perch rhabdovirus genomic RNA: Cut the tissue material from the California perch tissue to be inspected, place it in a sterilized mortar, add liquid nitrogen to grind it, transfer it to a centrifuge tube, add 1ml TRIzol reagent and mix evenly; add 0.2ml chloroform, shake for 10-15s, place at room temperature for 2-3min; centrifuge at 4°C for 15min at high speed (12000×g) to separate layers; gently suck out about 500ul of the upper aqueous phase, add 500ul of isopropanol, mix well, and let stand at room temperature for 10min , high-speed centrifugation (12000×g) at 4°C for 10 minutes, discard the supernatant to retain a small amount of white precipitate of RNA; add 1ml of 75% ethanol solution fo...

Embodiment 3

[0038] Embodiment 3 detection specificity experiment

[0039] The method of Example 1 is used to detect the DNA or RNA of mandarin fish and California perch rhabdovirus positive RNA and ISKNV, CyHV-2, CCV, WSSV, GCRV, Aeromonas hydrophila, Edwardsiella and other pathogens respectively.

[0040] Identification results such as figure 2 Shown: the amplification reaction was performed with the LAMP primer set of mandarin fish and California perch rhabdovirus. The positive RNA of mandarin fish and California perch rhabdovirus was amplified normally, and the nucleic acid (DNA or RNA) of the negative water control and other pathogens did not amplify, showing good results. specificity.

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Abstract

The invention discloses an LAMP detection primer group for Siniperca chuatsi-perch rhabdovirus, application of the LAMP detection primer group and a detection kit. The primer group comprises a pair ofouter primers, a pair of inner primers and a pair of loop primers; the outer primers comprise a sequence L-F3 and a sequence L-B3, the sequence L-F3 of the outer primers is shown in SEQ ID NO: 1, andthe sequence L-B3 of the outer primers is shown in SEQ ID NO: 2; the inner primers comprise a sequence L-FIP and a sequence L-BIP, the sequence L-FIP of the inner primers is shown in SEQ ID NO: 3, and the sequence L-BIP of the inner primers is shown in SEQ ID NO: 4; and the loop primers comprise a sequence L-LF and a sequence L-LB, the sequence L-LF of the loop primers is shown in SEQ ID NO: 5, and the sequence L-LB of the loop primers is shown in SEQ ID NO: 6. The invention further provides application of the primer group in preparation of a Siniperca chuatsi-perch rhabdovirus detection kitand the kit. The primer group and kit, provided by the invention, have the advantages of simplicity in operation, high detection speed, good specificity, high sensitivity, reliable result, and the like.

Description

technical field [0001] The invention relates to a set of LAMP detection primers and its application, a detection kit, in particular to a set of LAMP detection primers for mandarin perch rhabdovirus, its application, and a detection kit. Background technique [0002] Rhabdovirus (Rhabdovirus) is a kind of enveloped negative-strand RNA virus, which has strong pathogenicity and a wide range of hosts, and can infect mammals, birds, reptiles, fish, insects and plants, etc. variety of organisms. Fish rhabdoviruses are one of the most numerous groups of bony fish viruses. So far, nearly twenty species of fish rhabdoviruses have been isolated and identified, such as: mandarin fish rhabdovirus (Siniperca chuatsi rhabdovirus, SCRV), California perch Rhabdovirus (Micropterus salmoides rhabdovirus, MSRV), and they are usually associated with aquaculture epidemics and significant economic losses. [0003] Siniperca mandarin fish (California perch) and perch (California perch) are impor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844
Inventor 赵哲周国勤喻飞郝凯夏思瑶陆健陈树桥王绍绵
Owner HOHAI UNIV
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