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LAMP (loop-mediated isothermal amplification) primer group, kit and detection method for rapidly identifying russula vinosa

A detection method, the technology of poisonous russula, is applied in the field of LAMP primer set for rapid identification of poisonous russula, which can solve the problems such as difficult to meet the on-site rapid detection, and achieve the effect of improving amplification efficiency and reaction sensitivity

Active Publication Date: 2022-08-09
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reaction process of this technology relies on a temperature cycle instrument (PCR instrument), and the reaction time is about 2 hours. It can only be completed in the laboratory, and it is difficult to meet the needs of on-site rapid testing.

Method used

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  • LAMP (loop-mediated isothermal amplification) primer group, kit and detection method for rapidly identifying russula vinosa
  • LAMP (loop-mediated isothermal amplification) primer group, kit and detection method for rapidly identifying russula vinosa
  • LAMP (loop-mediated isothermal amplification) primer group, kit and detection method for rapidly identifying russula vinosa

Examples

Experimental program
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Effect test

Embodiment 1

[0054] According to the red mushroom ITS sequence included in NCBI GeneBank, including the ITS sequence containing 5.8S rDNA in the middle, download 10 described red mushroom ITS sequences from the above-mentioned database, and download and download red mushroom both belong to the subgroup of the genus Russula. R. subnigricans, R. japonica, R. densifolia, R. brevipes, R. nigricans, verdigris The ITS sequences of R. aeruginea and R. foetens, the above 7 species include species that are morphologically and sequence-similar to Russula vulgaris. After sequence homology comparison, the species are determined. Internally conserved, interspecies specific sequence fragments, in order to ensure the specificity of the Russula rutaformis primers, and the LAMP amplification primers are designed according to the determined sequence fragments. Considering that the existence of loop primers can improve the rate of amplification reaction, the present invention designs 3 primer sets, each prim...

Embodiment 2-1

[0059] Using primer set 1 in Example 1 as the amplification primer, the outer primer (F3 / B3), inner primer (FIP / BIP), and loop primer (LB) were each diluted to 10 μM, followed by F3, B3, FIP, BIP and LB were mixed in a volume ratio of 1.5:1.5:16:16:16 to obtain a primer mixture. Based on the total LAMP reaction volume of 10 μL, including 5 μL of LAMP master mix (consisting of DNA polymerase, base and MgSO) 4 Buffer composition); 1.09 μL primer mix; 2 μL Russula emetica genomic DNA template (obtained by pyrolysis, at a concentration of 10 ng / μL); 1 μL of SYB Green fluorescent dye; add ddH 2 O supplemented to 10 μL, all reagents were placed in a 0.2 mL centrifuge tube, and the mixed system was fully vortexed and mixed. The LAMP reaction was carried out in a real-time PCR instrument, and the condition was set to 62 °C, and the fluorescent signal was collected every 45 s for a total of 50 cycles. The reaction result is judged by the amplification S-shaped curve whether the ampli...

Embodiment 2-2

[0061] Same as Example 2-1, the difference is that Russula emetica is replaced by Russula nigricans.

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Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an LAMP (loop-mediated isothermal amplification) primer group, a kit and a detection method for rapidly identifying russule. The invention provides an LAMP (loop-mediated isothermal amplification) primer group for rapidly identifying russula vinosa. The LAMP primer group comprises a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP and a loop primer LB. The primer group disclosed by the invention is strong in specificity and high in sensitivity, can detect russula vinosa DNA with the concentration of 1pg / mu L, is successfully used for detecting a boiled mushroom mixture (the russula vinosa content is as low as 1%), has the characteristics of rapidness, sensitivity, specificity and visualization, and can be used for rapid identification, traceability and the like of russula vinosa poisoning.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a LAMP primer set, a kit and a detection method for rapidly identifying Russula rutae. Background technique [0002] Poisonous red mushroom (Russula emetica), also known as vomit red mushroom. The cap is 5-9 cm in diameter, light pink to coral red. In summer and autumn, it is scattered or grouped on the ground in the forest, and is widely distributed in the southwest of China. Poisonous red mushroom has a spicy taste, and it mainly causes acute gastroenteritis-type poisoning symptoms after eating. It can cause nausea, vomiting, diarrhea, abdominal pain, muscle twitching, rapid pulse, and death due to weak heart or blood circulation in severe cases. The toxicity of red mushrooms can be reduced to a certain extent in the process of heating and cooking, but there is still a high toxicity. [0003] Wild red mushrooms, also known as red mushrooms, true red...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12R2001/645C12Q2563/107C12Q2563/173Y02A50/30
Inventor 赵志勇赵兰馨周昌艳赵晓燕范婷婷鄂恒超李晓贝张艳梅董慧李旭娇
Owner SHANGHAI ACAD OF AGRI SCI
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