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Method for detecting microcystins with high throughput by using LAMP-LFD chip

A technology of LAMP-LFD and microcystin, applied in biochemical equipment and methods, specific-purpose bioreactor/fermenter, enzymology/microbiology device, etc., can solve the problem that microcystin cannot be applied in the field and other problems, to achieve effective distinction, no cross-contamination, and obvious results

Inactive Publication Date: 2015-01-14
NINGBO UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the above fields, the object of the present invention is to provide a method for high-throughput detection of microcystins by using a LAMP-LFD chip, so as to realize rapid, safe, specific, sensitive and simple on-site detection of microcystin genes. To overcome the problem that existing laboratories use cytotoxicity test method, chemical analysis method, ELISA and PPIA method to detect microcystin and cannot be applied in the field, so as to make up for the deficiency of the existing technology

Method used

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  • Method for detecting microcystins with high throughput by using LAMP-LFD chip
  • Method for detecting microcystins with high throughput by using LAMP-LFD chip
  • Method for detecting microcystins with high throughput by using LAMP-LFD chip

Examples

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Embodiment 1

[0030] Example 1: Sensitivity detection of microcystin primers by high-throughput LAMP-LFD detection method for microcystins in natural water bodies of the present invention

[0031] Select the original density as 2.4×10 5 cfu / μL of Microcystis aeruginosa925 genomic DNA was diluted 10 0 , 10 1 , 10 2 , 10 3 , 10 4 Double as the reaction template, adopt the high-throughput LAMP-LFD of the present invention to detect the sensitivity detection of the method for microcystin, the specific steps are as follows:

[0032] The concentration of LAMP primers in the 25 μL reaction system is 1.6 μmol / L for inner primers FIP and BIP, 0.2 μmol / L for outer primers F3 and B3, 0.8 μmol / L for loop primers LF and LB, 2 μL for DNA template, and 1 μL for fluorescent dye calcein Add the corresponding LAMP reaction solution to make up to 24 μL; add 1 μL of Bst DNA polymerase to each concentration gradient reaction tube, and incubate at 61°C for 40 minutes; after the LAMP reaction, observe the co...

Embodiment 2

[0035] Example 2: Specific detection of the method for detecting microcystins by high-throughput LAMP-LFD in natural water bodies of the present invention

[0036] Common cyanobacteria strains in 15 natural water bodies were selected for the specificity verification of the method of the present invention. Among them, 11 strains of toxin-producing cyanobacteria are Microcystis aeruginosa FACHB-905, Microcystis aeruginosa FACHB-924, Microcystis aeruginosaFACHB-925, Microcystissp. , Chroococcus sp.FACHB-193, Nostocsp FACHB-596, Aphanizomenonflos-aquae FACHB-1039, 4 strains of non-toxic cyanobacteria, Microcystis wesenbergii908, Microcystis elabens Kutz FACHB916, Oscillatoria agardhii FACHB920, Anabaenaflos-aquae FACHB2.

[0037] The specific operation is the same as that in Example 1, and the results show that 11 strains of toxin-producing cyanobacteria LAMP-LFD were detected to be positive, and 4 strains that did not produce microcystins were negative, and sterile water was used...

Embodiment 3

[0038] Example 3: Using the high-throughput LAMP-LFD method for detecting microcystins to continuously monitor field samples.

[0039] In order to further understand the actual application effect of this set of primers in the field, we compared the detection method of high-throughput LAMP-LFD detection microcystins with the current common rapid detection method (ELISA method) for microcystins, in order to illustrate The high-throughput LAMP-LFD detection method for microcystins is convenient, fast, sensitive and simple and practical for early warning monitoring of microcystins.

[0040] 1. Materials and methods

[0041] 1.1 Field sample collection

[0042] The three locations of Sun Lake, Moon Lake and Jiulong Lake in Ningbo were selected for three-month continuous sampling (the initial sampling time was April 15, and the interval between each sampling was about 15 days, and a total of 8 batches continued until July 30. times, three natural water bodies distinguish ten sampl...

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Abstract

The invention aims to provide a method for detecting microcystins with high throughput by using an LAMP-LFD chip. The method is capable of realizing quick, safe, specific, sensitive and convenient field detection on the microcystins, and solving the problem that existing cytotoxicity test methods, chemical analysis methods, ELISA and PPIA methods which are adopted in laboratories for detecting the microcystins cannot be applied to field detection, so that the defects in the prior art can be overcome. The method has the advantages that the reaction results can be directly determined by naked eyes and the like, is suitable for detection reactions of various reagents and the like, and is especially suitable for field and outdoor detection requirements. According to the method, a primer for LMAP amplification is designed by combining an interspecific homology region of a mcyE gene responsible for synthesizing D-Glu necessary to bioactivity expression of microcystins in a microcystins synthesis gene cluster, and the microcystins gene is detected by utilizing a developed LAMP-LFD chip integration device.

Description

technical field [0001] The invention belongs to the technical field of rapid detection of toxins in natural water bodies, and in particular relates to a method for high-throughput detection of microcystins by using a LAMP-LFD chip Background technique [0002] Microcystins (Microcystins) are a class of biologically active cyclic heptapeptide compounds, which are mainly produced by freshwater Microcystis aeruginosa. In addition, other species of Microcystis, such as M. viridis, M. wesenbergii, and some species of Anabaena, Nostoc, and Oscillatoria Or strains can also produce such toxins. Microcystin is the cyanotoxin that is most likely to appear after the occurrence of cyanobacteria blooms in natural water bodies, produces the largest amount, and causes the most serious harm. It often causes acute liver poisoning accidents in humans and animals. Therefore, it is of great significance to develop a fast, simple, and sensitive high-throughput rapid detection method for microc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12M1/00
CPCC12Q1/6844C12Q2531/119C12Q2563/131
Inventor 朱鹏严小军陈炯张煜夏小磊范建忠黄海龙姚香菊
Owner NINGBO UNIV