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A method and kit for rapid detection of golden algae by loop-mediated isothermal amplification

A ring-mediated isotherm and kit technology, applied in biochemical equipment and methods, microbiological determination/testing, DNA/RNA fragments, etc., to achieve the effect of solving sensitivity problems, easy operation and high sensitivity

Active Publication Date: 2020-09-29
GUOTOU BIO TECH INVESTMENT CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, LAMP technology has not been effectively used for the early monitoring of the important pollutant golden algae in large-scale microalgal cultures

Method used

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  • A method and kit for rapid detection of golden algae by loop-mediated isothermal amplification
  • A method and kit for rapid detection of golden algae by loop-mediated isothermal amplification
  • A method and kit for rapid detection of golden algae by loop-mediated isothermal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Design and synthesis of primers

[0033] (1) Primer design and screening: Several pairs of primers were designed and synthesized according to the gene sequence of Chrysophylla cytochrome oxidase I (CoI cytochrome oxidase I), and primers that could be used in subsequent experiments were screened based on the following conditions.

[0034] a. The GC content of the sequence, the sequence with a content between 40% and 65% is defined as a common sequence, the sequence with a content greater than 65% is defined as a GC-rich sequence, and the sequence with a content of less than 40% is defined as an AT-rich sequence;

[0035] b. The primer length is based on the GC content. Set different primer lengths, as shown in the following table:

[0036]

[0037] c. Set the melting temperature (Tm value) of different primers according to the different GC content of the sequence.

[0038]

[0039]

[0040] d. The distance between primers, the distance between F2 and...

Embodiment 2

[0051] Example 2: Construction and preparation of plasmid quantitative standards

[0052] step:

[0053] 1. Construction of plasmid standards

[0054] The CoI gene fragment containing the target gene sequence was cloned by PCR method, recombined into the vector pGEM-T, and the DNA sequence was determined. The constructed recombinant plasmid was used as a quantitative standard and named pGEMT-CoI.

[0055] 2. Preparation of Quantitative Standards

[0056] Plasmid pGEMT-CoI was extracted and purified with Omega plasmid extraction kit, and the concentration and quality of the plasmid determined by Nanodrop8000 and Avogadro constant were converted into copy number.

[0057] Calculated as follows:

[0058]

[0059] The average molecular weight of one base pair is 660g / mol;

[0060] The total length of the plasmid is the total length of the vector plus the length of the insert;

[0061] N: stands for Avogadro's constant (6.02×10 23 copies / mol).

Embodiment 3

[0062] Example 3: Extraction of Genomic DNA

[0063] Carry out cell counting to the culture solution of purely cultured Chrysophylla and Chlorella, and obtain the cell densities of the two. Take 2mL of cell culture solution and use High Pure Template Preparation Kit (Roche) to extract pure Chrysophylla DNA, use DNeasy Plant Mini Kit (QIAGEN) DNA extraction kit to extract the mixed DNA, and use Nanodrop8000 to check the concentration and quality of DNA. Determination. Select the DNA with the best concentration and quality and save it for later use.

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Abstract

The invention provides a rapid detection of gold algae method and a test kit based on the cyclo-mediated isothermal expansion. Gold algae CoI gene is firstly provided as a molecular marker and applied in the rapid detection of gold algae based on the cyclo-mediated isothermal expansion, and a group of cyclo-mediated isothermal amplification primer is further provided, its specificity isothermally expands the molecular marker through the cyclo-mediate; and finally the rapid detection of gold algae method based on the cyclo-mediated isothermal expansion is provided, the method uses the to-be-tested sample granules and a gene group DNA as the template, and uses the specific primer group to conduct cyclo-mediated isothermal expansion reaction. The primer is strong in specificity, high in flexibility, and has very good repeatability. The method can detect the existence of gold algae mitochondria CoI genes with the lowest concentration of 100 copies / MuL and the existence of gold algae cells with the lowest concentration of 1000 cells / mL, and the sensitiveness is increased by 1000 times compared to the traditional PCR technique, the sensitiveness problem is very well solved, and the method has significant meaning for the early stage monitoring of microalgae culture contaminants.

Description

technical field [0001] The invention relates to the technical field of microbial molecular detection, in particular to a method and a kit for rapidly detecting golden algae by using a loop-mediated isothermal amplification technology. Background technique [0002] One of the most polluting protozoa present in large-scale cultures of Chlorella, Chrysophytum, is a flagellate - Poterioochromonas sp. The flagellates have the characteristics of autotrophy and mixed nutrition, and are very easy to infect and swallow chlorella quickly. Once infected, the large-scale cultivation of chlorella can be completely defeated in a short period of time, thus bringing great harm to the large-scale cultivation of chlorella. to great harm. Therefore, it is very important to determine the method for the early rapid monitoring and molecular identification of Chrysophylla in Chlorella culture. [0003] At present, the research on Chrysophyta is mostly limited to its isolation and culture and its...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6893C12Q2531/119
Inventor 龚迎春王贤慧展雪玲胡强
Owner GUOTOU BIO TECH INVESTMENT CO LTD
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