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Detection kit and detection method for Enteromorpha compressa

A detection kit and technology of Enteromorpha, which is applied in the field of detection kits of Enteromorpha, can solve the problems of long experiment cycle and inaccuracy, and achieve the effect of simple operation

Inactive Publication Date: 2011-05-11
INSPECTION & QUARANTINE TECH CENT OF NINGBO ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Molecular identification is mainly based on the ribosomal gene transcriptional spacer sequence of algal nuclei and the ribulose-bisphosphate carboxylase / oxygenase large subunit gene sequence of chloroplast to construct a phylogenetic tree, and comprehensive morphological observation results to determine Determine the species, but this method also has the problem of a long experimental period, and the accuracy of this method is affected by many factors such as the sequence used for analysis, analysis method, etc., and sometimes inaccurate results may be obtained

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The detection kit of Enteromorpha, which includes the following tubes: 2.5 μL of 10×PCR reaction buffer, MgCl with a molar concentration of 25 mM 2 Solution 1.5 μL, molar concentration of 2.5 mM dNTP solution 2 μL, concentration of 5 U / μL Taq DNA polymerase solution 0.2 μL, molar concentration of 10 μM forward primer and reverse primer each 1 μL and double distilled water, forward primer The nucleotide sequence is: 5'-CGTTTTCGGA ACCGCCGGTG A-3', the nucleotide sequence of the reverse primer is 5'-GGCCAGGTCC ACGGCCCGCT CT-3', the detection kit can be used with algae with a final concentration of about 1ng / μL Add 1 μL of DNA extract (template DNA) to 25 μL with double distilled water to form a 25 μL PCR reaction system.

[0017] In the above example, the volume of each component of the detection kit is correspondingly doubled, and the volume of the template DNA is also doubled, supplemented with double distilled water to 50 μL, which is a 50 μL PCR reaction system.

Embodiment 2

[0019] Optimization of PCR reaction system

[0020] Optimization of the concentration of magnesium ions: optimize the concentration of magnesium ions according to the following PCR reaction system and cycle conditions. The 25 μL reaction system is: 2.5 μL 10×PCR reaction buffer, magnesium ions (25 mM) (selection volumes are 1, 1.5, 2 , 2.5, 3 μL), 2 μL dNTP (2.5mM), 1 μL detection of Enteromorpha forward primer Ucm6f (10 μM), 1 μL detection of Enteromorpha reverse primer Ucm2r (10 μM), 0.2 μL Taq DNA polymerase (5 U / μL), 1 μL template DNA (1ng / μL), and finally replenished to 25 μL with double distilled water; the reaction conditions were: 94°C, pre-denatured for 2 min; 94°C, denatured for 30 s, 60°C, annealed for 40 s, 72°C ℃, extended for 40 s, 34 cycles; 72 ℃, extended for 10 min; the results showed that the volume ratio of magnesium ions to reaction buffer was 1.5 μL: 2.5 μL.

[0021] Optimization of the concentration of Taq DNA polymerase: optimize the concentration of...

Embodiment 3

[0025] Three strains of Ulva kongus from different regions in my country U. pertusa , 2 strains of Enteromorpha marginatus U. linza , 2 Enteromorpha U. flexuosa , 6 Enteromorpha U. prolifera, 3 isolates of unidentified species of Ulva and 5 strains of Enteromorpha U. compressa A total of 21 samples were tested.

[0026] Extract the respective template DNA solutions according to the following DNA extraction methods: pick a single algae body, weigh 0.1-0.2 g of the algae body and put it into a 1.5ml first centrifuge tube, add 200 μL of 65 ° C water bath to preheat the pH8 .0 CTAB extract (containing 2% CTAB, 1.4 M NaCl, 20mM EDTA disodium and 100 mM Tris HCl), grind until homogeneous, then add 400μL CTAB extract preheated in 65℃ water bath , after inverting and mixing, place in a water bath at 65°C for 1 h, cool to room temperature, centrifuge at 12,000 rpm for 10 min, take the first supernatant to a second centrifuge tube, add 300 μL of Tris saturated phenol and 300 μL...

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PUM

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Abstract

The invention discloses a detection kit and detection method for Enteromorpha compressa. The detection kit for Enteromorpha compressa comprises 10 PCR (polymerase chain reaction) buffer solutions, an MgCl2 solution, a dNTP (deoxynucleotide triphosphate) solution, a Taq DNA (deoxyribonucleic acid) polymerase solution, a forward primer and a reverse primer in the volume ratio of 2.5:1.5:2:0.2:1:1, wherein the nucleotide sequence of the forward primer is 5'-CGTTTTCGGA ACCGCCGGTGA-3', and the nucleotide sequence of the reverse primer is 5'-GGCCAGGTCCACGGCCCGCTCT-3'. The detection method for Enteromorpha compressa comprises the following steps: extracting DNA to obtain a template DNA solution, wherein the template DNA solution and the solutions in the detection kit constitute a PCR system; carrying out PCR to obtain a PCR product, and carrying out agarose gel electrophoresis on the PCR product; and quickly and simply determining whether the sample is Enteromorpha compressa according to the fact whether a 330bp specific segment strip appears, and accurately separating the Enteromorpha compressa from other seaweed samples. The invention can be used for detecting Enteromorpha compressa mature algae with multiple distinguishing features, and can also be used for quickly distinguishing seedlings with fewer morphological features and Enteromorpha compressa algae with other growth morphologies.

Description

technical field [0001] The invention relates to a detection reagent, in particular to a detection kit and a detection method of Enteromorpha flatus. Background technique [0002] Enteromorpha belongs to the genus Ulva, which can be used for food or medicine, but a large number of multiplication will cause marine disasters such as green tides, resulting in outbreaks of harmful organisms and death of aquatic organisms due to hypoxia. The current identification methods of green algae of the genus Ulva include morphological observation, hybridization test identification and molecular developmental systematic identification. Morphological observation is based on morphological characteristics such as the shape of the thallus or filament, the shape of the cell, the size of the cell, the thickness of the thallus, the arrangement of the cells, the position of the chloroplast, the number of protein nuclei, etc., but the same species of algae There will be great changes in different l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 段维军郭立新朱水芳陈先锋
Owner INSPECTION & QUARANTINE TECH CENT OF NINGBO ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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