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Rapid quantitative qPCR detection method of lecane in alga culture

A quantitative detection and rotifer technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. problems, to achieve the effect of shortening the test time, high sensitivity, and strong specificity

Active Publication Date: 2017-05-31
GUOTOU BIO TECH INVESTMENT CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research on coelomus is relatively limited, only in its classification, in terms of diversity and community structure, but there is almost no research on the harm it causes to large-scale algae cultivation. In environmental conditions Dormant cysts are formed when it is not conducive to production and reproduction. It is difficult to observe their existence in a microscope for large-scale algae culture, and the sensitivity of traditional morphological observation is low. can be completed
However, direct DNA sequencing and polymerase chain reaction (PCR) have low detection efficiency. When the DNA content is very low, it cannot be detected, and false positives in the test results often affect the judgment of the actual situation.
And when the above method is used to detect coeloid rotifers, it shows that the existing quantity is already very large, and this existing quantity is enough to completely defeat the large-scale cultivation of algae

Method used

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  • Rapid quantitative qPCR detection method of lecane in alga culture
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  • Rapid quantitative qPCR detection method of lecane in alga culture

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Design primers, steps:

[0055] Molecular barcoding is often used to identify microbial contamination. At present, a gene that exists in all eukaryotes is mainly studied: a part of the cytochrome oxidase I (CoI cytochrome c oxidase I) gene. This part of the DNA sequence can accurately distinguish species differences and has the specificity of each species, just like the "ID card" number of the species. In the process of algae cultivation, there will be pollution of different kinds of microorganisms. In order to clarify the information of pollutants, for the first time, we took the CoI gene of coelomus as the preferred choice for monitoring.

[0056] Using 119 coelomus CoI gene sequences obtained in GenBank, and in

[0057] The 43 CoI gene sequences of protozoa obtained from GenBank were compared and analyzed by bioinformatics, and the sequence-specific regions were selected. Using Primer Primer 5.0 software, three pairs of PCR primers were designed. The target amplific...

Embodiment 2

[0076] Construction and preparation of plasmid quantitative standards

[0077] Experimental steps:

[0078] 1. Construction of quantitative standards

[0079] 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 plasmids were used as quantitative standards and named as PGEMT-CoI-1, PGEMT-CoI-2 and PGEMT-CoI-3.

[0080] 2. Preparation of Quantitative Standards

[0081] 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.

[0082] Calculated as follows:

[0083]

[0084] The average molecular weight of one base pair is 660g / mol;

[0085] The total length of the plasmid is the total length of the vector plus the length of the insert;

[0086] N: stands for Avogadro's constant (6.02×10 2...

Embodiment 3

[0088] Genomic DNA extraction

[0089] Experimental steps:

[0090] 1. Collection of Coelomus

[0091] After the coelomus obtained in the algae culture system was washed and starved for 72 hours, a single individual or multiple individuals were separated under a dissecting microscope, divided into 1.5ml EP (eppendorf) tubes, and then the separation was confirmed under a dissecting microscope number of individuals. Afterwards, it was quick-frozen in liquid nitrogen for 5 minutes, then placed in a warm bath at 95°C for 5 minutes, and repeated 3 times for later use.

[0092] 2. Genomic DNA Extraction

[0093] The extraction efficiency of coelomus DNA was compared with different DNA extraction kits. Three kits, DNeasyBlood&Tissue Kit (QIAGEN), DNeasy Plant Mini Kit (QIAGEN) and High Pure TemplatePreparation Kit (Roche), CTAB method and bead beading were used respectively. The method and the method described in the patent application titled "A Rapid Extraction Method of Single ...

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Abstract

The invention firstly provides applications of a lecane CoI gene as a molecular marker for a rapid quantitative qPCR detection method of harmful lecane in alga culture, and a DNA molecular marker and a specific primer group shown as SEQ ID No.1-3. The invention further provides a rapid quantitative qPCR detection method of harmful lecane in alga culture. The specific primer group is adopted to perform fluorescent quantitative PCR to genome DNA of to-be-detected samples. The technical effect is to find a molecular marker and design a specific marker, and lecane can be effectively identified under the condition of having other polluted protozoa in the process of alga culture. The detection sensitivity and specificity are greatly enhanced, the test time is shortened, the operation is simplified, fluorescent detection results are analyzed by computer software, the pollution caused by after-treatment processes of products can be avoided, and the rapid quantitative qPCR method is more objective, sensitive and accurate compared with conventional PCR.

Description

technical field [0001] The invention relates to the technical field of microbial molecular detection, in particular to a qPCR method for early monitoring of coelomus in large-scale cultivation of algae. Background technique [0002] Coeloid rotifers are an important polluting protozoan present in large-scale algae cultures, and they are a broad class of freshwater rotifers. At present, it has been found and exploded in the large-scale cultivation of Chlorella, Scenedesmus and Haematococcus. Once infected, the large-scale cultivation of algae can be completely defeated within 2 to 4 days, resulting in "soaking", thus affecting the large-scale cultivation of algae cause great harm. It is therefore crucial to establish methods for the early monitoring and molecular identification of coeloid rotifers present in algal cultures. [0003] At present, the research on coelomus is relatively limited, only in its classification, in terms of diversity and community structure, but ther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12N15/11
CPCC12Q1/6851C12Q1/6893C12Q2600/166C12Q2563/107C12Q2545/114C12Q2537/16
Inventor 龚迎春李焕楠胡强
Owner GUOTOU BIO TECH INVESTMENT CO LTD