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Method for detecting prymnesium parvum

A technology of Purinella and primer set, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of inability to quickly detect Purinella, expensive instrument experiment environment, etc., and achieve It is beneficial to detection and control, the detection results are clear and obvious, and the detection time is short

Inactive Publication Date: 2018-10-16
NINGBO UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods require expensive instruments and rigorous experimental environments
Moreover, these can only be performed by trained researchers and are therefore only suitable for laboratory studies and cannot be used for the rapid detection of P. pullenella in point-of-care testing (POCT)

Method used

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  • Method for detecting prymnesium parvum
  • Method for detecting prymnesium parvum
  • Method for detecting prymnesium parvum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: the method establishment of LAMP-LFD technology detection harmful Pulinna officinalis

[0044] Takara MiniBEST Universal Genomic DNA Extraction Kit Ver.50 was purchased from Bao Biological Engineering (Dalian) Co., Ltd. DNA amplification kit, Rongyan Biotechnology (China) Co., Ltd., and LFD detection test strips were purchased from Milenia Biotec GmbH company (Milenia GenLine HybriDetect MGHD1, Germany).

[0045] 1. Design of LAMP primers

[0046] After the applicant conducted a homology analysis on the ITS gene of Puerinia microbiota published in NCBI, and compared the sequence of the ITS gene of Puerinia microbiota with similar species, the applicant designed and screened the following 5 primers. The primer sequences are as follows Shown:

[0047] P.P-F3:5′-AACTTTTGAACGCAACTGGC-3′

[0048] P.P-B3:5′-GATGGCACAACGACTTGGTA-3′

[0049] P.P-FIP:5′-CAAGCTCGGATGTGCGCGAGGCCTGGGAGCATGTTTCT-3′

[0050] P.P-BIP:5′-GATCAAGGCTCGAGCGTCGCCCTACTAGCTGCACGGCA-3′

[...

Embodiment 2

[0061] Use the LAMP-LFD technology of the present invention to measure the sensitivity of harmful algae P.

[0062] 1. Refer to the DNA extraction kit instructions to extract the Purinella minor genomic DNA, and dilute the original concentration (30ng / μL) of the extracted Purlinella minor genomic DNA template by 10 times, respectively, as the reaction template.

[0063] 2. Using LAMP, LAMP-LFD and PCR method to detect Puerinia microbiota to compare the sensitivity.

[0064] 2.1 Use the above-mentioned gradient diluted genomic DNA as a PCR reaction template, and use the above-mentioned specific primers P.P-F3 and P.P-B3 to amplify. The reaction system is: 1 μL each of 20 μmol / L P.P-F3 and 20 μmol / L P.P-B3, Premix 25 μL of Taq enzyme (TaKaRa), 3 μL of Purinella minor genomic DNA template, and distilled water to make the system 50 μL. Optimized PCR reaction conditions: pre-denaturation at 95°C for 8min; 35 cycles of 95°C for 15s, 54°C for 30s, and 72°C for 20s; 72°C for 10min. ...

Embodiment 3

[0069] Use the LAMP-LFD technology of the present invention to measure the specificity of harmful algae P.

[0070] Select Prymnesium parvum, Pleurochrysis caterae, Phaeocystis globosa, Alexandrium tamarense, Karenia mikimotoi, highly toxic Karo sp. (Karlodinium veneficum), diatoms (Cylindrothecasp.), Gymnodinium sp., Heterosigma akashiwo, Prorocentrum donghaiense (Prorocentrum donghaiense) 10 common harmful algae as LAMP-LFD specific Test algae, in which double distilled water was used as negative control. Genomic DNA of algae was extracted, and the amplification products of LAMP were observed by LFD test strips and 2% agarose gel electrophoresis respectively. See the test results Figure 4-6 , indicating that the LAMP-LFD detection method of Puerinia microalgae provided by the present invention can ensure the specific detection of harmful algae Puerinia microbiota, and does not cross-react with other related algae.

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Abstract

The invention provides a method for detecting prymnesium parvum. 6 LAMP primers are designed, namely P.P-FIP, P.P-BIP, P.P-LF, P.P-LB, P.P-F3 and P.P-B3, wherein the P.P-FIP is a 5' terminal biotin labeled primer; the P.P-LB is a 5' terminal thiocyanate fluorescein FITC labeled probe. The method comprises the following steps: preparing an LAMP reaction system containing three pairs of primers andsample templates; amplifying the LAMP reaction system, then detecting with an LFD test paper strip and subsequently performing sensitivity and specificity evaluation. The method provided by the invention is more practical and convenient that an existing method for detecting the prymnesium parvum by traditional technology PCR, has high sensitivity and specificity, can be applied to an actual field,is beneficial to detection and control on the outbreak of the prymnesium parvum and takes precautions in advance. When being applied to the detection on the harmful alga prymnesium parvum, the methodprovided by the invention can effectively prevent the harmful alga prymnesium parvum from large-scale red tides, which is of great significance in the protection of marine and aquaculture in China.

Description

technical field [0001] The invention belongs to the technical field of harmful algae detection, and in particular relates to a detection method of the harmful algae Prymnesium parvum. Background technique [0002] The occurrence of harmful algal blooms (HABs) has increased in recent decades as a hazard to aquatic resources, human health and marine ecosystems. Purimynia minor is a harmful species of algae, and molecular studies have shown that Purimynia minor and Dinnoflagellate are two stages of the life cycle of the same species. Primella minor was first found in a coastal pond. At present, it has been widely reported in coastal and inland waters with medium and high salinity, and it has also been reported in coastal marine farms in my country. Puriminia minor can secrete hemolytic exotoxin and anatetoxin, which can dissolve the gill cells of fish, resulting in the death of fish and shellfish. P. smallpox has strong adaptability and is widely distributed all over the wor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/89
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2565/625
Inventor 朱鹏黄海龙党晨阳乔龙亮庞建虎徐继林严小军
Owner NINGBO UNIV