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A real-time quantitative PCR detection method for spores/gametes of Ulva feldspar, Ulva hard and Hemerocallis in sea cucumber culture water

A real-time quantitative technology for aquaculture water, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problem that it is difficult to determine the quantity or biomass, and the number of sporophytes and gametophytes of large moss algae cannot be achieved Detection and dynamic monitoring and other issues, to achieve the effect of good application potential, high sensitivity, simple and easy method

Active Publication Date: 2021-04-02
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, due to the small size (<10 μm) of germ cells such as sporophytes and gametophytes, it is difficult to distinguish them from other single-celled protists (such as autotrophic and mixed-trophic flagellates) under a light microscope, and only by traditional morphological observation It is difficult to determine its quantity or biomass, so the detection and dynamic monitoring of the sporophytes and gametophytes of macromoss algae in environmental samples cannot be realized

Method used

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  • A real-time quantitative PCR detection method for spores/gametes of Ulva feldspar, Ulva hard and Hemerocallis in sea cucumber culture water
  • A real-time quantitative PCR detection method for spores/gametes of Ulva feldspar, Ulva hard and Hemerocallis in sea cucumber culture water
  • A real-time quantitative PCR detection method for spores/gametes of Ulva feldspar, Ulva hard and Hemerocallis in sea cucumber culture water

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Detection of algae spores / gametes in different seasons in sea cucumber culture water

[0031] The water body samples were collected from the Dongfang Ocean Sea Cucumber Breeding Base in Yunxi Village, Yantai City. After being pre-filtered through a sieve with a pore size of 200 μm, they were slowly filtered onto a cellulose acetate membrane with a pore size of 0.22 μm (diameter: 47 mm; Millipore, USA). Soil extraction kit (Mpbio, USA) was used to extract DNA.

[0032] The right end of the 18S rDNA sequence and the ITS sequence region of Ulva ulva, Ulva hardina and Hemerocallis were obtained from the Genbank database to search for species-specific primers covering part of the ITS region or the full length of the ITS region, respectively. Species of the same genus or family with a sequence similarity higher than 90% were selected as references to search for specific sites. Use the website of Intergrated DNA Technologies (http: / / sg.idtdna.com / site) to conduct online eval...

Embodiment 2

[0036] Comparison of algae spores / gametes in sea cucumber culture waters with or without moss outbreaks in the same season

[0037] The classification and identification of macroalgae, the design and verification of ITS region-specific primers, and the optimization of quantitative PCR conditions are the same as in Example 1.

[0038] Using the specific primers for the ITS region of three algae (Ulva ulva, Ulva hardina, and Hemerocarpus) and optimized quantitative PCR conditions, comparing sea cucumber culture waters in December 2014, December 2015, and March 2015 (moss outbreak) Algal spore / gamete counts between March 2016 (no moss outbreak). The results showed that the number of spores / gametes of Ulva feldspar and Ulva hardina was lower than the detection limit; in December 2014, the ITS rDNA copy number of H. 7 copy / mL, while the copy number of ITS rDNA in Hecrophyllum sp. in December 2015 was only about 3.0×10 6 copy / mL; the copy number of ITS rDNA in Hemerophyllum was as...

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Abstract

The invention relates to the field of prevention, control and early warning for moss outbreak of holothurian culture water and specifically relates to a real-time quantitative PCR detection method for ulva linza, hard ulva and scytosiphon lomentaria spores / gametes in holothurian culture water. Ulva linza, hard ulva and scytosiphon lomentaria are the important species of the moss outbreak of holothurian culture pond, but the spores / gametes individuals thereof are smaller and are difficult to be detected according to a traditional method. Specific primers are designed for three large-scale algae ITS areas, the fluorogenic quantitative PCR condition is optimized and lastly the quantitative PCR method is utilized to monitor the dynamic conditions of the three large-scale alga spores / gametes in the culture water in real time. A result proves that the dynamic change of the ulva linza, hard ulva and scytosiphon lomentaria spores / gametes in holothurian culture water is more effectively matched with the moss outbreak rule. The method provided by the invention can be used for quickly, efficiently and high-sensitively monitoring the dynamic change of the three large-scale algae in the holothurian culture water and supplying early warning for the possible moss outbreak.

Description

technical field [0001] The invention relates to the field of prevention and control and early warning of moss outbreaks in sea cucumber culture waters, in particular to a dynamic monitoring method for spores / gametes of Ulva ulva, Ulva hardina and Hemerocybe spores in sea cucumber culture water. Background technique [0002] "Moss" is a common name for the bloom-prone macroalgal organisms in aquaculture water. In recent years, in the process of sea cucumber culture in ponds, there has been an increasing trend of large moss algal blooms. Large algal blooms compete with beneficial microalgae (diatoms, etc.) for limited resources such as light and nutrients, resulting in slow or even lack of growth of bait organisms, limiting the growth of sea cucumbers; algal blooms may be accompanied by the secretion of toxins, causing physiological stress on sea cucumbers and chemical poisoning; the filamentous structure of moss will trap sea cucumbers in it, affecting the movement and feedi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/156C12Q2561/113C12Q2545/114
Inventor 张倩倩缪栋张晓黎苏蕾包卫洋龚骏
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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