长棘海星(Acanthaster planci)幼体特异性PCR检测技术与应用*

doi:10.11978/2022011

Abstract

Abstract:

The outbreak of crown-of-thorns seastar (CoTS, Acanthaster planci) is one of the main causes to coral reef degradation in the South China Sea and the Indo-Pacific region. The density of CoTS larvae is an important indicator to determine whether the outbreak of CoTS adult population occurs or not. However, as the larvae are not visible and thus difficult to be distinguished, conventional investigation and microscope observation cannot effectively detect the CoTS larvae in natural seawaters. Therefore, it is needed to develop a sensitive and specific method for detecting CoTS larvae. This study established specific polymerase chain reaction (PCR) method to detect the CoTS larvae based on CoTS mitochondrial cytochrome oxidase subunit I gene (ApmtCOI), and this method was applied to detect the CoTS larvae in the coral reef around Qilianyu Island, Xisha. The results showed that the designed and screened four pairs of specific primers could succeed in amplifying ApmtCOI gene, and had no cross-reaction with Linckia laevigata, Culcita novaeguineae, Choriaster granulatus and Echinaster luzonicus. Moreover, the primer 2aooniF/2anooiR had the best specificity and higher sensitivity when annealing temperature was 58.5°C, which could detect the CoTS genomic DNA in pictogram grade. Furthermore, this method had succeeded in detecting the CoTS larvae in the Qilianyu Island of Xisha at the end of October, and we found that the distribution of CoTS larvae was not uniform. Therefore, this detection technology can be used as an effective method for monitoring CoTS larvae population in the future.

Key words: Acanthaster planci, cytochrome oxidase subunit I, polymerase chain reaction, larvae

CLC Number:

Q953

ZHANG Ying, YANG Litong, LIU Bing, ZHENG Fanyu, LUO Peng, CHEN Chang. Specific PCR detection for Acanthaster planci larvae and its application^*[J].Journal of Tropical Oceanography, 2022, 41(6): 125-131.

Figures/Tables 8

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References 26

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站位	坐标	地理位置	样品名
1	16°57.708′ N, 112°13.080′ E	西沙洲	XSZ
2	16°58.117′ N, 112°14.848′ E	赵述岛	ZS-1
3	16°58.315′ N, 112°16.300′ E	赵述岛	ZS-2
4	16°57.112′ N, 112°19.432′ E	中岛	ZD
5	16°56.794′ N, 112°19.801′ E	南岛	ND
6	16°56.086′ N, 112°20.221′ E	北沙洲	BSZ

序号	引物名称	引物序列(5'—3')	熔解温度(Tm)/℃		目的片段大小/bp	来源
1	COTS-F-1321	TCCGACTACCCGGACGCC TATAC	62.9	5985—6007	126	Doyle et al, 2017
1	COTS-R-1446	AGTGGT TCGCTGGGAAGTGAAGG	62.2	6008—6110	126	Doyle et al, 2017
2	2aooniF	ATTTGGGGCCTGAGCAGGAATG	60.6	4727—4748	301	Suzuki et al, 2016
2	2aooniR	GCCTCTTTCTACTCCGGCTGATG	60.2	5005—5027	301	Suzuki et al, 2016
3	AcanP-TF	GCACGATTTGTCTCTGCCAAA	56.1	3074—3094	785	本研究
3	AcanP-TR	AGTCCTTCTCTCGCCAGGT	58.4	3840—3858	785	本研究
4	COTS-F-69	GGCCTGAGCAGGAATGGTTGGAA	62.6	4733—4755	919	Uthicke et al, 2015
4	COTS-R-987	GCCTTGTAGCGTTGCCATTCACC	61.9	5629—5651	919	Uthicke et al, 2015

序号	引物名称	PCR 反应条件
1	COTS-F-1321 COTS-R-1446	95℃ 3min; 95℃ 15s, 60℃ 15s, 72℃ 30s, 34个循环; 72℃ 10min;
2	2aooniF 2aooniR	95℃ 3min; 95℃ 15s, 60℃ 15s, 72℃ 30s, 34个循环; 72℃ 10min;
4	AcanP-TF AcanP-TR	95℃ 3min; 95℃ 15s, 57℃ 15s, 72℃ 1min, 34个循环; 72℃ 10min;
5	COTS-F-69 COTS-R-987	95℃ 3min; 95℃ 15s, 57℃ 15s, 72℃ 1min, 34个循环; 72℃ 10min;

Specific PCR detection for Acanthaster planci larvae and its application^*

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Specific PCR detection for Acanthaster planci larvae and its application*

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Specific PCR detection for Acanthaster planci larvae and its application^*