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Development of a Blocking Primer to Inhibit the PCR Amplification of the 18S rDNA Sequences of Litopenaeus vannamei and Its Efficacy in Crassostrea hongkongensis

文献类型： 外文期刊

作者： Liu, Cong 1 ; Qi, Ren-Jie 1 ; Jiang, Jing-Zhe 1 ; Zhang, Ming-Qing 5 ; Wang, Jiang-Yong 1 ;

作者机构： 1.Chinese Acad Fishery Sci, South China Sea Fisheries Res Inst, Key Lab Aquat Prod Proc, Minist Agr & Rural Affairs, Guangzhou, Guangdong, Peoples R China

2.Shanghai Ocean Univ, Coll Fisheries & Life Sci, Shanghai, Peoples R China

3.Tianjin Agr Univ, Coll Fisheries, Tianjin, Peoples R China

4.Chinese Acad Fishery Sci, Shenzhen Base South China Sea Fisheries Res Inst, Shenzhen, Peoples R China

5.Guangzhou Univ, Sch Life Sci, Guangzhou, Guangdong, Peoples R China

关键词： blocking primer; PCR; eukaryotic microorganisms diversity; high-throughput sequencing; shrimp; oyster

期刊名称：FRONTIERS IN MICROBIOLOGY （ 影响因子：5.64； 五年影响因子：6.32 ）

ISSN： 1664-302X

年卷期： 2019 年 10 卷

页码：

收录情况： SCI

摘要： Diversity analyses of the eukaryotic microorganisms in the gut of marine animals is hampered by the presence of host DNA in the samples. PCR amplification of rRNA genes of eukaryotic microorganisms is inefficient with universal primers targeting 18S rRNA gene when the host DNA is dominant. In this study, we designed several blocking primers to inhibit PCR amplification of rRNA genes of the shrimp Litopenaeus vannamei, and tested their efficacy on the oyster Crassostrea hongkongensis. We first compared the intensity of PCR product bands obtained with and without the blocking primers. Then, one primer was selected for further verification using high-throughput sequencing. Our results showed that X-BP2-DPO was the most effective blocking primer in suppressing the host 18S amplification compared to nine other candidates. The inhibition rate was 99% for the amplification of shrimp rDNA, and 17% for the amplification of oyster rDNA. The concentration of the blocking primer in the PCR mixture was an important factor to be considered in the experimental design. The development of blocking primers provided a valid method to study the composition and characteristics of eukaryotic microorganisms in shrimp gut for a better understanding of its diets.

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