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

Cong Liu,Ming-Qing Zhang,Ren-Jie Qi,Jing-Zhe Jiang,Jiang-Yong Wang

doi:10.3389/fmicb.2019.00830

Abstract

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.

Highlights

A major focus of ecology is understanding trophic relationships and energy flows in natural environments, associated food web dynamics and changes in food webs due to disturbance, environmental change, invasions and extinctions
Considering the fact that the blocking primers have a strong inhibitory effect on shrimps (Figure 1A and Supplementary Figure S3A) and a relatively good inhibition on oysters (Supplementary Figures S3D, S4), we were concerned about the inhibition of the primers at different concentrations on the non-host taxa
Further Verification In order to further verify the effect of blocking primer, we evaluated X-BP2-Dual priming oligonucleotide (DPO) with another six repetitions of shrimp (Figure 7A) and oyster (Figure 7B) at the concentration of 0.6 μM

Summary

Introduction

A major focus of ecology is understanding trophic relationships and energy flows in natural environments, associated food web dynamics and changes in food webs due to disturbance, environmental change, invasions and extinctions. The identification of gut content species has been conducted using morphological methods (Paquin et al, 2014; Harms-Tuohy et al, 2016; Aguilar et al, 2017). DNA-based methods are perhaps the most promising ones, and are established as powerful tools for studying the diversity of eukaryotes (Symondson, 2002; King et al, 2008; Harms-Tuohy et al, 2016). The species diversity that could be detected by universal primers in samples is often compromised by high concentrations of the host DNA templates (Vestheim and Jarman, 2008). If the sample contains the host sequences at relatively high concentrations, the less concentrated sequences of other eukaryotes are often not amplified as PCR favors the dominant DNA types (Jarman et al, 2004; Deagle et al, 2005; Belda et al, 2017)

Methods

Results

Discussion

Conclusion