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Open Access Methodology article

More than fishing in the dark: PCR of a dispersed sequence produces simple but ultrasensitive Wolbachia detection

Daniela I Schneider1, Lisa Klasson2, Anders E Lind2 and Wolfgang J Miller1*

Author Affiliations

1 Laboratory of Genome Dynamics, Department of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Waehringerstrasse 10, Vienna 1090, Austria

2 Department of Molecular Evolution, Cell and Molecular Biology, Science for Life Laboratory, Biomedical Centre, Uppsala, Sweden

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BMC Microbiology 2014, 14:121  doi:10.1186/1471-2180-14-121

Published: 12 May 2014

Abstract

Background

Detecting intracellular bacterial symbionts can be challenging when they persist at very low densities. Wolbachia, a widespread bacterial endosymbiont of invertebrates, is particularly challenging. Although it persists at high titers in many species, in others its densities are far below the detection limit of classic end-point Polymerase Chain Reaction (PCR). These low-titer infections can be reliably detected by combining PCR with DNA hybridization, but less elaborate strategies based on end-point PCR alone have proven less sensitive or less general.

Results

We introduce a multicopy PCR target that allows fast and reliable detection of A-supergroup Wolbachia - even at low infection titers - with standard end-point PCR. The target is a multicopy motif (designated ARM: A-supergroup repeat motif) discovered in the genome of wMel (the Wolbachia in Drosophila melanogaster). ARM is found in at least seven other Wolbachia A-supergroup strains infecting various Drosophila, the wasp Muscidifurax and the tsetse fly Glossina. We demonstrate that end-point PCR targeting ARM can reliably detect both high- and low-titer Wolbachia infections in Drosophila, Glossina and interspecific hybrids.

Conclusions

Simple end-point PCR of ARM facilitates detection of low-titer Wolbachia A-supergroup infections. Detecting these infections previously required more elaborate procedures. Our ARM target seems to be a general feature of Wolbachia A-supergroup genomes, unlike other multicopy markers such as insertion sequences (IS).

Keywords:
Wolbachia; Drosophila; Glossina; Hybrid; High- and low-titer endosymbiont infection; Limit of detection; A-supergroup repeat motif (ARM)