Abstract
BackgroundDetection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci.ResultsIn this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe.ConclusionBy employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.
Highlights
Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction
When compared to DNA only oligonucleotide probes, it is seen that Locked Nucleic Acid (LNA) modified DNA oligonucleotide probes are 10 fold more sensitive when applied in techniques like northern analysis [11]
Comparing LNA and DNA probes to detect Portiera the primary bacterial endosymbiont of Bemisia tabaci While detecting Portiera we found LNA to be more sensitive than DNA oligonucleotide probes (Figure 1)
Summary
Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. Microscopic detection and localization of a specific DNA or RNA segment within single cells or a histological section has been made possible with the advent of the In-Situ Hybridization (ISH) technique. This technique relies principally on formation of Watson-Crick base pairing between the gene of interest and the applied complementary sequence to which the reporter molecule is attached [1]. The extra bridge in an LNA structure makes the ribose moiety inaccessible, thereby locking the structure to high binding affinity conformation [8,9] Such LNA nucleotides can be mixed with DNA or RNA residues during synthesis of oligonucleotide to enhance the hybridization specificity, sensitivity and duplex stability [8,10]. There are many reports that have identified and localized bacteria by targeting 16 S rRNA gene in whole mount or microtome section samples but till date there has been no report wherein LNA probes have been employed for bacterial detection by FISH in whole mount or microtome section of biological samples
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