LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent <it>in-situ</it> hybridization
<p>Abstract</p> <p>Background</p> <p>Detection of unculturable bacteria and their localization in the host, by fluorescent <it>in-situ</it> hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to t...
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| Format: | Article |
| Language: | English |
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BMC
2012-05-01
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| Series: | BMC Microbiology |
| Online Access: | http://www.biomedcentral.com/1471-2180/12/81 |
| _version_ | 1828499070627348480 |
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| author | Priya Natarajan Pandey Neeti Rajagopal Raman |
| author_facet | Priya Natarajan Pandey Neeti Rajagopal Raman |
| author_sort | Priya Natarajan |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Detection of unculturable bacteria and their localization in the host, by fluorescent <it>in-situ</it> 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 <it>Bemisia tabaci</it>.</p> <p>Results</p> <p>In 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 <it>Bemisia tabaci</it> as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, <it>Portiera</it> (and present in more numbers) while the other a secondary endosymbiont <it>Arsenophonus</it> (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.</p> <p>Conclusion</p> <p>By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing <it>Portiera</it> and <it>Arsenophonus</it> in the males of <it>B. tabaci</it>. 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.</p> |
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| institution | Directory Open Access Journal |
| issn | 1471-2180 |
| language | English |
| last_indexed | 2024-12-11T13:17:25Z |
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| series | BMC Microbiology |
| spelling | doaj.art-4814382b041b44faae8f42494df700b12022-12-22T01:05:58ZengBMCBMC Microbiology1471-21802012-05-011218110.1186/1471-2180-12-81LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent <it>in-situ</it> hybridizationPriya NatarajanPandey NeetiRajagopal Raman<p>Abstract</p> <p>Background</p> <p>Detection of unculturable bacteria and their localization in the host, by fluorescent <it>in-situ</it> 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 <it>Bemisia tabaci</it>.</p> <p>Results</p> <p>In 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 <it>Bemisia tabaci</it> as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, <it>Portiera</it> (and present in more numbers) while the other a secondary endosymbiont <it>Arsenophonus</it> (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.</p> <p>Conclusion</p> <p>By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing <it>Portiera</it> and <it>Arsenophonus</it> in the males of <it>B. tabaci</it>. 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.</p>http://www.biomedcentral.com/1471-2180/12/81 |
| spellingShingle | Priya Natarajan Pandey Neeti Rajagopal Raman LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent <it>in-situ</it> hybridization BMC Microbiology |
| title | LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent <it>in-situ</it> hybridization |
| title_full | LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent <it>in-situ</it> hybridization |
| title_fullStr | LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent <it>in-situ</it> hybridization |
| title_full_unstemmed | LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent <it>in-situ</it> hybridization |
| title_short | LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent <it>in-situ</it> hybridization |
| title_sort | lna probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent it in situ it hybridization |
| url | http://www.biomedcentral.com/1471-2180/12/81 |
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