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Optimization of a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the detection of bacteria and disclosure of a formamide effect

Paper ID Volume ID Publish Year Pages File Format Full-Text
23026 43413 2014 9 PDF Available
Title
Optimization of a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the detection of bacteria and disclosure of a formamide effect
Abstract

•Bacteria detection by fluorescence in situ hybridization was modelled.•Response surface methodology was used to optimize an universal probe.•Optimum stringency does not depend uniquely on the oligonucleotide probe.•Formamide can impair hybridization efficiency depending on peptidoglycan thickness.

Despite the fact that fluorescence in situ hybridization (FISH) is a well-established technique to identify microorganisms, there is a lack of understanding concerning the interaction of the different factors affecting the obtained fluorescence. In here, we used flow cytometry to study the influence of three essential factors in hybridization – temperature, time and formamide concentration – in an effort to optimize the performance of a Peptide Nucleic Acid (PNA) probe targeting bacteria (EUB338). The PNA-FISH optimization was performed with bacteria representing different families employing response surface methodology. Surprisingly, the optimum concentration of formamide varied according to the bacterium tested. While hybridization on the bacteria possessing the thickest peptidoglycan was more successful at nearly 50% (v/v) formamide, hybridization on all other microorganisms appeared to improve with much lower formamide concentrations. Gram staining and transmission electron microscopy allowed us to confirm that the overall effect of formamide concentration on the fluorescence intensity is a balance between a harmful effect on the bacterial cell envelope, affecting cellular integrity, and the beneficial denaturant effect in the hybridization process. We also conclude that microorganisms belonging to different families will require different hybridization parameters for the same FISH probe, meaning that an optimum universal PNA-FISH procedure is non-existent for these situations.

Keywords
PNA EUB338; PNA-FISH; Bacteria; Formamide; Peptidoglycan
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Optimization of a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the detection of bacteria and disclosure of a formamide effect
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 187, 10 October 2014, Pages 16–24
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us