Authors: F S Bitaraf I Rasooli S L Mousavi Gargari
Publish Date: 2016/01/14
Volume: 35, Issue: 3, Pages: 503-510
Abstract
Haemophilus influenzae type b Hib causes acute bacterial meningitis ABM in children with a mortality rate of about 3–6 of the affected patients ABM can lead to death during a period of hours to several days and hence rapid and early detection of the infection is crucial Aptamers the short singlestranded DNA or RNA with high affinity to target molecules are selected by a highflux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology SELEX In this study wholecell SELEX was applied for the selection of targetspecific aptamers with high affinity to Hib ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells Hib The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry The aptamers with higher binding affinity were cloned Four of 68 aptamer clones were selected for sequencing The dissociation constant Kd of the highaffinity aptamer clones 45 and 63 were 4710 and 2846 pM respectively These aptamers did not bind to other bacterial species including the seven meningitiscausing bacteria They showed distinct affinity to various H influenzae strains only These aptamers showed the highest affinity to Hib and the lowest affinity to H influenzae type c and to other meningitiscausing bacteria Clone 63 could detect Hib in patients’ cerebrospinal fluid CSF samples at 60 colonyforming units CFU/mL The results indicate applicability of the aptamers for rapid and early detection of infections brought about by HibThe authors wish to thank the College of Basic Sciences Shahed University for granting us permission to use its wellequipped laboratory We thank Mr Samani of Royan Institute for his assistance with the flow cytometry analysis We appreciate Hazrat Rasool Hospital Tehran for providing us with the CSF samples
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