Rapid detection of φX-174virus based on synchronous fluorescence of tryptophan.

Abstract

The development of rapid methods for the detection of virus particles based on their intrinsic fluorescence (the native auto-fluorescence that originates from the non-labeled analyte) is challenging. Pure viruses may be detected in filtered solutions, based on the strong fluorescence of the amino acid tryptophan (Trp) in their proteins. Nevertheless, Trp also exists in high quantities in the hosts and host cultivation media. In this work, we developed a new method for the detection of the naked φX-174virus. We show that a separation of φX-174from its Escherichia coli host (grown on the standard cultivation medium nutrient agar) by simple extraction and filtration is not sufficient for its detection based on the intrinsic fluorescence since ~ 70% of the Trp fluorescence is derived from impurities. We formulate a new cultivation medium with a very low Trp concentration. We apply synchronous fluorescence measurements to show that no Trp fluorescence is detected in the extract solution upon incubation of this medium substrate with ammonium acetate extraction buffer. Finally, we apply synchronous fluorescence to detect φX-174based on the spectral fingerprint of its native Trp content. Such a method is more rapid than usual traditional separation and detection methods which can take several hours and does not require any addition of labeling agents such as fluorescent dyes or antibodies for the detection. As other virus species contain Trp as one of the amino acids presents in their proteins, this method has the potential to apply to the detection of other viral species.