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L. Feiner, R.N. Van Gelder; Accelerated Detection and Identification of Ocular Pathogens Using PCR Coupled With Array Screening . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5060.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: PCR of ribosomal RNA can be performed upon ocular samples to determine if bacteria or fungi are present in a specimen. Identification of the specific isolate has traditionally been performed by cloning and subsequent sequencing, a costly and time–consuming endeavor. We show that labelled PCR reactions can be used as probes to identify the isolate in a few hours and at low cost. Methods: PCR of 16S ribosomal was performed on known bacterial isolates using universal primers tagged at each 5' end with digoxygenin. The products of these reactions were used as probes to screen nylon filters carrying several clinically relevant bacterial pathogens. Probe detection was performed using alkaline phosphatase coupled anti–digoxygenin antibody. Alkaline phosphatase activity was detected using the chemiluminesenct substrate CPSD. Results: As proof of principle, several bacterial isolates were used as mock pathogens. PCR products from each of these isolates were correctly identified in the array hybridization. Conclusions: PCR using universal primers coupled with hybridization to a panel of known pathogens permits rapid, sensitive, and specific identification of bacteria from ocular samples. This technique may be particularly valuable in polymicrobial infections when sequencing and subcloning might fail to detect all bacteria present in a specimen. Future studies will be aimed at applying microarray technology to further improve the number of pathogens in the array and accelerate the procedure.
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