Figure. Reverse transcriptase-polymerase chain reaction (RT-PCR) for enteroviral RNA from acute hemorrhagic conjunctivitis cases. Lane 1: pX174 DNA/Hae III marker; lane 2: negative control; lane 3: positive control (enteroviral RNA); lanes 4-6: clinical samples. For PCR, viral RNA was extracted by the guanidinium thiocyanate method (2) from 200 µl of clinical specimen. The RNA pellet was resuspended in 10 µl of diethyl pyrocarbonate treated water. A 5-µl volume was used for cDNA synthesis. The primers used for PCR amplification were selected from the highly conserved 5' noncoding region of enterovirus described by Rotbart et al. (3) and, using the same protocol, cDNA for PCR was synthesized by reverse transcription. Briefly, cDNA (10µ Cl) was amplified in a PCR mix volume of 50 µl, containing 100 ng of each primer, 250 µl of each dNTP, 1.5 mM MgCl2, 2.5 units Taq polymerase, and 5 µl of 10X PCR buffer. The tubes were subjected to 30 cycles at 94°C for 1 minute, 58°C for 1 minute, and 72°C for 2 minutes, respectively, followed by a final extension for 7 minutes at 72°C. A 154-bp sized band of the PCR-amplified product was visualized under UV illumination on a 2% agarose gel after ethidium bromide staining.
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