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Two methods for the extraction of RNA of vesicular stomatitis virus (VSV) Indiana1 and New Jersey and their simultaneous amplification by one-step polymerase chain reaction using reverse transcriptase were evaluated. A guanidine-thiocyanate-based RNA extraction (Qiagen RNeasy Mini Kit, Qiagen, Valencia, CA ) followed by column-based purification coupled with one-step RT-PCR proved to be a simple, safe, practicable, and reliable tool for rapid, highly sensitive, and specific differential diagnosis of both types of VSV in cell lysate and spiked tissue samples as compared with the tri-phasic extraction method (Tri-reagent method). When RNA was extracted either from VSV cell culture stock or from VSV spiked bovine lymph nodes by using Qiagen RNeasy Mini Kit, the detection limit in the multiplex RT-PCR was as low as 0.505 to 2.84 TCID(50) for VSV-IND and VSV-NJ, respectively. The multiplex RT-PCR consistently detected VSV-IND and NJ RNA in as little as 0.1-1.0  fg of total RNA from spiked BHK-21 cell suspension when Qiagen RNeasy mini kit was used. The multiplex RT-PCR assay was capable of detecting both types of VSV in a one-step reaction tube. The minimum sensitivity of this assay in various experiments was 0.1683 TCID(50) (IND), 0.0946 TCID(50) (NJ), and 0.057  fg (IND and NJ) per 2  µl PCR sample, which is significantly more sensitive than reported previously (0.28-2.8 TCID50/1  µl). So the present study improved the sensitivity of previously reported multiplex RT-PCR for the detection and differentiation of VSV-IND and VSV-NJ in a single assay., (© 2011 Wiley-Liss, Inc.)