Milk is seen as a chief source of protein and other biologically available nutrients for human beings. Pakistan, the fourth largest milk-producing country, is badly affected by the contagious transboundary apthoviral disease of ungulate animals; the foot and mouth disease (FMD) virus. FMD is endemic in Pakistan and has caused significant economic loss to the dairy industry in the form of a profound decrease in milk production and increased morbidity and deaths of dairy animals. Inclusively, the case fatality ratio of FMD was 15.11%. Of the seven FMDV serotypes, (O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3), three serotypes (O, A, and Asia-1) are endemic in Pakistan. Rapid and highly sensitive diagnostic tools are required for efficient control of this disease. Presently, FMD in the laboratory is diagnosed via ELISA and molecular approaches, i.e., RT-PCR. Serotype-specific RT-PCR analysis not only confirms ELISA serotyping results but can also be used for the screening of ELISA negative samples. Genotypically, FMDV serotype O has a topotype (Middle East–South Asia (ME–SA) and lineage PanAsia-2) that is reported frequently from different areas of Pakistan. Confirmed cases of serotype A and Asia-1 are also reported. The information gathered can be used for understanding the molecular epidemiology of FMD in Pakistan. Further studies on the molecular dynamics of FMD could be useful for ensuring the timely diagnosis of this deadly pathogen, which would ultimately be beneficial for the mass vaccination programs of FMD in Pakistan.

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 TCID₅₀ 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 TCID₅₀ (IND), 0.0946 TCID₅₀ (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. J. Clin. Lab. Anal. 25:95–99, 2011.