FMDV Samples
A total of 14 samples (vesicular fluid and tongue epithelium) were collected from suspected FMD affected cattle of different outbreak areas of four districts (Rangpur, Jessore, Laxmipur and Feni) of Bangladesh during the period from September to October 2009. The samples were preserved at -200C, Before collecting the samples all instruments were sterilized properly.
Selection and Synthesis of Primers for RT-PCR
The oligonucleotide primers for the detection of FMDV and FMDV serotypes were used from the 1D, 2B and 5'URT regions of the viral genome as described.
Extraction of RNA
Total RNA was extracted from clinical samples using RNeasy mini kit (Qiagen, Germany) as per the manufacturer's instruction The extracted RNA was stored at -200C. The RNA samples were evaluated both quantitatively and qualitatively using spectrophotometer and agarose gel electrophoresis (Spectronic R GeneticsrM New York, USA) respectively
FMD virus detection usinq RT-PCR
The Promega Access RT-PCR Kit, USA was used to perform the reverse transcriptase and the subsequent PCR in a single reaction tube.
FMD virus serotyping using mRT-PCR
A RT-PCR mix (50 µl) consisted of 10µl AMV/Tƒl 5X reaction buffer, 0.5 µl of each primer, 2µl 25mM MgSO4, 1µl dNTP mix, 1µl T DNA Polymerase (5u/µl), 1µl AMV Reverse Transcriptase (5u/µl), 5µl template DNA and 26µl nuclease free H20. 5pl nuclease free H20 was used instead of template DNA in case of negative control, The primer mix for multiplex mix (A-C-O-ASIA 1) was 6 pmol P33, 50 pmol P110, 6 pmol P40, 6 pmol P38, 6 pmol P74, 6 pmol P75, 6 pmol P76 and 6 pmol P77. The amplification was performed in an oil-free thermal cycler (Master Cycler Gradient, Eppendorf, Germany) using the thermal profile: (1) 45°C for 45 min; (2) 95°C for 1 min, (3) 95°C for 15 sec; (4) 60°C for l min; (5) 60°C for 6 min; repeating steps (3) and (4) for 35 cycles. After completion of cycling program the tubes were held at 4°C. Electrophoresis
The amplified RT-PCR and mRT-PCR products were electrophorese d in 2% agarose gel at 100 V for 30 min. and stained with ethidium bromide (0.5 Fe/ml). DNA molecular weight marker type 100bp DNA ladder (lnvitrogen, USA) was used and examined against UV light using an image documentation system (Spectronic R GeneticsrM New York, USA),