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Table 2 Primer pairs used in PCR for amplifying whole O/YM/YN/2000 genome
Fragment
Name
Sequence (5
´
-
3
´
)
Position
a
F1
TGGAAAGGGGGCGCTAGGTCT
1~22
S1
Rc
GGGGGGGGGGTGAA
361~374
F-ployC
CCCCCCCCCMTAARGYYYTACCGWC
-
3
374~408
S2
IRES4
CCTATTCAGGCGTAGAAGCTT
991~1011
F916
CACTGGTGACAGGCTAAGGATG
887~908
F2
OR2
GACTGGGTTGTCGAGGTCGTG
1974~1994
OF3
CCACCCTCCTCGAGGACCGC
1931~1950
F3
2BR
AGCTTGTACCAGGGTTTGGC
4081~4100
2BF
CAGATGCAGGAGGACATGTC
3970~3989
F4a
R6434
AGAGGCCAGGCATGGTGTC
6406~6424
F5404
GAAAGGCCAACACGAAGCAGC
5373~5393
F4b
OR4
TCCATGGCGTCAAGTCCGTCGACGC
6920~6944
OF5
GGGTTGATCGTTGACACCAGAGA
6610~6632
F5
R-Not I dT
18
GGGGCGGCCGCTT
18
Poly A
Note:
a
: Nucleotide positions corresponds to the nucleotide sequence of the FMDV O/YM/YN/2000
Pfu
polymerase (Takara, Dalian) were performed
according to the manufacturer’ s protocol. For PCR
amplification, PCR reaction mixtures was followed by
1 cycle for pre-denaturing 5 min at 95 , 30 cycles for
℃
amplification reaction at 94 for 30 sec, 58 (for S1
℃
℃
and S2 fragment) or 52 (for F2
℃
、
F3
、
F4a
、
F4b and
F5 fragment) for 30 sec, 72 for 1 min (for S1 and
℃
S2) or 3 min (for F2
、
F3
、
F4a
、
F4b and F5), and 1
cycle for final extension at 72
℃
for 10 min. The PCR
products were purified from agarose gel electroph-
oresis and sequenced directly by ABI-PRIS MTM
377XL DNA Sequencer in two companies, Sangon
Biotech (Shanghai) and Taihe Biotech (Beijing).
3.3 Sequence and phylogenetic analysis
FMDV reference sequences were online acquired
from the GenBank database of National Center for
Biotechnology Information (NCBI, http://www.ncbi.
nlm.nih.gov). The sequence data were assembled
using the program Assemble (Vector NTI 8.0 suite,
InforMax, North Bethesda, MD). Multiple sequence
alignments were performed by ClustalX multiple
sequence alignment program, version 1.83 (Thompson
et al., 1997). The phylogenetic tree was constructed by
neighbor-joining method in MEGA version 3.1 (Kumar
et al., 2004) and the reliability of the branching orders
was evaluated using the bootstrap test (n=1000).
Author contributions
XAG developed the laboratory protocols, sequenced the
genomes for the FMDV sequencing project, conducted the
phylogenetic analysis and wrote the paper. LE and YYQ
collected the clinical isolates, selected the subset for
sequencing, MHS prepared the viral RNA. ZMW and SCH
performed laboratory assurance and animal experimental test.
LDF managed development of bioinformatics software for
assembly. LHC and XAG contributed to overall project
management.
Acknowledgments
We do sincerely thank to Baoshan Vaccine Plant of Yunnan
Province for providing the necessary facilities to carry out this
work. This work was jointly supported by grants from Natural
Science Foundation of Yunnan Province (No.2008ZC070M)
and National Natural Science Foundation (No.31060343).
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