Molecular Pathogens 2012, Vol.3, No.1, 1
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16
Figure 7 Location of ASSVd in leaf tissues paraffin slice detected by
in situ
RT-PCR
Note: A, C, E and G: Positive staining of ASSVd in apple, peach, pear, apricot, respectively; B, D, F and H: Negative control with RT step
omitted in apple, peach, pear, apricot, respectively
viroids in these four kinds of fruit trees in the future.
3
Materials and Methods
3.1
Plant material and growth places
Tender leaves and shoots of apple, pear, apricot and
peach samples were collected from Yanqi, Hejing,
Bohu, Heshuo, Xinhe, Korla and Aksu in Xinjiang
province, China. Biological characteristics of ASSVd
samples were showed in table 3.
3.2
RNA extraction and RT-PCR detection
Total RNA of ASSVd isolated from each sample was
obtained as described previously (Zhao and Niu,
2006).
Then the first-strand cDNA was synthesized by
using reverse transcriptase (RT). Primers were
designed according to the reported sequences by The
National Center for Biotechnology Information of
USA (NCBI, GenBank accession number X17696)
(
Puchta et al., 1990): ASSVd
1
: 5′-
CAGCACCACAGG
AACCTCACGG-3′ (antisense, 10~32 sites); ASSVd
2
:
5′-
CTCGTCGTCGACGAAGG-3′ (sense, 80~97 sites);
ASSVd
3
:5′-
CCTTCGTCGACGACGA-3′ (antisense,
82
~97 sites), ASSVd
4
: 5′-
CCGGTGAGAAAGGAGCT
GCCAGCA-3′ (sense, 98~121 sites) and were
synthesized by Shanghai Biological Engineering
Technology & Service Corp. The antisense primer
ASSVd
1
/
ASSVd
3
was used as a primer for RT by
Moloney Murine Leukemiavirus Reverse Transcriptase
(
Shanghai Biological Engineering Technology & Service
Corp.) at 42
℃
for 1 h in 50 mmol/L Tris-Cl pH 8.3,
75
mmol/L KCl, 3 mmol/L MgCl
2
,
and 10 mmol/L
DDT. PCR was performed using a reaction mixture
containing 10 mmol/L dNTPs, 2 mmol/L Mg
2
+
,
antisense and sense (ASSVd
1
/
ASSVd
3
and
ASSVd
2
/
ASSVd
4
20
µmol/L), cDNA (3 µg) and 2.5 U
Taq
DNA polymerase (Shanghai Biological
Engineering Technology & Service Corp.). Samples
were amplified for 32 cycles as follows: denaturation
at 95
℃
for 3 min, 62
℃
for 45 s and 72
℃
for 60 s,
with the final extensions at 72
℃
for 7 min. The
products were denatured at 95
℃
(
or above) for 4 min
after removal from the thermal cycler. The amplified
products were separated by electrophoresis on 2%
(
w/v) non-denaturing agarose gel in 1×TAE buffer at
120
V for 40 min, and visualized by staining with
Gold Viewer Dye (Beijing Liuyi Factory, China). PCR
markers (Hua Mei, China) were used to determine the
sizes of the amplified fragments. Expected fragments
of about 330 nt were obtained through RT-PCR using
specific primers for ASSVd.
