Molecular Pathogens 2012, Vol.3, No.3, 12
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1
Results and Analysis
1.1
Total RNA extraction
The conventional method of fast detection of RNA is
high voltage electrophoresis, which can reduce the
degradation of the RNA in a relatively short time. Gel
electrophoresis analysis of the total RNA samples
showed that the total RNA we obtained was in a good
integrity and the value of OD
260
/
OD
280
was more than
1.80,
which displayed that it was suitable for RT-PCR
experiment (data was not shown here) (Figure 1).
1.2
RT-PCR amplification products
Using specific primers for ASSVd, RT-PCR was
carried out, and the expected fragments about 330 nt
were obtained in these four kinds of trees.
1.2.1
RT-PCR detection for apple ASSVd
The specific product can be amplified in 15 strains by
RT-PCR (Figure 2). The results showed as follows,
ASSVd were detected in cultivars of Ralls (Aa3, Aa7
and Aa15), in cultivars of Red Fuji (Ad17, Ad23), and
in cultivars of Starkrimson (Ae5, Ae23) in Yanqi
county; similarly, in cultivars of Ralls (Aa98, Aa117)
and Starkrimson (Ae35 ) in Korla region; in cultivars
of Ralls (Aa123, Aa149), Red Fuji (Ad79) and
Starkrimson (Ae86) in Heshuo regeion; as well as
Aa176 of Ralls in Aksu. However, ASSVd were not
detected in all sampling sites in Red delicious and
Golden delicious cultivars.
1.2.2
RT-PCR detection for pear ASSVd.
The specific products were amplified in 13 trains by
RT-PCR (Figure 3). The results showed that the
ASSVd were detected in cultivars of Korla pear, Pa2,
Pa14 and Pa27 and in cultivars of Apple pear, Pe3,
Pe17, in Yanqi county; similarly, in cultivars of Korla
pear, Pa39, Pa46, Pa52 and in cultivars of Apple pear,
Pe93, Pe97, Pe116, in Korla region; as well as Pa63 of
Korla pear and Pe136 of Apple pear in Heshuo
regeion. However, ASSVd were not detected in all
sampling sites in Jinfeng pear, Ya pear and Dangshan
pear cultivars.
1.2.3
RT-PCR detection for peach ASSVd
With the same approach, 11 strains were obtained
(
Figure 4). The results demonstrated that the ASSVd
were detected in cultivar of
P. persica Sieb. et Zucc
Figure 1 Total RNA for apple, pear, apricot and peach trees
Note: A5, P5, T5 and X5: Total RNA of leaves for apple, pear,
apricot and peach,respectively; A6, P6, T6 and X6: Total RNA of
phloems for apple, pear, apricot and peach,respectively
Figure 2 RT-PCR detection results for apple ASSVd
Note: Lane 1: Products amplified with primers ASSVd1 and
ASSVd2 primers from RNA of healthy apple leaf tissues; Lane 2:
Products amplified from RNA isolates from sample Aa3 using the
antisense primer ASSVd1 only; Lanes 3~17: Fragments amplified
using both ASSVd1 and ASSVd2 primers from RNA isolates from
apple samples Aa3, Aa7, Aa15, Ad17, Ad23, Ae5, Ae23, Aa98,
Aa117, Ae35, Aa123, Aa149, Ad79, Ae86 and Aa176,
respectively; Lane 18: DNA Marker
Figure 3 RT-PCR detection results for pear ASSVd
Note: Lane 1: Products amplified with primers ASSVd1 and
ASSVd2 primers from RNA of healthy pear leaf tissues; Lane 2:
Products amplified from RNA isolates from sample Pa2 using the
antisense primer ASSVd1 only; Lanes 3~15: Fragments amplified
using both ASSVd1 and ASSVd2 primers from RNA isolates from
pear samples Pa2, Pa14, Pa27, Pe3, Pe17, Pa39, Pa46, Pa52, Pe93,
Pe97, Pe116, Pa63 and Pe136, respectively; Lane 16: DNA Marker
(
Ta1, Ta4, Ta5, Ta9, Ta10, Ta12, Ta17, Ta21, Ta24,
Ta25 and Ta29) only in Yanqi county. And the ASSVd
were not detected in others sampling sites.
1.2.4
RT-PCR detection for apricot ASSVd
Similarly, we obtained 12 strains with specific product
from 300 apricot samples (Figure 5). The ASSVd were
detected in cultivar of apricot trees (X3, X7, X12,
