John M. S. Bartlett.pdf - Bio-Nica.info

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Table 2
Detection Rate of Hepatitis Virus Nucleic Acids in Serum.
Comparison of Conventional PCR and Direct PCR
HBV DNA HCV RNA HGV RNA
Conventional PCR Positive 121 56 32
Direct PCR-Positive 121 51 30
Agreement Rate 100% 91% 94%
Fig. 1. Efficiency of serum volume and diluent as a factor in HCV-PCR reaction. A different
amount of serum sample ranging in volume from 1 to 5 µL of serum was used. Each aliquot
of serum, except for the 5 µL of volume, was diluted with PBS or H 2 O to the final volume of
5 µL, respectively. M = 50-bp DNA ladder.
2. Perform the first PCR reaction by adding 45 µL of mastermix 1 to each 5-µL sample of
heat-inactivated serum from Subheading 3.1. Then, program the thermocycler to incubate
the samples for 50 min at 37°C for the initial RT step and then preheat at 95°C for 10 min
to activate AmpliTaq Gold followed by 50 cycles consisting of 94°C for 20 s, 55°C for 20 s,
and 72°C for 30 s using a Thermal Cycler. RT-PCR is performed with a one-step method
combined with cDNA synthesis reaction, followed by the PCR reaction in a single tube
(see Note 2). That is, for RNA of HCV, the first PCR is combined with the RT step
in the same tube containing 50 µL of a reaction buffer as shown above. To obtain an
automatic hot-start reaction, we use the AmpliTaq Gold DNA polymerase instead of
regular thermostable DNA polymerase.
3. For the second reaction, 2.5 µL (1/20 volume) of the first PCR product is added to a tube
containing second PCR buffer. The thermocycling for 50 cycles is performed as above, but
omitting the initial 50 minute incubation at 37°C and the annealing temperature is raised
to 60°C instead of 55°C for the second round of PCR.
4. The PCR products are electrophoresed on a 2% agarose gels staining with ethidium
bromide and evaluated under ultraviolet light. The sizes of PCR products are estimated
according to the migration pattern of 50-bp DNA ladder.
4. Notes
1. Examination of the optimal serum quantity reveals that 1 to 2 µL of serum give a readily
amplifiable band of HCV RNA but 3 µL of serum give a faint band and 4 to 5 µL of serum
do not yield any band (Fig. 1). This result is much better when PBS is used to dilute serum
than that obtained using H 2 O. It is similar even if this result is in the case of HBV.
2. The whole process of the direct RT-PCR can be completed within 6 h by combination with
the one-step amplification method and the second round PCR.
3. Detection rate of HCV RNA and HBV DNA by direct PCR are consistent with the results
obtained by conventional PCR (Fig. 2 and Table 2).