Background:
The reverse transcription-polymerase chain reaction (RT-PCR) technique for detection of HCV RNA has gained popularity recently due to its sensitivity. In a block-based thermal cycler, a major drawback of this technique is the time required
for
performing the reactions. In order to correct the shortcoming of the forementioned technique, a proposal of the protocol of HCV PCR to be performed in the capillaries using an air-based thermal cycling instruments is made.
Methods:
We determined the optimum reaction conditions by the investigation of the effect of difference in concentration of the components and in the conditions.
Results:
According to our protocol, the first HCV PCR (50 μL) is performed in (final concentration) 45mM Tris/HCl (pH 8.3), 2.5mM MgCl2, 500㎍/mL bovine serum albumin, 200μM dNTP, 50 pmol/50μL primers, 2.0U/50μL Taq polymerase with 5μL reverse
transcription product. The second PCR (10μL) is done with the same Tris/HCl, MgCl2, bovine serum albumin and dNTP as in the first PCR, and 2.5mM, 10pmol/10μL primers, 0.8 U/10μL Taq polymerase with 2μL first PCR product. For the first HCV
PTPCR, an
initial 120 sec denaturation at 94℃, and 35 cycles of 55℃for 15sec, 72℃for 20 sec, 94℃ for 15 sec were carried out, followed by a 180 sec elongatio at 72℃For the second PCR, the mixture was denatured at 94℃for 20sec, and 35 cycles of 55℃
for
1.5
sec, 72℃for 20 sec, 94℃for 1.0sec and postelongation at 72℃for 30 sec were carried out.
Conclusions:
An introduction of the protocol for the thermal cycling using air and thin glass capillary tube in HCV RT-PCR should bring a reduction in amplification time and cost.
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