Abstract:
Semi-quantitative PCR technique is applying to indirectly quantify the level of nucleic acid. It is
becoming more popular due to the limitation real-time quantitative PCR technology. The present
study was done to optimization of a semi-quantitative PCR (semi-qPCR) protocol to quantification
of sugarcane white leaf (SCWL) phytoplasma presence in the sugarcane tissues based on the
conventional PCR technology. Genomic DNA was extracted from SCWL infected leaves and
disease-free seedlings by CTAB protocol. A PCR program was performed with the DNA
concentration gradient to find the optimum template DNA concentration for subsequent
optimization of other parameters of the program. Well optimized primer pair SPP1/SPP2 and
DnhF/DnhR newly designed primer pair were used to molecular detection of SCWL phytoplasma
by conventional PCR instead of nested PCR. A PCR cycle gradient 25, 27, 29, 31, 33 and 35 was
performed to determine the peak of the linear phase of the amplification for each primer pair. The
PCR program was performed at 950C for 3 min initial denaturation followed by 25-35 cycles:
denaturation at 95 0C for 45, annealing at 53 0C for SPP1/SPP2 and 56 0C for DnhF/DnhR for 1 min
and extension at 72 0C for 45 seconds for SPP1/SPP2 and 30 sec for DnhF/DnhR primers. The
endpoint analysis was determined by quantification and analysis of DNA band intensities by
ImageJ software of the resolved DNA bands in 1.5% agarose gels. The template DNA concentration
250 ng/µL was given consistent and significant band intensity. The primer pair SPP1/SPP2 had
been amplified the target band for all replicates in all PCR cycles and band intensity was gradually
increased with the cycle number. Statistical analysis confirmed that cycle number 31 started the
plateau of the PCR program of SPP1/SPP2 primer pair. The primer pair DnhF/DnhR was capable
of producing the target band, however, poorly amplified the negative control also in the later stage
of the program. Hence, this primer pair cannot be used for the semi-qPCR. The SPP1/SPP2 primer
pair, 250 ng/µl template DNA with 31 cycles PCR program can be successfully used to quantify
the level of SCWL phytoplasma by conventional PCR instrument.
