Presently I'm using Lanciotti et al., protocol for detection of Dengue using SYBR green1 PCR and I need to extend it for quantification of copy number / viral load in patient serum samples. I know this is not reliable to use for quantification of viral load of Dengue as it gives somewhat big PCR product for 1st PCR (511bp) and also more than 200bps product for serotyping Nested PCR. Is there any established PCR protocol for quantification of viral load of Dengue as well as serotyping PCR like as Lanciotti et al., by using either one reverse or forward primer for all PCR product? I'm mainly focused on a cost-effective quantitative PCR method for SYBR green1.
Dear Yohan,
It;s not advisable to use SYBR green based methodology for any DNA quantification. I hope, you are well aware about the binding of SYBR with DNA. I appreciate your intention to find a cost effective methodology for quantification. However, by the time of publication and implication for routine diagnostic or research purpose it will give big headache. Despite of extreme optimisation any slight changes in master mix, primer concentration and SYBR concentration will reflect in your experiment. I think you can try with molecular beacons instead of other TaqMan or hydrolysis probe.
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