Effect of Lowering PCR Reaction Volumes and Increasing the Speed of DNA Amplification on the Sensitivity of PCR

ABSTRACT Recently, PCR is being reported more frequently with satisfactory results on the diagnosis of clinical infections. Widespread availability of PCR promises a sensitive and specific alternative, to traditional methods, but these benefits must be balanced against cost. Protocols using small volumes have described where the reaction taken place in capillary tubes, and small volumes work when specialized thermocycler used. Despite its benefits on increasing the speed and decreasing the cost, this technique needs special equipment and thermocycler to perform which may not be possible for most molecular diagnostic laboratories. In this study, we wanted to evaluate effects of reducing time and volumes of reagents and therefore increasing speed and lowering reagent costs by routine equipment and ordinary thermocycler. In this study, M. pneumoniae DNA was extracted by phenol Chloroform method after treating with proteinase K. PCR was performed in different volumes and various amplification, and PCR products were analyzed by gel agarose electrophoresis. Experiments performed with extracted M. pneumoniae DNA confirmed that volume of reaction mixture can be decreased from 100 to 10 ul. Obtained results also proved. amplification time can be decreased from 6.5 to 3.5 minute for each cycle without losing of the sensitivity. These finding indicate dropping of the cost up to one tenth and necessary amplification time to the half in comparison with conditions. In the next step 34 throat swabs were tested with both conditions. All specimens had same sensitivity for optimized and main conditions. Performing PCR in small volumes not only reduce the cost of the test, but also can remarkably increase speed of the DNA amplification.