Study on the effect of nine nucleic acid extraction lysing reagents on PCR amplification efficiency

Abstract

ObjectiveTo explore the maximum allowable concentration of six chemical reagents and three enzyme lysates in a polymerase chain reaction （PCR） amplification system and the differences of IGEPAL CA-630 cleaving different species.MethodsPrepare different kinds of working solutions， including six chemical reagents （IGEPAL CA-630， CTAB， SDS， EDTA， guanidine isothiocyanate， and NaOH） and three enzymes （lysozyme， lysostaphin， and protease K） with different concentrations. Add the working solutions to the fluorescent quantitative PCR system and then analyze the differences in Ct values. Streptococcus thermophilus， Staphylococcus aureus， and Escherichia coli were lysed with IGEPAL CA-630 solutions （1% and 0.1% with TE buffer）， TE buffer and deionized water， respectively， and the differences in PCR results were analyzed.ResultsIn the PCR system， the maximum allowable final concentrations of IGEPAL CA-630， CTAB， SDS， EDTA， guanidine isothiocyanate， and NaOH were 0.800‰， 0.256‰， 0.012 8‰， 0.8 mmol/L， 0.012 8 mol/L， and 0.64 mmol/L respectively. The maximum allowable concentrations of lysozyme， lysostaphin， and protease K were 0.512， 1.28， and 0.512 μg/mL. In the lysing of S. thermophilus and S. aureus with IGEPAL CA-630， the Ct values of TE solution， 0.1% and 1% IGEPAL CA-630 decreased successively， with no significant difference between the TE solution and deionized water. There was no significant difference in Ct values of E. coli treated with deionized water， TE solution， 0.1% and 1% IGEPAL CA-630.ConclusionThis study established the maximum allowable concentration of the above nine components in the PCR amplification solution. Additionally， it was found that increasing the concentration of IGEPAL CA-630 could enhance DNA extraction from gram-positive bacteria.

Keywords

• polymerase chain reaction
• lysing reagent
• chemical trial dosage form
• enzymatic
• species