Development and Comparison of Determination of Staphylococcus aureus in Different Processed Products of Angelicae Sinensis Radix Based on Real-time PCR Technique / 中国实验方剂学杂志

ABSTRACT

Objective:To quantitatively analyze the changes of Staphylococcus aureus in different processed products of Angelicae Sinensis Radix. Method:The real-time fluorescence quantitative polymerase chain reaction method （Real-time PCR） was established to quantitatively analyze S. aureus in Angelicae Sinensis Radix decoction pieces which bought from different producing areas， different enterprises and different storage time. The fluorescence quantitative reaction system was SYBR Premix Ex Taq Ⅱ of 10 μL， each of forward primer and reverse primer （10 μmol·L-1） of 0.8 μL， template/genome DNA of 1 μL， double distilled water of 7.4 μL. The reaction conditions of the fluorescence quantitative amplification curve were pre-denaturing for 30 s at 94 ℃， denaturing for 10 s at 94 ℃， annealing for 12 s at 60 ℃， extensing for 30 s at 72 ℃， cycling 45 times， single-point detection signal at 72 ℃. The melting curve was made from 72 ℃， and the step temperature of 0.5 ℃ was kept for 15 s to collect fluorescence. According to the results of Real-time PCR， representative samples were selected from Angelicae Sinensis Radix decoction pieces for comparison between plate counting method and Real-time PCR. Result:The content of S. aureus in different processed products was sorted by rank of raw Angelicae Sinensis Radix>soil-fried Angelicae Sinensis Radix>wine-processed Angelicae Sinensis Radix. The content of S. aureus was the lowest in the samples from Weiyuan area of Gansu province by comparing with other producing areas. Compared with the retail enterprises， the content of S. aureus in raw products and wine-processed products from production and sale enterprises was lower. Different storage time had certain effect on the content of S. aureus in raw products and wine-processed products， and the content of S. aureus increased with the increase of storage time. The detection results of plate counting method were 3-4 orders of magnitude lower than that of Real-time PCR. Conclusion:The established Real-time PCR is superior to plate counting method in specificity， sensitivity， reliability and reporting period， which can provide an effective method for rapid and accurate quantitative detection of S. aureus in different processed products of Angelicae Sinensis Radix.