بررسی امکان استفاده از Real time PCR برای اندازه گیری آمیکاسین به روش میکروبی

Abstract

IntroductionMicrobial assay is one of the most important biological methods for determination ofantibiotics assay. Potency evaluation of antibiotics, rapidity and ease of assay are themain privileges of the microbial method versus low precision and repeatability thatare the most important limitations of this method. The objective of the present studyis to evaluate the efficacy of real time-PCR technique in comparison withturbidimeteric method in microbial assay of amikacin.MethodsMicrobial determination of amikacin concentration was performed usingstaphylococcus aureus ATTC 29137 according to USP and final detection of theamount of microbial growth was performed with our developed taq-man quantitativePCR method and compared to standard spectrophotometric method. The standardcurves in different concentration for both methods were plotted and methodvalidation parameters of linearity, precision and accuracy were calculated usingstatistical procedures.Results and discussionTurbidimetric method for the determination of amikacin in distilled water usingstaphylococcus aureus ATTC 29137 was performed according to USP. The results ofthe turbidimetric method showed linearity with r2 of 0.993and precision and accuracyof 2/34- 6/91 (RSD) and 94/79- 112/1 % respectively. nuca gene of staphylococcusaureus was amplified and cloned in plasmid vector using InsTAcloning Kit(Fermentas) in order to use as a standard in real time-PCR determination of amikacin.According to the developed Real time-PCR assay, staphylococcus aureus amountswere quantified in the presence of different concentrations of amikacin andconsequently the plot of bacterial log concentrations versus amikacin logconcentrations showed linearity with r2 of 0.999 and precision and accuracy of 0.01-0.04 (RSD) and 94.2- 102.2 % respectively.ConclusionReal-time quantitative PCR assay was developed for absolute S. aureusquantification. In presence of different concentrations of amikacin the method wasused to determine amikacin concentrations. This method can provide a rapid, accurateand precise technique for measurement of amikacine potency in quality controllabroatories and it can be applied for microbial determination of the antibiotics withthe same test organism.Keyword amikacin, Staphylococcus aureus, real time-PCR, turbidimetric assayFilename أع‌ش‍ عاع‌کةکDirectory CUsersmohamadDesktopTemplateCUsersmohamadAppDataRoamingMicrosoftTemplatesNormal.dotmTitleSubjectAuthor mohamadKeywordsCommentsCreation Date 5/28/2011 111400 PMChange Number 10Last Saved On 6/5/2011 115200 PMLast Saved By mohamadTotal Editing Time 21 MinutesLast Printed On 6/8/2011 102600 AMAs of Last Complete PrintingNumber of Pages 2Number of Words 374 (approx.)Number of Characters 2,136 (approx.)