Evaluation of Internal Standards in a Competitive Nucleic Acid Sequence-Based Amplification Assay

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• 作者：Wan-Yu Lo ; Antje J. Baeumner
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：February 15, 2007
• 年：2007
• 卷：79
• 期：4
• 页码：1386 - 1392
• 全文大小：212K
• 年卷期：v.79,no.4(February 15, 2007)
• ISSN：1520-6882

文摘

An end-point quantitative nucleic acid sequence-basedamplification (NASBA) reaction with two exogenous internal standards for the detection of the model analyteE. coli clpB mRNA was developed and statisticallyanalyzed. Electrochemiluminescence was chosen as ahighly sensitive detection means allowing careful evaluation of the internal standards used. The two internalstandards examined had been designed previously usinga novel and rapid NASBA-based method. Initially, eachstandard was used separately in a NASBA reaction;subsequently, two internal standards were added into onereaction at different concentrations. The accuracy andprecision of the data obtained were analyzed using linearand multiple regression analysis. In the case of single-standard reactions, the accuracy was >95% and theprecision >98.5%. In the case of double-standard reactions, the accuracy increased to >97%. With a singleinternal standard, 3 orders of magnitude of target sequence could be quantified; using three different concentrations of one internal standard, the dynamic rangeincreased to 5 orders of magnitude. In both cases, adetection limit as low as 0.14 pg of target sequence wasobtained. In the case of double-internal standard reactions, a dynamic range with 5 orders of magnitude and adetection limit of 1.76 pg was determined. The high-performance quality of the internal standards was assumed to be in part due to the unique synthesis processusing two NASBA reactions rather than traditional cloningtechniques.