增修PCR方法技术，药典委公示聚合酶链式反应法标准草案

Core Viewpoint - The National Pharmacopoeia Commission has released a draft for the standard of the 1001 Polymerase Chain Reaction (PCR) method, which includes the addition of real-time quantitative PCR and revisions to conventional PCR methods [1][2]. Summary by Sections Introduction - The draft standard introduces new methods for quantitative detection using PCR, specifically adding real-time fluorescence quantitative PCR as a second method. It covers general requirements for instruments and reagents, measurement methods, and method validation requirements [2][6]. Revisions to Conventional PCR Method - The conventional PCR method has been revised to include: 1. Quality requirements for template DNA extraction from traditional Chinese medicine and biochemical drugs, including OD260/OD280 ratios for nucleic acid quality control. 2. Requirements for method applicability, including sequence, annealing temperature, types of restriction endonucleases, DNA polymerase concentration, and enzyme digestion time. 3. The addition of DNA characteristic sequences as quality control DNA markers [7][14]. Real-time Fluorescence Quantitative PCR Method - The real-time quantitative PCR method is designed for real-time detection of PCR amplification products, allowing for quantitative analysis of starting templates. It is applicable for specific nucleic acid components in traditional Chinese medicine, raw materials, intermediates, active pharmaceutical ingredients, and excipients [25][26]. - General requirements for instruments include continuous temperature control and the ability to excite and read fluorescence signals in real-time [25][26]. Method Validation Requirements - New methods must undergo validation, which includes defining experimental plans, analysis steps, and acceptable standards. Validation indicators are closely related to the intended use of the method, such as specificity for qualitative identification and accuracy, precision, linearity, and range for quantitative detection [29][30]. Laboratory Requirements - Laboratories must meet national biosafety management requirements and implement measures to prevent cross-contamination, such as functional area separation and pressure control [30].