Blok H J, Gohlke A M, Akkermans A D
Wageningen Agricultural University, The Netherlands.
Biotechniques. 1997 Apr;22(4):700-4. doi: 10.2144/97224st05.
A method for precise and accurate quantification of 16S rDNA has been developed that uses competitive PCR and the QPCR System 5000. The method is based on co-amplification of 16S rDNA sequences, along with an internal standard sequence, using only one set of conserved eubacterial primers. Co-amplified PCR products are rapidly identified and quantified by measuring the electrochemiluminescent signals from specific oligonucleotide reporter probes that are directed against a hypervariable 16S rDNA sequence. Because in the exponential phase of amplification the different target sequences and the internal standard sequence are amplified with the same efficiency, unknown amounts of a target sequence in a sample can be inferred by extrapolating against a standard curve that is generated for the internal standard sequence. This method provides a rapid, nonradioactive and reliable way to simultaneously quantify different specific 16S rDNA targets that are present in low numbers, and may thus be suitable for enumeration of specific target microorganisms in environmental samples.
已开发出一种使用竞争性聚合酶链反应（PCR）和5000型定量PCR系统对16S核糖体DNA（rDNA）进行精确和准确定量的方法。该方法基于仅使用一组保守的真细菌引物对16S rDNA序列与内标序列进行共扩增。通过测量针对高变16S rDNA序列的特异性寡核苷酸报告探针的电化学发光信号，快速鉴定和定量共扩增的PCR产物。由于在扩增的指数期，不同的靶序列和内标序列以相同的效率扩增，因此可以通过相对于为内标序列生成的标准曲线进行外推来推断样品中未知量的靶序列。该方法提供了一种快速、无放射性且可靠的方式，可同时对低数量存在的不同特定16S rDNA靶标进行定量，因此可能适用于环境样品中特定靶标微生物的计数。
