Soejima Takashi, Xiao Jin-Zhong, Abe Fumiaki
Functional Food Ingredients, Food Ingredients &Technology Institute, Morinaga Milk Industry Co., Ltd. 5-1-83, Higashihara, Zama, Kanagawa, 252-8583, Japan.
Next Generation Science Institute, Morinaga Milk Industry Co., Ltd. 5-1-83, Higashihara, Zama, Kanagawa, 252-8583, Japan.
Sci Rep. 2016 Jun 23;6:28000. doi: 10.1038/srep28000.
Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.
通常，聚合酶链反应（PCR）在DNA分离后进行。实时PCR（qPCR），在细胞膜较脆弱的哺乳动物细胞中也被称为直接qPCR，是一种常见技术，使用经初步煮沸以洗脱DNA的粗样品。然而，与在水中会立即破裂的哺乳动物细胞不同，将这种方法应用于具有坚固细胞壁的原核细胞时，可能导致检测效果不佳。我们在新开发的直接qPCR预混液中加入1.3 cfu的穆氏克罗诺杆菌，成功实现了PCR延伸，且未进行任何粗DNA提取（测试样品的检测限为1.6×10(0) cfu/ml，而主要针对粗提（煮沸）或经典DNA分离的检测限为1.6×10(3) cfu/ml）。我们发现，原核细胞中保留的染色体DNA可作为PCR模板，这与原位PCR的机制类似。阐明这种反应机制可能有助于开发用于直接qPCR的创新预混液，以检测具有坚固细胞壁的单个细菌中的基因，并可能在原核基因组学研究中带来众多新发现。
