Development of fluorescent adjacent hybridization probes and their application in real-time PCR for the simultaneous detection and identification of Fervidobacterium and Caloramator
Development of fluorescent adjacent hybridization probes and their application in real-time PCR for the simultaneous detection and identification of Fervidobacterium and Caloramator
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DOI：
10.1099/00207713-52-5-1837
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发表时间：
2002-09-01
影响因子：
2.8
通讯作者：
Patel, BKC
中科院分区：
文献类型：
--
作者：
Connolly, GR;Patel, BKC
细菌域成员的 165 rDNA 中由保守区和相邻上游可变区组成的一段核苷酸被确定为开发实时 PCR 相邻杂交测定的合适靶位点。设计并合成了针对保守区的单一通用荧光花青素 5 标记探针 CY5 1046+ 和针对可变区的两种 FITC 标记探针 Calo 和 Fervi，用于鉴定和区分嗜热厌氧菌 Caloramator 和 Fervidobacter。在 PCR 扩增过程中，探针 CY5 1046+ 和 Fervi 与 Fervidobacter 物种的 165 个 rDNA 靶位点同时杂交，导致荧光发射增加，并在 LightCycler 中连续监测。由于探针和靶序列的核苷酸碱基的组成变化而导致杂交探针解离温度 (T-m) 的差异，使得岛状发酵杆菌 DSM 5733(T) 能够与冈瓦纳发酵杆菌 ACM 5017(T) 和结节发酵杆菌 ATCC 35602(T) 区分开来。在 PCR 扩增过程中，探针 CY5 1046+ 和 Calo 与 Caloramator indicus ACM 3982(T) 的 16S rDNA 靶序列同时杂交也导致荧光发射增加。然而，由于 C. indicus ACM 3982(T) 的目标序列与其他三个 Caloramator 物种 Caloramator fervidus ATCC 43204(T)（以前称为 Clostridium fervidus）、Caloramator proteoclasticus DSM 10124(T) 和 Caloramator Coolhaasii DSM 12679(T) 的目标序列相同，因此需要在Tm 是不可能的，这使得 Calo 探针成为有用的属特异性探针。所设计的方法随后用于检测和鉴定来自我们之前未表征的培养物保藏和富集培养物中的 Fervidobacter 和 Caloramator 分离株。该测定方法便宜且灵活，因为只需要合成一组廉价的 FITC 探针来区分细菌域的其他成员，并且除了纯化的 DNA 之外，还可以使用通过简单的裂解煮沸方法制备的 DNA 模板。
A stretch of nucleotides consisting of a conserved region and an adjacent upstream variable region in the 165 rDNA of members of domain Bacteria was identified as a suitable target site for developing a real-time PCR adjacent hybridization assay. A single universal fluorogenic cyanin 5-labelled probe, CY5 1046+, targeting the conserved region, and two FITC-labelled probes, Calo and Fervi, targeting the variable region were designed and synthesized for the identification and differentiation of the thermophilic anaerobes Caloramator and Fervidobacterium. The simultaneous hybridization of probes CY5 1046+ and Fervi to the 165 rDNA target sites of Fervidobacterium species during PCR amplification resulted in an increase in fluorescence emission that was monitored continuously in a LightCycler. The differences in the temperature of disassociation of the hybridization probes (T-m) due to compositional variation in the nucleotide bases of the probe and target sequences enabled Fervidobacterium islandicum DSM 5733(T) to be differentiated from Fervidobacterium gondwanense ACM 5017(T) and Fervidobacterium nodosum ATCC 35602(T). The simultaneous hybridization of probes CY5 1046+ and Calo to the 16S rDNA target sequence of Caloramator indicus ACM 3982(T) during PCR amplification also resulted in an increase in fluorescence emission. However, as the target sequence of C. indicus ACM 3982(T) is identical to those of the other three Caloramator species, Caloramator fervidus ATCC 43204(T) (formerly Clostridium fervidus), Caloramator proteoclasticus DSM 10124(T) and Caloramator coolhaasii DSM 12679(T), further species discrimination on the basis of Tm will not be possible, making probe Calo a useful genus-specific probe. The devised method was subsequently used to detect and identify Fervidobacterium and Caloramator isolates from our previously uncharacterized culture collection and from enrichment cultures. The assay is cheap and flexible, as only a battery of inexpensive FITC probes for differentiating other members of domain Bacteria needs to be synthesized and DNA templates prepared by a simple lyse-boil method, in addition to purified DNA, can also be used.