Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR
Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR
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DOI：
10.1373/clinchem.2006.069286
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发表时间：
2006-11-01
影响因子：
9.3
通讯作者：
Tseng, Ching-Ping
中科院分区：
文献类型：
--
作者：
Cheng, Ju-Chien;Huang, Chien-Ling;Tseng, Ching-Ping
背景：广谱聚合酶链式反应为检测细菌感染提供了有价值的信息。方法：采用LightCycler实时荧光聚合酶链式反应(LightCycler)实时定量聚合酶链式反应(LightCycler)扩增16S rRNA基因，用LightCycler实时定量聚合酶链式反应扩增16S rRNA基因。用HR-I仪对扩增产物进行高分辨率熔融分析，以熔融图谱为分子指纹进行细菌种属鉴定。结果：25种细菌的16S rRNA基因均可用该方法扩增，扩增产物长度分别为216或217bp。在25种细菌中，我们通过I步后聚合酶链式反应高分辨熔融分析鉴定了11种细菌。通过测试细菌和参考细菌的PCR产物之间的异源双链形成获得的高分辨率融化图谱或通过针对16S rRNA基因的不同区域的第二次实时PCR来鉴定剩余的细菌物种。建立了一个高分辨率的熔融数据库和一个工作规程来鉴定这25个细菌物种。在验证实验中，当细菌种在高分辨率熔融数据库中时，准确率达到94%。结论：该方法不需要多重或杂交探针，为分子诊断实验室的细菌种鉴定提供了一种新的方法。(C)2006年美国临床化学协会。
Background: Broad-range PCR provides valuable information for detecting bacterial infections. This study assesses the combined use of broad-range real-time PCR and high-resolution melting analysis for rapid detection and identification of clinically important bacteria.Methods: We subjected 46 bacterial culture colonies representing 25 clinically important bacterial species to LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye. We performed high-resolution melting analysis of the PCR products with the HR-I instrument and used melting profiles as molecular fingerprints for bacterial species identification. We validated this method via assessment of 54 consecutive bacteria culture colonies obtained from a clinical microbiology laboratory.Results: The 16S rRNA gene of all 25 bacterial species was amplifiable by this method, with PCR product lengths of 216 or 217 bp. Of the 25 bacterial species, we identified 11 via a I-step post-PCR high-resolution melting analysis. The remaining bacterial species were identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species or by a 2nd real-time PCR targeting a different region of the 16S rRNA gene. A high-resolution melting database and a working protocol were established for identifying these 25 bacterial species. In the validation assay, a 94% accuracy rate was achieved when the bacterial species were in the high-resolution melting database.Conclusions: This assay requires no multiplexing or hybridization probes and provides a new approach for bacterial species identification in a molecular diagnostic laboratory. (c) 2006 American Association for Clinical Chemistry.