Specific and sensitive detection of H. pylori in biological specimens by real-time RT-PCR and in situ hybridization.
Specific and sensitive detection of H. pylori in biological specimens by real-time RT-PCR and in situ hybridization.
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通过实时 RT-PCR 和原位杂交特异性、灵敏地检测生物样本中的幽门螺杆菌。
DOI：
10.1371/journal.pone.0002689
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发表时间：
2008-07-16
期刊：
影响因子：
3.7
通讯作者：
Dubois A
中科院分区：
文献类型：
--
作者：
Liu H;Rahman A;Semino-Mora C;Doi SQ;Dubois A
由于幽门螺杆菌基因的广泛多态性和许多菌株中某些基因的表达被抑制，使得在生物标本中进行幽门螺杆菌的PCR检测变得困难。本研究的目的是(1)确定幽门螺杆菌16S rRNA序列的一个高度保守的结构域，同时也是该物种所特有的；(2)建立和验证检测幽门螺杆菌的特异和敏感的分子方法。我们结合使用了硅胶和分子方法来实现对生物介质中幽门螺杆菌的灵敏和特异的检测。我们对来自不同大陆的两个分离株进行了测序，并证明了幽门螺杆菌16S rRNA序列的546个碱基域在这些菌株和已发表的序列中是保守的。在这个保守的序列中，我们定义了一个229个碱基的结构域，该结构域在GenBank中的大多数H.Pylori菌株中是100%同源的，也是H.Pylori特有的。这个亚区被用来设计(1)一组高质量的RT-PCR引物和探针，它包含一个76bp的序列，并包括至少两个与其他螺杆菌sp.的错配。16S rRNA；(2)原位杂交反义探针。然后，通过使用患者和恒河猴的胃活检标本来验证这些方法的敏感性和特异性。幽门螺杆菌16S rRNA序列的这个特异区域在大多数幽门螺杆菌菌株中高度保守，可以在生物标本中对这种细菌进行特异的检测、鉴定和定量。
PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.
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影响因子：
6.7
作者：
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通讯作者：
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GRAHAM, DY