近日，國際著名雜志BMC Microbiology在線刊登了斯坦福大學(xué)和加利福尼亞大學(xué)研究者的共同研究成果“Molecular probe technology detects bacteria without culture”,，文章中,，研究者開發(fā)出了一種不用通過培養(yǎng)基培養(yǎng)而可以直接檢測出細(xì)菌的新的探針技術(shù)。
人類的微生物組計劃自實施開始就使用了宏基因組的方法在不同的地方以及人類機(jī)體中識別各種細(xì)菌,，因為很多種細(xì)菌是不能夠在培養(yǎng)基被培養(yǎng)出來得到鑒定的,，第二代DNA測序技術(shù)并不能夠滿足我們的目的，至少不能夠在個體調(diào)查和臨床診斷上帶來一定幫助,，這就為今后的生物信息學(xué)技術(shù)帶來了更大的挑戰(zhàn),，因此未來最大的挑戰(zhàn)就是在混合物中如何準(zhǔn)確檢測出每一種細(xì)菌的具體信息，而目前我們只是掌握了細(xì)菌的部分基因組序列信息,。
研究者在以前的研究中使用了一種分子倒位探針（molecular inversion probes,，MIP）通過大量的多重分子技術(shù)操作來檢測并識別細(xì)菌，而且這種MIP技術(shù)也用于發(fā)現(xiàn)和測定人類DNA中的單核苷酸多態(tài)性,，人類的基因組是二倍體,，而細(xì)菌的是單倍體，因此在分子技術(shù)探針的研究中我們便可以稍微簡化程序來進(jìn)行細(xì)菌的檢測,。
本文中研究者使用了Affymetrix GenFlex Tag16K來對每一個臨床樣品中的細(xì)菌進(jìn)行多通路檢測,，為了在臨床樣品的檢測中達(dá)到多樣性，研究者引入了一種分子探針來進(jìn)行獨(dú)立的檢測試驗,，這種探針是基于寡聚核苷酸連接分析和檢測技術(shù)進(jìn)行測序的（SOLiD）,，通過在每一個臨床樣品中加入特異的寡核苷酸標(biāo)記條碼，然后適當(dāng)處理后,，研究者將樣品和標(biāo)記條碼結(jié)合,，隨后進(jìn)行測序。研究者是首次使用分子探針技術(shù)來檢測臨床樣品中的細(xì)菌,，借助Tag4陣列,，作者引入了SOLiD測序技術(shù)來進(jìn)行更精確的檢測，這種新的檢測技術(shù)允許被處理過的樣品重新被結(jié)合以進(jìn)行多樣性的測序檢測,，達(dá)到更精確的結(jié)果,。（生物谷：T.Shen編譯）
doi:10.1186/1471-2180-12-29
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Molecular probe technology detects bacteria without culture
Richard W Hyman3,1,7*, Robert P St Onge3,1, Hyunsung Kim4, John S Tamaresis5, Molly Miranda3, Ana M Aparicio3, Marilyn Fukushima3,1, Nader Pourmand4, Linda C Giudice6 and Ronald W Davis3,1,2
Background Our ultimate goal is to detect the entire human microbiome, in health and in disease, in a single reaction tube, and employing only commercially available reagents. To that end, we adapted molecular inversion probes to detect bacteria using solely a massively multiplex molecular technology. This molecular probe technology does not require growth of the bacteria in culture. Rather, the molecular probe technology requires only a sequence of forty sequential bases unique to the genome of the bacterium of interest. In this communication, we report the first results of employing our molecular probes to detect bacteria in clinical samples.
Results While the assay on Affymetrix GenFlex Tag16K arrays allows the multiplexing of the detection of the bacteria in each clinical sample, one Affymetrix GenFlex Tag16K array must be used for each clinical sample. To multiplex the clinical samples, we introduce a second, independent assay for the molecular probes employing Sequencing by Oligonucleotide Ligation and Detection. By adding one unique oligonucleotide barcode for each clinical sample, we combine the samples after processing, but before sequencing, and sequence them together.
Conclusions Overall, we have employed 192 molecular probes representing 40 bacteria to detect the bacteria in twenty-one vaginal swabs as assessed by the Affymetrix GenFlex Tag16K assay and fourteen of those by the Sequencing by Oligonucleotide Ligation and Detection assay. The correlations among the assays were excellent.
