Dataset Information

Ultrasensitive Detection of Bacteria by Targeting Abundant Transcripts.

ABSTRACT: Molecular detection assays are increasingly becoming routine diagnostic techniques for bacterial infection; however, their sensitivities are restricted by the low concentrations of bacteria in clinical samples. Here, we report a new paradigm for ultrasensitive detection of bacteria. The principle of this approach is that by choosing highly transcribed genes as signature sequences and detecting both DNA and its RNA transcripts, assay sensitivity can be greatly improved. First, signature genes with abundant transcripts were screened by RNA-Seq. We confirmed that RT-PCR efficiently amplifies both DNA and RNA, while PCR amplifies only DNA. Unexpectedly, we found that the RNA extraction efficiency is relatively low, while simplified denaturation was more appropriate for transcript detection. For highly transcribed genes, RT-PCR consistently generated lower cycle threshold (Ct) values than those of PCR. The sensitivity of RT-PCR targeting abundant transcripts could detect quantities as low as one bacterium, which was not possible using PCR. Amplification of different genes among several other common bacteria also confirmed that transcript detection by RT-PCR is more sensitive than is DNA detection by PCR. Therefore, abundant transcript detection represents a universal strategy for ultrasensitive detection of bacteria.

SUBMITTER: Wang X

PROVIDER: S-EPMC4742959 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Ultrasensitive Detection of Bacteria by Targeting Abundant Transcripts.

Wang Xinhui X Li Xinran X Liu Shiwei S Ren Hang H Yang Mingjuan M Ke Yuehua Y Huang Liuyu L Liu Chao C Liu Bo B Chen Zeliang Z

Scientific reports 20160205

Molecular detection assays are increasingly becoming routine diagnostic techniques for bacterial infection; however, their sensitivities are restricted by the low concentrations of bacteria in clinical samples. Here, we report a new paradigm for ultrasensitive detection of bacteria. The principle of this approach is that by choosing highly transcribed genes as signature sequences and detecting both DNA and its RNA transcripts, assay sensitivity can be greatly improved. First, signature genes wit ...[more]

PMID: 26848029

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Project description:Foodborne pathogens, especially bacteria, are explicitly threatening public health worldwide. Biosensors represent advances in rapid diagnosis with high sensitivity and selectivity. However, multiplexed analysis and minimal pretreatment are still challenging. We fabricate a gold nanoparticle (Au NP)-amplified microcantilever array biosensor that is capable of determining ultralow concentrations of foodborne bacteria including Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, Listeria monocytogenes, Shigella, etc. The method is much faster than using conventional tools without germiculturing and PCR amplification. The six pairs of ssDNA probes (ssDNA1 + ssDNA2 partially complementary to the target gene) that originated from the sequence analysis of the specific gene of the bacteria were developed and validated. The ssDNA1 probes were modified with -S-(CH2)6 at the 5'-end and ready to immobilize on the self-assembled monolayers (SAMs) of the sensing cantilevers in the array and couple with Au NPs, while 6-mercapto-1-hexanol SAM modification was carried out on the reference cantilevers to eliminate the interferences by detecting the deflection from the environment induced by non-specific interactions. For multianalyte sensing, the target gene sequence was captured by the ssDNA2-Au NPs in the solution, and then the Au NPs-ssDNA2-target complex was hybridized with ssNDA1 fixed on the beam of the cantilever sensor, which results in a secondary cascade amplification effect. Integrated with the enrichment of the Au NP platform and the microcantilever array sensor detection, multiple bacteria could be rapidly and accurately determined as low as 1-9 cells/mL, and the working ranges were three to four orders of magnitude. There was virtually no cross-reaction among the various probes with different species. As described herein, it holds great potential for rapid, multiplexed, and ultrasensitive detection in food, environment, clinical, and communal samples.

Dataset Information

Ultrasensitive Detection of Bacteria by Targeting Abundant Transcripts.

Publications

Ultrasensitive Detection of Bacteria by Targeting Abundant Transcripts.

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