Bacteriophage amplification

Madonna, A. J. Van Cuyk, S. Voorhees, K. J. Detection of Escherichia coli using immunomagnetic separation and bacteriophage amplification coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Comm. Mass Spectrom. 2003,17, 257-263. 

A REVIEW OF ANTIBODY CAPTURE AND BACTERIOPHAGE AMPLIFICATION IN CONNECTION WITH THE DIRECT ANALYSIS OF WHOLE-CELL BACTERIA BY MALDI-TOF MS... 

Figure 14.10 Spectra showing the selective bacteriophage amplification of Salmonella in the presence of Shigella.

Pierce CL, Rees JC, Fernandez FM, Barr JR. Viable Staphylococcus aureus quantitation using N-15 metabolically labeled bacteriophage amplification coupled with a multiple reaction monitoring proteomic workflow. Mol Cell Proteomics. 2012 11 M111.012849. doi 10.1074/mcp. Mlll.012849-5. 

Rees JC, Pierce CL, Schieltz DM, Barr JR. Simultaneous identification and susceptibility determination to multiple antibiotics oi Staphylococcus aureus by bacteriophage amplification detection combined with mass spectrometry. Anal Chem. 2015 87 6769-77. 

Figure 14.9 Detection of decreasing levels of E. coli with bacteriophage (MS-2) amplification.

A majority of the work done in our laboratory has been conducted on E. coli and B. anthracis using MS2 and gamma-phage, respectively. Amplification results have been obtained from several other host bacteria and bacteriophages. Table 14.1 summarizes the materials studied. In all cases studied, no two bacteriophages contained the same proteins. 

The principles that govern the delivery of recombinant DNA in clonable form to a host cell, and its subsequent amplification in the host, are well illustrated by considering three popular cloning vectors commonly used in experiments with E. coli—plasmids, bacteriophages, and bacterial artificial chromosomes—and a vector used to clone large DNA segments in yeast. 

Figure 6. Amplification of RNA molecules by assays that are sequence- insensitive. The first assay (upper part) combines the polymerase chain reaction (PCR) of DNA templates with reverse transcription and transcription. Commonly used enzymes are TAQ-polym-erase, HIV reverse transcriptase and bacteriophage T7 RNA polymerase. The assay requires a temperature program applying higher temperatures for double strand dissociation. The second assay (lower part) shows the self-sustained sequence replication reaction (3SR) which can be carried out isothermally because double strand dissociation is replaced by enzymatic digestion of the RNA strand in the RNA-DNA duplex. The enzymes used are HIV reverse transcriptase, RNase H and T7 RNA polymerase.

The polymerase chain reaction is the prevalent method for DNA amplification. Much effort has been made to integrate PCR chambers on microchips to carry out amplifications of DNA molecules prior to their analysis. For instance, PCR was first achieved on a Si-based reaction chamber (25 or 50 pL) integrated with a polysilicon thin-film (2500-A-thick) heater for the amplification of the GAG gene sequence (142 bp) of HIV (cloned in bacteriophage M13) [997]. 

Strain specific lysis by bacteriophages can be used to release intracellular ATP or adenylate kinase (AK). At the 13 ISBC a paper is presented on identification of bacteria using bacteriophages. There are also several papers on bioluminescent realtime detection of nucleic acid amplification, which may be used for identification of bacteria. In these assays either pyrophosphate or AMP is converted to ATP as a measure of the amplification reaction. 

Recombinant luciferases and stable liquid ATP standards have increased the reliability of ATP biomass assays. Detection limits of ATP and ATP+AMP assays have reached the level of a single cell and can, combined with filtration techniques, reach 1 cell/mL. The assay of ATP+AMP is the most reliable method for hygiene monitoring. With immunocapture, bacteriophages or nucleic acid amplification combined with sensitive ATP reagents bacteria can be identified. In the near future we expect to see an abundance of user-friendly systems at acceptable prices. 

Barnes, W.M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216-2220. 

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