Detection of low-copy-number genomic DNA sequences in individual bacterial cells by using peptide nucleic acid-assisted rolling-circle amplification and fluorescence in situ hybridization

Abstract

An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes.

FIG. 1.

Major steps of a DNA-based assay for fluorescence in situ detection of short DNA sequences in a single bacterial cell. dNTPs, deoxynucleoside triphosphates.

FIG. 2.

Images of bacterial cells observed by using a fluorescence microscope in experiments performed according to the scheme presented in Fig. 1. The fluorescence signals were acquired separately using three filter sets (DAPI for DNA and Cy3 or fluorescein for the labeled RCA product). Each image is a superposition of two separate images, with DAPI and Cy3 or DAPI and fluorescein signals pseudocolored in blue and red or blue and green, respectively. (A) E. coli cells to which the probes corresponding to the 21-nt target site in the E. coli cold shock protein gene (csp) region, PNA1, PNA2, ODNcspG, and decR, were applied (Table 1). Virtually all cells displayed very bright spots. No such spots were observed in numerous negative control experiments in which any of the steps of the protocol given in Fig. 1 were omitted (see the supplemental material). (B) The same procedure as that described in the legend to panel A was carried out with a combination of all probes specific to E. coli (PNA1, PNA2, PNA3, PNA4, ODNcspG, ODNrpoN, ODNrnr, and decR) (Table 1) applied to B. subtilis cells. No signal was detected. (C) A combination of probes specific to B. subtilis (PNA4, PNA5, PNA6, PNA7, ODNserA, ODNyxjA, and decG) was applied to E. coli cells. No signal was detected. (D) B. subtilis cells to which the probes corresponding to the 23-nt target site in the B. subtilis phosphoglycerate dehydrogenase gene (serA) region (PNA5, PNA6, ODNserA, and decG) were applied.

FIG. 3.

(A) Images of bacterial cells observed by using a fluorescence microscope in experiments performed with a mixture of E. coli and B. subtilis cells to which a combination of probes specific to E. coli and B. subtilis, PNA3, PNA4, PNA7, ODNrnr, ODNyxjA, decR, and decG, were applied. E. coli and B. subtilis bacteria can be distinguished from each other in the mixture of both types of cells. (B) S. mutans cells to which the probes corresponding to the 22-nt target site in the S. mutans wall-associated protein gene (wapA), PNA8, PNA9, ODNwapA, and decG, were applied. Virtually all cells are colored green. No such spots were observed in various negative control experiments in which any of the steps were omitted (data not shown). The insert shows an enlarged image of several S. mutans cells. (C) Images of bacterial cells observed by using a fluorescence microscope in experiments performed with E. coli and S. mutans cells with the combination of probes specific to E. coli (rpoN and rnr) and S. mutans (hypP): PNA3, PNA4, PNA6, ODNrpoN, ODNrnr, ODNhypP, decR, and decG. The fluorescent signals were acquired separately using three filter sets with DAPI, Cy3, and fluorescein.