Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

Abstract

Background: PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology.

Results: Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics.

Conclusions: The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria.

Figure 1

Schematic representation of LDR applied to microbial diversity. A) Each microbial group of interest is identified by a Common Probe and a Discriminating Oligo. The common probe is phosphorylated on its 5' end and contains a unique cZip Code affixed to its 3' end. The discriminating oligo carries a fluorescent label (Cy3) on its 5' end, and a discriminating base at its 3' terminal position. The two probes hybridize adjacently to each other on the template DNA (PCR-amplified rDNA) and the nick between the two oligos is sealed by the ligase only if there is perfect complementarity at the junction. The reaction can be thermally cycled B) The presence of a microbial group is determined by hybridizing the content of a LDR to an addressable DNA Universal Array, where unique Zip Code sequences have been spotted.

Figure 2

Examples of probe design Final alignment between group-specific consensi that shows selected probes for bacilli (A) and pseudomonads (B).

Figure 3

LDR detection on the Universal Microarray with defined templates. Each row of the array corresponds to a group and contains ten-spot replicates. Deposition scheme: 1, Zip 3 (bacilli,) 2, Zip 5 (myxobacteria); 3, Zip 11 (cyanobacteria); 4, Zip 13 (universal); 5, Zip 15 (pseudomonads); 6, Zip 21 (actinomycetes). Zip sequences are from Barany et al (6). Panel A shows the results obtained after hybridization to Cy5 poly(dT)10. Panel B through H show the results of hybridization after LDR on 16S rDNA templates from bacilli (panel B), myxobacteria (panel C), cyanobacteria (panel D), pseudomonads (panel E), actinomycetes (panel F), C. perfringens (panel G) and E. coli (panel H). All the images were acquired setting both the Laser power and PMT gain to 85%

Figure 4

Sensitivity of LDR The reaction was performed using 100 fmol (panel A), 10 fmol (panel B), 1 fmol (panel C) of purified PCR product from P. putida DNA. All images were acquired setting both PMT gain and laser power to 85%.

Figure 5

LDR in presence of multiple DNA templates A) LDR on a mix of PCR products B) LDR on a PCR product obtained from amplification of a mix of genomic DNAs. For deposition scheme, refer to fig. 3. (Laser power and PMT gain were set to 80%).

Figure 6

LDR on environmental DNA Panels A and B refer to environmental sample 23; panels C and D to sample No. 46. (provided by Dr. Ventura) The templates used for LDR derived from cyanobacteria-specific amplification of 16S rDNA (panels A and C) or from amplification with universal primers (panels B and D).