Method For Geno-And Pathotyping Pseudomonas Aeruginosa

Abstract

The invention relates to a method for genotyping and pathotyping bacteria of the species Pseudomonas aeruginosa by means of hybridization assays on a biochip or microarray. The invention further relates to specific oligonucleotide probes, which can be employed within the scope of the detection method, as well as to biochips having such oligonucleotide probes.

Description

EXAMPLE

Within the scope of the present invention, a detection method was developed, by means of which genotyping and pathotyping Pseudomonas aeruginosa can be performed within six hours, starting from the bacteria on an agar plate. To this end, only basic laboratory methods, like for example PCR, and devices belonging to the basic equipment of a molecular-biological laboratory are required. A critical step herein is the PCR, in which more than 40 different sequences are amplified in parallel in the same reaction setup. In order to achieve this, in one embodiment of the method according to the present invention, 80 DNA primers have been optimized in such a way that they have about the same melting points and binding kinetics. Furthermore, the template nucleic acids were only amplified linearly, i.e. on one DNA strand, thus also minimizing the effects of minor kinetic differences. Said optimization allows the use of a multiplex PCR for target amplification.

With the DNA chip provided within the scope of the present invention it is thus possible to examine Pseudomonas aeruginosa quickly and easily in a routine diagnostic laboratory within one day and thus to be able to react quickly, for example, in case nosocomial propagation of said pathogen is suspected.

An experimental protocol is given in the following:

• • a) Preparation of the bacteria
    • taking up 2 inoculating loops of the bacterial culture (20 μl bacteria from an LB agar plate) in 1.5 ml H₂O
    • centrifuging (3,000×g, 6 min)
    • removing supernatant
    • washing pellet 4 times
    • resuspending in 5 mM EDTA
    • centrifuging (14,000×g, 5 min)
    • removing supernatant
    • resuspending pellet in 50 μl distilled H₂O

b) Polymerase chain reaction (PCR)

The bacterial DNA sequences to be examined are amplified using polymerase chain reaction (PCR).

Polymerase:	Terminator polymerase (New England Biolabs)
dNTPs:	2 mM dATP, dGTP, dCTP each
	1.5 mM dTTP
	0.5 mM biotin-dUTP (Roche)
Primers:	Mixture of two 21 bp oligonucleotides each per
	sequence to be detected. The primers have the same
	melting points and binding kinetics and bind on the
	same strand, about 100 bases upstream of the
	examined DNA sequence. The mixture used has a
	total concentration of oligonucleotides of 5 μmol/l.
	The sequences of the primers used are depicted
	in FIG. 17.
Reaction setup:	10 x reaction buffer	2.5	μl
	dNTP mixture	2.5	μl
	Primers	2.5	μl
	DMSO	1.2	μl
	Bacteria suspension	8.0	μl
	Terminator polymerase	0.5	μl
	Water	7.8	μl
		= 25	μl
Reaction procedure:	Start	96° C.	300 s
	40 cycles	60° C.	20 s
		72° C.	40 s
		96° C.	60 s
	End	10° C.

c) Hybridization assay

The oligonucleotide probes employed and the layout of the oligonucleotide probes on the nucleic acid chip according to the present invention are shown in FIGS. 18 to 21.

The chips are washed twice for 5 minutes with 500 μl of the hybridization buffer (6×SSPE/0.1% SDS/2% w/v Blocking Reagent (Roche)) in a thermomixer (30° C., 550 rpm).

20 μl of the PCR product are denatured together with 80 μl hybridization buffer in a heating block (96° C., 5 min) and cooled down on ice.

Said probe solution is applied onto the ArrayTube® chip (Clondiag) and incubated for one hour at 60° C. and 550 rpm (Thermomixer).

The probe solution is discarded and the DNA chip is washed:

• • 500 μl 2×SSC/0.01% Triton X-100 for 10 min at 30° C. and 550 rpm
  • 500 μl 2×SSC for 10 min at 20° C. and 550 rpm
  • 500 μl 0.2×SSC for 10 min at 20° C. and 550 rpm

The ArrayTube® chip is incubated with 100 μl of an horseradish streptavidin conjugate (1:100 dilution) for 15 min (30° C., 550 rpm) and subsequently washed:

• • 500 μl 2×SSC/0.01% Triton X-100 for 10 min at 30° C. and 550 rpm
  • 500 μl 2×SSC for 10 min at 20° C. and 550 rpm
  • 500 μl 0.2×SSC for 10 min at 20° C. and 550 rpm

For detection, 100 μl of a tetramethylbenzidine derivative (Medac, Wedel, Germany) are applied onto the chip and the result is evaluated by means of an AT reader (Clondiag) and the program IconoClust (Clondiag). The results for various strains of Pseudomonas aeruginosa are depicted in FIGS. 1 to 15.

d) Solutions

	10 x SSPE buffer	1.5 M	NaCl
		0.1 M	sodium phosphate
		0.01M	EDTA
		pH	7.4
	20 x SSC buffer	3.0 M	NaCl
		0.3 M	sodium citrate
		pH	7.0

FIGURES

FIGS. 1 to 15 show hybridized DNA chips, which were hybridized with different P. aeruginosa strains. Processing of the strains was performed according to the protocol described in the above.

FIG. 16 shows a laboratory reaction tube of typical shape and size.

FIG. 17 shows the nucleotide sequences of the primers used in the Example.

Oligonucleotide probes according to the present invention as well as the layout of the oligonucleotide probes on the nucleic acid chip according to the present invention are shown in the FIGS. 18 to 21.

Claims

1. Oligonucleotide for genotyping and pathotyping the species Pseudomonas aeruginosa with a nucleic acid sequence, selected from the group consisting of (all sequences in 5′→3′ direction):

2. Microarray device comprising a support element, on which oligonucleotide probes are immobilized on predetermined regions, for specifically detecting bacterial strains of the species Pseudomonas aeruginosa.

3. Device according to claim 2, characterized in that the device is a reaction tube having a shape and/or size typical for a laboratory reaction tube and having a support element, on which oligonucleotide probes are immobilized on predetermined regions, arranged on one of its base areas for specifically detecting bacterial strains of the species Pseudomonas aeruginosa.

4. Device according to claim 2, characterized in that the oligonucleotide probes are selected in such a way that they detect 30% to 70% of the population of Pseudomonas aeruginosa strains in each case.

5. Device according to claim 2, characterized in that the oligonucleotide probes are specific for nucleic acids having a base substitution compared to the sequence of the reference strain of Pseudomonas aeruginosa.

6. Device according to claim 2, characterized in that the oligonucleotide probes are specific for nucleic acids present in only one or few strains of the species Pseudomonas aeruginosa.

7. Device according to claim 2, characterized in that the oligonucleotide probes are specific for nucleic acids present in pathogenicity islets in the genome of Pseudomonas aeruginosa.

8. Device according to claim 2, characterized in that the oligonucleotide probes are specific for nucleic acids present in disease-associated genes like exoS and exoU.

9. Device according to claim 2, characterized in that the oligonucleotide probes are specific for nucleic acids contained in genes coding for flagella of Pseudomonas aeruginosa.

10. Device according to claim 2, characterized in that the oligonucleotide probes are selected from the oligonucleotides according to claim 1.

11. Method for specifically detecting bacterial strains of the species Pseudomonas aeruginosa in a sample, comprising the following steps: a) contacting the sample with a nucleic acid chip in a microarray device according to claim 2; andb) detecting the interaction between the oligonucleotide probes and the target nucleic acids contained in the sample.

12. Method according to claim 11, characterized in that the target nucleic acids contained in the sample are amplified before the detection.

13. Method according to claim 12, characterized in that the amplification is performed by means of multiplex PCR.

14. Method according to claim 13, characterized in that primers, which have similar melting points and/or similar binding kinetics, are used for the amplification.

15. Method according to claim 12, characterized in that the amplification is performed linearly.

16. Method according to claim 12, characterized in that the primers are selected with a nucleic acid sequence selected from the group consisting of (all sequences in 5′→3′ direction):

17. Use of the oligonucleotides according to claim 1 for specifically detecting bacterial strains of the species Pseudomonas aeruginosa.

18. A method for genotyping and pathotyping Pseudomonas aeruginosa, comprising the following steps: a) contacting the sample with a nucleic acid chip in a microarray device according to claim 2; andb) detecting the interaction between the oligonucleotide probes and the target nucleic acids contained in the sample.

19. A method for amplifying nucleic acids of bacterial strains of the species Pseudomonas aeruginosa, comprising the following steps: a) contacting the sample with a nucleic acid chip in a microarray device according to claim 2; andb) detecting the interaction between the oligonucleotide probes and the target nucleic acids contained in the sample.