Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosain sputum of cystic fibrosis patients

Table 1 Comparison of the sensitivity of different DNA-extraction protocols as assessed by means of conventional PCR combined with agarose gel electrophoresis and by real-time PCR on LightCycler using TaqMan probe

Molecular detection
Extraction	Protocol	Pretreatment	Last positive dilution
			PCR^a	Real-time^b
easyMAG	Generic 2.0.1	Proteinase K	6	8
easyMAG	Generic 2.0.1	None	5	7
easyMAG	Specific B	Proteinase K	5	7
easyMAG	Specific B	None	5	7
High Pure	Manual	Proteinase K	5	6
Detection by culture
McConkey Agar (MCA)			8^c
Cetrimide Agar (CA)			8^c
Cetrimide Broth with subculture on Blood Agar (CB)			8

^a Conventional PCR with primers PAO1 S and PAO1 A using the Veriti 96-Well Thermal Cycler.
^b Real-time PCR with primers PAO1 S and PAO1 A and TaqMan probe oprL TM using the LightCycler 1.5.
^c The initial inoculum was calculated by averaging the number of cfu at dilution 8 on MC and CA, i.e. 2.5 cfu/50 μl, multiplying with 20 to obtain the cfu/ml, i.e. 50 cfu/ml, multiplying with the dilution factor 1/3125000 to obtain the initial inoculum after dilution with Sputasol, i.e. 78 125 000 cfu/ml, and finally multiplying with factor 2 to obtain the original number of cfu/ml of sputum, i.e. 156 250 000 cfu/ml, or approx. 1.6 log8 cfu/ml.

ISSN: 1471-2180