Factors affecting the performance of 5' nuclease PCR assays for Listeria monocytogenes detection.
J Microbiol Methods
; 51(3): 361-8, 2002 Nov.
Article
in En
| MEDLINE
| ID: mdl-12223296
ABSTRACT
The design and operating parameters affecting the performance of 5' nuclease PCR (TaqMan) assays for the detection of Listeria monocytogenes was investigated. A system previously developed and based on the hlyA gene was used as a model [Appl. Environ. Microbiol. 61 (1995) 3724]. A series of fluorogenic probes labeled with a reporter and a quencher dye was synthesized to explore the effect of probe position and sequence content on the efficiency of probe hydrolysis. In addition, a series of PCR primer pairs that altered the distance between the upstream primer and the interceding probe was examined. The effects of various assay parameters were evaluated by measuring the ratio of the fluorescence intensity of the reporter dye over the quencher dye (deltaRQ). For a given probe sequence, the deltaRQ was typically lower if the 5' terminus was a G residue. Decreasing the probe concentration increased the deltaRQ, although this was at the expense of reproducibility in the assay readout. The distance between the upstream primer and the interceding probe has a significant effect on probe hydrolysis. Reducing the primer-probe distance from, for example, 127 to 4 nt increased the deltaRQ from 2.87 to 5.00. These general rules were used to develop a 5' nuclease PCR (TaqMan) assay with enhanced signal output, providing higher and more reproducible deltaRQ values for L. monocytogenes detection.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Bacterial Toxins
/
Polymerase Chain Reaction
/
Deoxyribonucleases
/
Listeria monocytogenes
Type of study:
Diagnostic_studies
/
Evaluation_studies
/
Prognostic_studies
Language:
En
Journal:
J Microbiol Methods
Year:
2002
Document type:
Article
Affiliation country:
Brasil