High-throughput Universal Probe Salmonella Serotyping (UPSS) by nanoPCR.
Salmonella enterica subsp. enterica serovar identification is of great importance with respect to outbreak monitoring and case verification. Therefore rapid, sensitive and cost efficient detection of Salmonella spp. is indispensable within microbiology labs. To amalgamate single tube isolate identification with Salmonella typing, we developed the high-throughput Universal Probe Salmonella Serotyping (UPSS) technique based on nano liter PCR. In comparison to the classical approach, where O- and H-antisera are applied, the UPSS relies on specific gene content amplification of Salmonella spp. by a universal TaqMan assay for all markers and identification of the specific amplicon pattern. To enable high-throughput technology we employed a chip format containing 1024 wells loaded by an automated liquid-handling system which allowed us to perform TaqMan PCR reactions in volumes of 100nL per well. Herein we present proof of principle of the UPSS method by the use of a test panel of 100 previously serotyped Salmonella isolates to successfully verify the usability, accuracy and feasibility of the newly developed UPSS approach. We found that the methodology of the UPSS technology is capable of unequivocally identifying 30 Salmonella serotypes on a single chip within 3 hours but can be highly parallelized by the use of multiple PCR machines. Therefore the UPSS method offers a robust and straightforward molecular alternative for Salmonella detection and typing that saves expensive chemistry and can be easily automated.
Mertes
Florian
F
Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany. mertes@molgen.mpg.de
Biens
Katja
K
Lehrach
Hans
H
Wagner
Martin
M
Dahl
Andreas
A
D000818:Animals; D015373:Bacterial Typing Techniques / Q000379:methods*; D005838:Genotype; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D017720:Molecular Epidemiology / Q000379:methods; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012480:Salmonella Infections / Q000382:microbiology; D012481:Salmonella Infections, Animal / Q000382:microbiology; D019779:Salmonella enterica / Q000145:classification* / Q000235:genetics*; D012703:Serotyping / Q000379:methods*; D013997:Time Factors
D015345:Oligonucleotide Probes