Simultaneous detection of listeria monocytogenes in chicken meat enrichments by PCR and reverse-transcription PCR without DNA/RNA isolation.
Environmental and food samples can be analyzed using PCR and reverse transcription (RT)-PCR techniques to discriminate between viable and nonviable cells of bacterial pathogens. Here, we describe the use of a commercial lysis buffer, initially designed for mammalian cells, that permits the rapid extraction of bacterial DNA and RNA. The buffer is an RT-PCR-compatible lysis solution in which RNA is stable and can be frozen for later use. RT-PCR is carried out directly after DNase I treatment of crude bacterial lysates using rTth polymerase for RT-PCR in a single tube. Untreated lysate is used for standard PCR. The procedure permits the amplification of either mRNA or DNA of Listeria monocytogenes at a level similar to that obtained with purified nucleic acids. Using lysates obtained with this buffer, nested PCR and RT-PCR assays detected low numbers of L. monocytogenes cells from artificially contaminated chicken meat samples. The simplicity of this system may foster the development of similar buffers specifically designed for bacteria to improve RNA detection methods that can be performed in parallel with DNA analysis. The use of a single buffer decreases the time needed for analysis, is amenable to automation and real-time assays, and might be adaptable to all bacteria and amplification methods.
Navas
Jaime
J
Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Carretera de La Coruña, Madrid, Spain.
Ortiz
Sagrario
S
Martínez-Suárez
Joaquin V
JV
D000818:Animals; D002645:Chickens; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000032:analysis; D005506:Food Contamination / Q000032:analysis*; D006801:Humans; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D008460:Meat / Q000382:microbiology*; D008461:Meat Products / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D012329:RNA, Bacterial