Real-time reverse-transcriptase polymerase chain reaction for the rapid detection of Salmonella using invA primers.
Recent outbreaks of Salmonella linked to fresh produce emphasize the need for rapid detection methods to help control the spread of disease. Reverse-transcriptase polymerase chain reaction (RT-PCR) can detect the presence of mRNA (shorter half-life than DNA) with greater potential for detecting viable pathogens. The chromosomally located invA gene required for host invasion by Salmonella is widely used for detection of this pathogen by PCR. Detection of Salmonella was undertaken by real-time RT-PCR (rt-RT-PCR) using newly designed invA gene primers to develop a sensitive and specific assay. Salmonella serovars Typhimurium and Enteritidis were grown (7.68 log(10) CFU/mL) in Luria-Bertani broth overnight at 37 degrees C, and RNA was extracted, followed by rt-RT-PCR with and without SYBR green I and agarose gel electrophoresis. All experiments were replicated at least thrice. Detection for both serovars using traditional RT-PCR was lower ( approximately 10(5) CFU/mL) than rt-RT-PCR (10(3) CFU/mL) by gel electrophoresis. Melt curve analysis showed melt temperatures at 87.5 degrees C with Ct values from 12 to 15 for up to 10(3) CFU/mL and improved to 10(2) CFU/mL after further optimization. Further, addition of RNA internal amplification control constructed using in vitro transcription with a T7 RNA polymerase promoter, to the RT-PCR assay also gave detection limits of 10(2) CFU/mL. Cross-reactivity was not observed against a panel of 21 non-Salmonella bacteria. Heat-inactivated (autoclaved) Salmonella showed faint or no detection by rt-RT-PCR or gel electrophoresis. This method has potential to be applied for the detection of Salmonella serovars in fresh produce and the simultaneous detection of foodborne viral (RNA viruses) and bacterial pathogens in a multiplex format.
D'Souza
Doris H
DH
Department of Food Science and Technology, The University of Tennessee, Knoxville, Tennessee, USA. ddsouza@utk.edu
Critzer
Faith J
FJ
Golden
David A
DA
D001419:Bacteria / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D005516:Food Microbiology; D005517:Foodborne Diseases / Q000517:prevention & control; D006358:Hot Temperature; D057230:Limit of Detection; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019779:Salmonella enterica / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D044366:Transition Temperature
D001426:Bacterial Proteins; D017931:DNA Primers; D012333:RNA, Messenger; C075518:invA protein, Bacteria