Rapid detection of Salmonella by polymerase chain reaction.
Salmonella enterotoxin gene (stn) was sequenced from Salmonella enterica serotypes: Typhimurium, Typhi, Paratyphi A and B. The sequences from all the four serotypes showed complete homology with the already reported stn gene sequence of the serotype Typhimurium. As a tool for detection of this organism, four pairs of oligonucleotide primers were designed to amplify different fragments of this important pathological marker. The protocols were standardized with serotype Typhimurium in such a way so as to complete the PCR reaction in 75-90 min. These primers were found to generate specific amplicons with all the serotypes of Salmonella tested. The PCR protocols were found to be highly specific as no amplifications, specific or non-specific, were found when reactions were run using non-Salmonella DNA as template. The employment of a nested PCR markedly increased the sensitivity of the assay system in natural water samples. The protocol described herein is highly sensitive as it detects less than 10 cells of Salmonella in 250 microl of blood and approx. 1 cell in 1 ml of water without any enrichment. For the validation of this protocol, 72 coded samples of 11% skimmed milk spiked with different pathogens were received from NICED, Kolkata and analyzed for the presence of Salmonella. Our procedures detected correctly the presence of Salmonella in nine samples. 50 samples of raw milk were subjected to this PCR after enrichment for 8 h and 6 samples were found positive for Salmonella. The study indicates that Salmonella enterotoxin (stn) gene is highly conserved and the protocol devised in this study can be used as rapid and reliable method for detection of Salmonella spp. in water, milk and blood samples.
Biotechnology Division, Regional Research Laboratory, Canal Road, Jammu Tawi 180001, India.
Syed
S
Riyaz-Ul-Hassan
Vijeshwar
V
Verma
Ghulam Nabi
GN
Qazi
D000818:Animals; D001769:Blood / Q000382:microbiology; D008892:Milk / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D012480:Salmonella Infections / Q000382:microbiology; D014871:Water Microbiology