Real-time PCR-based rapid and culture-independent detection of Salmonella in dairy milk--addressing some core issues.
In this study, methodologies were developed for cost-effective, rapid and user-friendly culture-independent detection of Salmonella in milk by real-time PCR. The SYBR Green-based real-time PCR assay was standardized with primers targeting the Salmonella enterotoxin gene (stn) that have been earlier used for its detection by conventional PCR. Inclusivity tests generated the specific amplifications with a Tm corresponding to 81 ± 0·5°C. The specificity of the reaction was evaluated with a panel of 36 non-Salmonella strains. Standard curves generated, with different number of cells of this organism in milk, depicted the detection of five cells with a CT value of 37·17 (SD 0·43). To make the assays user-friendly and suitable for field applications, protocols were also established for the immobilization of the SYBR Green reaction mixes in the reaction tubes. The immobilized master mixes were stable at 25°C for 4 months and at 8°C for over 6 months. Total DNA was prepared from 150 samples of full-fat dairy milk and subjected to real-time PCR detection wherein 31 samples tested positive for Salmonella. The time of analysis was <5 h.
Microbial Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Jammu Tawi, India. srhassan@iiim.ac.in
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Riyaz-Ul-Hassan
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Verma
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Qazi
D000818:Animals; D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D004768:Enterotoxins / Q000235:genetics; D008892:Milk / Q000382:microbiology*; D009930:Organic Chemicals; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D004768:Enterotoxins; D009930:Organic Chemicals; D011804:Quinolines; C082864:enterotoxin, Salmonella; C098022:SYBR Green I