Rapid Molecular Detection Of Salmonella From Produce Using Real Time Pcr
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Rapid Molecular Detection of Salmonella from Produce Using Real-time PCR
Recent outbreaks of Salmonella linked to fresh produce emphasize the need for rapid and sensitive assays to help control outbreaks. Reverse-transcriptase PCR (RT-PCR) detects the presence of mRNA (shorter half-life than DNA), with greater potential of detecting viable cells. Rapid real-time methods using fluorescent dyes and probes simultaneously detect and confirm the presence of target nucleic acid, eliminating the need for gel electrophoresis. The objective of this research was to rapidly detect Salmonella Typhimurium from spiked lettuce, tomatoes, and peppers using real-time RT-PCR. Washed and ultraviolet light treated lettuce (~25gram), tomato (~100g), and peppers (~130g) samples were inoculated with high (108 to 106 CFU) and low (103 to 101CFU) Salmonella Typhimurium overnight cultures. Samples were then rinsed or hand massaged with 225 ml 0.05 M glycine-saline buffer containing 0.05% Tween and 3% beef extract. Un-inoculated washed produce and sterile buffer were used as negative controls; with S. Typhimurium as a positive control. RNA was extracted from each sample using the Qiagen RNeasy Mini Kit. RT-PCR was carried out using a SYBR Green I RT-PCR kit with previously described Salmonella specific invA gene primers and an internal amplification control (IAC) to eliminate false negatives. Reaction conditions were RT at 50 degree Celsius/40min; PCR at 95 degree Celsius /45s, 58 degree Celsius /45s, 72 degree Celsius /45s for 40 cycles followed by melt temperature(Tm) analysis in a BioRad iCycler to determine specific invA product (~Tm=87.5 degree Celsius) and IAC (Tm=82 degree Celsius). To improve detection sensitivity of low inocula, spiked lettuce, tomatoes, and peppers were pre-enriched in buffered peptone water for 6 hours at 37 degree Celsius, followed by RNA extraction and RT-PCR detection. Each experiment was repeated twice. Real-time RT-PCR after 6-h pre-enrichment, Qiagen RNA extraction and the SYBR Green I kit gave Salmonella detection up to 103 CFU/25g from lettuce, 104 CFU/~130g from tomatoes, and 104 CFU/25g from peppers. Without enrichment, detection limits were 106 CFU/25g for lettuce, 107 CFU/25g for tomatoes, and 107 CFU/25g for peppers. Sensitive and rapid detection of Salmonella from spiked lettuce, tomatoes, and peppers could still be obtained within one day (~2 working shifts).
Detection of Non-Amplified Genomic DNA
Author: Giuseppe Spoto
language: en
Publisher: Springer Science & Business Media
Release Date: 2012-07-06
This book offers an overview of state-of-the-art in non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specific detection tools. Advanced materials play multiple roles in ultrasensitive detection. Optical and electrochemical detection tools are among the most widely investigated to analyze non amplified nucleic acids. Biosensors based on piezoelectric crystal have been also used to detect unamplified genomic DNA. The main scientific topics related to DNA diagnostics are discussed by an outstanding set of authors with proven experience in this field.
Molecular Detection of Human Bacterial Pathogens
As more original molecular protocols and subsequent modifications are described in the literature, it has become difficult for those not directly involved in the development of these protocols to know which are most appropriate to adopt for accurate identification of bacterial pathogens. Molecular Detection of Human Bacterial Pathogens addresses this issue, with international scientists in respective bacterial pathogen research and diagnosis providing expert summaries on current diagnostic approaches for major human bacterial pathogens. Each chapter consists of a brief review on the classification, epidemiology, clinical features, and diagnosis of an important pathogenic bacterial genus, an outline of clinical sample collection and preparation procedures, a selection of representative stepwise molecular protocols, and a discussion on further research requirements relating to improved diagnosis. This book represents a reliable and convenient reference on molecular detection and identification of major human bacterial pathogens; an indispensable tool for upcoming and experienced medical, veterinary, and industrial laboratory scientists engaged in bacterial characterization; and an essential textbook for undergraduate and graduate students in microbiology.