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Development and Application of Fluorescence PCR Methods for Rapid Detection of Salmonella
Author: ShiXiaoLu
Tutor: ShiXianMing;ZuoQingHua
School: Huazhong Agricultural University
Course: Microbiology
Keywords: Salmonella Fluorescent PCR Detect Molecular beacons Taqman-MGB
CLC: TS207.4
Type: Master's thesis
Year: 2003
Downloads: 678
Quote: 9
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Abstract
On a global scale, Salmonella is the most common cause of bacterial food poisoning pathogens. Traditional Salmonella detection methods usually take 4-7 days, and the operation is cumbersome and difficult to cope with the rapid diagnosis of food poisoning and food enterprises and administrative departments of the real-time monitoring of food safety. This study Salmonella rapid detection method proposed requirements. The current Salmonella rapid detection method using an immunological method, the method of nucleic acid hybridization and polymerase chain reaction (Polymerase Chain Reaction, or PCR), wherein the PCR method is becoming more widely used for the detection of Salmonella. However, conventional PCR detection methods common PCR product contamination, specificity and slow (need-staining rubber) and other defects. Fluorescent PCR detection technology is a newly developed technology, its application will fundamentally solve these problems. In this study, using molecular beacons technology and TaqMan-MGB technology, two specific fluorescent PCR detection methods for Salmonella, and initially used in clinical diagnostics and food contamination investigation. The experimental results are as follows: 1. Salmonella PCR for the detection system to establish (1) study the enrichment broth fluorescence PCR reaction, SC (selenite cystine) enrichment broth for the best training Salmonella enrichment broth. The establishment of a fast and easy sample preparation method. (2) According to the genus Salmonella specific gene invA designed four pairs of primers, and Rahn designed primers simultaneous detection of a large number of Salmonella and non-Salmonella strains, the choice of which a pair of specific strongest new primer. (3) between this pair of primers to select period oligonucleotide sequence ends with FAM and MGB tag, to optimize the reaction conditions, the establishment of Salmonella TaqMan-MGB detection method. (4) Q in this pair of primers to select another period of the oligonucleotide sequence, each end coupled 6bp oligonucleotides complementary to the formation of molecular beacons, optimizing the reaction conditions, the establishment of the Salmonella molecular beacon detection method. 2. The Salmonella PCR for the detection system evaluation (1) specificity: both fluorescent PCR method to detect Salmonella on 200 strains were positive; detected 200 strains of Salmonella spp were negative. (2) Sensitivity: Salmonella typhimurium the experiment strain (40 cycles). DNA extract, the method detection limit of TaqMan-MGB 69fg/PCR system, the method of molecular beacons the detection limit 346fg/PCR system; For the broth, TaqMan-MGB methods and molecular beacon detection limit are 2cfu/PCR system; For food samples, the detection limit of the TaqMan-MGB method and the method of molecular beacons are 2cfu/25g samples. (3) Immunity: The reaction from the interference of the other bacteria, food samples after treatment has no effect upon the reaction. (4) Repeatability: consistency of the results of the parallel experimental high repeatability. 3. The Salmonella PCR for the detection system (1) to conduct random checks of raw meat, cooked Meat Market in Shenzhen, 200 raw meat samples were positive, fluorescent PCR method and the traditional method 32, the fluorescent PCR positive while traditional method negative 33 copies. (2) of food practitioners anal swabs survey, 300 samples using fluorescence PCR method and the traditional method are 3 parts the positive, in line with the rate of 100%. (3) the rapid diagnosis of bacterial food poisoning: 45 samples with fluorescent PCR method and the traditional method were detected in 28 Salmonella-positive in line with the rate of 100%. In this study, the first time in the country established the dust plastic leek fish Weiwei plastic giant carp patrol through Cong nine tetrahedrite indistinct some once sleepy color fluorescence CR detection methods, and should be thrown while the passers-diagnosis the plastic villa Fish the gully Zheng's work record, able and Egypt Cong speed accurately detect Salmonella purpose of establishing fluorescent PCR method as the existing used in clinical inspection and food hygiene monitoring, raise a strong complement to existing seized tight corner house standard detection method, the degree of
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