2019
DOI: 10.1111/jam.14285
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Multiplex touchdown PCR assay to enhance specificity and sensitivity for concurrent detection of four foodborne pathogens in raw milk

Abstract: Aims The aim of this study was to develop a multiplex touchdown PCR (multiplex TD‐PCR) for rapid and simultaneous detection of four major foodborne pathogens to avoid mispriming and unwanted production during gene amplification. Touchdown PCR is the modified form of standard PCR, which enhances specificity, sensitivity. Methods and Results For this reason, a multiplex TD‐PCR assay with a pre‐enrichment step was developed to detect four foodborne pathogens namely Escherichia coli O157:H7, Listeria monocytogenes… Show more

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Cited by 21 publications
(7 citation statements)
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References 49 publications
(69 reference statements)
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“…Polymerase chain reaction (PCR) amplification was performed in a 50 µl reaction mixture containing 5 µl of 10× PCR buffer, 4 µl of MgCl 2 (25 mmol/L), 0.5 µl of each primer (10 µmol/L each), 30 ng of quantified template DNA measuring by Pico green, and 0.4 µl of Taq polymerase (5 U/µl; Fermentas). To increase specificity and sensitivity during gene amplification, the touchdown PCR was conducted in a thermocycler (Applied Biosystems Veriti Thermal Cycler, United States): denaturation at 94 • C for 5 min, 11 cycles of denaturation at 94 • C for 1 min, annealing at 65 • C for 1 min (temperature was decreased by 1 • C every cycle until 55 • C was reached), and extension at 72 • C for 1 min (Don et al, 1991;Moezi et al, 2019). Nineteen additional cycles were performed at an annealing temperature of 55 • C, followed by a final extension at 72 • C for 10 min.…”
Section: Dna Extraction Pcr Amplification and Illumina Sequencingmentioning
confidence: 99%
“…Polymerase chain reaction (PCR) amplification was performed in a 50 µl reaction mixture containing 5 µl of 10× PCR buffer, 4 µl of MgCl 2 (25 mmol/L), 0.5 µl of each primer (10 µmol/L each), 30 ng of quantified template DNA measuring by Pico green, and 0.4 µl of Taq polymerase (5 U/µl; Fermentas). To increase specificity and sensitivity during gene amplification, the touchdown PCR was conducted in a thermocycler (Applied Biosystems Veriti Thermal Cycler, United States): denaturation at 94 • C for 5 min, 11 cycles of denaturation at 94 • C for 1 min, annealing at 65 • C for 1 min (temperature was decreased by 1 • C every cycle until 55 • C was reached), and extension at 72 • C for 1 min (Don et al, 1991;Moezi et al, 2019). Nineteen additional cycles were performed at an annealing temperature of 55 • C, followed by a final extension at 72 • C for 10 min.…”
Section: Dna Extraction Pcr Amplification and Illumina Sequencingmentioning
confidence: 99%
“…Biochemical assays and molecular approaches are two major technologies for detecting bacteria and have made great contributions to the detection, identification, and control of spreading of foodborne pathogens (Umesha and Manukumar, 2018;Moezi et al, 2019). With this being said, however, biochemical assays are usually associated with the bacterium cultivation, morphological observation, and serologic confirmation tests, which are laborious and time-consuming and do not meet, nowadays, the needs for rapid detection (Zhao et al, 2014).…”
Section: Introductionmentioning
confidence: 99%
“…Therefore, efficient distinguishment and elimination of multiple bacteria are an urgent need for early diagnosis and clinical treatment of infectious diseases, especially high-risk population. Conventional techniques for bacterial detection, including plating and culturing, polymerase chain reaction (PCR), , enzyme-linked immunosorbent assay (ELISA), , etc., are restricted by the shortcomings of high cost, long operation time, and complicated experimental procedures. Over the past two decades, the conventional “lock-and-key” detection mode has been developed for bacterial detection. Although these methods have high sensitivity and good selectivity, most of them are only able to sense one specific bacteria .…”
Section: Introductionmentioning
confidence: 99%