Abstract
We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61–65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.
Highlights
Nucleic acid amplification is a valuable molecular tool in modern biology and medicine, and is widely applied in basic research, clinical diagnosis, forensic science, epidemiology, agriculture and many other fields[1]
The methodology presented in this study, multiple cross displacement amplification (MCDA), overcomes the technical difficulties posed by current isothermal amplification techniques
In order to demonstrate the mechanism of MCDA, the hlyA gene (GenBank GeneID: 223702383), which was specific for L. monocytogenes species, was chosen as a model DNA6
Summary
Nucleic acid amplification is a valuable molecular tool in modern biology and medicine, and is widely applied in basic research, clinical diagnosis, forensic science, epidemiology, agriculture and many other fields[1]. Loop-mediated isothermal amplification et al.) have been reported, which obviate the use of a thermocycling apparatus, can be real-timely detected and favor point-of-care (POC) diagnosis[4] These assays are still relatively complex protocols requiring rigorous optimization, the use of multiple enzymes (two or more) and/or special reagents, with no considerable improvement in their operativity, sensitivity and specificity[5]. The two strands from the CP1/C1 product yield stem-loop structures which can be re-annealed and stabilized by the base pair double helix formed between C1 and C1s These DNAs can be used as templates for further extension by amplification primers D1, C1 and cross primer CP1, forming CP1/D1 and CP1/C1 products (Cycle 1). We illustrate the optimization of the basic MCDA reaction and demonstrate an application of the MCDA assay
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
