Advantages And Disadvantages

One advantage of NASBA and TMA compared to PCR is that they are continuous, isothermal processes that do not require a thermocycler. The constant temperature maintained throughout the amplification reaction allows each step of the reaction to proceed as soon as an amplification intermediate becomes available. Thus, the exponential kinetic of the amplification process, which is caused by multiple transcription of RNA copies from a given DNA product, is intrinsically more efficient than DNA amplification methods, which are limited to binary increases per cycle.[11]

RNA is the genomic material of numerous viruses. The application of an RNA-based amplification technique offers advantages compared to a DNA-based amplification technique: no additional RT step is required, thus saving time and reducing the risk of contamination.

NASBA and TMA can be targeted at ribosomal RNA; thus they offer a diagnostic advantage because bacterial rRNA is present in multiple copies/cell and it also implies biological activity, whereas DNA is present at a much lower number of copies. Therefore the likelihood of initiating amplification is greater when rRNA is targeted instead of DNA. Furthermore, the assays could be used in viability studies.

There are also a number of disadvantages. The specificity of the reactions might be lower because the enzymes used are not thermostable, and so that the reaction temperature may not exceed 42°C without compromising the reaction. However, the specificity rate is increased by additional hybridization with target-specific probes. Finally, the length of the amplified RNA target sequence should be in the range of 120-250 nucleotides. Shorter and longer sequences are amplified less efficiently.

Furthermore, RNA integrity and amplification inhibitors are also the main causes of concern for NASBA, TMA, RT-PCR, and other RNA amplification procedures. Stability of RNA may be affected during collection, processing, and storage of specimens prior to its isolation. The addition of RNase inhibitors to the clinical specimens, such as guanidine thiocyanate, is required to preserve RNA integrity. Some studies suggest that quality control of buffers for storage of clinical material is critical when RNA is to be analyzed.[2]

To overcome part of this problem and to improve the reproducibility of the in-house developed NASBA standardized reagents, the ''NucliSens Basic Kit'' (bioMerieux) is now commercially available. It contains all the necessary reagents for 1) nucleic acid release and inactivation of RNases and DNAses; 2) silica-based extraction of nucleic acids; 3) NASBA reagents; and 4) reagents for electro-chemiluminescent detection including the ECL probe, and paramagnetic particles to link the capture probe for detection. The primers and the target-specific biotinylated capture probe are to be synthesized for each target.[12]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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