Dear Kardo

The answer to your question lies in the basic principle of NASBA itself. Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets. The complete amplification reaction is performed at the predefined temperature of 41°C (Fakruddin et al., 2013). Unlike amplification processes, such as PCR in which the initial primer level limits the maximum yield of product, the amount of RNA product obtained in NASBA exceeds the level of primers by at least one order of magnitude. The specificity of the reaction is dependent on thermolabile enzymes; hence the reaction temperature cannot exceed 42°C without compromising it. The NASBA enzymes (avian myeloblastosis virus reverse transcriptase (AMVRT), T7 RNA polymerase and RNase H) are not thermostable and thus can only be added after the melting step. The primers are not incorporated in the amplicon and thus labeled primers cannot be used for detection. Moreover, low temperature may increase the nonspecific interactions of the primers. Therefore, the length of the amplified RNA target sequence should be in the range of 120–250 nucleotides, shorter or longer sequences being amplified less efficiently (Loens et al., 2005). For more details I recommend you to read the following review article:

http://www.ijlpr.com/admin/php/uploads/40_pdf.pdf

ok thank you Sunil Kumar Sahu