The products of NASBA are single stranded and thus can be applied to detection formats that use probe hybridization without any denaturation step. in humans and is responsible for 15 to 20% of all cases of pneumonia (12) and a wide range of moderate to serious extrapulmonary complications (8, 18). In the past, diagnosis of contamination with this organism was usually based on serology because culture is slow and insensitive (14, 22). Therefore, nucleic acid amplification techniques have been introduced. PCR of fragments of the P1 gene or the 16S TG 100713 rRNA gene was shown to be considerably more sensitive than culture for the detection of (9, 17, 20, 39). Amplification methods often lack appropriate controls. A human -globin-specific amplification may be used to assess the presence of nucleic acids in the processed sample (1, 24, 31). For the detection of inhibitors, the use of an internal control (IC) to be amplified with the same primer set as the target sequence is straightforward since it avoids the use of different primer sets. ICs are now gradually being more widely used (10, 16, 19, 30, 41). Nucleic acid sequence-based amplification (NASBA; Organon Teknika, Boxtel, The Netherlands) is targeted at RNA. It makes use of the simultaneous enzymatic activities of avian myeloblastosis virus reverse transcriptase (AMV-RT), RNase H, and T7 RNA polymerase under isothermal conditions. One advantage of NASBA compared with PCR is that it is a continuous, isothermal process which does 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 kinetics of the NASBA process, which are caused by multiple transcription of RNA copies from a given DNA product, are intrinsically more efficient than DNA amplification methods, which are limited to binary increases per cycle (38). The products of NASBA are single stranded and thus can TG 100713 be applied to detection formats that use probe hybridization without any denaturation step. Furthermore, the detection of microorganisms by an rRNA-based amplification technique might be more sensitive than PCR because of the presence of multiple RNA copies, and it also implies biological activity. It may be a useful complement to culture in order to establish if the infection is Rabbit polyclonal to ZNF182 productive or to follow an antibiotic therapy. NASBA also has some disadvantages. NASBA is an RNA amplification procedure. RNA integrity and amplification inhibitors are the main causes of concern for NASBA, RT-PCR, and other RNA amplification procedures as well. The stability of the RNA may be affected during collection, processing, and storage of specimens TG 100713 prior to isolation. The addition of RNase inhibitors to the clinical specimens, such as guanidine thiocyanate (GuSCN), is required to preserve RNA integrity. The specificity of the reactions might be lower. The enzymes used are not thermostable, and the reaction temperature may not exceed 42C without compromising the reaction. However, the specificity is increased by additional hybridization with target-specific probes by enzyme-linked gel assay (ELGA), electrochemiluminescence detection, or even real-time detection. Furthermore, the length of the amplified RNA target sequence should be in the range of 120 to 250 nucleotides. Shorter and longer sequences will be amplified less efficiently. This might be more important for RNA amplification assays. The NASBA technique has already been successfully applied for the detection of human immunodeficiency virus type 1 (HIV-1) (21), human cytomegalovirus (13), citrus tristeza virus (23), human papillomavirus (36), human hepatitis C virus (34), malaria parasites (37), (25), (42), and (44) and for the detection and identification of and (43). We previously described the use of NASBA for the typing of strains and isolates (27). In the study described here we used the NASBA technique for the detection of RNA, constructed an IC for the assay, optimized the sample preparation procedure for detection of RNA in clinical specimens, and compared its performance with that of PCR on a number of clinical samples. MATERIALS.
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