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Molecular diagnostics: Nucleic acid sequence based amplification (NASBA).

The Organon Teknika NucliSens^® Basic Kit

The first isothermal transcription based amplification system was described by Guatelli et al., 1990. Modelled on retrovirus replication, it used the three key enzymes ribonuclease (RNAse) H, reverse transcriptase and a DNA-dependent RNA polymerase to produce complementary DNA then RNA copies of the original target. These functioned as templates for a series of transcription and reverse transcription reactions that was self-sustained and which amplified the original target some 10 million fold. Termed self-sustained sequence replication it was given the acronym 3SR [Chan & Fox, 1999]. Following refinement the method was patented by Organon Teknika (Cambridge) forming the basis of the Organon Teknika NucliSens^® Basic Kit. A variation on the enzymes used also led to the related Gen-Probe transcription mediated amplification (TMA) methodology, the basis of the chlamydial AMP-CT and Aptima Combo tests. A key improvement of NASBA over the original 3SR methodology was the addition of dimethylsulphoxide and elevation of the reaction temperature to 41C, which increased specificity, decreasing background. Further optimisation of reaction conditions for the TMA process enhanced RNAse activity of the other enzymes,  so that RNAse H could be omitted. Since RNA in clinical specimens is readily degraded by RNAse in the sample, the chaotropic agent guanidinium thiocyanate is used to release the nucleic acid and to prevent nuclease digestion. The target rRNA then binds to particles of activated silica [Boom et al., 1990].

Classically, the NASBA reaction mix consists of the relevant nucleotide triphosphates plus two primers complementary to the RNA region of interest.  Primer 1 has a target-binding region plus a promoter for T7 RNA polymerase. After annealing to the RNA target the primer is extended by retrovirus reverse transcriptase to form an RNA-cDNA hybrid. RNAse H digestion allows the second primer to bind to the cDNA upstream of the primer 1 region. Primer 2 is extended by the by the DNAse dependent DNA polymerase activity of the reverse transcriptase producing a second DNA strand which is extended by the T7 RNA polymerase promoter, leading to a double stranded DNA copy of the original RNA target, including, at one end, the T7 promoter, which is used by the T7 RNA polymerase to produce large amounts of RNA complementary to the original target. This then functions as a template for the cyclic phase of the reaction, in which primer 2 binds before primer 1. The whole process is a sustained isothermal reaction producing very high level amplification, typically of the order of 10⁹ fold [Chan & Fox, 1999]. 

For detection,  a biotinylated capture probe binds to the single stranded antisense amplicon, and the complex then binds on an streptavidin-coated magnetic bead. This whole complex is immobilised on an electrode by a magnet and a ruthenium labelled detection probe is hybridised to the target. When voltage is applied to the electrode, a voltage induced oxidation-reduction reaction occurs which generates an electrochemiluminescent light signal that can be measured by a photometer. For an excellent review of NASBA and its variants see: Chan & Fox, 1999. For a general review of nucleic acid amplification methods see Jungkind 2001.

[Comment: The Organon Teknika NucliSens^® Basic Kit is the Linux system of the nucleic acid amplification world :-) an open system for RNA amplification and detection generally. Organon Teknika produce the basic extraction, amplification and detection reagents, including a generic detection probe. The user provides the specific primers and probes. Thus the methodology may be adapted, with appropriate primers, to the detection of a whole range of different microorganisms.  Unlike other commercial kits, it has been adapted for research / diagnosis of C. pneumoniae infections [Coombes & Mahony, 2000]. Indeed the web site of the kit manufacturer contains more than 60 assay protocols [Mahony et al., 2001]. Given the large numbers of companies able to synthesise primers on demand for highly competitive prices, this is not just an option restricted to the research laboratory. This is a potentially attractive method for those who harbour 'do it yourself' instincts].  

Clinical trials.

There are surprisingly few papers on the use of the Organon Teknika NucliSens^® Basic Kit for the diagnosis of C. trachomatis infections, a situation which will surely change.

In their initial evaluation, Morre et al., 1996 compared different primer sets for their sensitivities for C. trachomatis detection by NASBA, and found that both the cryptic plasmid and omp1 (MOMP gene) targets had a detection limit of 1 inclusion-forming unit (IFU), while the 16S rRNA was a thousand fold more sensitive, permitting detection of 1 x 10^-3 IFU [Morre et al., 1996].  This compares very favourably with other nucleic acid amplification methods, including PCR and LCR [Mahony et al., 2001]. Indeed, for DNA amplification by PCR, the plasmid target had a detection limit of 10^-2 IFU, ten fold less sensitive than rRNA NASBA [Morre et al., 1996]. An internal standard was also developed to detect amplification inhibition by clinical samples. The 16S rRNA NASBA with internal controls was compared with a plasmid DNA PCR on 41 C. trachomatis-negative and 37 positive cervical swabs, as determined by antigen detection enzyme immunoassay. Additionally, 17 urine samples from the chlamydial antigen-positive women were also tested. Both the NASBA and PCR assays were able to detect C. trachomatis in all EIA-positive cervical scrapings, the corresponding urine samples, and two samples from the EIA-negative group. There was no evidence of amplification inhibition. The authors concluded that the method had considerable potential for the diagnosis of C. trachomatis genital tract infections in cervical samples and urine [Morre et al., 1996]. 

In a follow up paper, the same group used 16S rRNA NASBA and cryptic plasmid-based PCR to monitor the effect of antibiotic treatment on 25 women with genital C. trachomatis infection. C. trachomatis RNA was found in all 24 cervical smears taken before antibiotic treatment and in two smears taken one week after antibiotic treatment, but in no smears collected two weeks or more after treatment. In contrast, C. trachomatis plasmid DNA was found in all such specimens before treatment, and in 21 of 25; six of 21, and five of 20 smears respectively at one, two, and three weeks after treatment. Of the 61 urine samples investigated, C. trachomatis DNA and rRNA respectively were found in 15 out of 15 before treatment. One week after treatment four of 15 were C. trachomatis DNA positive (PCR) and only one was rRNA positive. These results suggested that NASBA was at least as sensitive as PCR, but the RNA target was better correlated than the DNA target with eradication of infection following treatment [Morre et al., 1998]. However, as has been pointed out for a different system, the correlation between cell viability and persistence of nucleic acids must be well characterized for a particular analytical situation before molecular techniques can be substituted for traditional culture methods [Birch et al., 2000].

Mahony et al., 2001 confirmed Morre and colleague's finding that the NASBA Basic Kit could detect 1 inclusion-forming unit of C. trachomatis, or one colony forming unit of N. gonorrhoeae, and 100 RNA molecules of 16S rRNA for both bacteria. To assess clinical performance, a total of 250 specimens for N. gonorrhoeae were tested for gonococcal infection by culture or NASBA and 96 specimens were tested for C. trachomatis by PCR and NASBA. The Organon Teknika NucliSens^® Basic Kit detected 139 of 142 N. gonorrhoeae culture-positive specimens and gave a negative result for 73 of 74 culture-negative specimens; a sensitivity and specificity of 97.9% and 98.7%, respectively. For C. trachomatis, the Basic Kit detected 24 of 24 PCR-positive specimens, gave a negative result for 71 of 72 PCR-negative specimens, with a sensitivity and specificity of 100 and 98.6%, respectively. A multiplex NASBA assay also detected infection with both organisms. It was considered that the NucliSens Basic Kit offered a versatile platform for the development of sensitive RNA detection assays for sexually transmitted diseases [Mahoney et al., 2001].

The inherent versatility of the NASBA Basic kit was also demonstrated by Coombes & Mahony 2000, who targeted it against the gene encoding the major outer membrane protein of C. pneumoniae, using a different, aequorin-based, bioluminescent assay that was linear over 8 log units of RNA copy number. The method could detect between 100 and 1000 RNA transcripts equivalent to some 0.2 IFU of C. pneumoniae [Coombes & Mahony 2000]. 

[This is a very sensitive, standardisable method for the detection of C. pneumoniae infection].

[MEW] July 2003

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References

Birch, L., Dawson, C. E., Cornett, J. H. & Keer, J. T. (2000). A comparison of nucleic acid amplification techniques for the assessment of bacterial viability. Letters in Applied Microbiology 33, 296 - 301.

Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheim-van Dillen, P. M. & van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28, 495 - 503.

Chan, A. B. & Fox, J. D. (1999). NASBA and other transcription-based amplification methods for research and diagnostic microbiology. Reviews in Medical Microbiology 10, 185 - 196. [Excellent review].

Coombes, B. K. & Mahony, J. B. (2000). Nucleic acid sequence based amplification (NASBA) of Chlamydia pneumoniae major outer membrane protein (ompA) mRNA with bioluminescent detection. Combinatorial Chemistry and High Throughput Screening 3, 315 - 327.

Guatelli, J. C., Whitfield, K. M., Kwoh, D. Y., Barringer, K. J., Richman, D. D. & Gingeras, TR. (1990). Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proceedings of the National Academy of Sciences of the U S A. 87, 1874 - 1878. Full article  

Jungkind, D. (2001). Molecular testing for infectious disease. Science 294, 1553 - 1555. [A general overview. Good diagrams but not detailed].

Mahony, J. B., Song, X., Chong, S., Faught, M., Salonga, T. & Kapala, J. (2001). Evaluation of the NucliSens Basic Kit for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genital tract specimens using nucleic acid sequence-based amplification of 16S rRNA. Journal of Clinical Microbiology 39, 1429 - 1435  Full article  

Morre, S. A., Sillekens, P., Jacobs, M. V., van Aarle, P., de Blok, S., van Gemen, B., Walboomers, J. M., Meijer, C. J. & van den Brule, A. J. (1996). RNA amplification by nucleic acid sequence-based amplification with an internal standard enables reliable detection of Chlamydia trachomatis in cervical scrapings and urine samples. Journal of Clinical Microbiology 34, 3108 - 3114. Full article

Morre, S. A., Sillekens, P. T., Jacobs, M. V., de Blok, S., Ossewaarde, J. M., van Aarle, P., van Gemen, B., Walboomers, J. M., Meijer, C. J. & van den Brule, A. J. (1998). Monitoring of Chlamydia trachomatis infections after antibiotic treatment using RNA detection by nucleic acid sequence based amplification. Molecular Pathology 51, 149 - 154.

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