R Schukkink

NASBATM isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection

Isothermal nucleic acid amplification of target RNA or DNA sequences is accomplished by the simultaneous enzymatic activity of AMV reverse transcriptase, T7 RNA polymerase and RNase H. Amplification factors of the nucleic acid sequence based amplification (NASBA) method range from 2 x 10(6) to 5 x 10(7) after 2.5 h incubation at 41 degrees C. During NASBA there is a major accumulation of specific single stranded RNA. RNA:DNA hybrid and double stranded DNA are also synthesized, although to a minor extent. The system is optimized for the detection of HIV-1 sequences in in vitro infected cells, blood and plasma. Detection levels are 10 molecules of HIV-1 in a model system with in vitro generated HIV-1 RNA as input and 5 infected cells on a background of 5 x 10(4) non-infected cells. Blood and plasma can also be used as the source of nucleic acid for detection of HIV-1 sequences using a specifically developed sample preparation method. Using NASBA it is possible to amplify specifically RNA or DNA from a pool of total nucleic acid, which permits the investigation of the expression of specific genes involved in pathogenesis of infectious agents. The combination of NASBA with a rapid and user-friendly nucleic acid extraction method makes the whole procedure suitable for large scale diagnosis of infectious agents (e.g. HIV-1).

Quantification of HIV-1 RNA in plasma using NASBAtm during HIV-1 primary infection

Quantification of HIV-1 viral RNA in plasma was achieved by competitive co-amplification of a dilution series of in vitro generated RNA using the nucleic acid sequence based amplification (NASBA) technology. This 1.5 kilobase in vitro RNA, comprising the gag and part of the pol region, differs only by sequence-randomization of a 20 nt fragment from the wild-type RNA, ensuring equal efficiency of amplification. In model systems the accuracy of this method is within one log. Application of the Q-NASBA to plasma samples of a patient with a primary HIV-1 infection shows good concordance of the HIV-1 RNA profile with the p24 antigen profile. However, the HIV-1 RNA determination is more sensitive than the p24 antigen determination. Peak values of HIV-1 RNA are around 10(8) RNA molecules per ml plasma at the moment of seroconversion. Quantitative nucleic acid detection methods, like Q-NASBA, allow the monitoring of HIV-1 RNA during the course of infection which might have predictive value for disease development.

Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique for nucleic acids (NA), was investigated for the species-specific identification of mycobacteria. A set of primers was selected from a highly conserved region of the 16S rRNA sequence of mycobacteria sandwiching a variable sequence to perform amplification of mycobacterial RNA. Species-specific probes for the M. tuberculosis complex, M. avium-paratuberculosis, M. intracellulare and M. leprae were hybridized in-solution with the amplified nucleic acids of 10 pathogenic mycobacteria and 11 closely related bacteria, as well as with human-derived NA in an enzyme-linked gel assay (ELGA). Each probe was shown to hybridize specifically to the amplified single-stranded RNA of the corresponding species. Thirty-two clinical isolates of M. tuberculosis strains from different parts of the world were correctly identified by NASBA using the M. tuberculosis-complex-specific probe. In combination with the ELGA, NASBA could identify mycobacteria rapidly, i.e. in less than 6 h. The relative simplicity and rapidity of this technique makes it an attractive tool for species-specific identification of mycobacteria.

Application of the NASBA nucleic acid amplification method for the detection of human papillomavirus type 16 E6-E7 transcripts

Using a human papillomavirus type 16 (HPV-16) E6-E7 specific primer set in a nucleic acid sequence-based amplification (NASBA) reaction, detection of HPV-16 transcripts was accomplished in a single enzymatic reaction at 41 degrees C. The NASBA reaction product was visualized either by Northern bolt analysis with an HPV-16 E6-E7-specific 32P-labelled oligonucleotide probe or by a non-radioactive enzyme-linked gel assay (ELGA). In combination with a rapid nucleic acid extraction procedure this method appears to be very suitable for the sensitive and specific detection of HPV-16 transcripts on small amounts of HPV-16-expressing cells of various sources, including cervical smears.

Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species

Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.

Development of NASBA®, a nucleic acid amplification system, for identification of Listeria monocytogenes and comparison to ELISA and a modified FDA method

NASBA, an isothermal nucleic acid amplification system was used for identification of Listeria monocytogenes. A primer set and a species-specific probe were selected from the 16S rRNA sequence. The probe was shown to hybridize specifically to the amplified single-stranded RNA of L. monocytogenes. No hybridization occurred with amplification product of L. seeligeri, L. innocua, L. ivanovii, and L. welshimeri. Detection sensitivity for the NASBA assay was determined at 10(6) cfu/ml. The possibility of using the NASBA assay for detection of L. monocytogenes in foods after a 2-day enrichment procedure was explored. NASBA was compared to a modified FDA method and ELISA for detection of L. monocytogenes artificially inoculated (1-100 cfu/25 g) in eight food products. False-negative results were obtained with the modified FDA method (6.75%). NASBA and ELISA were shown in this study to detect the pathogen with equal efficiency (no false negative or false positive results). Both methods allowed detection of less than 10 cfu/25 g within 3 days but ELISA can only be used for diagnosis of Listeria spp. while the NASBA procedure permitted specific identification of the human pathogen L. monocytogenes.

A sensitive and robust method for measles RNA detection.

The aim of this study was to compare measles RNA amplification methods and to develop and select the most rapid, sensitive and robust procedure. The use of hybrid capture for measles RNA isolation was evaluated, and three RNA amplification detection techniques were compared. These were: (a) reverse transcription followed by nested polymerase chain reaction (RT-PCR) with MMLV reverse transcriptase and Taq polymerase; (b) a combined RT-PCR reaction using rTth polymerase; and (c) NASBA. An internal positive control was also developed. The sensitivities of the detection methods were quantified by using a dilution series of a known amount of total RNA from measles-infected Vero cells or by calculation of the number of transcript molecules (produced from a recombinant plasmid containing an insert measles nucleoprotein DNA) present in each amplification reaction, respectively. The results indicated that hybrid capture followed by combined RT-PCR with rTth polymerase was the most reproducibly robust and sensitive protocol and could detect as few as 10(4) synthetic measles RNA transcripts added to tissue homogenates. However, NASBA proved to be the most sensitive method for measles RNA detection in water.

Comparison of the Nucleic Acid Amplification System NASBA® and Agar Isolation for Detection of Pathogenic Campylobacters in Naturally Contaminated Poultry

A total of 160 poultry products were examined for the presence of pathogenic campylobacters using the traditional agar isolation method and the nucleic acid amplification system NASBA®, both after a 24-h selective enrichment. Pathogenic campylobacters could be isolated from 92 of 160 (57.5%) samples using agar isolation, among which 79 (49.37%) were identified as Campylobacter jejuni , six (3.75%) as C. coli , five (3.12%) as C. lari , and two (1.25%) as unclassified. The NASBA® assay provides a specific and sensitive method for detection of these campylobacters. A total of 149 samples (93.12%) gave similar results for both the traditional isolation procedure on modified Campylobacter charcoal desoxycholate agar and the NASBA® enzyme-linked gel assay detection system. Two false-negative results were obtained with the agar isolation procedure. Nine false-positive results were reported when the NASBA® system was used. However, the high sensitivity of the NASBA® method and indications that in some cases the traditional isolation procedure failed (abundance of a contaminating noncampylobacter bacteria which grew on the Campylobacter selective media) raises doubt about the true nature of these false-positive results. The NASBA® detection assay offers a rapid and useful analytical method when screening for the presence of pathogenic campylobacters. The complete procedure, including 24 h of selective enrichment, required 32 h.

Detection, identification and semi-quantification of malaria parasites by NASBA amplification of small subunit ribosomal RNA sequences

We have adapted the Nucleic Acid Sequence Based Amplification method (NASBA), a molecular detection method for the amplification of specific RNA sequences, for the detection, identification and semi-quantification of malaria parasites. Primers and probes were selected based on the nucleotide sequence of the small subunit ribosomal RNA gene. NASBA enabled the detection of as little as 0.04 parasitized erythrocytes per μl blood. When applied to blood samples of patients, NASBA allowed the detection of malaria parasites in confirmed malaria patients. In addition, malaria parasites were detected in a number of patients with a history of suspected malaria. Semi-quantification over a wide parasite range was achieved by analysing serial end-point dilutions of RNA preparations. Semi-quantitative NASBA analysis of malaria parasites in follow-up samples of treated malaria patients showed a rapid decline of parasite levels in most patients and a low level of persistent parasites in some of the patients.

Influence of bacterial age and pH of lysis buffer on type of nucleic acid isolated

This study deals with the determination of the factors which influence isolation of RNA using a silica-based nucleic acid isolation protocol. Four bacteria were included in the investigation: Campylobacter jejuni, Escherichia coli, Listeria monocytogenes and Pseudomonas fluorescens. An increase of RNA yield could be obtained with all four strains at the exponential growth phase, reaching a maximum at the beginning of the stationary phase. Later the amount of RNA isolated gradually decreased. At the late stationary phase DNA was also isolated in case of Escherichia coli and Pseudomonas fluorescens. DNA was only obtained at this stage of the growth curve, probably because RNA contents of the cell decreases in the late stationary phase thus enabling DNA only now to bind to the silica used in the isolation procedure. In the exponential and stationary phase, when there is a competition between RNA and DNA to bind to the silica, there is a preferential binding of RNA over DNA to the silica. It was shown that a pH of 6.0 and 6.5 promoted isolation of RNA. At pH 8.0 and 8.5 large amounts of DNA were obtained from the Gram-negative bacteria. Thus, the silica based nucleic acid isolation method enables isolation of either RNA or DNA when taking into account the appropriate conditions of pH of the buffers and applying the corresponding incubation time of the bacterial culture. A preceding treatment of lysozyme and proteinase K was sufficient to accomplish lysis of the Gram-positive cell.