B. van Gemen

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.

Direct detection of potato leafroll virus in potato tubers by immunocapture and the isothermal nucleic acid amplification method NASBA

NASBA, an isothermal amplification method for nucleic acids, was applied to the detection of RNA of potato leafroll virus (PLRV) in a single enzymatic reaction at 41 degrees C. A set of primers was selected from the coat protein open reading frame sequence of PLRV to allow amplification of viral RNA. The NASBA reaction products were visualized after electrophoresis by ethidium bromide or acridine orange staining. The specificity of the amplification products was validated by Northern blot analysis with a PLRV-specific 32P-labelled oligonucleotide probe. The procedure was coupled to immunocapture of PLRV virions from tuber extracts by immobilized antibodies in microtubes. It was possible to discriminate readily by this method between uninfected and primarily PLRV-infected potato tubers. NASBA is suitable for the direct detection of PLRV in potato tubers from primarily infected plants, offering the potential to considerably simplify the inspection of seed-potatoes for virus infection.

Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato

Aims: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death. 
Methods and Results: It was demonstrated by RNase treatment of extracted nucleic acids from R. solanacearum cell suspensions that NASBA exclusively detected RNA and not DNA. The ability of NASBA to assess viability was demonstrated in two sets of experiments. In the first experiment, viable and chlorine‐killed cells of R. solanacearum were added to a potato tuber extract and tested in NASBA and PCR. In NASBA, only extracts spiked with viable cells resulted in a specific signal after Northern blot analysis, whereas in PCR, targeting 16S rDNA sequences, both extracts with viable and killed cells resulted in specific signals. In the second experiment, the survival of R. solanacearum on metal strips was studied using NASBA, PCR‐amplification and dilution plating on the semiselective medium SMSA. A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCR‐amplification resulted in positive reactions also long after cells were dead. 
The detection level of NASBA for R. solanacearum added to potato tuber extracts was determined at 104 cfu per ml of extract, equivalent to 100 cfu per reaction. With purified RNA a detection level of 104 rRNA molecules was found. This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 105 copies of rRNA. Preliminary experiments demonstrated the potential of NASBA to detect R. solanacearum in naturally infected potato tuber extracts. 
Conclusions: NASBA specifically amplifies RNA from viable cells of R. solanacearum even present in complex substrates at a level of 100 cfu per reaction. 
Significance and Impact of the Study: The novel NASBA assay will be particularly valuable for detection of R. solanacearum in ecological studies in which specifically viable cells should be determined.

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.

Use of the direct RNA amplification technique NASBA to detect factor V Leiden, a point mutation associated with APC resistance.

APC resistance is a common and strong hereditary risk factor for venous thrombosis. This plasma abnormality appears to be almost always caused by the same defect in the coagulation factor V gene (a G --> A transition at nucleotide 1691 leading to replacement of 506 Arg by Gln; factor V Leiden). Therefore, it is possible to consider a simple and specific genetic test as an alternative to a plasma APC resistance test that is compromised by treatment and other factors. We have investigated whether a new amplification procedure, NASBA, together with the detection procedure ELGA would provide a simple protocol for the nucleotide specific detection of the factor V mutation.

NASBA® factor V Leiden QL; a new genotype assay to detect the factor V Leiden mutation in anticoagulated whole blood

Summary NASBA® Factor V Leiden, a new RNA amplification test appeared to be reliable in detecting the Factor V Leiden point mutation in whole blood.

Qualitative and Quantitative Detection of Nucleic Acids of Infectious Agents by NASBA

Since the advent of the polymerase chain reaction (PCR; Saki 1988; Mullis & Faloona 1987), a number of other nucleic acid amplification techniques have been developed. One of the most important and well developed of these new technologies is the Nucleic Acid Sequence Based Amplification (NASBATM method (Kievits et al 1991). NASBATM utilizes the coordinated activities of AMV reverse transcriptase (RT), RNase H, and T7 RNA polymerase to amplify a specific nucleic acid target. The specificity of the reaction is determined by a pair of oligonucleotide primers, which are specific for the sequence of interest. One of these primers (designated P1) is synthesized so as to include the promoter for T7 RNA polymerase as a 5’ overhang. The reaction is conducted at constant temperature (41°C) and produces a single stranded RNA product which represents a 106-109 amplification of the original target sequence (figure 1). Although capable of amplifying both DNA and RNA target sequences, NASBATM is most suitable for the amplification of RNA. Thus, NASBATM has become an extremely powerful technique for the detection and quantification of retroviruses (particularly HIV-1).