Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes

BACKGROUND: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed METHODS: We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. RESULTS: For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10(7) molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. CONCLUSION: These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material

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 °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

Comparison of the nucleic acid amplification system NASBA(R) 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(R), 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. coil, five (3.12%) as C. lari, and two (1.25%) as unclassified. The NASBA(R) 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(R) 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(R) system was used. However, the high sensitivity of the NASBA(R) 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(R) 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 of Campylobacter jejuni added to foods by using a combined selective enrichment and nucleic acid sequence-based amplification (NASBA).

An assay to detect Campylobacter jejuni in foods that uses a short selective enrichment culture, a simple and rapid isolation procedure, NASBA amplification of RNA, and a nonradioactive in solution hybridization was studied. The presence of high numbers of indigenous flora affected the sensitivity of the assay. However, detection of C. jejuni was possible up to a ratio of indigenous flora to C. jejuni of 10,000:1. Interference by food components was eliminated by centrifugation following the enrichment step. Fourteen food samples artificially inoculated with C. jejuni (1 to 1,000 CFU/10 g) were analyzed with the NASBA assay and the conventional culture method with Campylobacter charcoal differential agar (CCDA). A few false-negative results were obtained by both NASBA (1.42%) and CCDA (2.86%) isolation. Yet the use of enrichment culture and NASBA shortened the analysis time from 6 days to 26 h. The relative simplicity and rapidity of the NASBA assay make it an attractive alternative for detection of C. jejuni in food samples

Identification of Campylobacter jejuni, Campylobacter coli and Campylobacter lari by the nucleic acid amplification system NASBA®

NASBA(R), an isothermal amplification technique for nucleic acids, was evaluated for the specific identification of Campylobacter jejuni, Camp. coli and Camp. lari. A set of primers and a probe were chosen from the 16S rRNA sequence of Campylobacter. The probe was hybridized in solution with the amplified nucleic acids of 12 Campylobacter species and nine other Gram-negative bacteria. The probe was shown to hybridize specifically to the amplified single-stranded RNA of Camp. jejuni, Camp. coli and Camp. lari in an enzyme-linked gel assay (ELGA). In a Camp. jejuni model system the combination of NASBA(R) and ELGA was able to detect ca 1000 rRNA molecules. The presence of an excess of Gram-negative bacteria did not influence the sensitivity of detection. A number of 6 cfu of Camp. jejuni, present in a total count of 4 x 10(6) cfu of Gram-negative bacteria, resulted in a positive hybridization signal

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 a 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