Detection of HIV-1 distribution in different blood fractions by two nucleic acid amplification assays.

A new amplification procedure, NASBA (nucleic acid sequence-based amplification), was used together with the polymerase chain reaction (PCR) to detect HIV-1 sequences in different blood fractions of both HIV-infected and uninfected samples. We tested whole blood, plasma, peripheral blood mononuclear cells (PBMCs), and platelets. No HIV-1 sequences were found in blood fractions of 37 uninfected persons either by PCR, reverse transcriptase-PCR (RT-PCR), or NASBA. We found that none of the infected plasma samples contained HIV-1 DNA sequences. However, a high percentage of these plasma samples was positive for HIV-1 RNA: 86% by NASBA and 80% by RT-PCR. The concordance on a sample-to-sample basis of NASBA and RT-PCR was 91%. Only 33% of the plasma samples was HIV-1 p24-antigen positive, demonstrating the superior sensitivity of amplification procedures. We found that almost all PBMC fractions were positive for HIV-1 (pro)viral sequences (99% HIV-1 DNA positive, 91% HIV-1 RNA positive). A large proportion of the platelet fractions contained HIV-1 RNA, as demonstrated by positive RT-PCR and NASBA results. We found an inverse relation between CD4+ T cell count and T cell reactivity on the one hand and detection of HIV-1 sequences by PCR, RT-PCR, and NASBA on the other hand in all blood fractions. Quantification of the HIV-1 PCR signal in PBMCs revealed an inverse relation of proviral titers with CD4+ levels. This finding supports earlier observations that clinical disease and low CD4+ cell counts are related to an increased viral burden.

NASBA 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).

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.

Quantification of HIV-1 RNA in plasma using NASBA 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.

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.

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.

TcA, the putative transposase of the C. elegans Tc1 transposon, has an N-terminal DNA binding domain.

Tc1 is a transposon present in several copies in the genome of all natural isolates of the nematode C.elegans; it is actively transposing in many strains. In those strains Tc1 insertion is the main cause of spontaneous mutations. The transposon contains one large ORF that we call TcA; we assume that the TcA protein is the transposase of Tc1. We expressed TcA in E.coli, purified the protein and showed that it has a strong affinity for DNA (both single stranded and double stranded). A fusion protein of beta-galactosidase and TcA also exhibits DNA binding; deletion derivatives of this fusion protein were tested for DNA binding. A deletion of 39 amino acids at the N-terminal region of TcA abolishes the DNA binding, whereas a deletion of 108 C-terminal amino acids does not affect DNA binding. This shows that the DNA binding domain of TcA is near the N-terminal region. The DNA binding capacity of TcA supports the assumption that TcA is a transposase of Tc1.

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.

Identification of Campylobacter jejuni, Campylobacter coli and campylobacter lari by the nucleic acid amplification system NASBAR.

NASBAR, 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 NASBAR 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.

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.

Assessment of mycobacterial viability by RNA amplification.

We investigated whether the presence of intact RNA is a valuable indicator of viability of mycobacteria with Mycobacterium smegmatis. M. smegmatis was exposed to various concentrations of rifampin and ofloxacin suspended in broth for different periods of time. The NASBA nucleic acid amplification system was used because of its rapid, sensitive, and specific detection of 16S rRNA. During drug exposure, the viability of the mycobacteria, expressed by the number of CFU, was compared with the presence of 16S rRNA as determined by NASBA and with the presence of DNA coding for 16S rRNA as determined by PCR. Both NASBA and PCR were shown to have a detection limit of approximately 5 x 10(2) CFU/ml. The intensity of the NASBA signal corresponded well with the number of CFU, and the lack of NASBA signal coincided with a loss of viability, which was reached after 3 days of exposure to bactericidal concentrations of both drugs. The presence of mycobacterial DNA, as determined by the intensity of the PCR signal, and the viability of M. smegmatis were not related, but an increase in the number of cells and intensity of PCR signal correlated well. Bacterial viability may thus be assessed by a rapid, sensitive, and specific, and semiquantitative technique by using NASBA. This system of viability testing provides the potential for rapid evaluation of drug susceptibility testing.

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.

Use of NASBA RNA amplification for detection of Mycobacterium leprae in skin biopsies from untreated and treated leprosy patients.

This study was performed to assess the value of NASBA RNA amplification of a 16S rRNA target for the detection of presumably viable Mycobacterium leprae in sections of skin biopsies from leprosy patients. The NASBA positivity rate was 90.4% (84/93) for untreated multibacillary (MB) patients [bacterial index (BI) > or = 2] and 16.7% (8/48) for the untreated paucibacillary (PB) patients (BI < 2). NASBA positivity showed a good concordance with the presence of solidly stained M. leprae [morphological index (MI)] in skin biopsies from leprosy patients, but no relationship could be demonstrated between the strength of the NASBA signals and the BI. Furthermore, the usefulness of the detection of 16S rRNA by NASBA to monitor the efficacy of leprosy treatment was investigated using an additional 154 biopsy specimens analyzed from 80 MB patients during the course of treatment. The NASBA positivity rate declined during treatment. A significant decrease was observed after only 1-3 months. These results favor the view that detection of RNA by NASBA may reflect the viability of M. leprae.