Biomedical subjects

R Schukkink

Publications and source records attributed to R Schukkink.

8 recordsLinked to original sources

PubMed1998

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.

Animals↗

PubMed1995

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.

Base Sequence↗

PubMed1995

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.

Bacteriological Techniques↗

PubMed1995

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.

Animals↗

PubMed1994

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.

Base Sequence↗

PubMed1993

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.

Base Sequence↗

PubMed1993

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.

Base Sequence↗

PubMed1991

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).

Base Sequence↗