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Biomedical subjects

J Lundeberg

Publications and source records attributed to J Lundeberg.

At least 91 records · Page 5Linked to original sources
PubMed1996

Quantification of HIV-1 using multiple competitors in a single-tube assay.

Methods for quantification of human immunodeficiency virus type 1 (HIV-1) based on competitive PCR and fragment analysis have been developed. Samples containing HIV-1 DNA and known amounts of three cloned competitors were co-amplified by PCR with semi-nested primers. The competitor DNAs contained the same long terminal repeat primer binding sequences as the wild-type DNA, but they are different in internal sequences and length. One of the inner primers was fluorescent-labeled to allow discrimination between the wild-type DNA and the three competitors by fragment analysis using a standard automated sequencer. A calibration curve using the peak area of the three competitors enabled accurate determination of target amount with minimal variations. The method presented here can be used for quantification of HIV-1 in clinical samples and will be useful for monitoring disease progression and treatment effects.

Base Sequence↗
PubMed1995

Dynamic analysis of heterogeneous hepatitis C virus populations by direct solid-phase sequencing.

In the present study, we used a semiautomated solid-phase direct sequencing method to analyze sequence diversity and variation of the hypervariable E2/NS1 region in the hepatitis C virus (HCV) genome in isolates from patients seropositive for HCV. A total of 24 isolates of various origins were sequenced. Six of the patients, not subject to any antiviral therapy, were monitored longitudinally, and rapid sequence variations were observed over a period of 14 months. The nucleotide change rate was found to be 0.1 to 0.2 nucleotide substitution per genome site per year. Furthermore, isolates from five of the patients were used for a comparative study of the direct solid-phase sequencing approach versus the frequently used approach of sequencing individual reverse transcriptase PCR clones. The advantage of direct solid-phase sequencing for studying dynamic changes in heterogeneous populations of HCV is discussed.

Amino Acid Sequence↗
PubMed1995

Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alpha-thiotriphosphate nucleotides.

Among the candidate cancer-prognostic genes is the p53 tumor suppressor gene, which, when mutated, plays an important role in the development of many types of cancers. To facilitate robust large-scale DNA analysis of microdissected tumor biopsies, we describe a multiplex/nested PCR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, involving a total of 14 primers. This approach reduces the required number of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification step. The HLA sequencing allows sample identification because the DQB1 locus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomated format. To improve the DNA sequence quality, we used 2'-deoxyribonucleoside 5'-O-1-thiotriphosphates in the sequencing reactions.

Autoanalysis↗
PubMed1994

Colorimetric detection of competitive PCR products for quantification of hepatitis C viremia.

A method based on competitive polymerase chain reaction (PCR) and colorimetric detection of the amplified products was developed to quantify hepatitis C virus (HCV) genomes. Serum samples were obtained from patients who were treated with interferon alpha (IFN-alpha). After reverse transcription of the HCV RNA, the cDNA was coamplified with a serially diluted cloned HCV competitor DNA using nested PCR. The competitor DNA consisted of the amplified region of the wild type HCV cDNA with an internal region substituted with the lac operator (lacO) sequence. The PCR products were quantitated specifically by a colorimetric solid-phase assay. The results suggest that the method is well suited for analysing the kinetics of the anti-HCV effects during IFN-alpha treatment. The quantification assay is simple, reliable and suitable for quantitating HCV genomes in a large number of clinical samples.

Base Sequence↗
PubMed1994

Serum hepatitis C virus RNA levels in chronic hepatitis C--importance for outcome of interferon alfa-2b treatment.

Sera from 39 out of 40 patients with chronic hepatitis C virus (HCV) infection who had been treated for 60 weeks with interferon alfa-2b proved initially HCV RNA positive by reversed transcriptase polymerase chain reaction (PCR). These patients were analysed for genotype and quantitatively for HCV RNA levels prior to treatment by using a competitive PCR method with colorimetric detection of the amplified products. HCV RNA levels were correlated to outcome of treatment, mode of acquisition, histology and HCV genotype. The median pretreatment HCV RNA level in sustained responders (n = 15) with eradication of the viremia and normalization of serum ALT levels lasting 24 weeks post treatment was significantly lower than that in the combined group of non-sustained responders (n = 9) and non-responders (n = 15), 2.52 x 10(5) vs 8.90 x 10(5) genome equivalents per ml serum, p < 0.0125, respectively. 10 out of 17 patients with HCV RNA levels lower than the median level (5.64 x 10(5) genome equivalents per ml serum) had a sustained response to interferon treatment versus only 5/22 with levels equal to or higher than the median level, p = 0.04. No significant pretreatment differences in median HCV RNA levels according to mode of acquisition, genotype, or liver histology prior to treatment were seen. It is concluded that a low pretreatment HCV RNA level seems to be indicative of a sustained response to interferon alfa-2b treatment, whereas a high level seems to be indicative of a non-sustained or non-response. In the individual patient, however, the levels varied widely irrespective of response category.

Adolescent↗
PubMed1994

Microtiter assay for colorimetric detection of in vitro amplified Chlamydia trachomatis sequences.

An integrated diagnostic method for colorimetric detection of polymerase chain reaction (PCR) products in a microtitre format is described. The amplified material is labelled by using modified PCR primers introducing a biotin molecule and a lac operator handle. Positive samples are identified, after immobilization onto streptavidin-coated microtitre wells, using a reporter fusion protein consisting of the Escherichia coli lac repressor and beta-galactosidase. The analysis of the PCR products is thus, from a practical point of view, identical with the enzyme-linked immunosorbent assay (ELISA) using the enzymatic activity of beta-galactosidase. Here, we show that the assay can be used for rapid and simple detection of Chlamydia trachomatis in an automated format. The reported analysis of 90 urethral patient samples, using this microtitre assay, indicates that this technique is more sensitive and more specific than culture technique.

Base Sequence↗
PubMed1994

Sequence-based analysis of the human p53 gene based on microdissection of tumor biopsy samples.

A procedure for direct sequencing of the human p53 gene based on micro-dissection of frozen tumor tissue stained with methylene blue without prior fixation is described. The approach, which is suitable for large-scale analysis, is based on solid-phase DNA sequencing of amplified genomic DNA and does not involve preparative electrophoresis, precipitations, extractions or centrifugations. Here we show that basal cell carcinoma tissue isolated from human skin can be analyzed in a semiautomated fashion using a robotic workstation and a laser fluorescent electrophoresis unit. The results of analyses of patient material displaying various immunohistochemical staining patterns using a p53-specific antibody are presented.

Base Sequence↗
PubMed1993

Human immunodeficiency virus type 1 and cytomegalovirus in saliva.

The aim of this study was to evaluate whether HIV-1 or cytomegalovirus (CMV) may contribute to oral lesions frequently found in patients with the acquired immunodeficiency syndrome (AIDS). Saliva samples from 63 HIV-1 positive patients and 21 healthy controls were tested for the presence of HIV-1 and CMV using the polymerase chain reaction (PCR) and virus isolation. CMV IgG titres in serum were also compared in the different groups. HIV-1 RNA, but not DNA, was detected in saliva from 15% (9 out of 59) of the HIV-infected patients. There was no correlation between the presence of HIV-1 RNA and oral symptoms in the patients. CMV DNA was detected significantly more frequently in samples from HIV-1 seropositive than from seronegative patients. CMV was detected in saliva from AIDS patients more often than in saliva from patients with mild or no symptoms. CMV IgG titres were also significantly higher in symptomatic than in asymptomatic patients. There was a significant correlation between the presence of CMV DNA and necrotizing gingivitis, and oral Kaposi's sarcoma in the patients, and also between these lesions and the stage of disease. This does not prove that CMV causes these oral lesions, but a direct or indirect role for CMV cannot be excluded.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Immobilization and recovery of fusion proteins and B-lymphocyte cells using magnetic separation.

A new approach to facilitate immobilization and affinity purification of recombinant proteins and selected human B lymphocytes has been developed. Using magnetic beads with attached DNA containing the Escherichia coli lac operator, fusion proteins comprising the DNA-binding lac repressor could be affinity-purified and recovered by gentle elution conditions, such as with a lactose analogue or by enzymatic means using either deoxyribonuclease (DNase) or restriction endonucleases. The results show for the first time that a DNA-binding protein can be used for affinity purification of fusion proteins as exemplified by the specific and gentle recovery of beta-galactosidase and alkaline phosphatase from bacterial lysates using immunomagnetic separation. The approach was further extended to cell separation by the efficient recovery and elution of human CD37 B lymphocytes from peripheral blood.

Alkaline Phosphatase↗
PubMed1993

Solid-phase gene assembly of constructs derived from the Plasmodium falciparum malaria blood-stage antigen Ag332.

A general method for solid-phase gene assembly on streptavidin-coated magnetic beads has been developed. The introduction of biotin in the 5'-end of the initiation oligonucleotide enables anchoring to the bead by means of the streptavidin-biotin interaction. The immobilization of one oligonucleotide enables controlled, stepwise annealing/ligation of successive 5'-phosphorylated oligonucleotides to rapidly build up predesigned gene constructs. In this report, we have assembled gene constructs of different lengths derived from the Plasmodium falciparum malaria blood-stage antigen Ag332. The encoded gene products were subsequently expressed in Escherichia coli using two parallel expression systems based on staphylococcal protein A and streptococcal protein G, respectively.

Amino Acid Sequence↗
PubMed1992

Colorimetric detection of Plasmodium falciparum and direct sequencing of amplified gene fragments using a solid phase method.

A rapid colorimetric assay for the detection of DNA from Plasmodium falciparum malaria is described, allowing direct sequencing of amplified fragments in the positive samples. The method is based on amplification by the polymerase chain reaction (PCR), with incorporation of biotin and a lac operator sequence in the amplified target DNA. The PCR product was immobilized on streptavidin-coupled magnetic beads, and detected by the specific binding of an Escherichia coli lac repressor beta-galactosidase fusion protein. Positive samples were subsequently treated with alkali to generate single stranded templates, which were used for solid phase genomic sequencing. As targets for amplification and sequencing we selected a region of the gene for the antigen Pf155/RESA and a region of the parasite dihydrofolate reductase gene (PfDHFR/TS). We show here that both of these gene targets can be used for specific detection of P. falciparum in patient blood samples. Genomic sequencing of five patient isolates revealed no variation in the Pf155/RESA gene fragment. In a comparison of this sequence with conserved protein domains, a marked similarity to the src homology region 3 was detected. A point mutation was found in the PfDHFR/TS gene fragment of one of the clinical samples, replacing Ser108 with Asn. This mutation has earlier been described in pyrimethamine and cycloguanile-resistant strains of P. falciparum.

Amino Acid Sequence↗
PubMed1992

Dynamic changes in HIV-1 quasispecies from azidothymidine (AZT)-treated patients.

The emergence of azidothymidine (AZT)-resistant human immunodeficiency virus (HIV) variants in clinical samples was studied by a direct genomic sequencing method. Sequential lymphocyte samples from four patients, who had been treated with AZT for up to 27 months, were shown to gradually accumulate multiple nucleotide changes, some of which are known to be associated with AZT resistance. Several samples were shown to contain mixtures of wild-type and mutated genomes, indicating gradual rather than sudden changes in the HIV-1 quasispecies. These results demonstrate for the first time that automated solid-phase DNA sequencing is a rapid and useful tool for investigation of antiviral drug resistance and suggest that DNA sequencing may be important in routine clinical diagnostics in the future.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Immunomagnetic recovery of Chlamydia trachomatis from urine with subsequent colorimetric DNA detection.

There is need for integrated systems that provide rapid and sensitive diagnosis of Chlamydia trachomatis. Here we describe for the first time that specific antibodies to a C. trachomatis surface protein can be used for magnetic recovery of bacteria directly from crude urine samples. The combination of immunomagnetic separation (IMS), where the bacterial cells are both purified and concentrated, with a colorimetric PCR assay makes the method sensitive and suitable for routine clinical screening in microtiter format. No filtrations, centrifugations, extractions, or electrophoretic techniques are required throughout the whole procedure. Using magnetic beads as the solid phase, the colorimetric detection of immobilized amplified nucleic acids (DIANA) also allow direct DNA sequencing of positive samples for more detailed analysis. Of the 27 urine samples analyzed, eight were found to be positive using this method. Only five out of these eight were determined to be positive by cell culture.

Adult↗
PubMed1992

Variations in the cytomegalovirus major immediate-early gene found by direct genomic sequencing.

An assay to detect and sequence DNA from human cytomegalovirus (HCMV) immediate-early gene region 1 has been developed; it involves in vitro amplification by the polymerase chain reaction and direct solid-phase sequencing of the amplified material. Urine samples from 16 patients tested positive for HCMV DNA in both a colorimetric assay for the detection of immobilized amplified nucleic acids and a standard polymerase chain reaction assay with agarose gel electrophoresis. Ten urine samples from healthy people tested negative in the same assays. Analysis of 106-bp fragments from seven patients and two laboratory HCMV strains (Ad 169 and Towne) demonstrated that the viral sequences were conserved in samples collected at different times from the same patient and in tissue-cultured samples. Two of the patient strains had variations in the amplified region, with a total of seven nucleotide substitutions yielding five amino acid alterations in the coding sequence.

Amino Acid Sequence↗
PubMed1992

Human immunodeficiency virus type 1 is present in the cerebrospinal fluid of a majority of infected individuals.

Cerebrospinal fluid (CSF) specimens from 63 patients with different severities of human immunodeficiency virus (HIV-1) infection, including asymptomatic virus carriers, were examined for the presence of HIV-1 by using polymerase chain reaction (PCR) and virus isolation. Polyadenylated RNA, presumably associated with virus particles, was extracted and reverse transcribed, and the pol region was amplified in a nested PCR. Virus could be detected in 90% of the CSF specimens examined by PCR, and data on isolation of virus from CSF were in agreement with these figures. In fact, when several CSF specimens from the same individual were studied, HIV-1 could be isolated from 80% of the patients. The presence of the viral RNA in CSF was independent of the clinical stage of infection and of neurological symptoms. These results show that the spread of HIV-1 to the brain represents an early event during infection and occurs in the majority of asymptomatic individuals.

Blood↗
PubMed1992

Persistence of azidothymidine-resistant human immunodeficiency virus type 1 RNA genotypes in posttreatment sera.

The rate of reversion from azidothymidine (zidovudine; AZT) resistance was studied by direct sequencing of human immunodeficiency virus type 1 (HIV-1) virion RNA in sera from four patients who discontinued long-term treatment. Before cessation of treatment, all four patients harbored HIV-1 with multiple mutations reported to confer AZT resistance. In three patients, slow reversions of these mutations starting after 9, 9, and 18 months were detected. The slow reversions indicate that AZT-resistant HIV-1 variants are likely to have an unaltered replicative capacity and pathogenic potential. Furthermore, there were discrepancies between the in vivo RNA sequences and the sequences of virus isolates, indicating that the isolation procedure may select for nonrepresentative virus variants.

Acquired Immunodeficiency Syndrome↗