Marjut Ranki

Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples

A method based on three-DNA-component, sandwich hybridization has been designed for the detection and quantitation of nucleic acids in crude samples using adenovirus DNA as a model. Two non-overlapping restriction fragments of adenovirus type 2 (Ad2) DNA were cloned into two vectors, the pBR322 plasmid and M13 phage. The recombinant plasmid DNA was immobilized onto nitrocellulose filters and the single-stranded recombinant phage DNA was labeled with 125I and used as a probe. When these two reagents were incubated under annealing conditions no radioactivity became filter-bound; only if denatured adenovirus DNA was added as the third reagent, it mediated the attachment of the radioactive probe to the filters. Hybridization efficiency was shown to be dependent on both the filter and probe DNA concentrations and on the hybridization conditions. When standardized, the assay is quantitative, and under the conditions used 0.2 ng of adenovirus DNA (8 X 10(-6) pmol) could be detected by an overnight incubation. The test is suitable for crude samples, e.g., solubilized cell extracts, without any purification steps. Less than 100 cells infected with Ad2 can be detected, implying that the assay could be applicable to virus diagnostics.

NOVEL TEST FOR RAPID VIRAL DIAGNOSIS: DETECTION OF ADENOVIRUS IN NASOPHARYNGEAL MUCUS ASPIRATES BY MEANS OF NUCLEIC-ACID SANDWICH HYBRIDISATION

Nucleic-acid sandwich hybridisation detected adenovirus in nasopharyngeal aspirates from children with acute respiratory infections within 20 h. The differentiation between subgroups of adenovirus (B and C) and the lack of false positives indicated the specificity of the method. The reference test was detection of an adenovirus protein (hexon) by means of radioimmunoassay. In nucleic-acid sandwich hybridisation the sample DNA mediates the binding of a labelled probe DNA fragment to a second DNA fragment bound on filter. The sample DNA itself is not fixed to a solid support, and the only pretreatment required is boiling in a detergent solution. The results indicate that the sandwich hybridisation is specific, quantitative, easy to do, and only marginally affected by the crude nature of the sample. It may therefore be a valuable addition to the range of rapid methods in microbial diagnosis.

Solubilized RNA replication complex from Semliki Forest virus-infected cells.

Abstract The postmitochondrial pellet from SFV-infected cells was incubated under in vitro RNA-synthesizing conditions in the presence of [ 3 H]UTP to label nascent chains in the RNA replication complex. The solubilized complex was purified by centrifugation in a glycerol gradient followed by a sucrose gradient in which it sedimented with a peak at 34 S. It contained double-stranded RNA which by mild ribonuclease treatment was converted to replicative forms I, II, and III, indicating that it was derived from replicative intermediates synthesizing 42 S and 26 S RNA. A similar structure was also isolated from infected cells labeled with [ 3 H]uridine early in infection. The only virus-specific proteins associated with the complex were ns70, ns72, and ns86. The presence of ns70 and ns72 was confirmed by tryptic peptide mapping.

Semliki Forest Virus Replication Complex Capable of Synthesizing 42S and 26S Nascent RNA Chains

A complex synthesizing Semliki Forest virus (SFV)-specific RNAs was purified from infected HeLa cells. During purification, the RNA-synthesizing complex was monitored by the presence of RNA chains synthesized during a 1 min pulse in vivo and the ability to synthesize 42S and 26S RNAs in vitro. Finally, the protein composition of the replication complex was analysed. Thirty to 40% of the pulse-labelled RNAs and 10 to 25% of the polymerase activity present in the postnuclear supernatant were recovered in smooth membranes. At this stage of purification single stranded 42S and 26S RNA were synthesized and released from the replication complex in vitro. After treatment of the smooth membrane fraction with Triton X-100 the replication complex was solubilized. When analysed by sucrose gradient centrifugation, the solubilized replication complex distributed heterogeneously. It had reduced RNA polymerase activity, but was still able to synthesize both 42S and 26S nascent RNA chains which were not released from RIs and RFs. The non-structural protein ns70 was the major virus-specified component associated with the replication complex.

Affinity-based collection of amplified viral DNA: application to the detection of human immunodeficiency virus type 1, human cytomegalovirus and human papillomavirus type 16.

We have devised a sensitive and convenient hybridization technique by combining the polymerase chain reaction (PCR) with affinity-based hybrid collection. In this method 5-biotinylated primers are used to introduce biotin residues into the DNA fragments during the amplification. The amplified DNA fragments are detected by liquid hybridization using a 32P- or 35S-labelled oligonucleotide as probe. For measurement the hybrids are collected on polystyrene microparticles or onto microtitre wells taking advantage of the biotinavidin interaction. The method is highly sensitive allowing the detection of 30 molecules of DNA. It involves few and simple operations, and is thus suitable for routine diagnostics. The applicability of the method to the detection of HIV-1 DNA from blood, HCMV DNA from urine and HPV-16 DNA from cervical scrapes was evaluated.

SEMLIKI FOREST VIRUS-SPECIFIC NONSTRUCTURAL PROTEIN IS ASSOCIATED WITH RIBOSOMES

Received 25 September 1979 1. Introduction Semliki Forest virus (SFV) 42 S RNA genome codes probably for eight different polypeptides. Four of these are nonstructural proteins with mol. wt 70 000 (ns70), 86 000 (ns86), 72 000 (ns72) and 60 000 (ns60). They are translated as a giant polyprotein in the above order [ 1,2]. At least three of them have been found in association with the RNA replication com- plex [3]. The structural proteins, capsid protein and envelope glycoproteins El, E2 and E3, are translated from a subgenomic 26 S mRNA as a 130 000 mol. wt polyprotein [4,5]. The amino-terminal capsid protein is cleaved from the growing polyprotein before the translation of the envelope proteins starts [S ,6]. The nascent envelope proteins penetrate the ER membrane, become glycosylated and are transported to the plasma membrane [ 5,6]. After cleavage the capsid protein remains associated with the polysomal ribosomes until it is transferred to the nucleocapsid (which consists of 42 S RNA and capsid proteins, [4,5]) or is released to the monosome pool after the translation of the polyprotein has been completed [7-91. Here we report that one of the nonstructural pro- teins, ns86, is also associated with ribosomes and can be crosslinked with the ribosomal RNAs by ultraviolet irradiation. 2. Materials and methods The origin and cultivation of the SFV prototype strain in HeLa and BHK21 cells have been described [3,8]. For labeling of ribosomal RNAs the cells were

Nucleocapsid and Envelope Protein of Semliki Forest Virus as Affected by Canavanine

SummarynCytoplasmic nucleocapsids of Semliki Forest virus were formed in BHK21 cells treated with canavanine under such conditions that virus RNA synthesis continued but no infectious virus was released. On the other hand, envelope protein synthesis was strongly inhibited. The canavanine nucleocapsids formed sedimented at 140s and contained 42s virus RNA and a protein indistinguishable from the normal protein by polyacrylamide gel electrophoresis. The canavanine nucleocapsids were not released from the cells.

Nucleic acid sandwich hybridization in adenovirus diagnosis.

The usefulness of viral diagnosis is critically dependent on the rapidity by which it is able to detect and identify virus taken from the site of infection. The methods suitable for rapid diagnosis so far available are mostly based on immunological detection of the virus and its antigenic components (for a review, see Halonen et al., this volume). An alternative to these techniques worth considering is provided by nucleic acid hybridization techniques to detect virus-specific nucleic acid sequences in the sample (for principles and applications see reviews by Bornkamm et al. and Pettersson et al., this volume).

Detection of Human Papillomavirus DNA by AffiProbe HPV-DNA Test Kit in Cervical Scrapes or Biopsies-Histopathologic Correlates

Objective: The aim of this study was to evaluate and compare the efficacy of punch biopsies and cervical scrapes in the detection of human papillomavirus (HPV) DNA from the cervix and compare the results with the histopathologic diagnosis. Methods: The specimens were collected simultaneously, and HPV DNA was detected using a liquid hybridization test. Results: Biopsies and scrapes were equally efficient, but each detected only two-thirds of all HPV-DNA-positive patients. Thus, the positivity rate increased when both tests were used. Overall, 13% of patients with normal histopathology, 38% of patients with benign atypia, and 66% of patients with squamous intraepithelial lesions (SIL) were HPV-DNA positive. HPV-DNA 16 was found in 54% of HPV-DNA-positive patients with SIL, in 20% of HPV-DNA-positive patients with atypia, and in none of patients with normal histopathology. Conclusions: The liquid hybridization test used in this study detects HPV DNA equally efficiently from both biopsies and scrapes. The test can be performed in 1 working day. However, the sensitivity of the test is low, and it only detects a limited number of HPV types.