Retrospective study of Gen-Probe rapid diagnostic system for detection of legionellae in frozen clinical respiratory tract samples.

The Gen-Probe Rapid Diagnostic System for legionellae, which uses 125I-labeled cDNA directed against the rRNAs of legionellae, was evaluated for its ability to detect members of the genus by using clinical specimens which had been frozen at -70 degrees C for 2 to 8 years. Culture and direct immunofluorescence (DFA) results obtained at the time of specimen collection were used to categorize samples. The specimens tested were 112 samples culture positive for legionellae and 230 samples negative on culture and DFA tests. They were tested in a blinded and randomized fashion. Results were expressed in terms of the ratio of counts per minute of the sample to the counts per minute of the provided negative control. A ratio of greater than or equal to 4.0 was picked for optimal specificity. Of the 112 previously positive specimens, 63 (57%) were positive by the probe assay, and of the 230 previously negative samples, 228 (99.1%) were negative. The 51 discrepant specimens were reexamined by culture and DFA testing if adequate amounts remained; this was possible for 34 specimens. On repeat culture, 22 of 33 previously culture-positive samples yielded legionellae and 11 were negative. Ten of the positive repeat cultures yielded two or fewer colonies per plate. One probe-positive but previously culture-negative sample was overgrown by contaminants on repeat culture. Reanalysis of data after exclusion of the 17 unavailable, 11 repeat culture-negative, and 1 unevaluable specimen gave a probe sensitivity of 74% and specificity of 100%. The Gen-Probe test is therefore specific and is of useful sensitivity.

Enrichment of measles virus-like RNA in the nucleocapsid fraction isolated from subacute sclerosing panencephalitis brains.

A procedure has been developed which facilitates the detection of measles virus RNA sequences in human brains. The procedure involves isolating subviral components (nucleocapsids) from brain tissues prior to RNA purification, followed by hybridization of these RNAs to cDNA synthesized from measles virus 50 S RNA template. Using these techniques we were able to obtain an RNA fraction which was manyfold enriched in measles virus-specific RNA, relative to unfractionated subacute sclerosing panencephalitis (SSPE) brain RNAs 70-100% of the measles virus-specific RNA present these SSPE brain samples were recovered in this enriched fraction.

Virus detection by nucleic acid hybridization: examination of normal and ALS tissues for the presence of poliovirus.

A nucleic acid hybridization assay was developed as a sensitive assay for the presence of poliovirus RNA in human tissue. The assay could detect the presence of an average of one poliovirus per 200 cells. A method for determining the extent of degradation of the tissue RNA was developed and used to show that a significant fraction of human central nervous system (CNS) autopsy material contains highly degraded RNA which is unsuitable for hybridization studies. A total of 15 different control and amyotrophic lateral sclerosis tissues were assayed for the presence of poliovirus-like RNA. Virus RNA was detected in one of the control tissues and in none of the ALS tissues.

Detection, sizing, and quantitation of polyadenylated ribonucleic acid in the nanogram-picogram range.

A method is described for using very high specific activity [3H]poly(deoxythymidylate) [[3H]poly(dT)] to detect, size, and quantiate subnanogram amounts of nonradioactive polyadenylated RNA. Short (approximately 100 nucleotides long) [3H]poly(dT) is hybridized to the poly(adenylate) [poly(A)] tracts in polyadenylated RNAs. The RNA may then be sized and quantitated by sucrose gradient analysis. The addition of the small [3H]poly(dT) molecules does not significantly alter the s values of RNAs. The amount of [3H]poly(dT) hybridized to polyadenylated RNA increases linearly with the amount of RNA. A room temperature hydroxylapatite (HA) method has also been developed to detect and quantitate poly(A)-containing RNA after hybridization to radioactive poly(dT). S-1 nuclease (S-1) analysis can also be used to measure the poly(A) content of polyadenylated RNA to less than nanogram RNA amounts. For both the S-1 and HA approaches, the amount of [3H]poly(dT) hybridized increases with the amount of RNA and the methods can detect to as little as 10(-12) g of polyadenylated RNA with [3H]poly(dT). Greater sensitivity is possible with higher specific activity poly(dT). The approaches presented here significantly extend the uses of radioactive homopolymers to detect, quantitate, and characterize RNAs containing complementary homopolymer tracts.

Presence of virus-specific DNA sequences in murine type C viruses.

Total nucleic acids prepared from a number of murine retroviruses have been shown to contain virus-specific DNA in addition to genomic RNA. This virus-specific DNA has been shown to be at least partially double stranded and to be present within the virus core particle. The DNA isolated from the virus is greatly enriched in virus-specific DNA relative to that from virus infected cells.

Characterization of the inhomogeneous DNA in virions of bacteriophage Mu by DNA reannealing kinetics.

The DNA of bacteriophage Mu has been studied to characterize a region of inhomogeneous sequence that occurs at one end of the molecule. The kinetics of reassocation of tracer amounts of labeled host DNA in the presence of Mu DNA show that Mu DNA contains a complete selection of host sequences. These host sequences are shown to be covalently attached to phage-specific sequences and are present at a concentration that accounts for the inhomogeneity observed in the electron microscope. The significance and possible function of the host DNA attachment is discussed.

Ribosomal RNA genes of Neurospora: isolation and characterization.

DNA sequences in Neurospora crassa that code for ribosomal RNA have been isolated in a highly purified state by DNA-RNA hybridization. About 100 separate ribosomal RNA genes exist in each nucleus. These genes are composed of repeated sequences that are present about 100 times per nucleus. The individual members of this group of DNA sequences are essentially identical.

Isolation and characterization of bacterial ribosomal RNA cistrons.

The DNA sequences which code for ribosomal DNA have been isolated and purified. The technique used has general application for RNA:DNA hybridization studies and enables the isolation of any gene for which sufficient gene product can be obtained. Experiments with isolated ribosomal RNA cistrons demonstrated that (a) the majority of the ribosomal cistrons are similar to one another; (b) the cistrons which are similar to one another are virtually identical to one another; (c) ribosomal cistrons of different bacterial species are closely related to one another.