Detection of Bordetella pertussis in clinical specimens by PCR and a microtiter plate-based DNA hybridization assay.

In order to improve detection of Bordetella pertussis in nasopharyngeal aspirates (NPAs) in our laboratory, a PCR-based assay was optimized, and a study was designed (i) to compare results obtained by PCR to those obtained by culture and (ii) to evaluate a novel microtiter plate-based DNA hybridization assay (PCR-plate) by comparing it to agarose gel electrophoresis (PCR-gel) for detection of the PCR product. DNA for the PCR was extracted with a guanidine thiocyanate buffer and used in a PCR mixture containing primers directed against a reiterated gene sequence in B. pertussis (Q. He, J. Mertsola, H. Soini, M. Skurnik, O. Ruuskanen, and M. K. Viljanen, J. Clin, Microbiol. 31:642-645, 1993). Of 96 NPAs submitted from a targeted study group, 23 were positive by culture, 27 were positive by PCR-gel, and 31 were positive by PCR-plate. All culture-positive specimens were also positive by PCR. Of nine patients with culture-negative-PCR-positive results, six had discharge diagnoses of pertussis. Thus, PCR with plate-based product detection is a sensitive method for the laboratory detection of B. pertussis in NPAs. Additional advantages of the plate assay include rapidity, objectivity in reading results, specificity, and the capability of being adapted to a high-volume, automated system.

Microplate-based DNA hybridization assays for detection of human retroviral gene sequences.

Nonisotopic, microwell-based DNA hybridization assays for the specific detection of human immunodeficiency virus type 1 (HIV-1) gag, human T-cell lymphotropic virus type I (HTLV-I) pol, and HTLV-II pol DNA sequences were evaluated. The performances of these detection kits (Gene Detective enzyme oligonucleotide assays; Cellular Products, Inc., Buffalo, N.Y.) were assessed by using clinical samples whose infection status were established by amplification by PCR and then liquid hybridization detection by using virus-specific probes. Peripheral blood mononuclear cell lysates from 59 HIV-1-, 35 HTLV-I-, and 19 HTLV-II-infected individuals and from 15 healthy blood donors were used as substrates for PCR amplification. The results of the study demonstrated a clinical sensitivity of 100%. In addition, the enzyme oligonucleotide assays were able to detect 1 to 10 proviral copies subsequent to PCR amplification, indicating an analytical sensitivity comparable to that of liquid hybridization.

Intragenic and intergenic recombination between temperature-sensitive mutants of vaccinia virus.

The frequency of recombination for a complete set of two-factor crosses between vaccinia virus mutations separated by distances of between 54 and 10692 bp was determined. The results show that in intragenic crosses there is a linear relationship between the recombination frequency observed and distances between the mutations of up to 700 bp. However, no length dependence of the recombination frequency in intergenic crosses with a distance between mutations of 328 to greater than 10000 bp is observed. We attribute this lack of dependence to the high rate of viral DNA interchange, which makes some step other than the cross-over event rate-limiting. Furthermore, we believe that the observed difference in recombination frequency between inter- and intragenic recombination is due to complementation between temperature-sensitive mutants at the permissive temperature.

Genetic and biochemical characterization of vaccinia virus genes D2L and D3R which encode virion structural proteins.

Polyclonal antisera raised against fusion proteins containing portions of the vaccinia virus D2L and D3R proteins were prepared. Immunoprecipitation of pulse-labeled infected cell extracts and Western blot analysis demonstrated that genes D2L and D3R encode 16.9- and 27-kDa proteins, respectively. Both are synthesized late during infection and there is no evidence for proteolytic processing of either protein. Western blots of purified virus and subvirion fractions showed that D2L and D3R are virion components, residing in a detergent-insoluble fraction, containing viral core structural proteins. Trypsin sensitivity experiments suggest that each is found in an equivalent position within the virus core. Pulse-chase analysis showed that both proteins exhibit biphasic stability in which an unstable nascent component is replaced by a stable form. This observation suggests that the stable component results from the insertion of D2L and D3R into an immature core structure. The DNA sequence of four ts mutants previously mapped to genes D2L and D3R is reported. Analysis of the ability of each mutant to synthesize and process viral proteins showed that protein synthetic patterns were indistinguishable from wild type, however, three of the four mutants were defective in the processing of the major virion structural precursor, p4a. Unlike the biphasic stability observed in wild-type infected cells, D2L and D3R were totally degraded in cells infected at 40 degrees with any of the four ts mutants. Stability of the D2L and D3R proteins, in cells treated with rifampicin, is unaffected which demonstrates that a block in morphogenesis is not directly responsible for the observed instability of the mutant proteins.