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The frequency of recombination for a complete set of two-factor crosses between vaccinia virus mutations separated by distances of between 54 and 10 692 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.

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.

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%k. 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.

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