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A nested primer-based polymerase chain reaction was constructed for the detection of Puumala virus RNA in patient samples. Puumala virus RNA was detected in cells from the urinary and the respiratory tracts and in peripheral blood mononuclear cells of patients with nephropathia epidemica. After inoculation with nephropathia epidemica patient material on Vero E6 cells and propagation for nine passages (4 months), Puumala virus RNA was detected at every passage. Hybridization under high-stringency conditions revealed that the overall nucleotide homology between the different patient isolates and the prototype strain (Puumala) is high. Using cDNA from Hällnäs B1 strain as a probe, hybridization occurred only under low-stringency conditions.

The aim of this study was to increase the sensitivity of an earlier version of an HIV-2 nested PCR assay based on primers in the gag, pol, LTR, and env regions. The assay was first optimized with regard to concentrations of dNTP, MgCl2, and primers, using a method that allowed optimization of all three parameters in only two test runs. We then designed and optimized new primer sets in the LTR, gag, and gag/pol regions that were based on more isolates than were the former (old) primer sets. Samples from 57 HIV-2 antibody-positive individuals were tested with the four old primer sets as well as with the three new primer sets. Five primer sets from this run (new gag, new gag/pol, old LTR, old env, and new LTR) were then tested with 35 more samples, giving a total number of 92 tested samples from HIV-2-infected individuals. At initial testing of the 92 samples a combination of primer sets from two different regions yielded a sensitivity ranging from 93.5 to 98.9%. After repeated testing the sensitivity ranged from 96.7 to 100% for the different primer combinations. The specificity was 100% for all primer sets except old LTR, which had a specificity of 97%. In conclusion, it is possible to create a more sensitive PCR assay by optimizing the different PCR parameters as well as by including primer sets based on more isolates.

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This study compared the performance of several in-house nested PCR systems and the Amplicor human immunodeficiency virus type 1 (HIV-1) PCR kit in the detection of HIV-1 DNA in Tanzanian samples prepared by two different methods. All six of the in-house primer sets evaluated had a higher sensitivity for HIV DNA detection in samples prepared by the Amplicor PCR sample preparation method than in those prepared by the Ficoll-Isopaque (FIP) density gradient centrifugation method. A sensitivity of 100% was achieved by combining two in-house primer sets. The sensitivity of the standard Amplicor HIV-1 PCR kit was only 59%, whereas a modified Amplicor HIV-1 PCR test had a sensitivity of 98%. Our data show that Tanzanian samples prepared by the Amplicor preparation method are more suitable for HIV-1 PCR testing than samples prepared by the FIP method. The modified, but not the standard, Amplicor HIV-1 PCR kit provides an alternative to the nested in-house PCR technique for the diagnosis of HIV infection.