New highly sensitive and accurate lyophilized real-time RT-PCR tests for early detection of avian influenza.

New lyophilized real-time reverse transcription (RT)-PCR avian influenza detection assays were designed and tested. The M-gene assay detects all avian influenza virus (AIV) subtypes, and the H5 and H7 specific assays can discriminate the AIV subtypes H5 and H7 of Eurasian origin. The assays are formulated in a lyophilized bead format containing an internal positive control to monitor inhibitors in the reaction. Fifty-six AIV cultured isolates covering all 16 hemagglutinin types and 44 positive swabs from an outbreak of AIV in turkeys (H5N1 highly pathogenic avian influenza) were used to determine analytical performance and diagnostic sensitivity of these veterinary assays. The lyophilized real-time RT-PCR assays were demonstrated to be more sensitive than the wet assays, being able to detect down to 4 to 16 molecules of synthetic target RNA compared to 16 to 80 molecules for the corresponding wet assays. The diagnostic sensitivity of the lyophilized M-gene assay was determined to be 97.7% (43/44), whereas concurrent testing of these samples with the wet assay was only 86.3% sensitive (38/44). Using a panel of 19 noninfluenza respiratory and enteric pathogens, the analytical specificity of the M-gene assay was shown to be 100%. High diagnostic specificity of the assays was also confirmed by testing 496 negative swab samples from a combination of wild bird species and poultry.

Analysis of insertions and deletions in the gag p6 region of diverse HIV type 1 strains.

Sequence variation in the gag p6 region in subtype B HIV-1 has been associated with changes in viral replication capacity and antiretroviral drug susceptibility. We examined sequence variation in the HIV-1 gag p6 region using plasma samples from 22 individuals with non-subtype B HIV-1 infection [subtypes A, C, D, F, and G, and circulating recombinant forms (CRFs) CRF01-AE and CRF02_AG]. An additional 105 gag sequences from the Los Alamos National Laboratory database were also analyzed. Extensive length variation was observed in the p6 gag region. Specific patterns of insertions and deletions were observed in different subtypes and CRFs, and no two subtypes or CRFs had the same general pattern. PTAP duplications were more common in subtype C than other strains (3 of 14 in subtype C vs. 2 of 113 in other strains, p = 0.004), and KQE duplications were seen only in subtype B. Further studies are needed to determine whether such genotypic differences influence viral replication capacity, antiretroviral drug susceptibility, or other phenotypic properties of these strains.

Evaluation of novel H1N1-specific primer-probe sets using commercial RT-PCR mixtures and a premixed reaction stored in a lyophilized format.

The recent emergence of a novel H1N1 influenza A virus in humans caused the first influenza pandemic of this century. Many clinical diagnostic laboratories are overwhelmed by the testing demands related to the infection. Three novel H1N1-specific primer-probe sets reported during the early phase of the pandemic were tested in three commercial real-time RT-PCR mixtures. The amplification efficiencies and detection limits of these assays were determined. A ready-to-use premixed RT-PCR stored in a lyophilized format was developed. The detection limits of the studied assays were highly variable, ranging from 1.68E-01 to 1.68E-05 TCID(50) per reaction. The detection limit of the lyophilized reaction mixture was found to be 1.68E-05 TCID(50) per reaction, but the amplification efficiency of the assay was lower than those deduced from the other assays. All respiratory samples from infected patients and all control nasopharyngeal aspirates were positive and negative, respectively, in the newly developed assay. The results highlighted that, to enhance the sensitivity of an assay, it is essential to evaluate a primer-probe set with different commercial RT-PCR assays. This study also demonstrated the feasibility of using lyophilized reaction mixtures for the molecular diagnosis of novel H1N1.