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Background: In China, various assays for human papillomavirus (HPV) genotyping are currently used for cervical cancer screening. However, a proficiency test system is not available for standardizing and evaluating assay performance. The aim of this study was to evaluate the performance of clinical laboratories for their ability to discriminate 9 HPV types with the proficiency panel. Methods: The panel of 24 samples included cloned genomic DNAs for HPV types 6, 11, 16, 18, 31, 33, 39, 51 and 52 at different concentrations, which were distributed to 76 clinical laboratories. Results reported by participants were compared with the reference results. Results: The samples containing 106 IU HPV-16/ml and 106 IU HPV-18/ml were (98.7 and 96.0%, respectively) identified correctly most often. For other high-risk HPV types, about 90% of data sets correctly identified samples containing 106 GE/ml of HPV-6, HPV-31, HPV-33 and HPV-52, while HPV-51, HPV-11 and HPV-39 in 106 GE/ml were correctly identified by only 42.7, 55.6 and 21.3% of laboratories, respectively. Conclusion: Our proficiency test system provided a traceable panel and showed that the differences in performance between laboratories were high, indicating that it is necessary for the laboratories to improve their operation and standardization of HPV genotyping.

Objective: To observe whether or not the small-interfering RNA (siRNA) that conjugated with human immunodeficiency virus type 1 (HIV-1) TAT47–57 peptides can enter Huh-7 cells and efficiently silence hepatitis C virus (HCV) infection in cell culture. Methods: siRNA targeting the highly conserved stem loop IV of the HCV 5′ untranslated region (5′UTR) was conjugated to TAT47–57 peptides via the crosslinker sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate, and then the conjugates were added to the Huh-7 cell culture. Firefly luciferase activity and HCV RNA were asssessed using a luciferase assay reagent and real-time reverse transcript polymerase chain reaction, respectively. Results: The expression of firefly luciferase in HCV replicons and the concentration of HCV RNA were downregulated by siRNA-TAT47–57, and siRNA-TAT47–57 mediated RNA interfering activity which was directly correlated with increasing concentrations of the siRNA-TAT47–57 conjugate used. Conclusion: Cell-penetrating peptides such as HIV-1 TAT are an effective method for the delivery of siRNA targeted at 5′UTR of HCV in mammalian cells.

Objective: Transmission of HIV-1 and diagnosis of infection in hospitals and public health settings remains a worldwide concern. HIV-1 detection is sometimes not possible using current commercial assays, probably due to mismatches between the selected primers and probes. Methods: By screening primers and probes, we developed a dual-specificity probe real-time reverse transcriptase-polymerase chain reaction (DSPrtRT-PCR) assay using dual-specific armored RNA as the internal control. The specificity and sensitivity were compared between the monospecificity probe real-time and DSPrtRT-PCR techniques. Results: The sensitivity of DSPrtRT-PCR improved significantly, with no effect on its specificity. The detection limit was 173 IU/ml. All the HIV-1 group M and group O could be detected. In clinical assays, 1,000 copies/ml of armored RNA was required as internal control. When applied to negative samples, 100% specificity was achieved. Among 60 samples from the tested patients, DSPrtRT-PCR demonstrated high sensitivity, accurately detecting 50 positives and 10 negatives that were confirmed by the COBAS AmpliScreen assay. Conclusion: DSPrtRT-PCR is a more efficient and effective viral assay with high sensitivity and specificity as compared to monospecificity probe PCR. It can be widely applied in blood donor screening and qualitative individual detection of HIV-1 RNA.

Objectives: To construct a one-plasmid expression system of the armored RNA containing long chimeric RNA byincreasing the number and affinity of the pac site. Methods: The plasmid pET-MS2-pac was constructedwith one C-variant pac site, and then the plasmid pM-CR-2C containing 1,891-bp chimeric sequences and two C-variant pac sites was produced. Meanwhile, three plasmids (pM-CR-C, pM-CR-2W and pM-CR-W) were obtained as parallel controls with a different number and affinity of the pac site. Finally, the armored RNA was expressed and purified. Results: The armored RNA with 1,891 bases target RNA was expressed successfully by the one-plasmid expression system with two C-variant pac sites, while for one pac site, no matter whether the affinity was changed or not, only the 1,200 bases target RNA was packaged. It was also found that the C-variant pac site could increase the expression efficiency of the armored RNA. The armored RNA with 1,891-bp exogenous RNA in our study showed the characterization of ribonuclease resistance and stability at different time points and temperature conditions. Conclusions: The armored RNA with 1,891 bases exogenous RNA was constructed and the expression system can be used as a platform for preparation of the armored RNA containing long RNA sequences.

Objectives: In the present study, we aimed to determine whether signal-to-cutoff (S/Co) ratios of reactive anti-HCV samples could be used as a basis for avoiding the need for supplemental testing in our study population. Methods: We analyzed 901 anti-HCV-positive sera from 8 institutions in China. The Ortho VITROS anti-HCV assay and Monolisa Plus anti-HCV version 2 were used as screening assays to detect anti-HCV antibodies. Recombinant immunoblot assay (RIBA) and quantitative tests for HCV RNA were performed to validate confirmed HCV infection status. Results: Receiver operating characteristic curve analyses demonstrated that 41.5% (114/275) of true-positive samples with S/Co ratios ≤3.0 would be missed and the negative predictive value was 63.9 and 87.06%, using real-time polymerase chain reaction (RT-PCR) and RIBA as supplemental testing, respectively. 29.8% (90/302) of those who tested positive by RIBA samples were missed when only RT-PCR was used as supplemental testing. Conclusions: We determined that very low anti-HCV levels (S/Co ≤3.0), as determined by chemiluminescence immunoassay, was not an appropriate marker to preclude the need for supplemental testing in our study population. A screening strategy employing a secondary HCV antibody assay using different HCV antigens from the first assay as the supplemental testing method should be studied further. The immunoblot assay, as a supplemental testing method, is still necessary.