Objective: To obtain the optimum of lentiviral library packaging based on CRISPR/cas9 (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9). Methods: Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence antibody (IFA) and enzyme linked immunosorbent assay (ELISA) were used to detect the lentivirus titers in condition of different ratio of packaging plasmids, different addition of lipofectamine 3000 reagent and different time points post-transfection. Then, high-throughput sequencing was performed to evaluate the representation and distribution of single guide (sg)RNAs in the library. Results: The lentivirus titer was the highest when the molar ratio of psPAX2∶pMD2.0G∶Lentivirus library was 2∶1∶1, and the optimum addition of Lipofectamine 3000 reagent was 10 μl, while the result of ELISA were correspondent to that of RT-PCR. The IFA result showed that the lentivirus titer was the highest at 60 h post-transfecion. The coverage of sgRNAs in the lentivirus library packaged with the optimum we obtained was 99.3%, and the read counts of sgRNAs was observed in a normal distribution. Conclusions The optimal lentivirus library packaging was obtained, and this can provide basis for CRISPR/cas9-based screening.

Objective:To develop a rapid nucleic acid detection method for Zika virus (ZIKV), Dengue virus (DENV), Yellow fever virus (YFV), Chikungunya virus (CHIKV) based on microfluidic fluorescence quantitative RT-PCR technologies, in order to achieve rapid diagnosis of these four viral infections.Methods:Four sets of specific primers and probes were designed targeting the NS1 gene of ZIKV, the NS5 gene of DENV, and YFV, the E1 gene of CHIKV, respectively. The sensitivity was evaluated using in vitro transcribed RNA of ZIKV, DENV, YFV and CHIKV, and the specificity were evaluated using other viral nucleic acid. ZIKV, YFV and CHIKV detection method were verified using simulated positive samples, and DENV detection method was verified using clinical patient samples, the result of which were also compared with the quantitative RT-PCR detection method . Results:The limit of detection (LOD) of ZIKV, DENV, YFV, and CHIKV microfluidic qRT-PCR method were 14.57 copies/μl, 94.27 copies/μl, 8.25 copies/μl, and 223.19 copies/μl, respectively, and the four detection method showed no cross-reactivity with other viral nucleic acids. The prepared ZIKV, YFV and CHIKV simulated positive samples were 100% detected, and the variation coefficient of Ct value measured at each concentration were all around 2%; the 20 clinical patient specimens of DENV infection were 100% detected, which is consistent with the result of fluorescent quantitative RT-PCR detection.Conclusions:The ZIKV, DENV, YFV, and CHIKV microfluidic quantitative RT-PCR detection method showed good sensitivity, specificity, and stability. The detection could be completed within 25 minutes, which could be used for laboratory detection and early diagnosis.

Objective: To explore the method of enhancing the ability to detect HCV infection and to provide the advice for practical work. Methods: A total of 487 samples from a village in Hebei Province were selected by cluster sampling. Serum samples were detected by anti-HCV and HCV-RNA assays. The results were compared between the two tests. The correlation of HCV-RNA quantification and its anti-HCV s/n value was analyzed. Results: The positive values of anti-HCV and HCV-RNA were 22.1% and 14.2%, respectively, which were lower than that of the parallel test (χ²=4.0000, P=0.0455, χ²=42.0000, P <0.0001). The HCV-RNA quantification of the samples was positively correlated with the anti-HCV s/n value, which means anti-HCV s/n value increased with HCV-RNA quantification. Conclusion The parallel test of anti-HCV and HCV-RNA can enhance the ability to detect HCV infection.