Methods of packaging lentivirus library for CRISPR/cas9 -based screening / 中华实验和临床病毒学杂志

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.

Parallel test of anti-HCV and HCV-RNA can enhance the ability to detect HCV infection / 中华实验和临床病毒学杂志

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.