OBJECTIVE:To establish a simple and rapid preparation technique of Rifampicin eye drops.METHODS:The calculating quantity of hydrochloric acid was used to dissolve Rifampicin,then the equimol quantity of potassium hydroxide was added to neutralize the acid and yield potassium chloride,whose quantity was designed according to the prescrip?tion.RESULTS:Neutralization reaction not only overcame the difficulty of Rifampicin's dissolution in water,but also avoided the irritation to eyes caused by using ethanol as solvent.CONCLUSION:The method is well-versed in design,simple in preparation and controllable in quality.

Objective: To evaluate the effectiveness of droplet digital PCR (ddPCR) assay for detection of neuraminidase (NA) H275Y mutations in influenza A H1N1 pdm09 virus. Methods: The primers and dual probes were designed based on the sequence of the H1N1 pdm09 NA gene fragment which contained 275 amino acid sites, and the annealing temperature of ddPCR assay was optimized to establish a method for detection of H275 drug-sensitive genes and H275Y drug-resistant genes in H1N1 pdm09 virus. The sensitivity of ddPCR assay and fluorescent quantitative PCR (qPCR) assay was compared using the detection limit, and the specificity of ddPCR and qPCR assays was compared for detection of 14 respiratory virus samples. In addition, 64 clinical samples and 5 influenza isolates were tested to calculate the abundance of H275Y mutations, and the mutation abundance of 5 influenza isolates was compared with next-generation sequencing results. Results: The optimal annealing temperature was 62.2 ℃. The detection limits of ddPCR assay were 5.28 (95%CI: 4.28-7.45) copies/reaction for H1N1 pdm09 H275 drug-sensitive plasmids and 6.51 (95%CI: 5.25-9.37) copies/reaction for H1N1 pdm09 H275Y drug-resistant plasmids, and the detection limits of qPCR assay were 5.70 (95%CI: 4.83-7.45) copies/reaction for H1N1 pdm09 H275Y drug-sensitive plasmids and 7.06 (95%CI: 5.92-9.40) copies/reaction for H1N1 pdm09 H275Y drug-resistant plasmids. Both ddPCR and qPCR assays detected H275 and H275Y drug-resistant plasmids in H1N1 pdm09 viral samples but did not detect H275 and H275Y drug-resistant plasmids in other 11 respiratory virus samples, and these two assays showed consistent results. Of the 64 clinical samples, ddPCR assay detected H275Y mutation in three pharyngeal swab specimens from a severe pneumonia patients infected with H1N1 pdm09 virus, and the greatest mutation abundance was detected in samples collected on day 4 post-treatment with oseltamivir phosphate (53.37%). ddPCR assay detected 0.63, 88.93%% and 1.27% H275Y mutation abundance in samples collected on days 2, 4 and 5 post-treatment with oseltamivir phosphate, and next-generation sequencing detected 89.46% H275Y mutation abundance in samples collected on day 4 post-treatment with oseltamivir phosphate; however, no H275Y mutation was detected in samples collected on days 2 or 5 post-treatment with oseltamivir phosphate. Conclusions ddPCR presents a higher sensitivity and specificity than qPCR assay for detection of H275Y mutations in H1N1 pdm09 virus, and presents a higher sensitivity than next-generation sequencing for detection of low-frequency mutations, which is effective for quantitative detection of H275Y mutations in the NA fragment of the H1N1 pdm09 virus.

Objective: To identify the avian influenza virus subtype from the avian and environmental samples using the Ion Torrent new-generation semiconductor sequencing technology and to establish a high-throughput sequencing method to identify unclassified influenza A virus. Methods: Virus RNA was extracted from the nine avian swab and environmental samples and real-time RT-PCR was carried out to detect universal fluA, H5N1, H7N9 and H9N2. The whole genome of influenza A virus was amplified by PathAmpFluA kit. Sequencing library was prepared using Next Fast DNA Fragmentation & Library Prep Set for Ion Torrent kit and high-throughput sequencing was done by Ion Torrent Personal Genome Machine(PGM). Data from the PGM was processed and quality evaluated using Ion TorrentSuite v3.0 software. Sequence assembly and influenza database blast were carried out by FluAtyping v4.0 and PathogenAnalyzer bioinformatics software to identify the influenza A virus subtype of these nine samples. Results: The results of real-time RT-PCR for universal fluA of these nine samples were positive but the results for H5N1, H7N9 and H9N2 were negative. Seven subtypes of influenza A virus were identified by high-throughput sequencing and bioinformatics analysis: six samples were H2N3, H5N6, H5N8, H7N1, H7N7, H11N3 subtype respectively and three samples were H6N6 subtype. Conclusions Avian influenza virus has many subtypes in the environment of Zhejiang province. Ion Torrent semiconductor sequencing technology is suitable for fast identification of unclassified influenza virus for avian influenza environment monitoring.

Objective:Using the Taqman low-density array (TLDA) to develop a detection method which can rapidly type influenza A virus and the subtypes of H1-H16 and N1-N9, which is suitable for rapid typing and identification of influenza A in influenza like cases and avian influenza samples.Methods:The primers and probes of influenza A, H1-16 subtypes, N1-9 subtypes were designed and pre-customized on the TLDA reaction chip; Optimize the TLDA annealing temperature; The sensitivity of TLDA was evaluated by 10 fold gradient dilution of influenza A virus nucleic acid of various subtypes and the digital PCR; The specificity of the TLDA was verified by influenza B, adenovirus, respiratory syncytial virus and other respiratory viruses. At the same time, a variety of influenza A viruses that have been clearly typed were selected to evaluate the specificity and effectiveness of TLDA; The TLDA was used to detect 16 samples of influenza like cases and 24 samples of external environment of avian influenza.Results:The best annealing temperature of TLDA was 60 ℃, the detection sensitivity of TLDA was between 1.52 and 8.00 copies/μl for H1N1, H3N2, H5N6, H7N9 and H9N2, there was no specific cross reaction with influenza B, avian influenza, adenovirus, respiratory syncytial virus and other respiratory viruses. A total of 12 influenza positive samples and 24 samples from environmental surveillance of avian influenza were detected by TLDA, all of the result of samples were correct in typing.Conclusions:The high-throughput detection method of influenza A virus based on TLDA has good sensitivity and specificity, and the result of clinical samples show that the TLDA is suitable for rapid detection of influenza like cases and avian influenza samples, especially for rapid subtype screening and identification of new influenza A virus that cannot be identified by conventional commercial kits.

Objective: The objective of this study was to establish a next generation sequencing (NGS) method for severe fever with thrombocytopenia syndrome virus(SFTS). Methods: SFTS virus RNA was extracted from the patient serum inoculated and isolated by Vero cells. Two methods of random primer sequencing and oligo(dT) beads selection sequencing were used for library construction. The libraries were built based on the best amplification and purification conditions. Whole genome sequencing was performed on NGS platform. Results: There were significant differences in data of 3 virus between the two methods.The sample was sequenced by random primer sequencing showed low coverage and depth. However, three samples were sequenced by oligo(dT) beads selection showed coverage was over 99% and depth was over 900.The alignment rate of database from NCBI was more than 90%. The initial detection quality of this method was 300ng RNA. Conclusions In this study, we used the method of oligo(dT) beads selection to build libraries, and established a SFTS virus genome detection based on next generation sequencing.

Aedes ; virology ; Animals ; China ; epidemiology ; Genetic Variation ; Genome, Viral ; Humans ; Multigene Family ; Viral Proteins ; genetics ; Zika Virus ; classification ; genetics ; isolation & purification ; Zika Virus Infection ; epidemiology ; virology