1.Clinical features of sporadic cases with H7N9 avian influenza in Taizhou city of Zhejiang province and characteristics of viral gene
Junyan LIU ; Shelan LIU ; Ling LIN ; Jin CHEN ; Qiupeng WANG ; Enfu CHEN ; Hongbiao LIANG ; Huazhong CHEN
Chinese Journal of Clinical Infectious Diseases 2016;9(6):491-495
Objective To examine the clinical features of sporadic patients with H7N9 avian influenzain Taizhou city of Zhejiang province and to characterize its viral genes.Methods Fifteen patients with H7N9 influenza infection confirmed by Zhejiang Provincial Centre for Disease Control and Prevention during January 201 4 and January 201 5 were included in the study.The basic diseases,poultry exposure history,clinical manifestation,laboratory examination,imaging features,treatment and outcome and viral gene sequencing were analyzed retrospectively.Results The first clinical symptoms were fever and cough in all patients,acuterespiratory distress syndrome(ARDS)occurred in 1 3 patients,the average time from onset to antiviral therapy was (7 ±2)d.Among 1 5 patients,9 survived and 6 died,including 2 died of multiple organ failure (MOF).The phylogenetic tree showed that there was highly homologous in hemagglutinin (HA)and neuraminidase(NA)genes between human H7N9 virus strains and poultry reference strains.The result of genetic sequencing indicated that human H7N9 virus strains had mutations at 226 (Q226L)sites in HA protein.Conclusions ARDS is likely to occur in patients with H7N9 viral infection,and early antiviral treatment usually leads to a good prognosis.With the occurrence of adaptive mutation in avian influenza virus H7N9,spread from poultry to the human beings may take place.
2.Efficient identification of gene knockout mutant mediated by CRISPR/Cas9 by CELⅠ crude extracts.
Wanbing LUO ; Qiupeng LIN ; Xiaoxia LI ; Zejiao ZHOU ; Huiyong YANG ; Rongyu DU ; Hongqing LI
Chinese Journal of Biotechnology 2017;33(5):775-784
CRISPR/Cas9, emerged as an efficient and powerful gene editing technology, has become the mainstream genome editing technology. Constructing mutants using CRISPR/Cas9 system is of great significance to the functional study and breeding application of useful genes. As the basis of the technology, a method for identification of mutation with efficiency and lower cost is needed. In this report, we studied the factors influencing mutation detected by CEL Ⅰ crude extracts, such as the amount of protein, enzyme incubation time, PCR buffers. Under the optimized conditions, we can integrate the mutation detection steps into one-tube reaction. We used this system to examine the mutation types and frequency of rice stn1 mediated by CRISPR/Cas9. We also used this method to identify different mutation types including homozygous, heterozygous and bi-allelic mutations. The accuracy of this method reached 100% verified by sequencing. Altogether, our results showed that using CELⅠ crude extracts was an efficient and low cost method for identification of CRISPR/Cas9 mediated mutation.