Development and application of a rapid scheme for detection of respiratory virus nucleic acid / 生物工程学报

This study aimed to develop a portable, accurate and easy-to-operate scheme for rapid detection of respiratory virus nucleic acid. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the effect of extraction-free respiratory virus treatment reagent (RTU) on viral nucleic acid treatment and the effect of ultra-fast fluorescence quantitative PCR instrument (FQ-8A) on nucleic acid amplification, respectively. RTU and FQ-8A were combined to develop a rapid detection scheme for respiratory virus nucleic acid, and the positive detection rate was judged by Ct value using a fluorescence quantitative PCR instrument, and the accuracy of the scheme in clinical samples detection was investigated. The results showed that RTU had comparable sensitivity to the automatic nucleic acid extraction instrument, its extraction efficiency was comparable to the other 3 extraction methods when extracting samples of different virus types, but the extraction time of RTU was less than 5 min. FQ-8A had good consistency in detection respiratory syncytial virus (RSV) and adenovirus (ADV) compared with the control instrument ABI-7500, with kappa coefficients of 0.938 (P < 0.001) and 0.887 (P < 0.001), respectively, but the amplification time was only about 0.5 h. The RTU and FQ-8A combined rapid detection scheme had a highly consistent detection rate with the conventional detection scheme, with a sensitivity of 91.70% and specificity of 100%, and a kappa coefficient was 0.944 (P < 0.001). In conclusion, by combining RTU with FQ-8A, a rapid respiratory virus nucleic acid detection scheme was developed, the whole process could be completed in 35 min. The scheme is accurate and easy-to-operate, and can provide important support for the rapid diagnosis and treatment of respiratory virus.

Construction of real-time polymerase chain reaction detection for infection-related cytokines of tree shrew / 生物医学工程学杂志

Tree shrew is a novel and high-quality experimental animal model. In this study, the real-time polymerase chain reaction methods were established to detect infection-related cytokines interleukin-6 (IL-6), IL-8, IL-10, IL-17A, interferon-γ (IFN-γ) and housekeeping gene glyceraldehyde-phosphate dehydrogenase ( ) of tree shrew. The results indicated that the establised methods had good specificity. The high point of the linear range of these reagents reached 1 × 10 copies, and the low points ranged from 10 copies (IL-6, IL-17A), 100 copies (IL-10, ) to 1 000 copies (IL-8, IFN-γ). In this interval, the linear correlation coefficient of each reagent was greater than 0.99. The lowest detectable values of IL-6, IL-8, IL-10, IL-17A, IFN-γ and were 8, 8, 4, 8, 128 and 4 copies, respectively. The results showed that the established detection methods had good specificity, sensitivity and wide linear range. The methods were suitable for detection of multiple concentration range samples, and could be used for the subsequent studies of tree shrew cytokines.

Construction of human adenovirus type 4 vector expressing enhanced green fluorescence protein / 中华微生物学和免疫学杂志

Objective To prepare human adenovirus type 4 (Ad4) vector expressing enhanced green fluorescence protein (EGFP). Methods This study used a previously prepared plasmid pBRAd4 containing the whole genome DNA of Ad4-GZ01 strain. The Ad4 genome E3 region of pBRAd4 was deleted and replaced with the EGFP expression frame by conventional molecular cloning method. Then the recombi-nant plasmid was transfected into AD293 cells to rescue recombinant virus which was identified by sequen-cing,SDS-PAGE and ELISA. The purified virions were injected to mice and the induced immune responses were detected by ELISA and microneutralization test. Results The recombinant Ad4 vector rAd4EGFP ex-pressing EGFP was obtained and could be recognized and neutralized by monoclonal antibody MN4b and an-tisera against Ad4. The Ad4-specific and EGFP-specific antibodies with high titers could be detected in mice immunized with rAd4EGFP. Conclusion Human Ad4 vector expressing EGFP was successfully obtained and could be used in research on vaccine development,drug evaluation and transgene vector.

Analysis of cystic fibrosis transmembrane conductance regulator gene mutations in Chinese children with cystic fibrosis / 中华实用儿科临床杂志

Objective To summarize the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation spectrum in Chinese children with cystic fibrosis(CF).Methods The data of Chinese children with CF reported in China national knowledge infrastructure,wanfang database,VIP journal database,PubMed were collected.The CFTR gene mutations of the patients retrieved and summarized,1 case diagnosed in the First Affiliated Hospital,Guangzhou Medical University were summarized.Inclusion criteria included:data from published literature,the cases reported were Chinese children with CF and with CFTR gene mutations.Exclusive criteria included:repetitive reports,undiagnosed patients,or patients without CFTR gene mutations.Results There were 58 Chinese children with CF,and 61 CFTR gene mutations were found.The CFTR gene mutations were ranked in order from more to less as the following:c.2909G→A (p.G970D) (9 times);1898 ±5G→T,which was not found in Caucasians,and c.263T→G(6 times respectively);c.3196 C--→T,c.1766 ± 5 G→T,c.3068 T→ G (5 times respectively);2215 insG,c.1666A→ G (4 times respectively);G2816A,c.293A→G,c.595C→T,c.326A→G (3 times respectively);c.3635delT,c.2907A→C,c.648 G→A (W216X),c.960_961insA (1092insA),c.1075C→T,c.1699G→T,c.2491-126T→C,c.3307delA and c.110 C→G were novel observation.△F508 was not found.Conclusions The most common CFTR gene mutation is c.2909G→A (p.G970D) in Chinese children with CF.△ F508 which is the most common mutation in Caucasian not found in Chinese children with CF.The gene mutation spectrum of CFTR in Chinese children with CF is significantly different from those in European and American countries.