Objective:To establish a fluorescent quantitative RT-PCR assay based on N_sgRNA of SARS-CoV-2 and preliminarily apply it on real samples.Methods:Recombinant plasmid, specific primers and probes of N_sgRNA were designed and synthesized based on Wuhan-Hu-1/2019_MN908947 and synthesis mechanism of subgenomic RNA (sgRNA). Using recombinant plasmid as amplification templates, the optimal reaction conditions and reaction system were screened according to the Ct value, fluorescence intensity, and shape of amplification curve and was evaluated for sensitivity, reproducibility, and specificity. Meanwhile, the possibility of practical application of the method was explored by testing 172 clinical samples and 256 municipal wastewater samples. Results:A qRT-PCR assay for N_sgRNA in SARS-CoV-2 was initially established. The detection limit of the assay was 20 copies/mL, and the variation coefficients of in-batch (0.002%~0.767%) and batch to batch repetition (0.016%~0.752%) were less than 1%. Only N_sgRNA recombinant plasmid was detected in the specificity assay. So the method is more highly sensitive, specific and reproducible. The RatiosgRNA/ gRNA of clinical samples HK.3, EG.5.1, JN.1 and their sub-lineages and their corresponding urban sewage samples in epidemic period were significantly different ( P<0.05). There is a strong correlation between the median of RatiosgRNA/ gRNA in clinical samples and sewage samples in the same period (correlation coefficient r=1.000, P=0.010). Conclusions:In this study, a qRT-PCR method for detecting N_sgRNA of SARS-CoV-2 was established and the method has the characteristics of higher sensitivity, stronger specificity and better repeatability, and it can be used to detect SARS-CoV-2 infectivity.

Objective:The SARS-CoV-2 has a high natural mutation rate, and dynamic monitoring of virus variants remains a key focus in current COVID-19 prevention and control efforts.Methods:In this study, a sensitive and rapid method for detecting SARS-CoV-2 omicron variant nucleic acid was established based on the BMD-PCR technology.Results:This method showed good specificity, and had no cross-reactivity with 11 common viruses transmitted via the respiratory and gastrointestinal tracts, and the limit of detection is 555 copies/ml. Compared with SARS-CoV-2 whole-genome sequencing result, among 50 samples with original Ct values ≤32 tested for the Omicron variant, 49 samples tested positive for the N679K mutation site using BMD-PCR Omicron variant detection, achieving a concordance rate of 98.00%. For 30 samples JN.1 lineage, 29 samples tested positive for the K356T mutation site using BMD-PCR JN.1 lineage detection, with a concordance rate of 96.67%. For 10 samples with original SARS-CoV-2 detection Ct values between 35 and 32, 7 samples tested positive for the N679K mutation site using BMD-PCR Omicron variant detection, Resultsing in a detection rate of 70.00%. For samples with SARS-CoV-2 nucleic acid detection Ct values>35, the detection rate for the N679K mutation site in the BMD-PCR Omicron variant was 20.00%.Conclusions:This method can serve as a high-throughput supplementary approach for the preliminary identification of SARS-CoV-2 variant genotypes.

Objective:To examine the near-complete genomic analysis of norovirus (NoV) subtype GⅡ.17 [P17] outbreaks in Beijing Chaoyang District from 2014 to 2024.Methods:Data and specimens related to outbreaks of the NoV aggregation in Beijing′s Chaoyang District from 2014 to 2024 were collected. The NoV was identified using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). Specimens with positive nucleic acid were amplified by standard PCR, whole genome sequencing and evolutionary analysis. Amino acid site variations were compared.Results:In Chaoyang District, from 2014 to 2024, a total of 637 aggregated outbreaks caused by the NoV infection were reported, of which 584 were successfully typed. The epidemic caused by the GⅡ.17 [P17] subtype accounted for 8.79% (56/637), which was the dominant epidemic gene subtype in 2014-2015, sporadic in 2016-2019, reappeared in 2022, and significantly increased in 2024 (27.27%, 24/88). Outbreaks caused by the GⅡ.17 [P17] subtype occurred mainly from October to December, with the main sites of occurrence in primary schools and kindergartens. This study yielded 53 near-complete genome sequences of the GⅡ.17 [P17] subtype from 46 incidents in Chaoyang District. The GⅡ.17 [P17] subtype sequences of Chaoyang District from 2014 to 2024 were segmented into three subgroups on the evolutionary tree, with sequences from 2014 to 2019, 2022 to April 2024, and May to December 2024 clustered into the d, e, and b subgroups, respectively. In the VP1 region′s P2 area, particularly at the HBGA binding site, subgroups b and e exhibited mutations in 22 and two sites, while subgroups b and e showed mutations in four and one sites, predominantly in the RdRp region.Conclusion:The outbreak caused by the NoV GⅡ.17 [P17] subtype in Chaoyang District from 2014 to 2024 continues, with a significant increase in 2024, and it becomes the dominant gene subtype from October to December. The sequence formation of the NoV GⅡ.17 [P17] subtype in Chaoyang District from January to April 2022 and from May to December 2024 shows two different evolutions, with specific mutation sites, requiring continuous monitoring of the NoV GⅡ.17 [P17] subtype.

Objective:To examine the near-complete genomic analysis of norovirus (NoV) subtype GⅡ.17 [P17] outbreaks in Beijing Chaoyang District from 2014 to 2024.Methods:Data and specimens related to outbreaks of the NoV aggregation in Beijing′s Chaoyang District from 2014 to 2024 were collected. The NoV was identified using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). Specimens with positive nucleic acid were amplified by standard PCR, whole genome sequencing and evolutionary analysis. Amino acid site variations were compared.Results:In Chaoyang District, from 2014 to 2024, a total of 637 aggregated outbreaks caused by the NoV infection were reported, of which 584 were successfully typed. The epidemic caused by the GⅡ.17 [P17] subtype accounted for 8.79% (56/637), which was the dominant epidemic gene subtype in 2014-2015, sporadic in 2016-2019, reappeared in 2022, and significantly increased in 2024 (27.27%, 24/88). Outbreaks caused by the GⅡ.17 [P17] subtype occurred mainly from October to December, with the main sites of occurrence in primary schools and kindergartens. This study yielded 53 near-complete genome sequences of the GⅡ.17 [P17] subtype from 46 incidents in Chaoyang District. The GⅡ.17 [P17] subtype sequences of Chaoyang District from 2014 to 2024 were segmented into three subgroups on the evolutionary tree, with sequences from 2014 to 2019, 2022 to April 2024, and May to December 2024 clustered into the d, e, and b subgroups, respectively. In the VP1 region′s P2 area, particularly at the HBGA binding site, subgroups b and e exhibited mutations in 22 and two sites, while subgroups b and e showed mutations in four and one sites, predominantly in the RdRp region.Conclusion:The outbreak caused by the NoV GⅡ.17 [P17] subtype in Chaoyang District from 2014 to 2024 continues, with a significant increase in 2024, and it becomes the dominant gene subtype from October to December. The sequence formation of the NoV GⅡ.17 [P17] subtype in Chaoyang District from January to April 2022 and from May to December 2024 shows two different evolutions, with specific mutation sites, requiring continuous monitoring of the NoV GⅡ.17 [P17] subtype.

Objective:To establish a fluorescent quantitative RT-PCR assay based on N_sgRNA of SARS-CoV-2 and preliminarily apply it on real samples.Methods:Recombinant plasmid, specific primers and probes of N_sgRNA were designed and synthesized based on Wuhan-Hu-1/2019_MN908947 and synthesis mechanism of subgenomic RNA (sgRNA). Using recombinant plasmid as amplification templates, the optimal reaction conditions and reaction system were screened according to the Ct value, fluorescence intensity, and shape of amplification curve and was evaluated for sensitivity, reproducibility, and specificity. Meanwhile, the possibility of practical application of the method was explored by testing 172 clinical samples and 256 municipal wastewater samples. Results:A qRT-PCR assay for N_sgRNA in SARS-CoV-2 was initially established. The detection limit of the assay was 20 copies/mL, and the variation coefficients of in-batch (0.002%~0.767%) and batch to batch repetition (0.016%~0.752%) were less than 1%. Only N_sgRNA recombinant plasmid was detected in the specificity assay. So the method is more highly sensitive, specific and reproducible. The RatiosgRNA/ gRNA of clinical samples HK.3, EG.5.1, JN.1 and their sub-lineages and their corresponding urban sewage samples in epidemic period were significantly different ( P<0.05). There is a strong correlation between the median of RatiosgRNA/ gRNA in clinical samples and sewage samples in the same period (correlation coefficient r=1.000, P=0.010). Conclusions:In this study, a qRT-PCR method for detecting N_sgRNA of SARS-CoV-2 was established and the method has the characteristics of higher sensitivity, stronger specificity and better repeatability, and it can be used to detect SARS-CoV-2 infectivity.

Objective:The SARS-CoV-2 has a high natural mutation rate, and dynamic monitoring of virus variants remains a key focus in current COVID-19 prevention and control efforts.Methods:In this study, a sensitive and rapid method for detecting SARS-CoV-2 omicron variant nucleic acid was established based on the BMD-PCR technology.Results:This method showed good specificity, and had no cross-reactivity with 11 common viruses transmitted via the respiratory and gastrointestinal tracts, and the limit of detection is 555 copies/ml. Compared with SARS-CoV-2 whole-genome sequencing result, among 50 samples with original Ct values ≤32 tested for the Omicron variant, 49 samples tested positive for the N679K mutation site using BMD-PCR Omicron variant detection, achieving a concordance rate of 98.00%. For 30 samples JN.1 lineage, 29 samples tested positive for the K356T mutation site using BMD-PCR JN.1 lineage detection, with a concordance rate of 96.67%. For 10 samples with original SARS-CoV-2 detection Ct values between 35 and 32, 7 samples tested positive for the N679K mutation site using BMD-PCR Omicron variant detection, Resultsing in a detection rate of 70.00%. For samples with SARS-CoV-2 nucleic acid detection Ct values>35, the detection rate for the N679K mutation site in the BMD-PCR Omicron variant was 20.00%.Conclusions:This method can serve as a high-throughput supplementary approach for the preliminary identification of SARS-CoV-2 variant genotypes.

Objective:To analyze the distribution and genetic characteristics of sporadic adult diarrhea virus in Chaoyang District, Beijing.Methods:Fecal samples from 177 adult patients with sporadic diarrhea were collected from 4 enteric outpatient clinics in Chaoyang District, Beijing from May to December 2019. Nucleic acid detection of Norovirus, Sappovirus, Rotavirus, Enteric Adenovirus and Astrovirus in the samples was performed by real-time quantitative PCR. The positive samples were amplified by RT-PCR/PCR and sequenced. The phylogenetic analysis was performed by neighbor-Joining (NJ) methods of Mega 6.0 software.Results:There were 60 of 177 (33.90%) adult sporadic diarrhea samples positive for enteric viral pathogens. Among them, 47 cases were infected with single virus, including 29 cases of Norovirus, 9 cases of Sappovirus, 8 cases of Astrovirus and 1 case of Enteric Adenovirus, in addition with 13 cases of multiple infections. None of rotavirus was detected. Partial sequences were successfully obtained for analysis, including 16 cases of GI Norovirus (7 subtypes and GI.3[P13] predominant), 10 cases of GII Norovirus (5 subtypes and GII.6[P7] predominant), 12 cases of Sappovirus (4 subtypes and GI.2 predominant), and 7 cases of Astrovirus (2 subtypes and AST-1 predominant).Conclusion:Norovirus, Astrovirus and Sappovirus are main pathogens among sporadic adult diarrhea in Beijing in 2019, and and different pathogenic gene subtypes show diverse characteristics.

Objective:To analyze the distribution and genetic characteristics of sporadic adult diarrhea virus in Chaoyang District, Beijing.Methods:Fecal samples from 177 adult patients with sporadic diarrhea were collected from 4 enteric outpatient clinics in Chaoyang District, Beijing from May to December 2019. Nucleic acid detection of Norovirus, Sappovirus, Rotavirus, Enteric Adenovirus and Astrovirus in the samples was performed by real-time quantitative PCR. The positive samples were amplified by RT-PCR/PCR and sequenced. The phylogenetic analysis was performed by neighbor-Joining (NJ) methods of Mega 6.0 software.Results:There were 60 of 177 (33.90%) adult sporadic diarrhea samples positive for enteric viral pathogens. Among them, 47 cases were infected with single virus, including 29 cases of Norovirus, 9 cases of Sappovirus, 8 cases of Astrovirus and 1 case of Enteric Adenovirus, in addition with 13 cases of multiple infections. None of rotavirus was detected. Partial sequences were successfully obtained for analysis, including 16 cases of GI Norovirus (7 subtypes and GI.3[P13] predominant), 10 cases of GII Norovirus (5 subtypes and GII.6[P7] predominant), 12 cases of Sappovirus (4 subtypes and GI.2 predominant), and 7 cases of Astrovirus (2 subtypes and AST-1 predominant).Conclusion:Norovirus, Astrovirus and Sappovirus are main pathogens among sporadic adult diarrhea in Beijing in 2019, and and different pathogenic gene subtypes show diverse characteristics.

OBJECTIVE:To optimize the formulation of ferulic acid ligustrazine (FATM) solid lipid nanoparticles (FATM-SLN),and conduct the quality evaluation. METHODS:Emulsification ultrasonic method was used to prepare FATM-SLN. Using particle size and entrapment efficiency as indexes,amount of glyceryl monostearate,egg yolk lecithin (PC),poloxamer 188 (P188),and sodium stearate as factors,single factor test and orthogonal test were used to optimize the formulation;and verifica-tion test was conducted. The appearance morphology,distribution of particle size,Zeta potential,stability and in vitro release de-gree of prepared FATM-SLN were investigated. RESULTS:The optimal formulation was as follows as FATM of 10 mg,glyceryl monostearate of 300 mg,PC of 200 mg,P188 of 200 mg,sodium stearate of 10 mg,and purified water of 20 mL. The prepared FATM-SLN showed spherical solid particles,appearance morphology was round,distribution of particle size was 40-800 nm,parti-cle size was 106.23 nm,polydispersity index was 0.254,Zeta potential was -34.8 mV,entrapment efficiency was 73.32%,drug loading was 1.20%;the appearance had no obvious changes within 10 d in 4 ℃(RSD<2%). The drug-release in 0.5-1 h was the fastest,the cumulative release degree reached to 60.47%;it tended to be stable after 8 h,the cumulative release degree reached to 93.46%,and drugs were basically released completely. CONCLUSIONS:FATM-SLN formulation is successfully optimized,and the prepared FATM-SLN has small particle size,high entrapment efficiency and good stability.

ObjectiveFrom the changes of expression of cold-inducible RNA-binding protein (CIRP) in rats with traumatic brain injury under mild hypothermia treatment with Shenfu decoction as a subsidiary, to speculate the mechanism of protective effect of the decoction on the injury.Methods Ninety Sprague-Dawley (SD) rats were divided into three groups by random number table: non-transfection control group, adenovirus mediated immune flourescent reverse transcription virus group (blank AD5-GFP transfection group) and adenovirus mediated immune flourescent reverse transcription virus carrying CIRP silent expression gene group (AD5-GFP-CIRP-SiRNA transfection group), 30 rats in each groups. Then, each group was subdivided into three subgroups: model group, traditional Chinese medicine (TCM) low and high dose groups, 10 rats in each subgroup. After the mild hypothermia treatment for 48 hours, in the TCM low dose group and high dose group, a dose of TCM 1 mL/kg and 5 mL/kg was injected via a tail vein into the rat respectively, while in the model group, 1 mL/kg normal saline was injected into the same vein, once a day for consecutive 2 days in all the groups. Before modeling in the blank AD5-GFP transfection group and AD5-GFP-CIRP-SiRNA transfection group, virus transfection models were reproduced at first by one-time intrathecal injection of 0.1 mL AD5-GFP and 0.1 mL (1×1010 pfu/mL) AD5-GFP-CIRP-SiRNA virus vector respectively, and in model group, 0.1 mL normal saline was given. The rat cortex, hippocampus and hypothalamus part were collected, the brain cell apoptosis was detected by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), the CIRP mRNA expression in the cortex, hippocampus and hypothalamus part was measured by reverse transcription-polymerase chain reaction (RT-PCR), the protein expressions of rat sarcoma protein Raf, Ras, extracellular signal-regulated kinase (ERK), phosphorylation ERK (p-ERK), mitogen activated protein kinase (MEK), p-MEK were determined by Western Blot.Results The brain tissue cell apoptosis indexes (AI) in the cortex, hippocampus and hypothalamus part in TCM low, high dose group of non-transfection control and blank AD5-GFP transfection group were lower than those in model group, and the expressions of CIRP mRNA were higher than those in model group, there were no significant differences in AI and CIRP mRNA in the cortex, hippocampus and hypothalamus between model, TCM low and high dose groups of AD5-GFP-CIRP-SiRNA transfection group, but AI was significantly higher and CIRP mRNA was significantly lower than that in corresponding subgroups of AD5-GFP transfection control group and blank AD5-GFP transfection group. Western Blot detection showed that: Raf/Ras, p-MEK/MEK protein expressions revealed no statistical significant differences in different parts of each group (allP > 0.05), the p-ERK/ERK protein expression in the cortex, hippocampus, and hypothalamus part was significantly lower in TCM low and high dose group than that in the model group of non-transfection control group and blank AD5-GFP transfection group, the degree of descent in the TCM high dose group being more significant (the cortex: non-transfection control group was 7.2±1.0 vs. 15.3±1.8, AD5-GFP transfection group was 8.1±0.7 vs. 16.2±1.5; hippocampus part: non-transfection control group was 6.6±0.8 vs. 14.7±2.0, AD5-GFP transfection group was 6.8±1.0 vs. 14.9±1.3; hypothalamus part: non-transfection control group was 9.4±1.1 vs. 12.7±1.7, AD5-GFP transfection group was 10.6±1.3 vs. 9.4±1.1, allP < 0.05). There were no significant statistical differences in p-ERK/ERK protein expression in above brain parts between AD5-GFP-CIRP-SiRNA transfection subgroups (allP > 0.05).Conclusions The Shenfu decoction used in rats with brain trauma under treatment of mild hypothermia is possibly by promoting CIRP over-expression, lowering ERK expression and inhibiting the initiation of signal transduction of the secondary transcription factor phosphorylation, thereby the neural cell apoptosis is decreased and play a subsidiary role of anti-apoptosis of mild hypothermia.