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Biomedical and Environmental Sciences 2022年第2期35卷 133-140页

作者： LI Jiang Shuai HAO Yan Zhe HOU Mei Ling ZHANG Xuan ZHANG xiao Guang cao yu xi LI Jin Ming MA Jing ZHOU Zhi xiang Faculty of environment and life Beijing University of TechnologyBeijing 100024China State Key Laboratory for Infectious Disease Prevention and Control National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China National Institute of Viral Disease Control and Prevention Beijing 100052China China Animal Health and Epidemiology Center Qingdao 266032ShandongChina

Objective To establish a sensitive,simple and rapid detection method for African swine fever virus(ASFV)B646L gene.Methods A recombinase-aided amplification-lateral flow dipstick(RAA-LFD)assay was developed in this study.Recombinase-aided amplification(RAA)is used to amplify template DNA,and lateral flow dipstick(LFD)is used to interpret the results after the amplification is completed.The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid.In addition,30 clinical samples were tested to evaluate the performance of the RAA assay.Results The RAA-LFD assay was completed within 15 min at 37°C,including 10 min for nucleic acid amplification and 5 minutes for LFD reading results.The detection limit of this assay was found to be 200 copies per reaction.And there was no cross-reactivity with other swine viruses.Conclusion A highly sensitive,specific,and simple RAA-LFD method was developed for the rapid detection of the ASFV.

关键词： African swine fever virus Recombinase aided amplification Lateral flow detection

Molecular Characterization of Full-length Genome of Japanese Encephalitis Virus Genotype V Isolated from Tibet, China

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Biomedical and Environmental Sciences 2014年第4期27卷 231-239页

作者： LI Ming Hua FU Shi Hong CHEN Wei xin WANG Huan yu cao yu xi LIANG Guo Dong State Key Laboratory for Infectious Disease Prevention and Control Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease

Objective To determine the molecular characterization of full-length genome of Japanese encephalitis virus （JEV） genotype V. Methods The full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis. Results The full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% （G I, KV1899） to 79.7% （G III, JaGAr01）, for the nucleotide sequences, and from 90.0%（G I, KV1899） to 91.8%（G III, JaGAr01） for the amino acid sequences. The open reading frame （ORF） of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides （encoded with a serine residue） were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3’-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V. Conclusion The molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.

关键词： Japanese encephalitis virus Genotype V Molecular characterization

A New Method for Ultra-sensitive P24 Antigen Assay Based on Near-infrared Fluorescent Microsphere Immunochromatography

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Biomedical and Environmental Sciences 2020年第3期33卷 174-182页

作者： WANG Qi HOU Mei Ling LIU Li Peng MA Jing ZHANG xiao Guang ZHOU Zhi xiang cao yu xi Beijing University of Technology College of Life Science and BioengineeringBeijing 100124China Beijing Runbio Biotechnology Development Co. LtdBeijing 100106China National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and PreventionBeijing 102206China

Objective To develop a rapid,highly sensitive quantitative method for detecting P24 antigen based on near-infrared fluorescent microsphere immunochromatography.Methods First,we prepared a lateral flow assay test strip,and labeled the detection antibody using a fluorescent microsphere.Second,we optimized the antibody labeling conditions.Third,we optimized the detection conditions.Fourth,we created a working curve.Fifth,we conducted a methodological assessment of the established fluorescent microsphere immunochromatography method.Sixty-six clinical samples were tested,and we compared the established fluorescent microsphere immunochromatography with the quantitative ELISA method.Results According to the working curve,the detection limit of the method is 3.4 pg/mL,and the detection range is 3.4 pg/mL to 10 ng/mL.The average intra-assay recovery was 99.6%,and the Coefficient of Variation(CV)was 5.4%–8.6%;the average inter-assay recovery was 97.3%,and the CV was 8.5%–11%.The detection rate of fluorescent microsphere immunochromatography was higher than ELISA method,and had a good correlation with ELISA.Conclusion The P24 antigen quantitative detection method based on near-infrared fluorescent microsphere immunochromatography has the advantages of rapid detection,high sensitivity,and wide detection range;thus,it is suitable for early clinical diagnosis and continuous monitoring of AIDS.

关键词： Fluorescent microsphere immunochromatography HIV P24 antigen POCT

A New Method for Blood NT-proBNP Determination Based on a Near-infrared Point of Care Testing Device with High Sensitivity and Wide Scope

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Biomedical and Environmental Sciences 2017年第6期30卷 426-431页

作者： ZHANG xiao Guang SHU Yao Gen GAO Ju WANG Xuan LIU Li Peng WANG Meng cao yu xi ZENG Yi National lnstitute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing 102206 China Beijing Runbio Biotechnology Development Co. Ltd Beijing 100106 China No.2 People's Hospital of Dali City Dali 671003 Yunnan China Department of Laboratory Medicine Beijing Hospital National Center of Gerontology Beijing 100730 China

Objective To develop a rapid, highly sensitive, and quantitative method for the detection of NT-proBNP levels based on a near-infrared point-of-care diagnostic （POCT） device with wide scope. Methods The lateral flow assay （LFA） strip of NT-proBNP was first prepared to achieve rapid detection. Then, the antibody pairs for NT-proBNP were screened and labeled with the near-infrared fluorescent dye Dylight-800. The capture antibody was fixed on a nitrocellulose membrane by a scribing device. Serial dilutions of serum samples were prepared using NT-proBNP-free serum series. The prepared test strips, combined with a near-infrared POCT device, were validated by known concentrations of clinical samples. The POCT device gave the output of the ratio of the intensity of the fluorescence signal of the detection line to that of the quality control line. The relationship between the ratio value and the concentration of the specimen was plotted as a work curve. The results of 62 clinical specimens obtained from our method were compared in parallel with those obtained from the Roche E411 kit. Results Based on the log-log plot, the new method demonstrated that there was a good linear relationship between the ratio value and NT-proBNP concentrations ranging from 20 pg/mL to 10 ng/mL. The results of the 62 clinical specimens measured by our method showed a good linear correlation with those measured by the Roche E411 kit. Conclusion The new LFA detection method of NT-proBNP levels based on the near-infrared POCT device was rapid and highly sensitive with wide scope and was thus suitable for rapid and early clinical diagnosis of cardiac impairment.

关键词： NT-proBNP Near-infrared POCT device Lateral flow assay

Real-time RT-PCR Assay for the Detection of Culex flavivirus

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Biomedical and Environmental Sciences 2015年第12期28卷 917-919页

作者： cao yu xi HE xiao xia FU Shi Hong HE Ying LI Hao GAO xiao Yan LIANG Guo Dong WANG Huan yu State Key Laboratory of Infectious Disease Prevention and Control(SKLID) Department of Viral EncephalitisNational Institute for Viral Disease Control and PreventionChinese Center for Disease Control and Prevention Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases

Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.

关键词： PCR Real-time RT-PCR Assay for the Detection of Culex flavivirus RT time

TaqMan Real-time RT-PCR Assay for Detecting and Differentiating Japanese Encephalitis Virus

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Biomedical and Environmental Sciences 2018年第3期31卷 208-214页

作者： SHAO Nan LI Fan NIE Kai FU Shi Hong ZHANG Wei Jia HE Ying LEI Wen Wen WANG Qian Ying LIANG Guo Dong cao yu xi WANG Huan yu Department of Viral Encephalitis National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing 102206 China State Key Laboratory of Infectious Disease Prevention and Control Beijing 102206 China State key laboratory for genetic engineering and molecular virology National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing 102206China School of Public Health Shandong University Jinan 250012 Shandong China Office of laboratory management National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing 102206 China

Objective To detect Japanese encephalitis virus（JEV） rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction（RT-PCR） detection system was developed.Methods By aligning the full-length sequences of JEV（G1-G5）, six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.Results With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/μL. The coefficients of variation of this real-time RT-PCR were all 〈 2.8%. The amplification efficiency of this method was between 90% and 103%.Conclusion A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.

关键词： Japanese encephalitis virus Genotype TaqMan real-time RT-PCR

Real-time RT-PCR Assay for the detection of Tahyna Virus

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Biomedical and Environmental Sciences 2015年第5期28卷 374-377页

作者： LI Hao cao yu xi HE xiao xia FU Shi Hong Lyu Zhi HE Ying GAO xiao Yan LIANG Guo Dong WANG Huan yu National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention

A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

关键词： PCR Real-time RT-PCR Assay for the detection of Tahyna Virus time RT

The Relationship between Japanese Encephalitis and Environmental Factors in China Explored Using National Surveillance Data

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Biomedical and Environmental Sciences 2018年第3期31卷 227-232页

作者： HUANG xiao xia YAN Lei GAO xiao Yan REN yu Huan FU Shi Hong cao yu xi HE Ying LEI Wen Wen LIANG Guo Dong WANG Shi Wen WANG Huan yu Office for Disease Control and Prevention National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and Prevention Harmacoepidemiology Asia-Pacific unit MSD R&D(China) Co.Ltd. Department of Viral Encephalitis National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and Prevention State Key Laboratory for Infectious Disease Prevention and Control Chinese Center for Disease Control and Prevention Institute of Remote Sensing and Digital Earth Chinese Academy of Sciences

Japanese encephalitis（JE）is a serious public health issue.This study was undertaken to better understand the relationship between JE distribution and environmental factors in China.JE data from 2005 to 2010 were ret... 详细信息

关键词： Japanese encephalitis Environmental factors Landscape

Neutralizing Antibody Titer Test of Ebola Recombinant Protein Vaccine and Gene Vector Vaccine pVR-GP-FC

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Biomedical and Environmental Sciences 2018年第10期31卷 721-728页

作者： YANG Ren ZHU Ying MA Jing HAO Yan Zhe WANG Xuan HOU Mei Ling LIU Li Peng FAN Li yun cao yu xi ZHANG xiao Guang LI xiao Jing National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and PreventionBeijing 102206 China Qingdao Municipal Center Jor Disease Control and Prevention Qingdao 266033Shandong China Qingdao Institute of Preventive Medicine Qingdao 266033 Shandong China

Objective In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. Methods A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. Results Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1：105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. Conclusion An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.

关键词： Ebola virus Recombinant subunit vaccine DNA vaccine Neutralizing antibody

A Centralized Report on Pediatric Japanese Encephalitis Cases from Beijing Children's Hospital,2013

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Biomedical and Environmental Sciences 2016年第12期29卷 902-908页

作者： LI Jiu Wei GAO xiao Yan WU yun FU Shi Hong TAN xiao Juan cao yu xi ZHANG Wei Hua YIN Zun Dongs HE Ying LI Yi xing LIANG Guo Dong XU Wen Bo FANG Fang WANG Huan yu Neurology Department Beijing Children's Hospital Affiliated to Capital Medical UniversityBeijing 100045China Department of Viral Encephalitis National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China State Key Laboratory for Infectious Disease Prevention and Control(SKLID) Chinese Center for Disease Control and PreventionBeijing 202206China Department of Polio National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China National Immunization Programme Chinese Center for Disease Control and PreventionBeijing 100050China

Fifteen pediatric cases of suspected Japanese encephalitis （JE） were reported in Beijing Children＇s Hospital during the late summer of 2013. The clinical manifestations in most cases included high fever, seizures, and abnormal magnetic resonance imaging findings. Twelve of 15 cases were laboratory-confirmed as JE cases by pathogen identification. Epidemiological investigations showed that five of the 12 laboratory-confirmed patients had an incomplete JE vaccination history. Follow-up investigations after discharge indicated that seven laboratory-confirmed JE patients without JE vaccinations had relatively poor prognoses, with an average Modified Rankin Scale （MRS） score of 2.6 when compared with the other five laboratory-confirmed, JE-vaccinated patients with an average MRS score of 0.5. The observation of pediatric JE cases among those with a history of JE vaccination warrants further attention.

关键词： Japanese encephalitis Pediatric Prognoses