Objective:To establish a quick on-site emergency detection method for severe fever with thrombocytopenia syndrome virus (SFTSV), dengue virus (DENV), and hantaan virus (HTNV).Methods:This research was based on the traditional TaqMan fluorescent probe technology, using the domestic rapid one-step quantitative RT-PCR kit, combined with the Magnetic induction cycler (Mic) qPCR instrument. The detection limit, specificity and repeatability of this method were evaluated by simulated samples, other virus infected samples and normal human blood samples.Results:Compared with the traditional RT-PCR assay, the required time of this method was greatly shortened, and the detection can be completed within 35 minutes. The limit of quantitation for SFTSV, DENV and HTNV are less than 100copies/PCR. No nonspecific amplification was found in the simulated negative samples and other virus infected samples. All the simulated positive sample for verification could be detected, and coefficient of variation Ct value of each group was less than 4%. Conclusions:The rapid fluorescence quantitative RT-PCR assays have certain application prospects for on-site emergency detection, and provide important technical supports and new directions for the prevention and control of common hemorrhagic fever viruses.

目的 建立新型冠状病毒(SARS-CoV-2)荧光型RT-RAA快速检测方法.方法 以体外转录SARS-CoV-2的RNA为模板,利用单链DNA结合蛋白、重组酶和DNA聚合酶,在40.5℃恒温下,快速完成orf1ab基因和S基因序列片段的扩增,并用已知新冠核酸阳性和其他呼吸道病毒感染患者咽拭子标本进行初步评价.结果 该研究建立的方法分别检测SARS-CoV-2两种基因的所需时长均在20 min以内,检测2种基因的灵敏度均为2拷贝数/反应管,特异性为100％,2种引物的最低检出限3次重复实验扩增反应起峰时间一致,曲线形态接近.结论 该研究建立的方法灵敏性高,特异性强,重复性良好,临床标本检测结果符合率高,且不需要昂贵的仪器,适用于现场快速检测.

目的 建立斑点热群立克次体(SFGR)TaqMan实时荧光定量PCR(qPCR)快速检测方法 .方法 根据日本斑点热立克次体外膜蛋白A(ompA)基因序列设计特异性引物和探针,建立基于TaqMan探针的实时荧光定量PCR检测方法,对其特异性、灵敏性、重复性进行检测,用建立的此检测方法和常规的巢式PCR方法同时对临床收集的80份患者血标本进行检测并比较两者结果 .结果 成功建立了检测SFGR的实时荧光定量PCR方法,标准曲线的循环阈值与模板拷贝数均呈良好的线性关系(r>0.99),且在检测SFGR核酸样本时具有良好的灵敏度、特异性和重复性.结论 该研究建立了基于TaqMan探针检测SFGR的实时荧光定量PCR检测方法,为临床实验室快速确诊SFGR疾病提供了快速、有效的技术手段.

2015年以来,寨卡病毒(Zika virus,ZIKV)感染被报告和小头症之间存有关联,已有多个国家和地区报告ZIKV感染证据,我国持续面对ZIKV输入风险.为发展适用于现场的快速高效的ZIKV核酸检测方法,选取ZIKVNS1片段保守区设计特异性引物探针,经优化建立了多种功能蛋白介导的ZIKV RNA恒温快速扩增检测方法.利用所制备的ZIKV NS1片段体外转录RNA作为参考品,并与实时荧光定量RT-PCR检测结果进行比较.同时利用ZIKV细胞培养物中加入健康人血清制备的模拟临床标本进行初步验证.结果 显示,建立的RNA恒温快速扩增检测方法可100％检出100拷贝/μL体外转录RNA,可在15 min以内判读,大大低于PCR的常规检测时间;用该方法检测其他相关病毒,无交叉反应.病毒滴度在(104～107 PFU/mL)之间的模拟临床病人标本可达到100％检出.本研究建立的ZIKV RNA恒温扩增快速检测方法快速、灵敏、特异,适用于ZIKV现场应急检测,为ZIKV的防控提供了重要的技术支撑.

Objective:To develop a rapid nucleic acid detection method for Zika virus (ZIKV), Dengue virus (DENV), Yellow fever virus (YFV), Chikungunya virus (CHIKV) based on microfluidic fluorescence quantitative RT-PCR technologies, in order to achieve rapid diagnosis of these four viral infections.Methods:Four sets of specific primers and probes were designed targeting the NS1 gene of ZIKV, the NS5 gene of DENV, and YFV, the E1 gene of CHIKV, respectively. The sensitivity was evaluated using in vitro transcribed RNA of ZIKV, DENV, YFV and CHIKV, and the specificity were evaluated using other viral nucleic acid. ZIKV, YFV and CHIKV detection method were verified using simulated positive samples, and DENV detection method was verified using clinical patient samples, the result of which were also compared with the quantitative RT-PCR detection method . Results:The limit of detection (LOD) of ZIKV, DENV, YFV, and CHIKV microfluidic qRT-PCR method were 14.57 copies/μl, 94.27 copies/μl, 8.25 copies/μl, and 223.19 copies/μl, respectively, and the four detection method showed no cross-reactivity with other viral nucleic acids. The prepared ZIKV, YFV and CHIKV simulated positive samples were 100% detected, and the variation coefficient of Ct value measured at each concentration were all around 2%; the 20 clinical patient specimens of DENV infection were 100% detected, which is consistent with the result of fluorescent quantitative RT-PCR detection.Conclusions:The ZIKV, DENV, YFV, and CHIKV microfluidic quantitative RT-PCR detection method showed good sensitivity, specificity, and stability. The detection could be completed within 25 minutes, which could be used for laboratory detection and early diagnosis.

目的 对从我国临床患者血液中新发现和分离出的日本斑点热立克次体安徽120株ompB基因及其编码蛋白进行系统的结构与功能预测,为深入了解其在立克次体致病机制中的作用、研发斑点热临床诊断试剂和疫苗提供理论基础.方法 基于日本斑点热立克次体安徽120株ompB基因及其编码的氨基酸序列,应用生物信息学方法预测其信号肽、跨膜区、疏水性等性质和可能的B细胞抗原表位.结果 日本斑点热立克次体安徽120株ompB基因在3297～3311处存在连续15个碱基的缺失,除此之外,该分离株还有4个碱基突变.该基因编码的氨基酸数目为1651个,蛋白质分子量为167.69 ku,共由20种氨基酸组成,其中含量较多的氨基酸是Gly;理论等电点pI为5.15,消光系数为58805或58680,蛋白不稳定指数为7.15,脂肪指数为88.86,总平均亲水性(GRAVY)为0.06,为疏水性蛋白;Sig-nal peptide(Sec/SPI)Likelihood为0.5677,有信号肽序列;存在一个Pfam区域和自转运体区,分别与细菌黏附宿主和蛋白质转运有关;蛋白二级结构中以无规卷曲为主,其次为β-折叠和 α-螺旋,分别占到53.73％、30.83％和15.45％.同源模建蛋白的三级结构中,第1308～1651氨基酸部分在蛋白表面形成桶状结构,可能具有丝氨酸蛋白酶的作用;蛋白分子内抗原表位较丰富,共有159个可能的抗原表位,预测分数最高(0.94)的为1269～1284位序列.结论 成功分析和预测了日本斑点热立克次体安徽120株ompB基因及其编码蛋白的组成、蛋白质的二级结构、三级结构和B细胞抗原表位,为研究日本斑点热立克次体的致病机制、临床诊断试剂和亚单位疫苗的研发提供了理论支持.