新型冠状病毒抗原快速检测研发现状及展望*

新型冠状病毒肺炎疫情的全球大流行,对全球公共健康、社会和经济运转造成了重大影响。在药物研发迟滞及疫苗有效性未得到充分验证的情况下,对人群进行大规模的快速筛查,寻找潜在的感染者(尤其是轻症和无症状患者),并进行集中隔离,切断传播途径和保护易感人群是首要的任务。因此对于SARS-CoV-2感染,早期诊断尤为重要。总结现有市场上的新冠病毒抗原快速检测产品,对全球抗原快速检测市场进行分析,概述其研发的动向并展望了我国在新冠抗原检测新方法、新技术方面的自主创新能力。     

Development and evaluation of a point-of-care test with a combination of EZ-Fast DNA extraction and real-time PCR and LAMP detection: evaluation using blood samples containing the bovine leukaemia DNA

Along with progress in globalization of society, the spread of infectious diseases has accelerated worldwide. The deployment of highly sensitive genetic tests is essential for early diagnosis and early containment of potential outbreaks and epidemics, as well as routine surveillance, although tedious and expensive nucleic acid extraction steps represent a major drawback. Here we developed a simple and rapid DNA extraction method, named as an EZ-Fast kit, applicable to the field setting. The kit does not require advanced laboratory equipment or expensive DNA extraction kits and achieves crude DNA extraction within 10 min at extremely low cost and can easily be performed in field settings. When combined with real-time PCR and LAMP analyses, the performance of the POCT, using 183 bovine blood samples, was similar to that of the existing DNA extraction method: 92·5% (135/146) (real-time PCR) and 93·7% (133/142) (LAMP) diagnostic sensitivities, and 100% diagnostic specificities. The developed POCT provides a powerful tool to facilitate on-site diagnosis in a field setting.     

Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy

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Engineered Ultra-High Affinity Synthetic Antibodies for SARS-CoV-2 Neutralization and Detection

The Covid-19 pandemic is a centenarial global catastrophe. Similar events are likely to be recurring with more frequency in the future. The inability to control the virus’ impact is caused by many factors, but the lack of a technology infrastructure to detect and impede the virus at an early stage are principal shortcomings. Using phage display mutagenesis, we have generated a cohort of high performance antibody fragments (Fabs) that can be used in a sensitive point of care (POC) assay and are potent inhibitors (IC₅₀-0.5 nM) to viral entry into cells. The POC assay is based on a split-enzyme (β-lactamase) complementation strategy that detects virus particles at low nM levels. We have shown that this assay is equally effective for detecting other viruses like Ebola and Zika. Importantly, its components can be freeze dried and stored, but becomes fully active when rehydrated.     

新型冠状病毒胶体金抗原快速检测试剂的研制及性能评价*

目的:建立新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)胶体金抗原快速检测试剂的制备方法,并对检测试剂的性能指标进行评价。方法:采用柠檬酸三钠还原法制备胶体金溶液,用鼠抗核衣壳蛋白(nucleocapsid protein, NP)单克隆抗体及二硝基苯酚-牛血清白蛋白(DNP-BSA)作为标记抗体,硝酸纤维素膜上分别包被鼠抗核衣壳蛋白单克隆抗体和兔抗DNP多抗作为检测线和质控线制备免疫胶体金试纸条;对试剂最低检出限、交叉反应性、加速稳定性及临床诊断特异性和灵敏度进行性能评价。结果:检测热灭活培养物的最低检出限为2.0×10² TCID₅₀/mL;测试16种常见呼吸道病原体高浓度样本均无交叉反应;试剂盒50℃加速破坏8周稳定。临床及健康人群鼻咽拭子样本测试,诊断灵敏度为96.67%(29/30),特异性为99.23%(129/130),总符合率为98.75%(158/160);一致性检验Kappa值为0.959 0,P<0.05。结论:SARS-CoV-2胶体金抗原快速检测试剂检测灵敏度和特异性高,检测速度快,操作便携,无需设备,肉眼观察,可作为现有核酸检测法的补充手段,用于新型冠状病毒的早期筛查。     

新型冠状病毒(SARS-CoV-2)SNV分型检测技术

新型冠状病毒肺炎(COVID-19)的全球大流行对整个人类社会造成了重大影响,人类面临着财政刺激、金融压力、债务重整等挑战。在特效治疗药物与方法出现之前,大规模的人群筛查隔离成为现在疫情治理的最有效方法。然而,这一次的新冠病毒(SARS-CoV-2)展示出了极高的遗传变异性,截至2022年3月31日统计突变率超过了2.3‰,迄今为止高传染性的新病毒株不断出现,被世界卫生组织正式警告的变异株就达到了7个。因此,在接下来的病毒防控与研究中,不但需要检测SARS-CoV-2,更需要精准、实用的单核苷酸变异(single nucleotide variation, SNV)基因分型技术,特别针对大规模人群筛查中,不仅需要获得SRAS-CoV-2的信息,还需要精准快速区分具有更高传染性与毒性的变异株感染。对病毒的感染和突变机制进行了简要介绍,并着重对现有主要的SARS-CoV-2 SNV分型技术进行了分类综述,希望为新型检测技术的开发提供参考。     

医疗服务分档评估指标体系的测试

医疗机构医疗服务具有差别性,原有的医疗服务定价方式已不能完全体现不同级别医疗机构医疗服务价值。医疗服务分档定价方式拉开了不同级别医院的差距,体现了医务人员的技术劳务价值,减少医疗资源的浪费,与以往的医疗服务价格定价方式相比,具有明显的优越性。前期课题组初步建立了医疗服务分档评估指标体系,为了解医疗服务分档评估指标体系在非营利性医疗机构中的运用情况,分别选取三级、二级、一级医院共4家非营利性医疗机构进行测试。测试结果与课题前期研究设想基本吻合。     

Evaluation of four hematology and a chemistry portable benchtop analyzers using non-human primate blood

Background  Near patient testing (NPT) and point-of-care testing (POCT) using portable benchtop analyzers has become necessary in many areas of the medical community, including biocontainment.
Methods  We evaluated the Beckman AcT diff, Abaxis Vetscan HMII (two instruments), Abbott Cell-Dyn 1800, and Abaxis Vetscan VS2 for within-run precision and correlation to central laboratory instruments using non-human primates blood.
Results  Compared with the central laboratory instruments, the Beckman AcT diff correlated on 80%; the HMII instruments on 31% and 44%, the CD1800 on 31%, and the VS2 on 71% of assays. For assays with published manufacturers precision guidelines, the AcT diff met all nine, the HMII instruments met one and six of six, and the CD 1800 met one of six.
Conclusions  Laboratories using NPT/POCT must test their individual instruments for precision and correlation, identify assays that are reliable, and exclude or develop supplemental procedures for assays that are not.     

GICA和CLIA联合检测SARS-CoV-2特异性抗体的检测策略研究

评价胶体金免疫层析法(GICA)与化学发光法(CLIA)联合检测对降低新型冠状病毒(SARS-CoV-2)特异性抗体假阳性的效果。收集2020年1月22日至2020年3月5日就诊于川北医学院附属医院及南充市中心医院的19例SARS-CoV-2确诊患者不同时段的血清33份,55例非SARS-CoV-2、其他病原体感染及自身免疫性疾病患者的血清55份,采用GICA和CLIA分别对血清SARS-CoV-2 IgM、IgG进行检测,并对结果进行分析。GCIA检测SARS-CoV-2 IgM、IgG的敏感性分别为100.0%、94.74%,与CLIA(92.86%和100.0%)比较没有差异(P>0.05);GCIA检测SARS-CoV-2 IgM、IgG的特异性分别为70.91%、74.55%,明显低于CLIA的特异性(98.18%和89.09%)(P<0.01);两种方法检测SARS-CoV-2 IgM、IgG结果具有一致性(P<0.001),Kappa值分别为0.434,0.406;ROC曲线分析发现,GCIA检测SARS-CoV-2 IgM、IgG的AUC分别为0.855、0.846,明显低于CLIA(0.955和0.945)(P<0.05)。两种方法联合检测SARS-CoV-2 IgM、IgG的敏感性分别为92.86%、94.74%,特异性分别为100.0%、100.0%;ROC曲线分析显示,联合检测SARS-CoV-2 IgM、IgG的AUC分别为0.964、0.974,高于两种方法的单独检测。GICA和CLIA联合检测能有效提高SARS-CoV-2 IgM和IgG的检测特异性,值得临床推广应用。     

SARS-CoV-2中和性纳米抗体的原核表达及中和活性检测

纳米抗体(nanobody,Nb)作为目前已知的能与目标抗原结合的最小单位抗体,在生物医药、临床研究等方面具有良好的应用前景。根据大肠杆菌密码子偏好性优化合成严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome-coronavirus 2,SARS-CoV-2)中和性纳米抗体H11-D4基因,将其克隆到pET28a表达载体上后,转化至大肠杆菌感受态细胞Rosetta(DE3)进行诱导表达,通过镍柱纯化、质谱分析、Western Blot鉴定H11-D4的表达情况并使用中和试验验证其中和活性。研究结果显示,纳米抗体H11-D4可成功在大肠杆菌中表达,最佳诱导条件为IPTG终浓度1.0 mmol·L^-1,37℃诱导5 h。H11-D4抗体的分子量大小约为17.9 kD,与预测值相符。经镍柱纯化后,产量为25.16 mg·L^-1。透析复性后利用TritonX-114快速有效地去除了内毒素,中和试验成功验证了H11-D4的中和活性(IC50)为171.1 nmol·L^-1,研究结果可为...     

Testing for group effect in a 2 x k heteroscedastic ANOVA model

It is natural to want to relax the assumption of homoscedasticity and Gaussian error in ANOVA models. For a two-way ANOVA model with 2 x k cells, one can derive tests of main effect for the factor with two levels (referred to as group) without assuming homoscedasticity or Gaussian error. Empirical likelihood can be used to derive testing procedures. An approximate empirical likelihood ratio test (AELRT) is derived for the test of group main effect. To approximate the distributions of the test statistics under the null hypothesis, simulation from the approximate empirical maximum likelihood estimate (AEMLE) restricted by the null hypothesis is used. The homoscedastic ANOVA F -test and a Box-type approximation to the distribution of the heteroscedastic ANOVA F -test are compared to the AELRT in level and power. The AELRT procedure is shown by simulation to have appropriate type I error control (although possibly conservative) when the distribution of the test statistics are approximated by simulation from the constrained AEMLE. The methodology is motivated and illustrated by an analysis of folate levels in the blood among two alcohol intake groups while accounting for gender.