用于新型冠状病毒检测的核酸等温扩增技术研究进展

核酸检测作为新型冠状病毒肺炎(COVID-19)筛查诊断和病情监测的主要手段,在疫情防控中发挥了重要作用。虽然实时荧光定量PCR被认为是新型冠状病毒(SARS-CoV-2)核酸检测的金标准,但其依赖荧光定量PCR仪且扩增检测时间较长,难以实现现场快速检测。因此许多基于核酸等温扩增的SARS-CoV-2检测方法相继诞生。等温扩增对仪器温控要求不高,通过与微流控芯片和可视化检测技术结合,可进一步简化操作、降低成本,为SARS-CoV-2现场快速筛查提供有力的技术支撑。本文围绕已报道的SARS-CoV-2等温扩增检测方法原理、检测性能及优缺点进行探讨,为进一步发展SARS-CoV-2现场快速检测平台提供参考。     

Application of a miniature biochip using the molecular beacon probe in breast cancer gene BRCA1 detection

We report for the first time the application of a biochip using the molecular beacon (MB) detection scheme. The usability of this biochip novel detection system for the analysis of the breast cancer gene BRCA1 is demonstrated using molecular beacon probes. The MB is designed for the BRCA1 gene and a miniature biochip system is used for detection. The performance of the biochip-MB detection system is evaluated. The optimum conditions for the MB system for highest fluorescence detection sensitivity are investigated for the detection system. The detection of BRCA1 gene is successfully demonstrated in solution and the limit of detection (LOD) is estimated as 70 nM.     

3种检测吡咯喹啉醌的方法比较

目的:研究和比较3种检测吡咯喹啉醌(PQQ)的方法,确定各种方法的特点和适用范围。方法:设计和改进了3种检测PQQ的方法,分别为活性电泳法、光谱法和NBT-Gly法,探讨其检测限和线性范围、精密度、样品检测和加样回收率。结果:活性电泳法专一性好,具有很高的灵敏度,可检测到12.6 ng/mL PQQ,可靠但准确性较差;NBT-Gly法操作简便,可用于大量样品的检测,但重复性不佳;光谱法精密度较好,但样品中存在吸光物质时对检测结果影响较大。结论:活性电泳法、光谱法和NBT-Gly法均可用于PQQ的检测,活性电泳法适于培养上清等复杂样品的粗略定量,NBT-Gly法适于大量样品的检测,光谱法适于纯度较高的PQQ的定量。     

基于CRISPR/Cas原理的转基因产品检测技术研究进展

随着转基因产品商业化种植面积不断增加、国际贸易日趋频繁,对转基因生物安全管理提出了更高的要求。转基因产品检测技术作为安全评价的关键环节,逐渐引起了各国政府的关注。目前,针对转基因产品的快速检测方法层出不穷,但这些检测方法对于设备、试剂和专业的实验人员均有较高的要求。因此,为了有效支撑转基因相关产业的发展和管理,亟需建立一种高灵敏度、高特异性及高效的转基因检测技术。基因组编辑技术是近年来迅速发展的一类遗传修饰技术,其代表技术——CRISPR/Cas技术,更是极大地推动了生物技术的发展。CRISPR/Cas技术除了被应用于基因编辑领域,也逐渐被应用于核酸分子检测领域。基于此,以转基因产品检测技术为立足点,从CRISPR/Cas的检测原理、检测效果等技术层面分析了CRISPR/Cas检测技术发展的必然性,并对其在转基因产品检测上的应用前景进行展望,旨在为我国转基因产品快速检测和有效监管工作提供资料,对于保障我国转基因产品贸易的顺利进行具有重要意义。     

The influence of abundance on detectability

Plant and animal survey detection rates are important for ecological surveys, environmental impact assessment, invasive species monitoring, and modeling species distributions. Species can be difficult to detect when rare but, in general, how detection probabilities vary with abundance is unknown. We developed a new detectability model based on the time to detection of the first individual of a species. Based on this model, the predicted detection rate is proportional to a power function of abundance with a scaling exponent between zero and one that depends on clustering of individuals. We estimated the model parameters with data from three independent datasets: searches for chenopod shrub species and coins, experimental searches for planted seedlings, and frog surveys at multiple sites in sub‐tropical forests of eastern Australia. Analyses based on the detection time and detection probability suggest that detection rate increases with abundance as predicted. The model provides a way to scale detection rates to cases of low abundance when direct estimation of detection rates is often impractical.     

An overview of transducers as platform for the rapid detection of foodborne pathogens

The driving advent of portable, integrated biosensing ways for pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques. The miniaturization and automation of integrated detection systems present a significant advantage for rapid, portable detection of foodborne microbes. In this review, we have highlighted current developments and directions in foodborne pathogen detection systems. Recent progress in the biosensor protocols toward the detection of specific microbes has been elaborated in detail. It also includes strategies and challenges for the implementation of a portable platform toward rapid foodborne sensing systems.     

利用表面等离子共振传感器检测苏云金芽孢杆菌cry1F蛋白

目的：建立检测苏云金芽孢杆菌（Bt）crylF蛋白的表面等离子共振（SPR）传感器方法。方法：采用SPR检测技术,利用生物分子相互作用分析原理,在金表面修饰特异性单克隆抗体,对crylF蛋白的检测进行研究。结果：该方法可以较好地检测到crylF蛋白,最低检测限可达10ng／mL,并且具有很好的特异性。结论：SPR检测方法的重复性较好,灵敏度高,目前可用于crylF蛋白的定性检测,为crylF蛋白及其他Bt蛋白的检测提供了新方法,在检测转Bt基因植物方面具有广阔的应用前景。     

Non-radioactive detection of single-copy DNA-DNA hybrids

A new procedure for non-radioactive detection of single-copy DNA-DNA hybrids combines an existing non-radioactive labeling and detection kit with a new substrate AMPPD for the enzyme alkaline phosphatase. The main advantages of this procedure are the possibility to reuse the blots easily and the much shorter detection time compared to radioactive detection methods.     

Analysis of mutagenic imidazo[4,5-f]quinolines and -quinoxalines (IQ compounds). Comparison of electrochemical and ultraviolet detection

Two synthetic imidazoquinolin-2-amines (IQ and MeIQ) and two imidazoquinoxalin-2-amines (MeIQx and 4,8-DiMeIQx), all known potent mutagens, have been separated by reversed-phase HPLC and detected by two methods - UV detection and electrochemical (EC) detection. The limits of detection were found to be 2.5 pmoles for UV detection and 0.5-1.5 pmoles for electrochemical detection.     

人乳头瘤病毒检测方法研究进展

人乳头瘤病毒（Human papilloma viruses,HPV）是少数几种明确与人类癌症相关的病毒之一,其持续感染是导致宫颈癌发生的重要危险因素。HPV的检测对宫颈癌的筛查及治疗宫颈病变药物的有效性评价有重要作用,因此国内外对HPV的检测方法不断革新。纵观近年来HPV检测方法,主要依赖于分子生物技术检测HPV的DNA、RNA、癌蛋白等,但各种检测方法的原理、适用范围及临床意义均不相同。本文将就目前常用的HPV检测技术及试剂盒进行简要介绍,并对各方法的优缺点、灵敏度及特异性进行分析,以期为临床选择合适的检测方法提供参考。     

新城疫液相芯片快速检测方法的建立

目的：建立可检测新城疫病毒（Newcastle disease virus,NDV）的液相芯片快速检测技术。方法：用DNAStar软件对GEN-BANK中NDV的NP基因进行序列分析设计NDV特异性探针并标记生物素,利用该探针与荧光编码微球偶联后与抽提的NDV病毒RNA的RT-PCR产物杂交反应,用液相芯片检测仪（Liquichip 200）检测荧光信号建立了NDV快速液相芯片检测方法。结果：检测结果显示,该法具有较好的特异性,不与H5AIV和H9AIV反应;检测灵敏度达到150个EID50;该法与鸡胚病毒分离法检出NDV的符合率达到97.1%。结论：初步建立了检测NDV的液相芯片技术,为进一步搭建NDV全新快速高通量检测平台奠定了基础,也为其他同类病毒的快速高通量检测提供了借鉴和经验。     

Comparison of three methods for beat-to-beat-interval extraction from continuous blood pressure and electrocardiogram with respect to heart rate variability analysis.

In recent years the analysis of heart rate variability (HRV) has become a suitable method for characterizing autonomous cardiovascular regulation. The aim of this study was to investigate the differences in HRV estimated from continuous blood pressure (BP) measurement by different methods in comparison to electrocardiogram (ECG) signals. The beat-to-beat intervals (BBI) were simultaneously extracted from the ECG and blood pressure of 9 cardiac patients (10 min, Colin system, 1000-Hz sampling frequency). For both data types, slope, peak, and correlation detection algorithms were applied. The short-term variability was calculated using concurrent 10-min BP and ECG segments. The root mean square errors in comparison to ECG slope detection were: 1.74 ms for ECG correlation detection; 5.42 ms for ECG peak detection; 5.45 ms for BP slope detection; 5.75 ms for BP correlation detection; and 11.96 ms for BP peak detection. Our results show that the variability obtained with ECG is the most reliable. Moreover, slope detection is superior to peak detection and slightly superior to correlation detection. In particular, for ECG signals with higher frequency characteristics, peak detection often exhibits more artificial variability. Besides measurement noise, respiratory modulation and pulse transit time play an important role in determining BBI. The slope detection method applied to ECG should be preferred, because it is more robust as regards morphological changes in the signals, as well as physiological properties. As the ECG is not recorded in most animal studies, distal pulse wave measurement in combination with correlation or slope detection may be considered an acceptable alternative.     

A Novel Dictionary Based Computer Vision Method for the Detection of Cell Nuclei

Cell nuclei detection in fluorescent microscopic images is an important and time consuming task in a wide range of biological applications. Blur, clutter, bleed through and partial occlusion of nuclei make individual nuclei detection a challenging task for automated image analysis. This paper proposes a novel and robust detection method based on the active contour framework. Improvement over conventional approaches is achieved by exploiting prior knowledge of the nucleus shape in order to better detect individual nuclei. This prior knowledge is defined using a dictionary based approach which can be formulated as the optimization of a convex energy function. The proposed method shows accurate detection results for dense clusters of nuclei, for example, an F-measure (a measure for detection accuracy) of 0.96 for the detection of cell nuclei in peripheral blood mononuclear cells, compared to an F-measure of 0.90 achieved by state-of-the-art nuclei detection methods.     

A compact CMOS biochip immunosensor towards the detection of a single bacteria

Recent use of biological warfare (BW) agents has led to a growing interest in the rapid and sensitive detection of pathogens. Therefore, the development of field-usable detection devices for sensitive and selective detection of BW agents is an important issue. In this work, we report a portable biochip system based on complementary metal oxide semiconductor (CMOS) technology that has great potential as a device for single-bacteria detection. The possibility of single-bacteria detection is reported using an immunoassay coupled to laser-induced fluorescence (LIF) detection. Bacillus globigii spores, which are a surrogate species for B. anthracis spores, were used as the test sample. Enzymatic amplification following immunocomplex formation allowed remarkably sensitive detection of B. globigii spores, and could preclude a complicated optical and instrumental system usually required for high-sensitive detection. Atomic force microscopy (AFM) was employed to investigate whether B. globigii spores detected in the portable biochip system exist in single-cell or multicellular form. It was found that B. globigii spores mostly exist in multicellular form with a small minority of single-cell form. The results showed that the portable biochip system has great potential as a device for single-particle or possibly even single-organism detection.     

Detection of mutations in human DNA

Efficient methods for the detection of mutations are of fundamental importance in research and in diagnostics. By detection of a DNA sequence alteration that cosegregates with a clinical phenotype in an affected family, the gene at fault may be identified and assigned a function. Mutation detection methods are also a rate-limiting factor for the clinical application of DNA diagnostics. Currently a large number of techniques are in use to scan for new mutations and to distinguish among previously established sequence variants. Here, some of the problems connected with mutation detection are discussed together with principles on which current and future mutation detection assays can be based.     

大肠杆菌变种的两种分子生物学检测方法分析

目的:对大肠杆菌的一种重要的变种--肠出血性大肠杆菌O157-H7的几种检测方法进行比较研究.方法:以自动免疫磁珠收集系统(AIMS)、自动酶标免疫测试系统(VIDAS)与传统常规的分离方法进行对比分析.结果:运用自动免疫磁珠收集系统(AIMS)方法对80份可能含有肠出血性大肠杆菌O157-H7的实样进行检测,检出份数为6份,检出率为7.5％,而且在一周之内可以全部对上述检出实样进行鉴定.AIMS法能够检出浓度在10cfu/mL模拟实样之中的肠出血性大肠杆菌O157-H7,然后将此法与CHROMagar 0157琼脂板相结合,其效果则更为明显.而自动酶标免疫测试系统(VIDAS)与传统与的分离方法则检测的效果不佳,检出率为0.自动免疫磁珠收集系统(AIMS)检测方法与自动酶标免疫测试系统(VIDAS)、传统与的分离方法在检出率方面存在显著的统计学差异,P＜0.01.结论:运用自动免疫磁珠收集系统(AIMS)结合CHROMagar 0157琼脂板对出血性大肠杆菌O157-H7进行检测,检出率较高、灵敏度较高且快速便捷,可以用于O157-H7外环境检测与食品污染源的实际调查之中,应该对其加以广泛地推广并应用.     

Using hierarchical models to compare the sensitivity of metabarcoding and qPCR for eDNA detection

Environmental DNA (eDNA) sampling—the detection of intra- or extra-cellular DNA in environmental samples—is a rapid and sensitive survey method for detecting aquatic species. Single-species detection methods (typically based on PCR or LAMP) have been shown to be more sensitive for detecting target species than multi-species detection methods, such as metabarcoding. However, previous studies have generally only compared these two eDNA detection approaches for a single target species and have used different methodological and statistical approaches. Here we present a comparison of single- and multi-species eDNA detection methods, drawing on two published case studies (one fish, one amphibian) and two new extensive datasets on a freshwater mammal (the platypus). To ensure consistent conclusions regarding the sensitivity of each eDNA method, we use the same hierarchical site occupancy-detection model for each dataset, incorporating uncertainty at the site, water sample, and technical replicate level. Overall, qPCR achieved higher detection probabilities than metabarcoding across species and datasets. However, differences in sensitivity between detection methods varied depending on methodological decisions concerning what constitutes a true positive detection (i.e., qPCR and metabarcoding thresholds). The decision as to which eDNA detection method to use should always be influenced by the study aims, but our results suggest that single-species detection methods based on qPCR may be preferable when the aim is to achieve a high detection probability for target species.     

The use of NASBA for the detection of microbial pathogens in food and environmental samples

The isothermal amplification method nucleic acid sequence-based amplification (NASBA), which amplifies RNA, has been reported as useful for the detection of microbial pathogens in food and environmental samples. Methods have been published for Campylobacter spp., Listeria monocytogenes and Salmonella enterica ser. Enteritidis in various foods and for Cryptosporidium parvum in water. Both 16S rRNA and various mRNAs have been used as target molecules for detection; the latter may have advantages in allowing specific detection of viable cells. Most of the methods to detect pathogens in foods have employed enrichment in nutrient medium prior to NASBA, as this can ensure sensitivity of detection and encourage the detection of only viable target cells. Although a relatively recent method, NASBA has the potential for adoption as a diagnostic tool for environmental pathogens.     

织物电极的皮肤-电极接触阻抗测量方法分析

随着可穿戴式健康监测技术的发展,新型心电传感器-织物电极成为人们关注的热点,本文对织物电极的皮肤-电极接触阻抗测量方法进行了综述。首先介绍了织物电极的概念,分析了织物电极的皮肤-电极电化学界面、皮肤-电极电化学界面的等效电路和简化电路模型,得出了皮肤-电极接触阻抗的计算公式;其次,将皮肤-电极接触阻抗的测量方法归纳为直接测量法、参比测量法和模拟皮肤测量法三类,讨论了它们的测量原理和优缺点。本文认为需将模拟皮肤测量法和真实皮肤测量法有机结合,才能有效评价织物电极的阻抗特性,为织物电极的性能评价和心电信号采集电路的设计提供重要依据。最后,本文对织物电极待解决的问题进行了分析讨论。     

电化学免疫传感器在传染病快速检测中的应用

传染病的快速检测是传染病预防控制的重要环节,其中现场快速检测对于及时有效控制传染病疫情尤为关键。相比于传统检测方法,电化学免疫传感器具有操作简单、快速、灵敏、准确、设备可小型化等优势,可用于传染病快速检测。简要综述了近年来电化学免疫传感器在传染病快速检测中的应用研究进展,重点阐述了该类传感器在现场检测中的主要贡献和不足之处,以及免疫磁分离技术与电化学传感检测相结合在传染病快速检测方面的优势。