Development of an Integrated Disposable Device for SARSCoV-2 Nucleic Acid Extraction and Detection

Objective To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods We designed,developed,and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection.The precision of the liquid transfer and temperature control was tested.A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction(RT-PCR).The entire process,from SARS-CoV-2 nucleic acid extraction to amplification,was evaluated.Results The precision of the syringe transfer volume was 19.2±1.9μL(set value was 20),32.2±1.6(set value was 30),and 57.2±3.5(set value was 60).Temperature control in the amplification tube was measured at 60.0±0.0℃(set value was 60)and 95.1±0.2℃(set value was 95)respectively.SARS-Cov-2 nucleic acid extraction yield through the device was 7.10×10^(6) copies/mL,while a commercial kit yielded 2.98×10^(6) copies/mL.The mean time to complete the entire assay,from SARS-CoV-2 nucleic acid extraction to amplification detection,was 36 min and 45 s.The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL.Conclusion The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test(POCT).

Ultrathin metal-organic framework nanosheets (Cu-TCPP)-based isothermal nucleic acid amplification for food allergen detection

The rapid and accurate detection of peanuts and soybeans allergen is important to the food safety. In this study, Cu-TCPP nanosheet, a kind of ultra-thin metal-organic framework(MOF)was synthesized and applied in loop-mediated isothermal amplification(named Cu-TCPP@LAMP), which can inhibit the non-specific amplification by absorbing and precise temperature releasing of single primer. As thus, Cu-TCPP@LAMP can achieve high sensitivity and specific amplification of the target gene. As a result, peanut and soybean allergens genes contained in food were successfully detected with a favorable detection sensitivity(5 ng/μL for peanuts and 10 ng/μL for soybeans)and reliable repeatability(The coefficient of variation was 3.38% for peanuts and 3.33% for soybeans). Moreover, the established method was utilized for detection of several commercial products, and had a high consistency with the standard method. Apart from food allergens, this novel assay can be widely used in other areas, such as pathogen detection, tumor nucleic acid detection and so on.

A brief review of novel nucleic acid test biosensors and their application prospects for salmonids viral diseases detection

Viral diseases represent one of the major threats for salmonids aquaculture.Early detection and identification of viral pathogens is the main prerequisite prior to undertaking effective prevention and control measures.Rapid,sensitive,efficient and portable detection method is highly essential for fish viral diseases detection.Biosensor strategies are highly prevalent and fulfill the expanding demands of on-site detection with fast response,cost-effectiveness,high sensitivity,and selectivity.With the development of material science,the nucleic acid biosensors fabricated by semiconductor have shown great potential in rapid and early detection or screening for diseases at salmonids fisheries.This paper reviews the current detection development of salmonids viral diseases.The present limitations and challenges of salmonids virus diseases surveillance and early detection are presented.Novel nucleic acid semiconductor biosensors are briefly reviewed.The perspective and potential application of biosensors in the on-site detection of salmonids diseases are discussed.

Recent advances and perspectives of nucleic acid detection for coronavirus

The recent pneumonia outbreak caused by a novel coronavirus(SARS-CoV-2)is posing a great threat to global public health.Therefore,rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments,saving people's lives and preventing epidemics.It is important to establish a quick standard diagnostic test for the detection of the infectious disease(COVID-19)to prevent subsequent secondary spread.Polymerase chain reaction(PCR)is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity.Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler opera-tion.A variety of improved or new approaches also have been developed.This review summarizes the currently available detection methods for coronavirus nucleic acid.It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coro-navirus infection.

Development of Nucleic Acid Sequence-Based Amplification Assay for Detection of Macrobrachium rosenbergii Nodavirus

A nucleic acid sequence-based amplification（NASBA）assay was established for the detection of Macrobrachium rosenbergii Nodavirus（MrNV）.The specific primers were designed according to the high conserved region of RNA2 sequence of MrNV.The 224 bp specific amplification product was obtained in positive sample determined with 3%agarose gel electrophoresis,while no product was generated from shrimp infected with other viruses including DNA viruses（IHHNV,WSSV）and RNA viruses（TSV,IMNV,YHV）.The detecting limit of the assay was 8pg nucleic acid,which is more sensitive than that of PCR method.

Clinical application of combined detection of SARS-CoV-2-specific antibody and nucleic acid

BACKGROUND The global outbreak of human severe acute respiratory syndrome coronavirus(SARS-CoV)-2 infection represents an urgent need for readily available,accurate and rapid diagnostic tests.Nucleic acid testing of respiratory tract specimens for SARS-CoV-2 is the current gold standard for diagnosis of coronavirus disease 2019(COVID-19).However,the diagnostic accuracy of reverse transcription polymerase chain reaction(RT-PCR)tests for detecting SARS-CoV-2 nucleic acid may be lower than optimal.The detection of SARS-CoV-2-specific antibodies should be used as a serological non-invasive tool for the diagnosis and management of SARS-CoV-2 infection.AIM To investigate the diagnostic value of SARS-CoV-2 IgM/IgG and nucleic acid detection in COVID-19.METHODS We retrospectively analyzed 652 suspected COVID-19 patients,and 206 non-COVID-19 patients in Wuhan Integrated TCM and Western Medicine Hospital.Data on SARS-CoV-2 nucleic acid tests and serum antibody tests were collected to investigate the diagnostic value of nucleic acid RT-PCR test kits and immunoglobulin(Ig)M/IgG antibody test kits.The j2 test was used to compare differences between categorical variables.A 95%confidence interval(CI)was provided by the Wilson score method.All analyses were performed with IBM SPSS Statistics version 22.0(IBM Corp.,Armonk,NY,United States).RESULTS Of the 652 suspected COVID-19 patients,237(36.3%)had positive nucleic acid tests,311(47.7%)were positive for IgM,and 592(90.8%)were positive for IgG.There was a significant difference in the positive detection rate between the IgM and IgG test groups(P<0.001).Using the RT-PCR results as a reference,the specificity,sensitivity,and accuracy of IgM/IgG combined tests for SARS-CoV-2 infection were 98.5%,95.8%,and 97.1%,respectively.Of the 415 suspected COVID-19 patients with negative nucleic acid test results,366 had positive IgM/IgG tests with a positive detection rate of 88.2%.CONCLUSION Our data indicate that serological IgM/IgG antibody combined test had high sensitivity and specificity for the diagnosis of SARS-CoV-2 infection,and can be used in combination with RT-PCR for the diagnosis of SARS-CoV-2 infection.

Improved Nucleic Acid Spot Hybridization Technique for Detection of Potato Spindle Tuber Viroid(PSTVd)

Potato spindle tuber viroid(PSTVd)disease is one of the major diseases that threatens potato production.Therefore,an advanced,rapid and sensitive detection technology is needed to detect the disease for better control.In order to establish an easier nucleic acid spot hybridization(NASH)method,some studies were tried as the followings:(1)the pre-hybridization step of nucleic acid spot hybridization(NASH)was omitted compared with ordinary way;(2)RNA extraction(phenol extraction and Ames buffer extraction)methods were compared;(3)fixed RNA by UV lamp and oven compared with UV cross-linker;(4)hybridized the RNA in shaking incubator and so on.The results showed that RNA extracted by Ames buffer was more effective than by the phenol extraction method.Besides,the result of hybridization without pre-hybridization step was better than that with 1.5 h of pre-hybridization.The more important discovery was that the shaking incubator could replace the hybridization oven and the ordinary UV lamp could replace the UV cross-linker.After a long term repeated research and testing,a new hybridization system that could rapidly detect the PSTVd by improved NASH technique merely using common instruments and equipment was established.

Consistency Analysis of Detection Results of Two Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kits

Objective: The objective of the study is to verify the clinical validity of the following kits with the comparative experimental analysis and evaluate whether their performance can meet the clinical requirements, i.e. Class III in vitro diagnostic reagent “Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kit (PCR-Fluorescence Probe Method)” of Daan Gene Co., Ltd. (Daan kit for short) and “Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kit (Fluorescence PCR Method)” of Wuhan Biot Gene Co., Ltd. (Biot kit for short). Method: In the study process, the samples were divided into positive and negative groups according to the control test results, and the clinical application performance of Daan kit and Biot kit was evaluated by comparing their test results. Results: The results show that two kits indicate the same test results, i.e. 26 positive and 107 negative samples in a total of 133 male urethral discharge samples, and 32 positive and 238 negative samples in a total of 270 female cervical secretion samples. Conclusion: It can be concluded from the clinical test that Daan and Biot Herpes Simplex Virus (HSV) Type II Nuc- leic Acid Test Kits are reliable, accurate, safe, convenient for use, stable and high-value in the clinical application.

Analysis of diagnosis and treatment of traditional Chinese medicine for COVID-19 nucleic acid

Since the outbreak of coronavirus disease 2019(COVID-19),the author has been involved in the treatment of COVID-19 in the first line.It is found that some patients had a long viral nucleic acid negative time,and even the course of the disease lasted more than 40 days.After a thorough investigation of the root causes,it is found the four possible causes were:"Blood stasis syndrome"is difficult to remove;"Damp heat"sticky greasy;Stubborn basic diseases;weak constitution.All the patients treated with traditional Chinese medicine have turned negative,and now the typical cases were analyzed and summarized in order to provide reference for the traditional Chinese medicine treatment and scientific research of the patients with COVID-19 who were difficult to turn negative.

水产品过敏原及其检测技术概述

水产品因鲜美的味道和丰富的营养而深受消费者青睐。水产品属于联合国粮农组织和世界卫生组织认定的过敏食物,其在加工、运输、贮藏过程中有可能受到外来过敏原的污染,由此引起的食品安全问题日益严峻,严重制约了行业的发展。明确水产食品中的过敏原,并利用适当的检测技术进行检测、监控,有利于预防水产食物过敏疾病的发生。本文概述水产食品中的主要过敏原,以及基于基因水平的核酸检测技术、蛋白水平的免疫检测技术及质谱检测技术研究进展,为丰富水产品过敏原及其检测手段提供理论依据。

早期香蕉枯萎病Foc4双探针核酸纸基检测传感器研制

为实现对早期香蕉枯萎病4号生理小种(fusarium oxysporum f.sp.cubense 4, Foc4)的准确检测,该研究提出一种基于胶体金的双探针纸基传感器。该传感器将2种不同粒径的胶体金分别与检测探针和信号增强探针结合,利用DNA探针代替抗原和抗体,形成双探针体系,通过增加信号增强探针降低检测限。基于目标序列与双探针体系的碱基互补配对形成“金标探针-目标序列-T线探针”复合物并在传感器的测试区被捕获,10 min内形成肉眼可见的目标产物,通过分析测试条带得到光强度峰面积并代入标准曲线中,实现Foc4定量检测。试验结果表明,双探针纸基传感器的检测限达到0.001 nmol/L,是传统纸基传感器的100倍,提高了检测灵敏度;在0.001~1 000.000 nmol/L范围内,Foc4浓度与测试线光强度峰面积呈线性关系;使用高浓度非互补探针进行试验干扰,发现非互补序列对检测效果基本无影响,表明该传感器具有较好的特异性;香蕉叶片Foc4检测的平均回收率为77.6%~102.3%,相对标准偏差为7.4%~7.7%。与传统形态学观察等检测方法相比,该研究提出的双探针核酸纸基检测传感器可以及时、快速、准确地判断早期Foc4的存在,具有良好的推广性和实际应用价值。

人乳头瘤病毒L1基因分型国家参考品的升级换代研制

目的 对人乳头瘤病毒(HPV)L1基因分型国家参考品升级换代,以提高基于L1检测靶区的HPV核酸(分型)试剂的质量。方法 研制包含了34种不同型别HPV L1的质粒样品。经商业化核酸检测试剂复核验证,分装组成国家参考品。采用荧光PCR法进行量值标定,并溯源至WHO第一代HPV16 DNA国际参考品。结合不同实验室的协作标定研究和适用性验证结果,确定参考品的质量标准,并进一步考察参考品的稳定性。结果 国家参考品包括34种不同型别HPV样本和5种HPV阴性病原体,型别上囊括了HPV68a和HPV68b亚型。34种不同型别HPV DNA含量为6.26~7.08 Log10 IU/mL,并要求各试剂准确性应能检出其检测范围内的所有型别且分型正确,其检出限应不高于104 IU/反应。结论 成功建立了新一代HPV L1基因分型国家参考品,用于评价L1检测靶区的HPV核酸检测试剂的质量。

核酸提取试剂盒对饲料中牛羊源性成分检测的影响

实时荧光聚合酶链反应法(荧光PCR法)是目前饲料中牛羊源性成分的定性检测中常用方法之一。为了熟练操作和掌握这个方法,使得实验室工作人员在做饲料中牛羊源性成分定性这类检测时能有一个清晰的认识,通过核酸提取试剂盒的选取及平行实验比对,对此进行了阐释。

基于核酸杂交反应的荧光生物传感器的制备及在黄曲霉毒素B_1检测中的应用

采用核酸适配体作为特异性识别元件,SYBR Green I(SGI)荧光染料为信号输出单元,构建了黄曲霉毒素B_1(AFB_1)生物传感器,并对试验条件进行了优化。优化的试验条件如下:适配体互补链与适配体的物质的量比为1.5,SGI加入量为10μL,适配体双链与SGI的作用时间为2 min,适配体与AFB_1作用时间为14 min。结果表明,在AFB_1质量浓度为0.1~1 000μg·L^(-1)时,荧光强度变化量与其质量浓度对数呈线性关系,检出限(3S/N)为0.081μg·L^(-1)。对实际玉米样品进行加标回收试验,回收率为95.2%~105%,测定值的相对标准偏差(n=7)均小于6.0%。与其他适配体传感器进行比较,该方法所构建的荧光适配体传感器对AFB_1的检测具有操作简便、检测范围宽、灵敏度高、特异性强、成本低廉等优点,适合现场快速测定。

基于蔬菜核酸检测实验的探究性实验教学设计

以蔬菜中维生素C积累相关基因的检测为例,探索将操作难度相对较高的RNA相关的核酸检测实验应用于本科实验教学中。实践结果表明,学生们可顺利掌握蔬菜中核糖核酸(RNA)的提取、反转录合成互补DNA(cDNA)、PCR扩增检验目的基因表达等RNA相关的实验操作技能,达到了预期的教学目标。本实验项目的实施促进了学生们对多门学科知识的融会贯通,有助于其提高发现问题和解决问题的能力并形成探索精神和创新思维。这对培养具有创新创业实践能力的复合型人才具有重要的作用。

不同核酸提取方法对HBV-DNA检测性能验证情况分析

目的评估2种乙型肝炎病毒(HBV)-DNA提取方法及2家检测试剂的性能,有助于选择优化提取试剂和检测试剂。方法2023年4月采用达安全自动核酸提取仪提取法(磁珠法)和手工提取法(一步法),并用达安和圣湘2种HBV-DNA试剂检测,对其进行精密度、正确度、线性范围、检出限及抗干扰能力等性能进行验证和评价。结果达安全自动核酸提取仪提取达安试剂检测、手工提取达安试剂检测和手工提取圣湘试剂检测在精密度、正确度、线性范围、检出限方面验证结果均达标;达安全自动核酸提取仪提取圣湘试剂检测在低值检测中变异系数大于5%,最低检测限验证不合格;抗干扰能力方面,全自动核酸提取仪提取的2.0 g/dL血红蛋白浓度的样本用达安和圣湘试剂检测结果均不受影响。手工提取甘油三酯浓度达3000 mg/dL的样本用达安和圣湘试剂检测的结果均不受影响。结论不同厂家的提取和检测试剂避免混用,达安和圣湘试剂对HBV-DNA定量检测的结果均符合要求。

基于功能化荧光氧化石墨烯的CuNCs标记生物传感器对癌胚抗原检测的价值研究

构建基于功能化荧光氧化石墨烯(graphene oxide,GO)的荧光铜纳米簇(Fluorescent copper nanoclusters,CuNCs)标记生物传感器的荧光分析法,用于灵敏检测癌胚抗原(Carcinoembryonic antigen,CEA),分析荧光猝灭效率与CEA浓度的相关性。适配体寡核苷酸探针(aptamer)与CEA特异性结合,形成CEA-aptamer-GO-CuNCs复合物,随着CEA浓度的增加,CuNCs的荧光强度能够被有效淬灭,绘制CEA浓度与荧光猝灭效率标准曲线,通过测定待测样本的荧光猝灭效率而定量CEA浓度。本次研究建立了检测CEA的aptamer-GO-CuNCs标记生物传感器的荧光分析法,最佳孵育时间为15 min,孵育温度为37℃,CuSO4溶液最佳浓度为300μmol/L,线性范围为0.05~50ng/mL,检出限(S/N=3)为0.01ng/mL。本荧光分析法可准确检测人血清样本中CEA浓度,且检测灵敏、特异性均较好,可为临床上癌症早期临床筛查、诊断以及治疗、预后评估等提供指导。

河流弧菌核酸检测试剂国家参考品的建立

目的 建立河流弧菌核酸检测试剂国家参考品并制定质量标准。方法 收集并筛选河流弧菌核酸阳性和阴性临床分离株样本,建立河流弧菌核酸检测试剂国家参考品。使用不同试剂对参考品进行协作标定,并对参考品稳定性和均匀性进行考核。结果 河流弧菌核酸检测试剂国家参考品由20份样本组成,包括8份阳性参考品P1~P8、10份阴性参考品N1~N10、1份检测限参考品L和1份重复性参考品R,L经菌落计数法测定浓度为1×10^(8)CFU/mL。国家参考品的质量标准为阳性符合率8/8,阴性符合率10/10,检测限要求1∶104稀释或更高浓度时应均为河流弧菌阳性,重复性要求对稀释后的两个浓度水平的Ct值的变异系数(CV,%)均不大于5.0%。经过随机抽样检测,参考品均匀性符合要求,在2~8℃冷藏条件、室温放置7天均未影响参考品稳定性。结论 本研究研制了河流弧菌核酸检测试剂国家参考品,并制定了其质量标准,可用于相关定性检测试剂的质量评价。

常见食源性病原菌的快速检测技术研究进展

食源性病原菌广泛存在于自然环境中,可通过肉类、海产品、奶制品等多种媒介传播,引起食源性疾病的发生。目前检测食源性病原菌的方法主要以平板培养辅以生化鉴定和血清型鉴定为主,检测的周期较长,效率较低,因此建立快速、精准的食源性病原菌检测方法对常见病原菌的现场检测及疾病的快速诊断尤为重要。本文对近年来常见食源性病原菌的快速检测方法进行了概述,主要包括免疫学检测手段、分子生物学检测方法和生物传感器检测技术,比较其优缺点,着重介绍了可以实现现场检测与实时检测的新兴技术:核酸-微流控检测技术,以期为常见食源性病原菌的预防与检测提供相关理论参考。