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.

基于危害分析及关键控制点体系的核酸检测实验室质量控制研究

目的通过危害分析及关键控制点(hazard analysis and critical control point,HACCP)体系系统化控制核酸检测实验室质量,以期能够实现核酸检测实验室质量的提升,为有效制定核酸检测质量控制对策提供借鉴。方法根据HACCP体系初步探究核酸检测实验室质量控制,重点分析HACCP体系在核酸标本接收、准备试剂、提取标本核酸、核酸扩增、结果判读、核酸检测结果报告环节制定的监控程序、验证措施以及纠偏措施等重要控制点。结果通过分析2021年12月-2022年12月的检测数据,结果显示,各批次核酸检测阳性质控品均满足判定规则,每一批次的实验均处于在控状态。对HACCP体系下实验室数据进行统计,计算其标准差、均值以及变异系数,对箱型图绘制后,展开离群值检验,结果发现,均值没有产生离群值。结论核酸检测实验室中应用HACCP体系,能够有效控制核酸检测实验室质量,全面掌控核酸检测实验室重要控制点,实现实验室生物安全水平与质量的提升,保证核酸检测实验室质控的有效性与科学性,继而有效控制核酸检测实验室质量。

Current testing strategies for hepatitis C virus infection in blood donors and the way forward

Screening tests for blood donations are based upon sensitivity, cost-effectiveness and their suitability for high-throughput testing. Enzyme immunoassay (EIAs) for hepatitis C virus (HCV) antibodies were the initial screening tests introduced. The ”first generation“ antibody EIAs detected seroconversion after unduly long infectious window period. Improved HCV antibody assays still had an infectious window period around 66 d. HCV core antigen EIAs shortened the window period considerably, but high costs did not lead to widespread acceptance. A fourth-generation HCV antigen and antibody assay (combination EIA) is more convenient as two infectious markers of HCV are detected in the same assay. Molecular testing for HCV-RNA utilizing nucleic acid amplification technology (NAT) is the most sensitive assay and shortens the window period to only 4 d. Implementation of NAT in many developed countries around the world has resulted in dramatic reductions in transfusion transmissible HCV and relative risk is now < 1 per million donations. However, HCV serology still continues to be retained as some donations are serology positive but NAT negative. In resource constrained countries HCV screening is highly variable, depending upon infrastructure, trained manpower and financial resource. Rapid tests which do not require instrumentation and are simple to perform are used in many small and remotely located blood centres. The sensitivity as compared to EIAs is less and wherever feasible HCV antibody EIAs are most frequently used screening assays. Efforts have been made to implement combined antigen-antibody assays and even NAT in some of these countries.

A fully automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing

The outbreak of virus-induced infectious diseases poses a global public-health challenge.Nucleic acid amplification testing(NAAT)enables early detection of pandemic viruses and plays a vital role in preventing onward transmission.However,the requirement of skilled operators,expensive instrumentation,and biosafety laboratories has hindered the use of NAAT for screening and diagnosis of suspected patients.Here we report development of a fully automated centrifugal microfluidic system with sample-in-answer-out capability for sensitive,specific,and rapid viral nucleic acid testing.The release of nucleic acids and the subsequent reverse transcription loop-mediated isothermal amplification(RT-LAMP)were integrated into the reaction units of a microfluidic disc.The whole processing steps such as injection of reagents,fluid actuation by rotation,heating and temperature control,and detection of fluorescence signals were carried out automatically by a customized instrument.We validate the centrifugal microfluidic system using oropharyngeal swab samples spiked with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)armored RNA particles.The estimated limit of detection for armored RNA particles is 2 copies per reaction,the throughput is 21 reactions per disc,and the assay sample-to-answer time is approximately 70 min.This enclosed and automated microfluidic system efficiently avoids viral contamination of aerosol,and can be readily adapted for virus detection outside the diagnostic laboratory.

Positive SARS-Cov-2 test in a woman with COVID-19 at 22 days after hospital discharge:A case report

Background:In a few discharged patients with coronavirus disease 2019(COVID-19),the nucleic acid test shows positive results again.Whether this is due to relapse of the disease,reinfection by the virus,or a false-positive result at hospital discharge is worth exploring.Case presentation:A woman with COVID-19 was discharged from the hospital after integrative treatment with traditional Chinese and Western medicine because she met the discharge standards.However,she obtained positive results on a nucleic acid test 22 days later.Conclusion:Based on this positive test result in a discharged patient with COVID-19,anal tests and coronavirus antibody tests should be combined with throat swab tests to further develop the diagnosis and discharge standards for patients with COVID-19.

Turnaround times for molecular testing of pediatric viral cerebrospinal fluid samples in United Kingdom laboratories

BACKGROUND Rapid molecular testing has revolutionized the management of suspected viral meningitis and encephalitis by providing an etiological diagnosis in<90 min with potential to improve outcomes and shorten inpatient stays.However,use of molecular assays can vary widely.AIM To evaluate current practice for molecular testing of pediatric cerebrospinal fluid(CSF)samples across the United Kingdom using a structured questionnaire.METHODS A structured telephone questionnaire survey was conducted between July and August 2020.Data was collected on the availability of viral CSF nucleic acid amplification testing(NAAT),criteria used for testing and turnaround times including the impact of the coronavirus disease 2019 pandemic.RESULTS Of 196/212(92%)microbiology laboratories responded;63/196(32%)were excluded from final analysis as they had no on-site microbiology laboratory and outsourced their samples.Of 133 Laboratories included in the study,47/133(35%)had onsite facilities for viral CSF NAAT.Hospitals currently undertaking onsite NAAT(n=47)had much faster turnaround times with 39 centers(83%)providing results in≤24 h as compared to those referring samples to neighboring laboratories(5/86;6%).CONCLUSION Onsite/near-patient rapid NAAT(including polymerase chain reaction)is recommended wherever possible to optimize patient management in the acute setting.

高海拔地区血液筛查核酸检测系统性能验证结果评价

目的 对西藏那曲地区(高海拔)的血液筛查核酸检测系统进行性能验证,以评估高海拔地区核酸检测的能力,进一步提升血液安全水平。方法 采用多种方法对核酸检测系统的分析灵敏度、重复性、防交叉污染能力以及不同系统间的比对等进行评估。结果 西藏那曲地区核酸检测系统对高浓度的HBV DNA和HIV-1 RNA标本的检出率均达到100%,中浓度标本的检出率超过90%;PROBIT分析结果显示,在HBV DNA和HIV-1 RNA的最低检出限(LOD)分别为8.29 IU/mL(95%CI, 5.88~20.55 IU/mL)和40.52 IU/mL (95%CI, 30.26~85.92 IU/mL),HCV RNA 2a型97.14 IU/mL (95%CI, 71.00~182.67 IU/mL),均低于说明书声明的最低检出浓度。在重复性分析中,系统的重复性达到100%。交叉污染性能验证结果显示,系统具有良好的抗交叉污染能力。通过对HBV DNA低浓度标本的重复检测和低海拔地区的多系统检测结果比对,检测结果的一致性分别为77.78%(14/18)和77.27%(17/22),结果显示那曲地区核酸检测系统与其他系统存在一定的差异。结论 该核酸检测系统在高海拔地区的血液筛查中表现出良好的性能。高海拔地区血液筛查核酸检测系统性能验证方面的研究结果与低海拔地区基本一致,为提升高海拔地区血液安全提供了可靠依据。

The point-of-care-testing of nucleic acids by chip, cartridge and paper sensors

Point-of-care nucleic acid testing(POCNAT) has played an important role in the outbreak of infectious diseases(e.g., COVID-19) over recent years. POCNAT aims to realize the rapid, simple and automatic detection of nucleic acid. Thanks to the development of manufacturing technology, electronic information technology, artificial intelligence technology, and biological information technology in recent years, the development of the POCNAT device has led to significant advancement. Instead of the normal nucleic acid detection methods used in the laboratory, some novel experimental carriers have been applied, such as chips, cartridges and papers. The application of these experimental carriers has realized the automation and integration of nucleic acid detection. The entire process of nucleic acid detection is normally divided into three steps(nucleic acid extraction, target amplification and signal detection). All of the reagents required by the process can be pre-stored on these experimental carriers, without unnecessary manual operation. Furthermore, all of the processes are carried out in this experimental carrier, with the assistance of a specific control device. Although they are complicated to manufacture and precise in design,their application provides a significant step forwards in nucleic acid detection and realizes the integration of nucleic acid detection. This technology has great potential in the field of point-of-care molecular diagnostics in the future. This paper focuses on the relevant content of these experimental carriers.

Evaluation of factors contributing to variability of qualitative and quantitative proficiency testing for SARS-CoV-2 nucleic acid detection

The pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has led to unprecedented social and economic disruption.Many nucleic acid testing(NAT)laboratories in China have been established to control the epidemic better.This proficiency testing(PT)aims to evaluate the participants’performance in qualitative and quantitative SARS-CoV-2 NAT and to explore the factors that contribute to differences in detection capabilities.Two different concentrations of RNA samples(A,B)were used for quantitative PT.Pseudovirus samples D,E(different concentrations)and negative sample(F)were used for qualitative PT.50 data sets were reported for qualitative PT,of which 74.00%were entirely correct for all samples.Fortytwo laboratories participated in the quantitative PT.37 submitted all gene results,of which only 56.76%were satisfactory.For qualitative detection,it is suggested that laboratories should strengthen personnel training,select qualified detection kits,and reduce cross-contamination to improve detection accuracy.For quantitative detection,the results of the reverse transcription digital PCR(RT-dPCR)method were more comparable and reliable than those of reverse transcription quantitative PCR(RT-qPCR).The copy number concentration of ORF1ab and N in samples A and B scattered in 85,223,50,and 106 folds,respectively.The differences in the quantitative result of RT-qPCR was mainly caused by the non-standard use of reference materials and the lack of personnel operating skills.Comparing the satisfaction of participants participating in both quantitative and qualitative proficiency testing,95.65%of the laboratories with satisfactory quantitative results also judged the qualitative results correctly,while 85.71%of the laboratories with unsatisfactory quantitative results were also unsatisfied with their qualitative judgments.Therefore,the quantitative ability is the basis of qualitative judgment.Overall,participants from hospitals reported more satisfactory results than those from enterprises and universities.Therefore,surveillance,daily qualitiy control and standardized operating procedures should be strengthened to improve the capability of SARS-CoV-2 NAT.

Development and evaluation of a thermostatic nucleic acid testing device based on magnesium pyrophosphate precipitation for detecting Enterocytozoon hepatopenaei

Enterocytozoon hepatopenaei(EHP)infection has seriously affected prawn culture globally.The symptoms of the infection are not apparent,and traditional detection methods are time consuming and low in accuracy.We developed a new onsite rapid testing device(size 18.8×16.7×6.6 cm^(3))for EHP based on magnesium pyrophosphate precipitation and facilitated by loop mediated isothermal amplification(LAMP).The design and fabrication of the device enables efficient light absorbance.The device has a highly sensitive detector,high-precision thermal controller,and humanized touch screen.The temperature control precision of the device is 0.2-0.3℃ at 60℃,63℃,and 65℃.The coefficients of variation values(CVV)of the luminous power in one channel at light on and off were found to be 0.0097 and 0.0014,respectively,within 1 h.The CVV of the background,luminous power,and values of eight PCR tubes filled with pure water were all less than 5%.In the EHP experiment,eight samples(including seven positive and one negative)confirmed the effectiveness of the device,and four positive and four negative samples verified whether cross-contamination exists.Among them,the rise time of the curve was about 15 min.These results assert that the developed device exhibits enhanced stability and uniformity and has excellent performance with high sensitivity,good specificity,and low testing time.Moreover,the optimal and minimum absorbance range was 555-655 nm for monitoring the production of LAMP.

医疗机构新冠核酸采集信息管理系统的设计与应用

在新冠疫情常态化防控下,医疗机构职工的日常核酸采集成为必须,但院内的职工情况较为复杂。为加强院内职工的核酸采集信息管理,设计并开发了院内职工核酸检测信息采集管理系统,实现动态监控全院职工是否完成当日核酸检测。系统基于PowerBuilder平台开发,包含人员信息登记、核酸混管检测编号等5个功能模块,大大提升了院内核酸采集的效率及管理水平。

Determination of the predictive factors for diagnostic positivity of nucleic acid amplification tests for diagnosing pulmonary tuberculosis

Background:Tuberculosis(TB)remains a major threat to human health,and TB diagnostic methods remain unsatisfactory.Nucleic acid amplification tests(NAATs)show higher sensitivity compared with culture for the diagnosis of pulmonary TB(PTB).However,NAATs are expensive and cannot be easily implemented outside major medical centers.To improve the sensitivity of NAATs for PTB diagnosis,we investigated the predictive factors that might optimize NAAT utilization.Methods:A total of 1263 patients with suspected PTB were enrolled for evaluation.The sensitivity,specificity,and accuracy of methods including smear-microbiology,culture of Mtb and NAAT for Mycobacterium tuberculosis(Mtb)detection in sputum and bronchoalveolar lavage fluid samples were compared.Odds ratios and 95%confidence intervals were used to assess variables that might be associated with positive NAAT results for sputum and bronchoalveolar lavage fluid from patients with suspected PTB.Results:NAAT showed higher sensitivity for Mtb detection(61.1%)when compared with smear(9.0%)and Mtb culture(47.8%).We found that an elevated erythrocyte sedimentation rate,the presence of cavities,and positive interferon-𝛾release assay(IGRA)results were indicative of positive Mtb detection by NAAT.Moreover,individuals who had all three of these characteristics showed an 86%diagnostic positivity for PTB from Mtb detection by NAAT.Conclusions:Our study suggests that an elevated erythrocyte sedimentation rate,a positive IGRA result,and the presence of pulmonary cavities are helpful factors for predicting positive Mtb detection by NAAT.Patients with the three positive clinical markers should undergo NAAT for Mtb detection because they are the most likely individuals to be bacteriologically confirmed as having TB.

2种核酸筛查系统应用于病毒核酸检测的比较

目的通过2种不同核酸筛查系统的应用与比较,进一步探讨核酸检测(nucleic acid testing,NAT)在输血工作中的作用和意义。方法采用两种核酸筛查系统对相同数量的献血者标本进行HBV DNA、HCV RNA和HIV RNA检测,对其有效拆分率和核酸阳性检出率进行统计分析,比较二者的差异性,并对部分阳性献血者进行追踪。结果罗氏核酸筛查系统和科华核酸筛查系统各检测121 019例,其中罗氏系统共检20 170个pool,混检阳性pool130个,经拆分有反应性pool 81个,反应性标本为84例,pool的混检阳性率、有效拆分率、标本阳性检出率分别为0.64%、62.31%、0.069%;上海科华核酸筛查系统共检测15 128个pool,混检阳性pool 116个,经拆分有反应性pool40个,反应性标本为42例,pool的混检阳性率、有效拆分率、标本阳性率分别为0.77%、34.48%、0.035%。对17例酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)阴性,NAT阳性的献血者追踪结果显示1例为HIV"窗口期"感染,1例为HCV"窗口期"感染,另外15例ELISA和NAT再检结果为阴性。结论罗氏筛查系统与科华筛查系统均能在酶免阴性的标本中检出一定数量的核酸阳性标本,但前者的灵敏度、特异性和NAT阳性检出率均明显高于后者。开展核酸检测能够进一步保障输血安全,但同时也存在个别的假阳性。

cobas s201核酸检测系统使用前性能验证

目的在cobas s201核酸检测系统投入使用前,验证其操作性能和检测性能是否可达到预期要求,确保检测系统性能与厂商声明的技术性能相匹配,可满足实验室的实际需求。方法通过通量测试、压力测试和试剂稳定性测试3个方面评估核酸检测系统的操作性能,通过设备间性能比对验证和分析灵敏度验证,评估检测系统的检测性能。设备间性能比对验证中分别在3台cobas s201核酸检测系统上使用6混样模式对比对血清盘进行检测,采用Kappa检验评估3套核酸检测设备检测结果的一致性;分析灵敏度验证使用cobas Taq Screen MPX 2.0检测试剂,采用WHO HIV-1、HCV和HBV标准株制备梯度浓度的分析灵敏度血清盘,对血清盘进行单人模式检测,采用Probit回归分析计算95%检出限(LOD),与试剂说明书的分析灵敏度进行比对。结果每套核酸检测系统24 h可完成1 440份标本的6混样模式检测,3套核酸检测系统在3 d压力测试运行期间均无故障发生。cobas Taq Screen MPX 2.0检测试剂室温放置24 h候仍可检测出弱阳性的HIV-1、HCV和HBV样本。在核酸检测系统间的性能比对验证中,3套核酸检测系统检测结果与血清盘一致。cobas Taq Screen MPX 2.0检测试剂HIV-1、HCV和HBV的95%检出限分别是34.58 IU/m L[(27.77-48.77)IU/m L]、16.38 IU/m L[(10.15-92.22)IU/m L]、1.32 IU/m L[(1.12-1.71)IU/m L]。结论 cobas s201核酸检测系统的通量、长时间运行下的设备状态和试剂稳定性都符合实验室需求,不同设备间不存在检测性能间的差异,检测系统的分析灵敏度与厂商声明无显著差别,可达到实际检测的需要。

基于EWMA质控图和泊松分布的核酸检测系统稳定性预警机制的建立

目的建立Procleix TIGRIS联检的稳定性预警机制,用以监测日常检测工作的稳定性。方法收集2017年1月—12月Procleix TIGRIS核酸检测系统外部质控品的HIV/HCV/HBV联检S/CO值。对数据进行正态分布检验,判断是否可以使用基于数据正态分布的统计学质量控制方法。应用指数加权移动平均(EWMA)质控图对外部质控品检测S/CO值进行分析,验证该方法对核酸检测稳定性的预警作用。使用泊松分布公式对核酸检测中反应性样本的检出率进行计算,建立阳性率异常的早期预警。结果HIV、HCV和HBV外部质控品联检S/CO值均不符合正态分布。EWMA质控图对外部质控品核酸联检S/CO值的波动敏感,可显示出不同试剂批号间的微小系统性差别。当阳性检出率在4.9‰时,169例标本中反应性标本数目>3的概率为1.02%,500例标本中反应性标本数目>5的概率为3.88%,均属于小概率事件,可根据此计算结果建立核酸联检反应性标本数目异常界限。结论应用EWMA质控图和泊松分布可建立核酸联检的早期预警机制,利于加强核酸日常检测过程的稳定性监控。

石家庄地区2012-2014年献血人群NAT检测情况分析

目的为进一步提高血液安全性,降低输血感染风险,分析在石家庄地区献血者中开展血液传染病毒核酸检测(NAT)的必要性和可行性。方法应用浩源核酸检测系统,对经ALT及两遍HBs Ag、抗-HCV、抗-HIV、梅毒螺旋体ELISA检测结果均呈非反应性的287 728份标本,进行HBV DNA、HCV RNA和HIV-1 RNA-3项联合NAT筛查,初筛采用8人份混样法,对检出某项有反应性的混样池进行单人份拆分检测。结果 ELISA检测非反应性的287728份标本,共检测42 472个混样池,检出209个HBV DNA反应性混样池,混样池阳性率为4.92‰;拆分结果为84例HBV DNA阳性,检测阳性率为0.29‰,所有标本中无HCV RNA和HIV-1 RNA的检出。结论核酸检测可缩短血液病毒的检测"窗口期"并有效降低隐匿性乙型肝炎病毒感染(OBI)的发生几率,进一步提高血液安全性。

不同核酸检测系统常用性能监控指标比较

目的通过对3家核酸检测(NAT)系统常用关键性能监控指标的回顾性比较和分析,为血站NAT系统的评价和选择提供支持。方法对2015年1月—2019年3月核酸检测情况和3家NAT系统的混检/联检阳性率、拆分/鉴别阳性率、无效结果率、试剂使用率等常用性能监控指标进行统计分析比较。结果 2015年1月—2019年3月间共检测核酸标本496 795份,单核酸阳性(酶免阴性NAT阳性)率0.13%。3家NAT系统单核酸阳性率分别为0.06%、0.09%、0.23%;拆分/鉴别阳性率分别为66.74%、55.43%、57.82%;无效结果率分别为1.18%、1.13%、3.06%;试剂使用率分别为141.61%、152.59%、125.15%;除2家混样NAT系统的无效结果率和拆分阳性率差异无统计学意义外,其余指标各系统两两比较差异均有统计学意义。结论核酸检测能进一步提高血液安全,但不同NAT系统检测能力存在差异,各血站应结合实际情况评价和选择适合的NAT系统。

全自动多重检测试剂同时检测HBV DNA、HCV RNA和HIV-1 RNA

目的寻找一种有效实用的全自动多重检测试剂同时检测HBVDNA,HCVRNA和HIV-1RNA系统MPLC-COBAS。方法笔者采用ACROMETRIX公司的NACTMHBVDNA,HCVRNA及HIV-1RNA核酸对照品,卫生部临床检验中心的HBVDNA,HCVRNA质控对照品,检测该系统的灵敏度;同时采用我中心酶免法(ELISA)初筛检结果阳性,经中和实验确认HBsAg阳性标本10份,经RIBA确认抗-HCV阳性标本3份,经WesternBlot确认抗-HIV阳性标本3份,检测该系统的阳性符合率;检测大量临床ELISA筛检结果为阴性的标本来检测其特异性,并且每次实验设立内对照,阴阳性对照以进行临床标本检测。结果该系统的检测灵敏度(95%可信限)为HBV病毒为24-60IU/mL,HCV病毒为78-125IU/mL,HIV-1RNA病毒为45-67IU/mL,阳性符合率为100%,假阳性率为0.13%。结论全自动多重检测试剂同时检测HBVDNA,HCVRNA和HIV-1RNA系统MPLC-COBAS用于检测临床标本是有效实用的,可进一步加大临床标本检测数量,将该系统更好地用于酶联阴性结果的大规模筛查。

核酸检测实验室质量监控指标的探讨

目的分析华益美血液筛查核酸检测系统数据,探讨应用实验室质量监控指标监控NAT过程。方法统计分析2017年3月—2018年7月核酸检测系统数据。结果共计检测238 300人份标本(29 809个pools),拆分阳性率为45.26%(105/232),其中有9个NAT混检阳性pools各拆分出来2份NAT阳性标本,NAT阳性率为0.48‰(114/238 300);NAT总的pools结果无效率为1.31%(393/30 202),批结果无效率0.46%(2/435),扩增曲线异常引起结果无效类型占比最多47.07%(185/393)。NAT共用9个试剂批号,试剂消耗率97.60%(33 917/34 752),试剂有效利用率85.78%(29 809/34 752);仪器运行故障率0.69%(3/435);参与室间质评结果符合率为98.09%(103/105)。结论通过实验室质量监控指标的统计和分析能有效反映核酸检测实验室运行情况,有助于质量体系的持续改进。