Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection

BACKGROUND: As we enter the post-genome sequencing era and begin to sift through the enormous amount of genetic information now available, the need for technologies that allow rapid, cost-effective, high-throughput detection of specific nucleic acid sequences becomes apparent. Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can greatly reduce the time, cost and labor associated with single reaction detection technologies. METHODS: The Luminex xMAP system is a multiplexed microsphere-based suspension array platform capable of analyzing and reporting up to 100 different reactions in a single reaction vessel. This technology provides a new platform for high-throughput nucleic acid detection and is being utilized with increasing frequency. Here we review specific applications of xMAP technology for nucleic acid detection in the areas of single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, HLA DNA typing and microbial detection. CONCLUSIONS: These studies demonstrate the speed, efficiency and utility of xMAP technology for simultaneous, rapid, sensitive and specific nucleic acid detection, and its capability to meet the current and future requirements of the molecular laboratory for high-throughput nucleic acid detection.     

Silicon-based biosensors for rapid detection of protein or nucleic acid targets

BACKGROUND: We developed a silicon-based biosensor that generates visual, qualitative results or quantitative results for the detection of protein or nucleic acid targets in a multiplex format. METHODS: Capture probes were immobilized either passively or covalently on the optically coated surface of the biosensor. Intermolecular interactions of the immobilized capture probe with specific target molecules were transduced into a molecular thin film. Thin films were generated by enzyme-catalyzed deposition in the vicinity of the surface-bound target. The increased thickness on the surface changed the apparent color of the biosensor by altering the interference pattern of reflected light. RESULTS: Cytokine detection was achieved in a 40-min multiplex assay. Detection limits were 4 ng/L for interleukin (IL)-6, 31 ng/L for IL1-beta, and 437 ng/L for interferon-gamma. In multianalyte experiments, cytokines were specifically detected with signal-to-noise ratios ranging from 15 to 80. With a modified optical surface, specificity was also demonstrated in a nucleic acid array with unambiguous discrimination of single-base changes in a 15-min assay. For homozygous wild-type and homozygous mutant samples, signal-to-noise ratios of approximately 100 were observed. Heterozygous samples yielded approximately equivalent signals for wild-type and mutant capture probes. CONCLUSIONS: The thin-film biosensor allows rapid, sensitive, and specific detection of protein or nucleic acid targets in an array format with results read visually or quantified with a charge-coupled device camera. This biosensor is suited for multianalyte detection in clinical diagnostic assays.     

荧光定量PCR法检测HTLV核酸方法的建立

目的建立荧光定量PCR (FQ-PCR)法从血浆标本中检测HTLV核酸,用于献血者HTLV筛查。方法在沈阳军区总医院和安徽省血液中心总共收集献血者标本3 043份,用自配核酸提取试剂提取HTLV核酸,用FQ-PCR法检测HTLV核酸。同时用ELISA方法进行anti-HTLV检测,如果ELISA呈反应性,进一步用免疫印迹实验进行确证,如果免疫印迹实验结果为不确定,间隔4周后追踪献血者,以确定是否为真阳性,以验证FQ-PCR检测方法的可行性。结果对留取的献血者标本进行扩增,所有扩增结果均为阴性。用ELISA方法对这些标本进行HTLV抗体检测,共有48份标本ELISA检测呈反应性(包括灰区0.5=S/CO 1.0),对这48份标本做确证实验,有16份标本出现条带,但结果均为不确定。间隔4周后对这16位献血者进行追踪,共追踪到11位献血者,再次留取标本进行检测,结果均为阴性。分别选择7种不同拷贝数的假病毒核酸进行最低检出限的确定实验,最低检出限可以达到1.56 Copies/mL。结论本研究建立的FQ-PCR方法可以检测HTLV核酸,可以用于献血者的HTLV核酸筛查。     

Non-viral nucleic acid delivery methods

Introduction: Delivery of nucleic acid-based molecules in human cells is a highly studied approach for the treatment of several disorders including monogenic diseases and cancers. Non-viral vectors for DNA and RNA transfer, although in general less efficient than virus-based systems, are particularly well adapted mostly due to the absence of biosafety concerns. Non-viral methods could be classified in two main groups: physical and vector-assisted delivery systems. Both groups comprise several different methods, none of them universally applicable. The choice of the optimal method depends on the predefined objectives and the features of targeted micro-environment.

Areas covered: In this review, the authors discuss non-viral techniques and present recent therapeutic achievements in ex vivo and in vivo nucleic acid delivery by most commonly used techniques while emphasizing the role of ‘biological particles’, namely peptide transduction domains, virus like particles, gesicles and exosomes.

Expert opinion: The number of available non-viral transfection techniques used for human therapy increased rapidly, followed by still moderate success in efficacy. The prospects are to be found in design of multifunctional hybrid systems that reflect the viral efficiency. In this respect, biological particles are very promising.     

艰难梭菌核酸检测方法应用进展

艰难梭菌是一种广泛存在于人和动物肠道中的厌氧革兰阳性芽孢杆菌。 近年来,由于抗生素滥用艰难梭菌感染人数大量增加,发病率和病死率急剧上升,准确的诊断对于最佳治疗手段和预防措施来说至关重要。 因此高灵敏度与特异度、省时省力的核酸检测方法逐渐引起重视。 本研究就目前最新通过美国食品药品监督管理局批准以及其他经典的实验室自建艰难梭菌核酸检测方法的应用进行介绍和比较,为艰难梭菌的检测提供参考。     

一种快速核酸提取试剂在流感病毒检测中的应用评价

目的 探讨一种快速核酸提取试剂在流感病毒检测中的适用性及有效性。方法 选取流感病毒H3亚型、乙型Yamagata系细胞株和甲型H1N1型鸡胚株各1株,对其倍比稀释液及2015年北京市流感病原学监测阳性的67份咽拭子样本,同时采用快速提取法及离心柱提取法提取病毒核酸,经实时荧光PCR法进行定性、定量检测,检测结果采用SPSS 19.0软件进行统计分析。结果 快速提取法提取细胞培养液、鸡胚培养液中流感病毒核酸的最低检测限高于离心柱法10倍,咽拭子样本则高100倍;快速提取法提取核酸后检测到的病毒载量均低于离心柱法,其中对含有病毒量较高的毒株检测到的病毒载量低于离心柱法10倍,而对病毒量较少的咽拭子样本,则低100倍;快速提取法稳定性优于离心柱法;提取后的核酸-80℃保存10 d后冻融复测,快速提取法核酸损失量大于离心柱法。67份离心柱提取法检测阳性咽拭子样本使用快速提取法提取核酸后检测,两种方法阳性符合率总计为92.54%,其中10～102拷贝/ml组,阳性符合率为76.92%,102～103拷贝/ml组,阳性符合率为92.59%,103拷贝/ml组,阳性符合率为100%。结论 快速提取法具有稳定性高、易于操作、不易污染的优势,适用于细胞培养液、鸡胚培养液等病毒含量较高的大量样本中流感病毒核酸提取,适于检测样本量大、人手不足、设备欠缺的基层实验室使用;但-80℃冻融后核酸损失量较大,不宜反复冻融后使用;传统的离心柱法对于病毒含量较少的咽拭子样本及核酸需被反复冻融使用时具有优势。     

Comparison of nucleic acid extraction methods for the detection of Mycoplasma pneumoniae

Four nucleic acid extraction procedures (2 automated and 2 manual) were compared for their efficiency at isolating Mycoplasma pneumoniae DNA. Oropharyngeal swabs from healthy volunteers were spiked with varying amounts of M. pneumoniae, extracted, and tested using real-time polymerase chain reaction. Our data indicate that both automated extraction methods consistently outperform the manual procedures.     

抗菌药物敏感性试验快速检测新技术

抗菌药物敏感性试验(AST)是一种体外定性或定量测定抗菌药物抑制或杀灭细菌能力的实验。随着耐药病原体的增多和耐药机制的变异,传统药敏试验逐渐无法满足临床快速诊治的需求,因此基于待测病原体表型、基因型及其他耐药机制的药敏快检技术正在不断发展。本文论述了AST快速检测技术的发展现状及存在的问题,所有新技术对临床诊治指南的指导意义均应进行多方面评估和规范化。     

Thin film biosensor for rapid visual detection of nucleic acid targets.

BACKGROUND: We have developed a silicon-based biosensor that generates a visual signal in response to nucleic acid targets. METHODS: In this system, capture oligonucleotide probes are immobilized on the surface of the biosensor. Interaction of the capture probes with a complementary target and a biotinylated detector oligonucleotide allows initiation of formation of an organic thin film on the biosensor. Thin film formation is completed by enzymatic activity of peroxidase conjugated to an anti-biotin antibody. Peroxidase catalyzes deposition of an insoluble product onto the silicon surface, generating a uniform thin film. The increased thickness on the surface alters the perceived color of the biosensor through changes in the interference patterns of reflected light from the surface, causing a color change from gold to purple. RESULTS: The biosensor results may be evaluated by direct visual inspection or quantified by ellipsometry. Results are obtained in 25 min with a detection limit of 5 pmol/L (150 amol/sample). Selectivity of the biosensor is demonstrated by discrimination of single nucleotide mismatches. Multitarget arrays are also analyzed with the thin film biosensor, and the system is capable of detecting targets from human serum and urine. CONCLUSIONS: The biosensor surface is inexpensive to produce, and the assay format is simple and rapid. The thin film biosensor is adaptable to a wide variety of nucleic acid detection applications, including rapid diagnostic testing for infectious disease panels, antibiotic resistance panels, or allelic discrimination of specific genetic markers.     

新型冠状病毒核酸临床检测要点及经验

2019年12月以来,新型冠状病毒感染引起的肺炎病例数快速攀升。因患者体内病毒核酸的存在是目前唯一的确诊依据,而目前临床上普遍反映核酸检测阳性率不够理想,故对病毒核酸检测的实验室条件、检验人员及检测过程中各个环节都有较高要求。本文着重阐述了新型冠状病毒核酸检测各个环节中的检测要点、最新的行业规范及共识内容,同时探讨临床检测中的疑难问题,以期为新型冠状病毒所致疾病的准确诊断提供理论依据。     

Triggers for switching from minipool testing by nucleic acid technology to individual-donation nucleic acid testing for West Nile virus: analysis of 2003 data to inform 2004 decision making

BACKGROUND: Concern about West Nile virus (WNV) transfusion-transmitted infections missed by minipool (MP) nucleic acid testing (NAT) has prompted consideration of the use of individual-donation (ID) NAT. Strategies were investigated for the application of limited ID-NAT capacity in 2004. STUDY DESIGN AND METHODS: Patterns of WNV MP-NAT-reactive donations tested by the Blood Systems Laboratory each week for 79 blood centers from June 29 to November 23, 2003 (196 MP-NAT repeat-reactive [RR] donations among 801,697 units), were analyzed. ID-NAT initiation strategies were developed consisting of counts of RR donations and/or weekly RR rates, together with three ID-NAT discontinuation strategies, and ID testing burden was assessed based on these combined start and stop strategies. RESULTS: The effectiveness, reported as the percentage of MP-RR donations that would trigger ID-NAT based on each initiation strategy, ranged from 57 to 100 percent. The addition of a 1- or 2-week no-yield requirement for ID discontinuation substantially increased testing burden. Combined strategies resulted in projected ID-NAT of between 10 and 50 percent of donations for a 10- to 20-week period. For this organization, the most feasible ID-NAT initiation strategy was 2 MP-reactive donations and a weekly rate of 1 in 1000, which had an effectiveness of 81 percent and led to peak weekly ID-NAT of 20 to 25 percent of donations depending on the discontinuation rule. CONCLUSION: This new approach of targeted ID-NAT based on ongoing monitoring of MP-NAT yield may prove to be a rational policy for agents like WNV that cause seasonal and regional epidemics.     

不同核酸提取方法和新型冠状病毒核酸检测试剂室间质评结果分析

目的 分析2种不同核酸提取方法和2种新型冠状病毒核酸检测试剂检测室间质评样本的结果,进行性能比较.方法收集2020年江苏省临床检验中心和国家卫生健康委临床检验中心发放的新型冠状病毒核酸检测室间质量评价样本共15份,用2种常用核酸提取方法(磁珠法、核酸释放剂法)、2个厂家实时荧光定量PCR核酸扩增检测试剂检测,进行质评物...     

应用PCR方法快速检测布鲁氏菌核酸的研究进展

本文主要就近年应用PCR方法快速诊断人畜布鲁氏菌病(布病)的进展情况进行阐述,包括用各种布鲁氏菌基因保守序列设计引物的常规PCR、不同类型检测样本的常规PCR方法对布病的快速诊断的评述;荧光定量PCR的原理、方法;各种荧光定量PCR方法在布病快速诊断中的应用及其未来应用前景的展望。     

Role of blood and nucleic acid testing in diagnosis of thyroid carcinoma

Measurement of representative tumor markers of thyroid carcinoma such as serum thyroglobulin, CEA, calcitonin, plays only a limited role in diagnosis of thyroid malignancies. Preoperative diagnosis of follicular carcinoma is quite difficult by means of cytological examinations. In some recent studies, some promising molecular targets in diagnosis of follicular carcinoma were reported. Among them, loss of expression of trefoil factor 3 (TFF3) mRNA is a marker of poorly differentiated thyroid tumors including follicular carcinoma and tumors do not express TFF3 mRNA may be considered to be a subject of surgical resection. To establish clinical tests to diagnose micro papillary carcinoma with poor prognosis, a new hypothesis of thyroid carcinogenesis, the fetal cell carcinogenesis, might be considered.