The microbial detection array combined with random Phi29-amplification used as a diagnostic tool for virus detection in clinical samples

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples.     

Microarray Multiplex Assay for the Simultaneous Detection and Discrimination of Influenza A and Influenza B Viruses

In this study, we present a microarray approach for the typing of influenza A and B viruses, and the subtyping of H1 and H3 subtypes. We designed four pairs of specific multiplex RT-PCR primers and eight specific oligonucleotide probes and prepared microarrays to identify the specific subtype of influenza virus. Through amplification and fluorescent marking of the multiplex RT-PCR products on the M gene of influenza A and B viruses and the HA gene of subtypes H1 and H3, the PCR products were hybridized with the microarray, and the results were analyzed using a microarray scanner. The results demonstrate that the chip developed by our research institute can detect influenza A and B viruses specifically and identify the subtypes H1 and H3 at a minimum concentration of 1 × 10² copies/μL of viral RNA. We tested 35 clinical samples and our results were identical to other fluorescent methods. The microarray approach developed in this study provides a reliable method for the monitoring and testing of seasonal influenza.     

Simultaneous detection of major enteric viruses using a combimatrix microarray

Various enteric viruses including norovirus, rotavirus, adenovirus, and astrovirus are the major etiological agents of food-borne and water-borne disease outbreaks and frequently cause non-bacterial gastroenteritis worldwide. Sensitive and high-throughput detection methods for these viral pathogens are compulsory for diagnosing viral pathogens and subsequently improving public health. Hence, we developed a sensitive, specific, and high-throughput analytical assay to detect most major enteric viral pathogens using “Combimatrix” platform oligonucleotide probes. In order to detect four different enteric viral pathogens in a sensitive and simultaneous manner, we first developed a multiplex RT-PCR assay targeting partial gene sequences of these viruses with fluorescent labeling for the subsequent microarray. Then, five olignonucleotides specific to each of the four major enteric viruses were selected for the microarray from the oligonulceotide pools targeting the specific genes obtained by multiplex PCR of these viruses. The oligonucleotide microarray was evaluated against stool specimens containing single or mixed viral species. As a result, we demonstrated that the multiplex RT-PCR assay specifically amplified partial sequences of four enteric viruses and the subsequent microarray assay was capable of sensitive and simultaneous detection of those viruses. The developed method could be useful for diagnosing enteric viruses in both clinical and environmental specimens.     

Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

Background

Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae), Alphavirus (Togaviridae), Orthobunyavirus (Bunyaviridae), and Phlebovirus (Bunyaviridae).

Methodology/Principal Findings

The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012.

Conclusions/Significance

We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish public health priorities, detect disease outbreaks, and evaluate control programs.     

Enteric Viruses of Humans and Animals in Aquatic Environments: Health Risks, Detection, and Potential Water Quality Assessment Tools

Waterborne enteric viruses threaten both human and animal health. These pathogens are host specific and cause a wide range of diseases and symptoms in humans or other animals. While considerable research has documented the risk of enteric viruses to human health from contact with contaminated water, the current bacterial indicator-based methods for evaluation of water quality are often ineffectual proxies for pathogenic viruses. Additionally, relatively little work has specifically investigated the risk of waterborne viruses to animal health, and this risk currently is not addressed by routine water quality assessments. Nonetheless, because of their host specificity, enteric viruses can fulfill a unique role both for assessing health risks and as measures of contamination source in a watershed, yet the use of animal, as well as human, host-specific viruses in determining sources of fecal pollution has received little attention. With improved molecular detection assays, viruses from key host groups can be targeted directly using PCR amplification or hybridization with a high level of sensitivity and specificity. A multispecies viral analysis would provide needed information for controlling pollution by source, determining human health risks based on assessments of human virus loading and exposure, and determining potential risks to production animal health and could indicate the potential for the presence of other zoonotic pathogens. While there is a need to better understand the prevalence and environmental distribution of nonhuman enteric viruses, the development of improved methods for specific and sensitive detection will facilitate the use of these microbes for library-independent source tracking and water quality assessment tools.     

Enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools.

Waterborne enteric viruses threaten both human and animal health. These pathogens are host specific and cause a wide range of diseases and symptoms in humans or other animals. While considerable research has documented the risk of enteric viruses to human health from contact with contaminated water, the current bacterial indicator-based methods for evaluation of water quality are often ineffectual proxies for pathogenic viruses. Additionally, relatively little work has specifically investigated the risk of waterborne viruses to animal health, and this risk currently is not addressed by routine water quality assessments. Nonetheless, because of their host specificity, enteric viruses can fulfill a unique role both for assessing health risks and as measures of contamination source in a watershed, yet the use of animal, as well as human, host-specific viruses in determining sources of fecal pollution has received little attention. With improved molecular detection assays, viruses from key host groups can be targeted directly using PCR amplification or hybridization with a high level of sensitivity and specificity. A multispecies viral analysis would provide needed information for controlling pollution by source, determining human health risks based on assessments of human virus loading and exposure, and determining potential risks to production animal health and could indicate the potential for the presence of other zoonotic pathogens. While there is a need to better understand the prevalence and environmental distribution of nonhuman enteric viruses, the development of improved methods for specific and sensitive detection will facilitate the use of these microbes for library-independent source tracking and water quality assessment tools.     

基因芯片技术检测重要人兽共患病病毒方法的建立

为了建立能对25种重要人兽共患病病毒进行筛查及鉴定用的基因芯片技术,本实验首先设计针对每种病毒的寡核苷酸探针并进行探针特异性的生物信息学验证.然后探索病毒核酸随机扩增方法,优化杂交动力学条件,建立本芯片标准的数据处理分析方法.最后用细胞培养的病毒和模拟临床标本验证芯片的敏感性与特异性．结果表明,锚定随机PCR扩增法适合于本芯片病毒核酸的扩增;芯片杂交前用0.25% NaBH4进行封闭,最优杂交条件为51 ℃,2 h及50%甲酰胺浓度;芯片具有较好的敏感性及检测特异性．初步结果表明,本实验所建立的基因芯片技术可应用于对25种重要人兽共患病病毒进行筛查及鉴定．     

新城疫病毒3种检测方法的研究比较

目的:利用3种方法对新城疫(Newcastle disease virus, NDV)病毒进行检测并对这3种检测方法的优缺点做出比较。方法:分别将NDV强毒F48E9和弱毒Lasota接种SPF鸡胚后,获取尿囊液。利用双抗夹心ELISA法、悬液芯片系统以及RT-PCR进行检测。通过对制备的针对新城疫病毒的抗体4D9和6C4蛋白浓度测定后,选择6C4进行生物素标记,将4D9作为固相捕获抗体,利用生物素-链霉亲和素放大系统构建双抗夹心检测体系。通过对Genebank上已发表的新城疫强弱毒F基因进行电脑分析后,设计一组针对NDV强弱毒的通用型引物,分别对强弱毒进行RT-PCR并检测其检出限。结果:ELISA法对NDV强弱毒尿囊液的检出灵敏度为1:160,但操作繁琐,耗时长;液相芯片对强弱毒尿囊液的检出限为1:160和1:320,然而和ELISA相比,操作较为方便,但仪器设备昂贵。RT-PCR对强弱毒RNA检出限分别为259pg和14pg,与前两种方法相比,PR-PCR在核酸水平上对病毒进行检测,理论上灵敏度较高,但是所需试剂、设备昂贵,且实验人员还需一定的技能培训。     

Simultaneous Detection and Identification of Enteric Viruses by PCR-Mass Assay

Simultaneous detection of enteric viruses that cause similar symptoms (e.g. hand, foot and mouth disease) is essential to the prevention of outbreaks and control of infections. In this study, a novel PCR-Mass assay combining multiplex polymerase chain reaction (PCR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed and used for simultaneous detection of eight distinct human enteric viruses. Enteric viral isolates and standard viral RNAs were examined to determine the sensitivity and specificity of the PCR-Mass assay. Clinical performance was evaluated with a total of 101 clinical specimens from patients suspected of having hand, foot and mouth disease (HFMD). The results were compared to those of previous analyses using real-time RT-PCR. The identification of specific viruses and clinical specimens shows that the PCR-Mass assay performed as well as or better than standard methods with respect to indicating the presence of multiplex pathogens in a single specimen.     

Linear amplification of catalyzed reporter deposition technology on nylon membrane microarray

The application of microarray analysis to gene expression from limited tissue samples has not been very successful because of the poor signal qualityfrom the genes expressed at low levels. Here we discussed the use of catalyzed reporter deposition (CARD) technology to amplify signals from limited RNA samples on nylon membrane cDNA microarray. When the input RNA level was greater than 10 microg, the genes expressed at high levels did not amplify in proportion to those expressed at low levels. Compared to conventional colorimetric detection, the CARD method requires less than 10% of the total RNA used for amplification of signal displayed onto a nylon membrane cDNA microarray. Total RNA (5-10 microg), as one can extract from a limited amount of specimen, was determined to produce a linear correlation between the colorimetric detection and CARD methods. Beyond this range, it can cause a nonlinear amplification of highly expressed and low-abundance genes. These results suggest that when amplification is needed for any applications using the CARD method, including DNA microarray experiments, precaution has to be taken in the amount of RNA used to avoid skew amplification and thus misleading conclusions.