Dataset Information

Microarray-in-a-tube for detection of multiple viruses.

ABSTRACT:

Background

The detection of multiple viruses is important for pathogenic diagnosis and disease control. Microarray detection is a good method, but requires complex procedures for multiple virus detection.

Methods

We developed a novel PCR assay, the microarray-in-a-tube system, which integrates multiple PCR processes and DNA microarrays for multiple virus detection. A 5 x 5 oligonucleotide microarray for detecting 4 respiratory tract viruses (severe acute respiratory syndrome-associated coronavirus, influenza A virus, influenza B virus, and enterovirus) with inner controls was arranged on the inner surface of a specially designed Eppendorf cap with a flat, optically transparent window.

Results

We were able to perform all detection processes in the encapsulated system without opening the cap. The 4 viruses were successfully amplified by one-step reverse transcription-PCR in the encapsulated tube. After the PCR process, the microarray-in-a-tube was inverted, and the fluorescence-labeled PCR products were directly hybridized on the microarray. Hybridization signals were obtained with an ordinary fluorescent microscope. The sensitivity of the system for virus detection reached 10(2) copies/microL. With the help of inner controls, the system provided reliable results without false negatives and false positives.

Conclusions

The microarray-in-a-tube system is a rapid, labor-saving tool for multiple virus detection with several advantages, such as convenience, prevention of cross-contamination of the PCR products, and potential for multiple-gene detection.

SUBMITTER: Liu Q

PROVIDER: S-EPMC7108471 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Publications

Microarray-in-a-tube for detection of multiple viruses.

Liu Quanjun Q Bai Yunfei Y Ge Qinyu Q Zhou Shixin S Wen Tian T Lu Zuhong Z

Clinical chemistry 20061207 2

<h4>Background</h4>The detection of multiple viruses is important for pathogenic diagnosis and disease control. Microarray detection is a good method, but requires complex procedures for multiple virus detection.<h4>Methods</h4>We developed a novel PCR assay, the microarray-in-a-tube system, which integrates multiple PCR processes and DNA microarrays for multiple virus detection. A 5 x 5 oligonucleotide microarray for detecting 4 respiratory tract viruses (severe acute respiratory syndrome-assoc ...[more]

PMID: 17158198

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Project description:Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 10(3) virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant virus. The microarray described in this work should help in understanding the etiology of gastroenteritis in humans and animals.

Project description:BackgroundArthropod-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 findingsThe 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/significanceWe 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.

Dataset Information

Microarray-in-a-tube for detection of multiple viruses.

Background

Methods

Results

Conclusions

Publications

Microarray-in-a-tube for detection of multiple viruses.

Similar Datasets

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