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Development of magnetic bead based sample extraction coupled polymerase spiral reaction for rapid on-site detection of Chikungunya virus.

ABSTRACT: The molecular detection system has evolved over last two decades and is rapidly replacing the conventional confirmatory techniques in diagnostic virology. However the major limitation in implementation of available molecular detection assays is the non availability of field deployable nucleic acid isolation platform coupled with gene amplification technique. The rapid and early molecular detection is crucial for employing effective measure against many viral infections. The re-emergence of chikungunya virus (CHIKV) has led to epidemics since 2004 in several parts of the world including India. The main association of CHIKV with severe arthritis and long-lasting arthralgia and closely mimics symptoms of Dengue and Zika virus infection requiring laboratory confirmation. In this study, a simple magnetic bead based ribonucleic acid extraction method was optimized, which was coupled with isothermal polymerase spiral reaction (PSR) technique for early and rapid detection. Subsequently, the polymerase spiral reaction reagents were converted to dry down format that led to a rapid user friendly field compatible sample processing to answer method for rapid and onsite detection of Chikungunya virus. Both the methods were evaluated with a panel of clinical samples. The sensitivity of the assays were compared with available commercial viral RNA extraction platform and qRT-PCR. The in-house nucleic acid extraction system based on magnetic bead followed by dry down RT-Polymerase Spiral Reaction assay was found to be highly sensitive with 10 copies of RNA as limit of detection in CHIKV clinical specimens. With respect to other closely related viruses no cross reactivity was observed. This novel methodology has the potential to revolutionize the diagnosis of infectious agents in resource limited settings around the world.

SUBMITTER: Sharma S

PROVIDER: S-EPMC7363856 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Development of magnetic bead based sample extraction coupled polymerase spiral reaction for rapid on-site detection of Chikungunya virus.

Sharma Shashi S Pardasani Deepak D Dash Paban Kumar PK Parida Manmohan M Dubey Devendra Kumar DK

Scientific reports 20200715 1

The molecular detection system has evolved over last two decades and is rapidly replacing the conventional confirmatory techniques in diagnostic virology. However the major limitation in implementation of available molecular detection assays is the non availability of field deployable nucleic acid isolation platform coupled with gene amplification technique. The rapid and early molecular detection is crucial for employing effective measure against many viral infections. The re-emergence of chiku ...[more]

PMID: 32669639

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Project description:Objective: A Tag Array chip was used to detect plasmids of different template concentration, and then analyzed for sensitivity and specificity. Drug resistance genes from tuberculosis clinical specimens were detected, giving comparative phenotypic resistance results to explore the feasibility and value of clinical applications. Methods: Twenty-four strains of Mycobacterium Tuberculosis (MTB) having sequence differences in extracted plasmids of mutant strains. The plasmid was diluted into different concentrations, and then was performed to analyze the sensitivity and specificity of the chip system. A total of 427 clinical specimens (including spinal tuberculosis and pulmonary tuberculosis) were collected from patients who came from seven hospitals. Design, optimization and preparation of the chip detection system, sequencing and phenotypic drug susceptibility results were analyzed to evaluate the sensitivity and specificity of the gene chip. Results: In the template, concentrations of 1 × 103 copies/?L and above were consistent with sequencing results in the mutant. The sensitivity and specificity in spine Tuberculosis specimen of rifampicin (RFP) were 94.40 and 92.86%; isoniazide (INH) were 92.37 and 87.50%; ethambutol (EMB) were 61.36 and 89.29%; fluoroquinolones (FQS) were 79.41 and 92.86%; streptomycin (SM) were 90.18 and 89.29%; second line drugs (SLD) were 77.61 and 83.93%. In Pulmonary Tuberculosis specimen, the sensitivity and specificity respectively were RFP: 92.74%; 93.75%; INH: 91.26%; 87.50%; EMB: 54.17%; 89.58%; FQS: 84.87%; 93.75%; SM: 86.73%; 85.42%; SLD: 80.9%; 91.67%. The RFP, INH, FQs and SM resistance genes was highly sensitive and specific: however, for detection of amikacin (AMK), capreomycin (CPM), kanamycin (KM), specificity was higher, but sensitivity was lower. Sensitivity for the detection of a mutation in the eis promoter region could be improved. Conclusion: Tag Array chip can achieve rapid, accurate detection of tuberculosis resistance, which has important clinical significance and feasibility.

Dataset Information

Development of magnetic bead based sample extraction coupled polymerase spiral reaction for rapid on-site detection of Chikungunya virus.

Publications

Development of magnetic bead based sample extraction coupled polymerase spiral reaction for rapid on-site detection of Chikungunya virus.

Similar Datasets

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