Development of A Sensitive Method for Novel Virus Detection
Date Issued
2009
Date
2009
Author(s)
Lu, Yu-Tsung
Abstract
In the past decades, the infections caused by newly emerging viruses increased rapidly. For the specimens containing potential unknown pathogens, in addition to the electron microscopy examination, microbe-microarray hybridization is usually used to identify the causative pathogens. To further increase the detection rate, several pre-analysis amplification methods were included for amplification of the nucleic acids of causative pathogens, such as the representational difference analysis (RDA), sequence-independent single primer amplification (SISPA), or random PCR reaction. However, the detection sensitivity is still quite limited. The specific aim of this study is thus set to the development of a method for efficient pre-amplification, which can help sensitive detection of causative unknown pathogens. Part I is to collect the clinical specimens suspected to contain unknown pathogens. Part II is to develop and optimize the pre-amplification method based on the whole genome amplification (WGA) platform. Part III is to evaluate the feasibility of the optimized pre-amplification method for the collected clinical specimens. In total, 676 clinical samples suspected with unknown pathogens from routine diagnostic lab were processed for cytopathic effect (CPE) validation by virus culture. Only 2 samples showed clear CPE until 4th passage, but without specific virus antigen detected. In addition, 10 primary specimens were collected from suspected cases of unknown infectious disease, at the acute phase. To set up the method for efficient pre-amplification, two approaches were used (1) using micrococcal nuclease to eliminate any non-virus nucleic acid, and (2) applying WGA platform to increase the sensitivity, respectively. The results from nuclease showed that most of the host genome (at least >99.7%) could be eliminated by the treatment, with the virus genome in the virions remained intact. To evaluate the efficiency of the WGA platform in amplification of viral genome, the coronavirus JHM strain was used for our testing. Two assays were included for the evaluation, the multiple strand displacement amplification and genomeplex amplification assay. We found that multiple strand displacement amplification was more suitable for closed circular form DNA, while linear form DNA was amplified more efficiently by PCR-based Genomeplex amplification. In general, the amplification efficiency depends on the length and the titer of viral genome analyzed. By using coronavirus JHM strain as the target, the sensitivity of the optimized pre-amplification step included in the amplification procedure were as low as 103copies in 400ul specimen and it could be efficiently amplified (> 104 fold). The feasibility of its general clinical application will be further pursued in different types of viruses, including the DNA and RNA viruses with different genome sizes. We hope that the method could be applied for the specimens suspected to contain unknown infectious pathogens in the near future.
Subjects
Novel Virus
Detection
Sensitive
Whole Genome Amplification
SDGs
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