A new combination of RNA-mediated DNA ligation and on-chip elongation for detecting viral RNA.
We describe a novel method that combines RNA-mediated DNA ligation and on-chip elongation for detecting viral RNA. These virus species-specific detection probes (DPs) were designed to match sequences of the "target" virus and then chemically synthesized into 2 parts. If the target virus exists, 2 parts of the DP can be ligated by T4 DNA ligase. The ligated DP was hybridized to its corresponding capture probe (CP) on a DNA array. Then, an on-chip DNA polymerization (including Cy3-dUTP) was performed using the ligated DP as a template and the CP as a primer, which resulted in a reporting fluorescent signal. If the target virus does not exist in a sample, no fluorescence signal is produced. Four common tobacco viruses, tobacco mosaic virus, cucumber mosaic virus, potato virus Y, and potato virus X in single and mixed infections were successfully detected by this method. The sensitivity of the detection limit of this assay is 10 times higher than that of ELISA. The minimum dilution detection limit of this assay was 10(-4) (infected sap/healthy sap). The method has the potential to detect any viral RNA from animal, germs, or fungi where the sequence is known.
Li
TongXiang
T
School of Life Sciences, Lanzhou University, Lanzhou, China.
Xie
ZhongKui
Z
Wang
YaJun
Y
Li
Bo
B
Zhang
YuBo
Y
An
LiZhe
L
D017799:Cucumovirus / Q000235:genetics / Q000302:isolation & purification; D011088:DNA Ligases / Q000378:metabolism*; D015342:DNA Probes; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D018515:Plant Leaves / Q000821:virology; D016133:Polymerase Chain Reaction; D017863:Potexvirus / Q000235:genetics / Q000302:isolation & purification; D017800:Potyvirus / Q000235:genetics / Q000302:isolation & purification; D012328:RNA Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014026:Nicotiana / Q000821:virology*; D014027:Tobacco Mosaic Virus / Q000235:genetics / Q000302:isolation & purification; D014773:Virology / Q000379:methods
D015342:DNA Probes; D012367:RNA, Viral; D011088:DNA Ligases