Volume 21, Number 8—August 2015
Genomic Assays for Identification of Chikungunya Virus in Blood Donors, Puerto Rico, 2014
|Collection date, 2014
||Prototype CHIKV real-time assay on Panther system, dilution†‡
|Initial testing, undiluted
|Reactive/total no. tested||1/1||3/3||3/3||3/3||3/3||2/3||0/3||NT||NT|
|Estimated copies/mL||2.9 × 105|
|Reactive/total no. tested||1/1||3/3||3/3||3/3||3/3||3/3||2/3||NT||NT|
|Estimated copies/mL||7.6 × 105|
|Reactive/total no. tested||1/1||3/3||3/3||2/2||3/3||3/3||3/3||3/3||3/3|
|Estimated copies/mL||9.1 × 107|
*CHIKV, chikungunya virus; NT, not tested.
†For the real-time CHIKV, transcription-mediated amplification assay, plasma samples (0.5 mL) were tested on the fully automated Panther system which performs magnetic target specific capture, amplification, and real-time detection in the presence of an internal control. During the target capture step, the hybridized target is captured onto magnetic micro-particles that are separated from the specimen in a magnetic field. Wash steps remove extraneous components from the reaction tube. Target amplification occurs by using 2 enzymes, MMLV (Moloney murine leukemia virus) reverse transcription and T7 RNA polymerase. Detection is achieved using single-stranded fluorescent labeled nucleic acid probes that are present during the amplification of the target. The time for the fluorescent signal to reach a specified threshold is proportional to the starting CHIKV RNA concentration. The primers, detection probes, and target capture oligonucleotides hybridize to highly conserved regions of CHIKV RNA genome and were designed to detect all 3 major CHIKV lineages. The cutoff for reactive reactions was set by the investigators at 1,000 relative fluorescent units. Estimated copies per mL were calculated relative to the emergence time of the emitted fluorescence of a calibration curve generated by logarithmic dilution of a CHIKV in vitro synthetized transcript.
‡Dilutions were performed in defribrinated, pooled plasma, passed through a 0.2-μm filter, dialyzed to approximate a human serum profile, delipidated for clarity/stability, and prescreened as nonreactive for CHIKV.
§CHIKV-positive donor reported postdonation fever and joint pain at 2 d postdonation.