Development of a method for concentrating and purifying SARS coronavirus RNA by a magnetic bead capture system.
Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel coronavirus, which has been designated the SARS coronavirus (SARS-CoV). To date, molecular assays for the detection of SARS-CoV has focused mainly on reverse transcriptase-PCR (RT-PCR) analysis of specimens. However, RT-PCR assays currently available have low sensitivity during the early stage of the disease in which the viral load in specimens is very low. A method for concentrating and purifying SARS-CoV RNA by a magnetic bead capture system was developed and followed by an RT-PCR assay in this study with the goal of improving the sensitivity of the RT-PCR method. This approach takes advantage of the cooperative interaction between adjacently hybridized oligonucleotides. A capture probe was covalently coupled to magnetic beads and a second probe, which anneals adjacent to the capture probe site, was prehybridized in solution to the target. It was shown that, when applied to SARS RNA samples, the sensitivity of nucleic acid capture RTPCR was about 10-fold greater than routine RT-PCR. This nucleic acid capture system was effective in improving the sensitivity of the RT-PCR, due to enriching and purifying SARS-CoV RNA. The method will be helpful for the early detection of the SARS-associated coronavirus.
Hei
Ai-Lian
AL
National Center for Clinical Laboratory, Beijing Hospital, Beijing, People's Republic of China.
Cai
Jian-Ping
JP
D017931:DNA Primers; D042241:Early Diagnosis; D006801:Humans; D008280:Magnetics; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis / Q000821:virology; D019809:Streptavidin
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D019809:Streptavidin