Real-time quantitative fluorescent reverse transcriptase-PCR for detection of severe acute respiratory syndrome-associated coronavirus RNA.

Aim: SARS-associated coronavirus (SARS-CoV) has been confirmed as the pathogen for severe acute respiratory syndrome (SARS). The aim of our study was to construct a sensitive and specific real-time quantitative fluorescent (QF) reverse transcriptase (RT)-PCR method for the detection of SARS-CoV RNA.
Methods: Stored blood specimens from 44 patients with confirmed SARS were used along with blood samples from two sets of controls, 30 healthy volunteers who had no contact with SARS patients, and 30 healthy doctors and nurses who had contact with SARS patients but were without symptoms of SARS. Two pairs of primers were synthesized by the Shanghai Sangon Company according to SARS-CoV BJ01 strain sequence (AY278488), and then a pair of primers were designed and compared with a pair of primers published by WHO.
Results: Using serial dilutions of SARS-CoV, the 44 blood samples from SARS patients specimens were tested. Using a 0.01% dilution of SARS-CoV, all 44 clinical samples tested positive in our assay. In comparison, using a 0.1% dilution of SARS-CoV, 26 of the 44 samples tested positive using the WHO primers. In the QF-RT-PCR assay, there was a linear amplification from 100 copies to 10(8) copies of the control RNA per RT-PCR and at least 10 copies, and sometimes even 1 copy, of target RNA tested positive in our assay.
Conclusion: The primer we developed is sufficiently sensitive and specific to diagnose symptomatic SARS-CoV infections and for monitoring virus load.