Sensitive and quantitative detection of severe acute respiratory syndrome coronavirus infection by real-time nested polymerase chain reaction

Abstract

A quantitative, real-time, nested polymerase chain reaction (PCR) method, combining the high sensitivity of nested PCR with time-saving real-time instrumentation, was developed for large-scale screening for severe acute coronavirus (SARS) coronavirus. Forty-six clinical specimens were analyzed by this method, and results were compared with those obtained by conventional, single-round, real-time reverse-transcriptase PCR (RT-PCR) performed in parallel. Of the 17 positive results, 2 identified by our method were not detected by single-round, real-time RT-PCR, which suggests that real-time nested PCR has the potential for increased sensitivity, leading to earlier detection of SARS.

Figure 1

A, Results of the real-time nested PCR with indicated starting RNA copy number in each assay. ⋄, <1 RNA copy; □, 10-fold dilution of the control RNA sample used for the single-copy RNA test; NC, negative control, in which input RNA was replaced by deionized water. B, Linear amplification by real-time nested PCR. The starting viral RNA copy number was plotted against the threshold cycle number of each dilution and fitted with a linear regression model.

Table 1

Comparison of results of real-time nested PCR and single-round RT-PCR test for clinical samples from 46 patients.