Rapid Identification of Emerging Pathogens: Coronavirus
We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp., including the severe acute respiratory syndrome-associated coronavirus (SARS-CoV). We show that this method could identify and distinguish between SARS and other known CoV, including the human CoV 229E and OC43, individually and in a mixture of all 3 human viruses. The sensitivity of detection, measured by using titered SARS-CoV spiked into human serum, was ≈1 PFU/mL. This approach, applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens, is capable of automated analysis of >900 PCR reactions per day.

Nucleic acid tests for infectious diseases are primarily based on amplification methods that use primers and probes designed to detect specific organisms. Because prior knowledge of nucleic acid sequence information is required to develop these tests, they are not able to identify unanticipated, newly emergent, or previously unknown infectious organisms. Thus, the discovery of new infectious organisms still relies largely on culture methods and microscopy, which were as important in the recent identification of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) as they were in the discovery of HIV 2 decades ago.

Broad-range polymerase chain reaction (PCR) methods provide an alternative to single-agent tests. By amplifying gene targets conserved across groups of organisms, broad-range PCR has the potential to generate amplification products across entire genera, families, or, as with bacteria, an entire domain of life. This strategy has been successfully used with consensus 16S ribosomal RNA primers for determining bacterial diversity, both in environmental samples and in natural human flora. Broad-range priming has also been described for detection of several viral families, including CoV, enteroviruses, retroid viruses, and adenoviruses. The drawback of this approach for epidemiologic applications is that the analysis of PCR products for mixed amplified samples requires sequencing hundreds of colonies per reaction, which is impractical to perform rapidly or on large numbers of samples. New approaches to the parallel detection of multiple infectious agents include multiplexed PCR methods and microarray strategies. Microarray strategies are promising because undiscovered organisms might be detected by hybridization to probes designed to conserved regions of known families of bacteria and viruses.

We present an alternative approach for rapid, sensitive, and high-throughput detection of infectious organisms. We use broad-range PCR to generate amplification products from the broadest possible grouping of organisms, followed by electrospray ionization mass spectrometry and base composition analysis of the products. The base compositions of strategically selected regions of the genome are used to identify and distinguish organisms in the sample. Enhanced breadth of priming is achieved through the use of primers and probes containing 5-propynyl deoxycytidine and deoxyuridine nucleotides that offer increased affinity and base pairing selectivity. Positioning the 5-propynyl primidine-modified nucleotides at highly conserved positions enables priming at short consensus regions and significantly increases the extent to which broad groups of organisms can be amplified.