Rapid identification of emerging infectious agents using PCR and electrospray ionization mass spectrometry

Abstract

Newly emergent infectious diseases are a global public health problem. The population dense regions of Southeast Asia are the epicenter of many emerging diseases, as evidenced by the outbreak of Nipah, SARS, avian influenza (H5N1), Dengue, and enterovirus 71 in this region in the past decade. Rapid identification, epidemiologic surveillance, and mitigation of transmission are major challenges in ensuring public health safety. Here we describe a powerful new approach for infectious disease surveillance that is based on polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry (ESI-MS) for accurate mass measurements of the PCR products, and base composition signature analysis to identify organisms in a sample. This approach is capable of automated analysis of more than 1,500 PCR reactions a day. It is applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens and will facilitate rapid characterization of known and emerging pathogens.

Figure 1

Automated PCR/ESI‐MS biosensor. Shown in this view are key modules including amplicon purification (desalting), plate stackers, sample injection, and the mass spectrometer. Precise molecular weight determinations of amplicons yield unambiguous base compositions that are used to uniquely “fingerprint” each pathogen. The automated system is capable of analyzing more than 1,500 PCR reactions in 24 h.

Figure 2

Mass spectra of calibrant and SARS coronavirus (SARS CoV) samples are shown. Both strands were amplified and thus there are two major peaks for both calibrant and SARS. The two templates were observed to be at a relative abundance ratio of approximately 1:1.5. From the input number of calibrant nucleic acid molecules (3 × 10⁴), we estimated that SARS RNA was present in the sample at ∼4.5 × 10⁴ genome copies.