Limitations of TaqMan PCR for detecting divergent viral pathogens illustrated by hepatitis A, B, C, and E viruses and human immunodeficiency virus

Abstract

Recent events illustrate the imperative to rapidly and accurately detect and identify pathogens during disease outbreaks, whether they are natural or engineered. Particularly for our primary goal of detecting bioterrorist releases, detection techniques must be both species-wide (capable of detecting all known strains of a given species) and species specific. Due to classification restrictions on the publication of data for species that may pose a bioterror threat, we illustrate the challenges of finding such assays using five nonthreat organisms that are nevertheless of public health concern: human immunodeficiency virus (HIV) and four species of hepatitis viruses. Fluorogenic probe-based PCR assays (TaqMan; Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.) may be sensitive, fast methods for the identification of species in which the genome is conserved among strains, such as hepatitis A virus. For species such as HIV, however, the strains are highly divergent. We use computational methods to show that nine TaqMan primer and probe sequences, or signatures, are needed to ensure that all strains will be detected, but this is an unfeasible number, considering the cost of TaqMan probes. Strains of hepatitis B, C, and E viruses show intermediate divergence, so that two to three TaqMan signatures are required to detect all strains of each virus. We conclude that for species such as hepatitis A virus with high levels of sequence conservation among strains, signatures can be found computationally for detection by the TaqMan assay, which is a sensitive, rapid, and cost-effective method. However, for species such as HIV with substantial genetic divergence among strains, the TaqMan assay becomes unfeasible and alternative detection methods may be required. We compare the TaqMan assay with some of the alternative nucleic acid-based detection techniques of microarray, chip, and bead technologies in terms of sensitivity, speed, and cost.

FIG. 1.

Flowchart outlining our computational approach for finding potential signatures from DNA sequence data.

FIG. 2.

Phylogenetic tree describing the DNA sequence relationships among the HIV strains used in these analyses and constructed by using the dnapars and drawgram programs in the PHYLIP package. For those strains that share TaqMan assay results with the strain with genomic identity (gi) number 11095910 (enlarged text, fifth from right), the numbers of (potentially overlapping) assays that the pair shares are indicated. The strain with genomic identity number 11095910 shares TaqMan signatures with phylogenetically distant strains and does not necessarily share signatures with many of the more closely related strains, nor do the numbers of signatures shared follow any distinct pattern. Thus, phylogenetic proximity does not directly relate to whether strains share potential TaqMan signatures.

FIG. 3.

Comparison of the TaqMan assay, bead-based assays, and GeneChips for pathogen detection in terms of costs per reaction (A), numbers of assays per reaction (B), the cost per assay if the maximum number of assays per reaction are performed (the value for GeneChips is $0.002) (C), start-up costs (D), sensitivity in a pure sample (number of genome copies) (E), and speed of detection after the DNA is prepared, that is, the amplification and detection steps (F).