A real-time fluorescence polymerase chain reaction assay for the identification of Yersinia pestis using a field-deployable thermocycler

Military Medicine, Oct 2003 by McAvin, James C, McConathy, Marlana A, Rohrer, Andrew J, Huff, William B, Et al

Real-time fluorescence polymerase chain reaction is a microbial identification method that can provide rapid and accurate results using a field-deployable thermocycler, the RAPID ("ruggedized" advanced pathogen identification device). A Yersinia pestis-specific TaqMan assay required approximately 75 minutes and achieved a sensitivity of 100 fg of Y. pestis genomic DNA (20 genome equivalents). Specificity testing against a genomic DNA cross-reaction panel comprised of 22 bacterial species encountered in the respiratory tract resulted in no false positives. No cross-reaction occurred with human genomic DNA.

Introduction

Quick and accurate identification assays are crucial in response to biological agents in warfare and bioterrorist ats. Biological agents that are currently of the greatest threat are Bacillus anthracis, variola major (smallpox virus), and Yersinia pestis.1 The plague bacillus, Y. pestis, has an ominous history. During three major pandemics over the past 1,500 years, it is estimated to have caused 100 to 200 million deaths with mortality rates as high as 50% to 60% during several epidemics.2 During a recent plague outbreak in Surat, India, dozens of deaths from the bacillus were confirmed by serology; however, the loss of life and suffering attributed to medical facilities overwhelmed by a panic-stricken population will never be known.3 The economic loss was in the billions. The threat as a biological weapon is significant; it is estimated that more than 50% of infected individuals will die without early antibiotic therapy, and the potential societal impact is staggering.4-6 Quick and accurate identification assays ensure implementation of antibiotic therapy and countermeasure responses in a time-critical manner as well as facilitate the dissemination of public awareness information to assuage alarm.

Molecular assays that can be implemented in the field are inherently valuable because identification can be achieved with enhanced sensitivity and specificity and identification is not diminished with nonviable organisms.7 A collaborating laboratory provided primer and fluorogenic TaqMan hybridization probe sequences designed to establish a high throughput polymerase chain reaction (PCR) assay for the identification of Y. pestis. Although the high throughput assay system achieved excellent sensitivity and specificity, it does not allow for rapid identification in a field environment. Our laboratory has adapted the high throughput TaqMan assay to a field-deployable assay platform, the RAPID ("ruggedized" advanced pathogen identification device).8-11 The TaqMan probe is a dual fluorophore-labeled, oligonucleotide specific to the PCR product, amplified Y. pestis DNA, generated by primer extension of the organism's DNA template.12 Increasing fluorescence represents the quantity of DNA target sequence amplified over the duration of the PCR. By implementing a standard curve, linear regression analysis quantifies the number of DNA target sequences in an unknown sample.

The time required for a conventional laboratory analysis is unacceptable in situations in which a biological weapon has been used against fielded troops or in any bioterrorism situation. The need for accurate in situ identification systems for biowarfare agents is paramount.13 This article describes a highly sensitive and specific, real-time, fluorogenic PCR assay for the detection of Y. pestis using a field-deployable thermocycler.

Materials and Methods

Assay sensitivity was determined with Y. pestis genomic DNA obtained from a collaborating laboratory. Identification of the strain is undisclosed. Specificity testing was conducted with human genomic DNA (Roche Molecular Biochemicals, Mannheim, Germany) and genomic DNA was isolated from bacterial cultures prepared from laboratory stocks (Clinical Microbiology Laboratory, Air Force Institute for Environment, Safety, and Occupational Health Risk Analysis, Epidemiology Surveillance Division, Brooks Air Force Base, Texas). Genomic DNA was isolated from overnight cultures using the MagNA Pure LC System and MagNA Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics, Mannheim, Germany).14,15

Bacterial organisms commonly cultured from the respiratory tract were selected and recovered from frozen cultures, restreaked to single colonies on agar plates, cultured under conditions appropriate for each species, and identification confirmed with conventional biochemical analyses and the Vitek Automicrobic system (Biomerieux-Vitek, Hazelwood, Missouri). A single colony of each organism was transferred with a sterile loop to liquid culture media, was incubated for 18 to 48 hours, and 200 [mu]L of each liquid culture was pipetted into the MagNA Pure LC sample cartridge. All postloading processing was completed in a closed system by automated robotics with preformatted reagents and a nucleic acid isolation matrix. Cell lysis and nucleic acid stabilization was completed with buffer containing guanidinium thiocyanate and proteinase K. Nucleic acid bound to the surface of magnetic glass particles was isolated from other cellular components by washing and eluting with a low-salt buffer.