Comparison of multiplex PCR hybridization-based and singleplex real-time PCR-based assays for detection of low prevalence pathogens in spiked samples☆
Introduction
It has been more than two decades since the introduction of PCR (Mullis et al., 1986; Shampo and Kyle, 2002). Although this technology was implemented almost immediately in research laboratories, it has only been within the past decade that new diagnostic PCR-based technologies are increasingly being integrated into clinical microbiology laboratory practice (Sobel et al., 2008). These technologies enable the detection and quantification of pathogens with increased sensitivity and speed. The implementation of PCR-based nucleic acid tests in blood donor screening and for diagnosing infectious diseases has substantially improved the ability to obtain rapid, actionable information (Brittain-Long et al., 2011; Roth et al., 2012). There are numerous competitive nucleic acid based technologies that have been utilized for the detection and characterization of microorganisms. These technologies can be separated into several broad categories such as direct hybridization, nucleic acid amplification, and a variety of methods for post-amplification analysis such as sequencing, melt-curve analysis and others. Several methods combine the sensitivity of end-point PCR amplification with hybridization-based methodology, allowing the detection of multiple pathogens in one reaction (multiplex) which increases the throughput of the testing platform without compromising assay performance. This work is aimed at comparing the performance of multiplex end-point TEM-PCR coupled with hybridization-based detection to singleplex RT-PCR, for detection of low prevalence pathogens spiked into human matrix such as whole blood.
The development of novel molecular devices for detection of emerging low prevalence pathogens such as Francisella tularensis and Babesia microti is hindered by the lack of clinical samples necessary to conduct clinical sensitivity studies required for U.S. Food and Drug Administration (FDA) clearance or approval (FDA/CDRH, 2014). The use of appropriate specimen matrix for assessment of analytical sensitivity is part of the regulatory requirements for laboratory developed tests or in vitro diagnostics (FDA/CDRH, 2014). Although sample transport medium or another simulated matrix can be used for demonstration of analytical sensitivity, analytical performance of molecular assays should be tested using pathogen-free clinical matrices (CLSI, 2008, FDA/CDRH, 2014). In addition to comparison of analytical sensitivity for multiplex and singleplex PCR tests, we compared human blood matrix collected from patients with fever or other signs of disease to blood collected from healthy donors because the composition of symptomatic blood can have an impact on analytical performance of both molecular methods that is different from the performance when testing healthy blood. The utilization of both testing platforms was recently demonstrated in work to standardize procedures for spiking low prevalence pathogens in human matrices (Dong et al., 2016). As in the first study, the crucial importance of this work is not so much the novelty of the methods described, but the standardization and validation of the methods for use by other investigators. By utilizing these standardized methods evaluation of new technology can be effectively compared to previously established platforms. This study expands upon previously described standardized procedures by developing methods for producing E. coli, F. tularensis, and B. microti mock specimens.
Section snippets
Materials and methods
The production of mock specimens for all three organisms shared a common work flow (Fig. 1). The organisms were cultured, aliquoted and cryopreserved. DNA extracted from the aliquots was serially diluted and tested with RT-PCR to determine the limiting dilution at which the sample could be detected, which was the basis for quantification of the cryopreserved aliquots used for spiking into blood.
Testing negative control blood specimens
The performance of two amplification methods, singleplex real time PCR (RT-PCR) and Target Enriched Multiplex PCR (TEM-PCR) was evaluated based on percentage of positive samples detected (a measure of sensitivity) and percentage of true negative samples classified correctly in these assays. Coded samples were tested and results were compared after completion of testing. The quantitative output of each assay that indicates positive detection of the pathogen (the cut off) was set in both
Discussion
RT-PCR and multiplex PCR are commonly used methods for detecting low abundance pathogens. Singleplex RT-PCR pathogen detection is considered to be a gold standard methodology for high sensitivity, reproducibility and ease of use. To reduce the burden of clinical validation for low abundance pathogens, mock specimens can be created from human matrices and cultured pathogens. However, spiking should be done by standardized methods to obtain results that can be compared appropriately (Dong et al.,
Conclusion
The standardized methods of preparing mock (spiked) clinical specimens for evaluating diagnostic devices intended to test for low prevalence pathogens have effectively demonstrated the equivalence of a proprietary multiplex platform to individual real-time PCR. The equivalence between TEM-PCR, optimized to screen for the presence of Babesia microti, Escherichia coli and Francisella tularensis, and individual real-time PCR assays for each pathogen over a three-log range of pathogen
Acknowledgements
The Francisella tularensis culture was initiated from a colony gratefully obtained from Dr. Karen Elkin's laboratory (FDA/CBER). We wish to acknowledge that this work was supported by NIAID through an Interagency Agreement AAI13005-002 awarded to CBER/FDA. Thanks to Karen Elkins, PhD and Rene Reese, PhD for reading and commenting on the manuscript.
References (20)
- et al.
Development of real-time PCR assay for differential detection and quantification for multiple Babesia microti-genotypes
Parasitol. Int.
(2011) - et al.
Kary B. Mullis—Nobel Laureate for procedure to replicate DNA
- et al.
The evolution of molecular genetic pathology: advancing 20th-century diagnostic methods into potent tools for the new millennium
J Mol Diagn.
(2008) - et al.
A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood
J. Mol. Diagn.
(2005) - et al.
Collaborative study for the characterization of a chikungunya virus RNA reference reagent for use in nucleic acid testing
Vox Sang.
(2015) - et al.
Access to a polymerase chain reaction assay method targeting 13 respiratory viruses can reduce antibiotics: a randomised, controlled trial
BMC Med.
(2011) Validation of laboratory-developed molecular assays for infectious diseases
Clin. Microbiol. Rev.
(2010)Verification and Validation of Multiplex Nucleic Acid Assays; Approved Guideline
(2008)- et al.
Microbiological Methods
(2004) Using the Student's t-test with extremely small sample sizes
Pract. Assess. Res. Eval.
(2013)
Cited by (0)
- ☆
Our contributions are an informal communication and represent our own best judgment. These comments do not bind or obligate FDA.