Lab Matters Summer 2017 | Page 30

from the bench

By Michael Perry, MS, MSEd, laboratory supervisor, New York State Department of Health, Wadsworth Center and Tyler Wolford, MS, senior specialist, Laboratory Response Network

Laboratory Response Network for Biological Threats Preparedness( LRN-B) laboratories receive environmental and clinical specimens for screening on a daily basis. Advancements in technologies have allowed for faster, more accurate detection of biological threat agents and emerging pathogens and a greater understanding of these organisms. But before new technologies such as real-time polymerase chain reaction( PCR), next generation sequencing( NGS) and Matrix Assisted Laser / Desorption Ionization- Time of Flight Mass Spectrometry( MALDI-TOF MS) can be implemented, the instruments and accompanying assays must be extensively validated to ensure that they will function as claimed by the manufacturers. The Biodefense Laboratory at the Wadsworth Center has led several verification studies to demonstrate reproducibility of assays and instruments across LRN-B laboratories.

Assay Validation

As with any assay used to govern public health decision making, a critical performance element of diagnostic assays is the determination of the limit of detection. The Wadsworth Center Biodefense Laboratory has undertaken multiple studies aimed at determining the sensitivity of assays for the detection of bacterial and viral agents such as variola virus. Recently the Biodefense Laboratory validated a variola virus PCR assay which uses a fluorescent labeled probe to monitor the quantity of doublestranded product that is produced during

PCR, providing LRN-B laboratories with qualitative detection of variola DNA in clinical specimens. Understanding the limit of detection as well as the limitations of these assays is critical for administrative decision making by federal, state and local emergency management and public health laboratories.

Recent assay validations include:

• Evaluation of biothreat agent viability with three MALDI-TOF MS preparation methods to determine if specimen preparation methods were capable of inactivating biothreat agents prior to analysis. This provided insight into the risks associated with analyzing agents on a MALDI-TOF MS.

• Evaluated the MALDI-TOF MS as an alternative method for the detection of Clostridium botulinum neurotoxin. This method allows for faster and more sensitive detection of the toxin.

• Collaborated with CDC on whole genome sequencing of select agents including Clostridium botulinum and Brucella species.

• Determined clinical specificity of LRN real-time Polymermase Chain Reaction( rt-PCR) assays by testing specimen extracts to demonstrate that human specimens will not generate false positive results, thereby increasing confidence in positive results.

• Evaluation of an internal extraction control for rt-PCR by evaluating both commercially available extraction kits as well as a Wadsworth Center in-house developed extraction control.

Michael Perry from the Wadsworth Center loading a plate for real-time Polymermase Chain Reaction( rt-PCR) testing. Photo courtesy of the New York State Department of Health – Wadsworth Center

• Compared a Lateral Flow Assay( LFA) and Time Resolved Fluorescence( TRF) assays for the detection of ricin toxin to determine a range of detection for LFA and whether it was a suitable alternative to the LRN-B approved TRF assay.