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 .