APHL 2022 POSTER ABSTRACTS
Two multiplex PCR panels with five primer and probe sets each have been designed to be tested sequentially . The first PCR panel differentiates whether a tested clinical specimen is a member of the Vibrio genus or of four additional genera that commonly cause foodborne illness : Listeria , Salmonella , Shigella and Yersinia . If the first PCR panel indicates that a tested clinical isolate is a Vibrio , a second multiplex PCR reaction is used to determine if the bacterium is one of five Vibrio species that commonly cause disease : Vibrio alginolyticus , Vibrio cholerae , Vibrio fluvialis , Vibrio parahaemolyticus and V . vulnificus . For the PCR panels , the oligonucleotide primer sets and TaqMan probes for Vibrio detection were designed to amplify sequences that are highly conserved among the Vibrio genus or among specific Vibrio species but are absent in other genera or species , respectively . Validation of the PCR primer and probe sets were performed individually and in multiplex on five clinical isolates of each Vibrio species . Future directions include adapting the PCR method for the detection of Vibrios on contaminated seafood and within clinical specimens , quantifying the detection limit of the method and testing the ability of the method to detect mixtures of Vibrio species .
Presenter : Michaela Eickhoff , New Jersey Department of Health , Public Health and Environmental Laboratories , mjeickhoff2 @ gmail . com
Analysis of Pesticides in Fruit Juices by Modified QuEChERS Extraction and Analyzed by High Performance Liquid Chromatography Quadrupole Time of Flight
D Wene , S O ’ Leary , A Lin , CH Yu and Z Fan , New Jersey Public Health and Environmental Laboratories
The New Jersey State Public Health and Environmental Laboratories ( PHEL ) was the recipient of an FDA grant to enhance public health by developing a method to test pesticides in juices . The method will enhance public health by providing a new method for the New Jersey public health laboratories . Recent studies report concerns in finding increasing quantities of pesticides in juices . Pesticides can be extremely harmful to humans . Carbamates and organophosphates negatively affect the nervous system . Research data supports findings that other groups cause cancer as well as detrimentally affect the endocrine system . This method was adapted from a liquid chromatography ( LC ) triple quad mass spectrometer ( QqQ MS ) method to detect pesticides in marijuana based on literature . The method was setup to detect harmful pesticides regulated in the US as well as pesticides banned in the US because fruit juices can be imported from other countries . Juices from Spain and the UK have relatively high level of pesticides compared to the US . Presently there are limited regulations set by the US government to monitor fruit juices for pesticides . The aim of the research is to develop a method to test for pesticides in apple and grape juices utilizing a quadrupole time-of-flight ( Q-ToF ) instrument . The method was optimized for ToF instrumentational analysis and utilizes ultra highpressure liquid chromatography ( UHPLC ) to separate compounds . The extraction process was modified from the standard QuEChERS method . Experiments were performed on the Q-ToF instrument to determine optimal gas flows and temperatures , voltages on the capillary and nebulizer pressure . UHPLC conditions were optimized as well as modifications with the mobile phase . A fully comprehensive method was developed and validated . The method will be able to detect all pesticides at levels below ten parts per billion , though most are more sensitive . All limits of detection are between 0.023 and 3.23 ppb . The laboratory was able to develop the method for 64 pesticides in multiple brands of apple and grape juices . Baseline separation was achieved for all compounds . The limits of quantitation , between 0.07 and 9.7 ppb , were below tolerances set by the US government . Linearity was achieved with all pesticides using six calibration points and a coefficient of determination greater than 0.995 . Spike recoveries were performed utilizing seven spikes . Spikes recoveries were evaluated and results support a recovery range of 60-130 %. Reproducibility was within 22 %. Interference determination was analyzed for three different apple and grape consumer brands with five extractions for each brand . All interferences were below the lowest standard . The validated method will be utilized by to run samples of fruit juices for any agency that requests testing . The results of development and validation will be discussed in the poster .
Presenter : Daniel Wene , New Jersey Public Health and Environmental Laboratories , daniel . wene @ doh . nj . gov
The Limit of Detection of the BioFire ® FilmArray ® Gastrointestinal Panel for the Foodborne Parasite Cyclospora cayetanensis
A Peterson 1 , T Richins 1 , K Houghton 2 , M Mishina 2 , S Sharma 2 , Y Qvarnstrom 2 , V Cama 2 ; 1 Oak Ridge Associated Universities , Oak Ridge Institute for Science and Education , 2 Centers for Disease Control and Prevention
Cyclosporiasis is a foodborne diarrheal illness caused by the parasite Cyclospora cayetanensis . Since 2018 , > 1000 laboratory confirmed cases of cyclosporiasis have been reported annually in the United States . Diagnosis of cyclosporiasis has primarily relied on parasite specific methods , though new detection tools are now available including syndromic panels such as the BioFire ® FilmArray ® Gastrointestinal ( GI ) panel , a molecular multiplex panel that tests for 22 unique gastrointestinal pathogens in a single stool sample , including detection of C . cayetanensis .
In 2021 , the Centers for Disease Control and Prevention ( CDC ) received 251 specimens from patients diagnosed with cyclosporiasis using the BioFire ® FilmArray ® GI panel for genotyping through our novel workflow . However only 71 % of these successfully genotyped , raising questions about potential differences in the limit of detection ( LOD ) of the BioFire ® FilmArray ® GI panel in comparison to the CDC genotyping workflow . The published BioFire ® FilmArray ® GI panel LOD is 180 genome equivalents ( GEq )/ mL , though it is unclear how this relates to the number of C . cayetanensis oocysts present in a clinical sample . Thus , we sought to determine the LOD of the BioFire ® FilmArray ® GI panel in terms of numbers of C . cayetanensis oocysts . We used a BD FACSAria™ II Cell Sorter to sort specific numbers of C . cayetanensis oocysts previously purified from three different human stool sources . We sorted 50 , 40 , 30 , 20 , 10 , 5 and 1 oocyst ( s ) from each of the sources in triplicate , for a total of nine samples at each of the seven values . We added 200 µ L of negative stool matrix in Cary-Blair to the sorted oocysts and ran samples on a BioFire ® FilmArray ® Torch instrument according to the manufacturer ’ s instructions . We found that the BioFire ® FilmArray ® GI panel consistently detected ≥20 C . cayetanensis oocysts ( 9 / 9
Food Safety