P1-22 Development of PCR Amplification Methods Based on Cyclospora cayetanensis Mitochondrial Genomes

Monday, July 10, 2017
Exhibit Hall (Tampa Convention Center)
Mauricio Durigan , U.S. Food and Drug Administration–CFSAN, Office of Applied Research and Safety Assessment , Laurel , MD
Gopal Gopinath , U.S. Food and Drug Administration , Laurel , MD
ChaeYoon Lee , U.S. Food and Drug Administration , Laurel , MD
Hediye Cinar , U.S. Food and Drug Administration–CFSAN, Office of Applied Research and Safety Assessment , Laurel , MD
Sonia Almeria , U.S. Food and Drug Administration–CFSAN, Office of Applied Research and Safety Assessment , Laurel , MD
Helen Murphy , U.S. Food and Drug Administration–CFSAN, Office of Applied Research and Safety Assessment , Laurel , MD
Alexandre da Silva , U.S. Food and Drug Administration–CFSAN, Office of Applied Research and Safety Assessment , Laurel , MD
Introduction:  A method to detect Cyclospora cayetanensis in produce was recently validated for regulatory testing by the FDA. This method, which relies on a sample preparation step followed by a TaqMan assay designed using the parasite’s 18S rDNA, detects approximately 10 C. cayetanensis oocysts in produce. Recent advances in genome sequencing of C. cayetanensis include a repository for genome assemblies created in collaboration with NCBI (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA316938), which is being populated with organelle as well as nuclear genome sequences. These novel genetic markers must be evaluated to advance the development of diagnostic detection of C. cayetanensis.

Purpose: We designed PCR primers based on C. cayetanensis mitochondrial genome sequences and compared them with the validated qPCR method for the detection of C. cayetanensis in produce.

Methods:  A total of eight PCR primers that produced amplicons ranging from 182 to 538 base pairs were designed. Specificity was tested using DNA extracted from Eimeria acervulina, cilantro, and raspberries. Sensitivity was evaluated using DNA extracted from cilantro and raspberries seeded with 10, 20, and 200 oocysts of C. cayetanensis. Unseeded cilantro and raspberries were used as negative controls.

Results:  All PCR assays from unseeded samples were negative. Two of the eight PCRs amplified C. cayetanensis as well as E. acervulina. The remaining six PCRs amplified C. cayetanensis only; furthermore, one of the PCRs detected as few as 10 oocysts in produce, comparable to the detection limit of the regulatory method.

Significance:  This investigation reports the first C. cayetanensis molecular detection method designed using mitochondrial genome sequences. This unique tool can be used to confirm the results of the qPCR C. cayetanensis method. Studies such as this, support public health and the FDA mission by developing secondary confirmatory methods to support findings requiring regulatory actions.