Honors College Thesis

 

Development of Simple, Rapid, and Sensitive qPCR Assay for the Detection of Pseudocapillaria tomentosa in Danio rerio Tissue Samples Public Deposited

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  • Pseudocapillaria tomentosa is a nematode that parasitizes Danio rerio, causing major disease that affects the economic commodities of fish industries and destroys experimental results at research facilities. There is a critical need for a rapid and sensitive diagnostic assay to test for P. tomentosa in D. rerio populations. PCR primers were designed for a small subunit ribosomal DNA (SSU rDNA) of P. tomentosa based on DNA sequence alignment with closely related capillarid nematode Pseudocapillaria xenopi. The SSU rDNA was amplified using PCR, then the 97-bp amplicon was purified and sequenced. A fluorescent reporter probe was created using the SSU rDNA sequence unique to P. tomentosa. Using the probe, a quantitative PCR (qPCR) assay was developed to detect the SSU rDNA of P. tomentosa sequence in isolated total DNA from minimally- to moderately-infected D. rerio samples (infection as determined through microscopic analysis of tissue). Positive Cycle Threshold (CT) values averaged 30.43±2.52 for moderately-infected D. rerio samples and 31.57±0.33 for minimally-infected D. rerio samples, indicating strong detection of P. tomentosa from both sample types. The specificity of this assay was validated by using negative control DNA from non-infected D. rerio. Pseudocapillaria tomentosa eggs analyzed had negative CT values. Therefore, a rapid and sensitive diagnostic assay was created for testing of P. tomentosa in D. rerio populations through the use of tissue samples. Key Words: Pseudocapillaria, zebrafish, quantitative PCR, nematode, sequencing
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