CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei.
Biotechnol Rep (Amst)
; 27: e00485, 2020 Sep.
Article
en En
| MEDLINE
| ID: mdl-32577410
ABSTRACT
Enterocytozoon hepatopenaei (EHP) is a parasite that infects pacific whiteleg shrimp, Penaeus vannamei, causing growth retardation and uneven size distributions that lead to severe losses in shrimp productivity. Routine monitoring is crucial to timely prevention and management of EHP, but field-deployable diagnostic kits for EHP are still scarce. Here, we proposed the use of recombinase polymerase amplification (RPA) and CRISPR-Cas12a fluorescence assay, henceforth RPA-Cas12a, for detection of EHP. Targeting ptp2 gene, RPA-Cas12a could detect as few as 50 copies of DNA and showed no reactivity with closely related microsporidia. The entire procedure could be performed at a temperature close to 37⯰C within 1â¯h. Naked eye visualization was possible with UV/blue-light excitation or lateral flow detection. Thus, RPA-Cas12a is a rapid, sensitive and specific detection platform that requires no sophisticated equipment and shows promise for on-site surveillance of EHP.
CRISPR, clustered regularly interspaced short palindromic repeats; CRISPR-Cas12a; Cas, CRISPR-associated protein; EHP, Enterocytozoon hepatopenaei; Eca, Enterospora canceri; Enterocytozoon hepatopenaei; FB, FAM-ssDNA-Biotin reporter; FQ, fluorescent-quencher reporter; Her, Hepatospora eriocheir; IHHNV, infectious hypodermal and hematopoietic necrosis virus; LFD, lateral flow dipstick; Lateral flow detection; NTC, no-template control; PAM, protospacer adjacent motif; RPA; RPA, recombinase polymerase amplification; RPA-Cas12a, RPA coupled with Cas12a cleavage assay; SWP-PCR, nested PCR targeting swp; WSSV, white spot syndrome virus; ptp2, polar tube protein 2; swp, spore wall protein
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Biotechnol Rep (Amst)
Año:
2020
Tipo del documento:
Article
País de afiliación:
Tailandia