Establishment of rapid visual detection methods for infectious hypodermal and hematopoietic necrosis virus based on RPA and CRISPR-Cas12a
After screening and optimization,two visual rapid detection methods,the RPA-Cas12a fluorescent detection and lateral flow dipstick,for infectious hypodermal and hematopoietic necrosis virus(IHHNV)were established by designing and synthesizing multiple sets of recombinase polymerase amplification primers and specific CRISPR RNA based on the conserved regions of the IHHNV gene se-quences.The results demonstrated that both visual detection methods could detect IHHNV after a con-stant temperature reaction at 37℃for 40 min.There was no cross-reactivity with white spot syndrome virus,Enterocytozoon hepatopenaei and decapod iridescent virus 1.The method exhibited high sensiti-vity,with a detection limit of 10 copies/μL.When applied to test 10 positive clinical samples of IHH-NV,the conformity rate was 100%.Overall,the established fluorescence and paper strip visual detec-tion methods are simple to operate,highly sensitive,and suitable for regular monitoring and prelimi-nary screening of IHHNV in shrimp farming facilities or basic laboratories.
infectious hypodermal and hematopoietic necrosis virusrecombinase polymerase am-plificationCRISPR-Cas12adetection method
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