African swine fever (ASF) is one of the most severe infectious diseases
of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a
system was established in one tube for the detection of the ASFV p72
gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporters
and CRISPR-derived RNA (crRNAs) were screened and selected for the
CRISPR detection system. In combination with LAMP amplification assay,
the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of
p72 gene per reaction. Furthermore, this method displays no
cross-reactivity with other porcine DNA or RNA viruses. The performance
of the LAMP-CRISPR assay was compared with real-time qPCR tests for
clinical samples, a good consistency between the LAMP-CRISPR assay and
real-time qPCR was observed. In the current study, a LAMP coupled with
the CRISPR detection method was developed. The method shed a light on
the convenient, portable, low cost, highly sensitive and specific
detection of ASFV, demonstrating a great application potential for
monitoring on-site ASFV in the field.
New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters
