Reverse transcription–enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains

Abstract

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets, and is associated with high mortality, thus causing severe economic losses in the pig industry. Currently, although attenuated vaccines are commonly used in commercial pig farms in China, they do not completely protect against all mutated wild-type strains. Existing nucleic acid assays have high sensitivity and specificity, but the complexity of the assay process and expensive instrumentation hinder disease detection. Here, reverse transcription–enzymatic recombinase amplification (RT-ERA) was combined with the CRISPR-Cas12a system to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The protocol used crRNA and RT-ERA amplification primers against open reading frame 3 (ORF3), followed by Cas12a/crRNA complex detection of predefined target sequences at 37 °C for 30 min, thus producing results visible to the naked eye under LED blue light. The assay is highly sensitive and specific, detecting as few as two copies of the target gene per test and showing no cross-reactivity with other porcine pathogens. Overall, this integrated RT-ERA pre-amplification and Cas12a/crRNA cleavage assay is a practical tool for reliable and rapid detection of PEDV for diagnostic differentiation. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03716-7.

Visual detection and differentiation of porcine epidemic diarrhea virus wild−type strains and attenuated vaccine strains using CRISPR/Cas13a-based lateral flow strip

“…All samples were used to confirm the applicability of PEDV-specific lateral flow in clinical diagnosis. The results were compared with those obtained with qRT-PCR described previously, which was run in parallel for the above clinical samples ( Yang et al., 2021 ).…”

Point-of-Care Tests for Rapid Detection of Porcine Epidemic Diarrhea Virus: A Systematic Review and Meta-Analysis

“…Alternate novel isothermal amplification techniques such as RT-LAMP and RT-RPA are rapid and do not require expensive equipment such as a thermal cycler, and hence can be performed in a point-of-care context or resource-poor laboratories. In our current meta-analysis, 7 of 13 studies with NAIA-based POCT used an RT-LAMP assay [ 29 , 30 , 33 , 35 , 37 , 40 , 41 ] and 6 studies adopted an RT-RPA assay to detect PEDV [ 31 , 32 , 34 , 36 , 38 , 39 ]. In general, the sensitivities of nucleic acid tests are in the order of real-time RT-PCR followed by RT-LAMP, RT-RPA, and conventional RT-PCR [ 53 ].…”

“…Additionally, RT-LAMP and RT-RPA techniques incorporating with devices such as strip [ 39 , 40 ] and microfluid chip [ 30 , 35 ] have made the assays more convenient and suitable for use in point-of-care testing for PEDV. Notably, CRISPR-based isothermal assay has shown great promise due to its high sensitivity, specificity, and reliability in the development of next-generation POC molecular diagnostic technologies for PEDV [ 32 ].…”

Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a Mediated Multiplexable and Portable Detection Platform for GII Genotype Porcine Epidemic Diarrhoea Virus Rapid Diagnosis

“…The differences in ORF3 may serve as markers to differentiate PEDV attenuated vaccine from wild-type strain ( Liu et al, 2019 ). Yang et al (2021) established a reverse transcription–enzymatic recombinase amplification method coupled with CRISPR/Cas12a against ORF3 gene, which can specifically detect PEDV variant strains. In the present study, the S gene of the variant and vaccine strains was compared and analyzed.…”