LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2

Abstract

Porcine circovirus 2 (PCV2) is the main pathogen of porcine circovirus-associated disease (PCVAD), which can cause considerable economic loss to the pig industry. The diagnosis of PCVAD is complicated and requires a series of clinical, pathological, and virological methods. Therefore, a rapid, highly sensitive, on-site, and visual diagnostic approach would facilitate dealing with the spread of PCV2. In this study, we intended to establish a new and effective PCV2 detection method through combining the no specific equipment requirement advantage of loop-mediated isothermal amplification (LAMP) with the property of clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system possessing the huLbCas12a collateral cleavage activity able to cleave single-stranded DNA fluorophore quencher probe sensor (designed as LAPM-CRISPR). Following a series of optimizations of its reaction conditions, this LAMP-CRISPR-based PCV2 detection could be conducted in constant temperature equipment, with the result reflected in a direct visual readout way. This established PCV2 detection approach presented fine sensitivity, rapidity, specificity, and reliability, as demonstrated by a low detectable limit of 1 copy/μL, completed within an hour, no cross-reaction with main porcine DNA or RNA viruses like PCV1, PCV3, and PEDV, and a 100% coincidence rate with that of the quantitative PCR (qPCR) method in the evaluation of 30 clinical blood samples, respectively. Therefore, this novel method makes rapid, on-site, visual, highly sensitive, and specific detection of PCV2 possible, facilitating the prevention of this pathogen in the field.

Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2)

“…In this study, a prototype for a duplex LAMP-LFD assay relevant to Technology Readiness Level 3 (TRL 3) was developed for simultaneous detection of PEDV and PCV 2 in the same specimens. According to the LOD, the duplex LAMP-LFD was 10-times more sensitive than PCR-based AGE and LAMP-based AGE, which correlates with previous reports [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ]. The high analytical specificity of the duplex LAMP-LFD resulted from primers and DNA probes that were uniquely designed from the conserved regions on the spike and ORF genes for PEDV and PCV2, respectively.…”

Visual and label-free ASFV and PCV2 detection by CRISPR-Cas12a combined with G-quadruplex

“…The detection sensitivity of EvaGreen real-time PCR combined with melting curve analysis could detect 5.0 copies/μL ( 35 ). LAMP-coupled CRISPR-Cas12a can be detected with a low detectable limit of 1 copy/μL of PCV2 ( 36 ). In this study, the detection sensitivity of ASFV and PCV2 was 10 2 copies and 10 3 copies, respectively, which are lower than the above detection methods.…”

Epidemiology and diagnosis technologies of human metapneumovirus in China: a mini review

“…Among these, the CRISPR-Cas12a system is considered a powerful tool for detecting nucleic acids in vitro [41]. In addition, the sensitivity and robustness of the CRISPR-Cas12a detection system could be greatly improved by combining with simultaneously reverse transcription and isothermal amplification technologies, such as LAMP [42], RAA [43], and recombinase polymerase amplification (RPA) [44]. Moreover, the CRISPR-Cas12a detection result can be observed via the naked eye when combined with lateral flow (LF), thus the established technology might be applied in these pointof-need places, such as airports and customs centers [45].…”