Detection of Clostridium perfringens Using Novel Methods Based on Recombinase-Aided Amplification Assay-Assisted CRISPR/Cas12a System

Abstract

Clostridium perfringens is a highly versatile pathogen of humans and animals. Rapid and sensitive detection methods for C. perfringens are urgently needed for the timely implementation of control. In this study, to provide novel promising methods for the detection of C. perfringens, two rapid, sensitive, and instrument-free C. perfringens detection methods based on recombinase-aided amplification (RAA) assay and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (CRISPR/Cas12a) system were developed depending on fluorescence signal (RAA-CRISPR/Cas12a-FL) and lateral flow strip (RAA-CRISPR/Cas12a-LFS), respectively. The limit of detection of the RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12a-LFS methods is 2 copies and 20 copies of C. perfringens genomic DNA per reaction, respectively, and the whole process can be completed in 1 hr. Moreover, these two methods show no cross-reactivity with nontarget bacteria, which were used as a negative control to evaluate the specificity of two developed methods in the detection of C. perfringens and have 100% consistent with real-time polymerase chain reaction tests for 12 clinical samples collected from 2 Chinese Milu at Beijing Milu Ecological Research Center and 6 spiked samples from human blood and stool. Overall, the constructed C. perfringens detection methods, RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12a-LFS, have great potential as a novel detection scheme for the early diagnosis of C. perfringens infection in humans and animals.

Novel methods for the rapid and sensitive detection of Nipah virus based on a CRISPR/Cas12a system

“…The specificity and sensitivity of RAA-CRISPR/Cas12a-based detection methods are mainly influenced by the choice of the RAA primer pair and crRNA sequence. 41,42 To develop an RAA-CRISPR/Cas12a-based NiV detection method with high specificity and sensitivity, we designed seven RAA primer pairs and five crRNAs according to the principle mentioned in Materials and Methods, and then screened the optimal combination of RAA primer pair and crRNA from those 10 combinations shown in Fig. 2A.…”

“…The Cas12a-mediated trans-cleavage reaction was performed following procedures similarly to our previous studies. 40,41 Briefly, 10 µL of 200 nM Cas12a (NEB ENGLAND Bio Labs Inc., USA) and 10 µL of 200 nM crRNA were preincubated for 20 min at 37 °C, and then 20 µL of Cas12a-crRNA complex, 10 µL of 500 nM ssDNA-FQ (5′-/6-FAM/TTATT/BHQ1/-3′) and 2 µL of RAA products were mixed and rapidly incubated at 37 °C for 30 min. The results of RAA-CRISPR/Cas12a-FQ assays were detected with a UV torch or multifunctional microplate reader (λ ex : 490 nm and λ em : 522 nm).…”