INTRODUCTION
The global burden of Brucella infection in livestock is substantial, and conservative estimates are that 300 million of the 1.4 billion worldwide cattle population are infected with the organism (de Figueiredo, Ficht, Rice-Ficht, Rossetti, & Adams, 2015) It threatens the breeding industry and human health and seriously affects the import and export trade of livestock and meat, causing huge economic loss and social burden. Even in developed countries, Brucella infection fails to be ultimately eradicated because the infected wildlife can infect persistently and spread to domestic animals (Al Dahouk & Nöckler, 2011; Godfroid, 2018). Currently, serological detection is the primary method to diagnose brucellosis, which depends on the detection of the infected livestock-specific antibodies, indirectly proving the existence of this pathogen. Because the history of Brucella  exposure and animal immune system, immune response, and the resulting patterns of the antibodies generation and dynamics are variable among individuals, thus can not be the direct evidence to prove the existence of the pathogen (Al Dahouk & Nöckler, 2011). RBT is a card agglutination test with simple operation, rapidity, and high sensitivity. It is suitable for field assay and is usually used as a screening test for brucellosis (Díaz, Casanova, Ariza, & Moriyón, 2011). Nonetheless, Brucella has cross-reacting antigens withYersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis easily emerging false positives. Therefore, the serological test of brucellosis is not the objective, direct diagnostic evidence (Yagupsky, Morata, & Colmenero, 2019).
Moreover, RBT cannot detect Brucella of infected livestock during the window period. PCR is a practical method that can amplify nucleic acid for detecting sample infected microorganisms (Wang & Cui, 2020; Lazcka, Del Campo, & Muñoz, 2007), and PCR has a high sensitivity (Kaden, Ferrari, Alm, & Wahab, 2017). Whereas PCR-based methods have many limitations, such as expensive instruments, matching standard reagents, relying on high-standard laboratories and professional technicians, and the diagnostic standards are not yet unified. Developing countries and rural areas with high morbidity of brucellosis have difficulties implementing PCR as they lack laboratory equipment and professional technicians. Furthermore, PCR is time-consuming and not suitable for rapid on-site screening (Yang & Rothman, 2004). Collectively, finding faster and more effective methods for field nucleic acid assay is urgent.
In recent years, RNA-based guide CRISPR/Cas nucleases have shown great promise in nucleic acid detection with high sensitivity and rapidity. This system has been transformed into an efficient gene-editing tool widely applied in gene editing in eukaryotes (Jinek et al., 2012). Lately, scientists have found that some class II Cas proteins, such as Cas12a (Cpf1), which have the activity of accessory cleaving single-stranded DNA (ssDNA) and applying the CRISPR-Cas system to the nucleic acid assay field (Chen et al., 2018). Zhang et al. discovered the CRISPR-Cpf1 system (Zetsche et al., 2015), which is mediated by a single Cas protein with cleavage activity similar to the CRISPR-Cas9 system in 2015. Compared to Cas9, Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, recognizing T-rich PAM (protospacer-adjacent motif), and Cpf1 introduces a staggered DNA double-stranded break with a 4 or 5-nt 5’ overhang. While Cas12a, guided by crRNA, binds to the target sequence and cleaves the target double-stranded DNA, it shows the activity of arbitrary cleavage of single-stranded DNA in the system; According to this discovery, Doudna et al. developed a diagnostic system named DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) in 2018 (Chen et al., 2018), this system can quickly and instantly detect trace amounts of DNA in samples. The DETECTR is a nucleic acid detection system that combines isothermal amplification, CRISPR-Cas12a, crRNA, and fluorescent reporter groups, with which human papillomavirus (HPV) has been successfully detected. The visualization is essential for the wild application of nucleic acid assay. The same year, Zhang et al. developed ”SHERLOCKv2” (Specific High-sensitivity Enzymatic Reporter un-LOCKing Version 2) (Gootenberg et al., 2018), which uses lateral flow strips by observing color changes with naked eyes to interpret the results. Several CRISPR-Cas-based methods have been developed to detect and diagnose infectious and non-infectious diseases (such as cancer) (Wang et al., 2018; Pomeroy et al., 2020; Otten & Sun, 2020) Compared to PCR, DETECTR and SHERLOCKv2 provide another level of ultrasensitive detection method (Mustafa & Makhawi, 2021). Recombinase Polymerase Amplification (RPA) is a new nucleic acid isothermal amplification technology developed in 2006 (Piepenburg, Williams, Stemple, & Armes, 2006), which can realize exponential amplification of template DNA in a short time at an isothermal temperature. Therefore, RPA can get rid of the dependence on the PCR instrument. Just like PCR, RPA also showed non-specific amplification (Li, Macdonald, & von Stetten, 2018). Thus, by combining the RPA with the CRISPR-Cas12a system, the single-stranded DNA containing a reporter group is added to the system, target fragment amplification through RPA, Cas12a recognizes and binds to the amplification product, then cleaves the single-stranded probe within the system to release the fluorescent reporter group, target DNA can be detected by capturing the fluorescent signal. The nucleic acid detection test strip adopts a chromatographic double-antibody sandwich method to detect the probes cleavaged by Cas12a. When designing probes, one end of ssDNA is labeled with biotin and the other with 6-carboxyfluorescein (6-FAM) or fluorescein isothiocyanate (FITC). The target fragment ofBrucella DNA is amplified through isothermal amplification (e.g., RPA, LAMP, RAA), then the amplification products and the labeled ssDNA with the fluorescent report group are cleaved by Cas12a simultaneously. As a result, the test strip can detect the fluorescent signal to identify the Brucella infection. The method has the characteristics of high sensitivity and strong specificity, which provides a new way for earlier and rapid home-based on-site diagnosis ofBrucella .
This study refers to the principle of DETECTR and SHERLOCKv2 system, combining CRISPR-Cas12a system and RPA, through observing lateral flow strip color to assay the result, building Brucella rapid CRISPR/CAST package. The package not only has the advantages of simplicity, high sensitivity, and strong specificity, the process ofBrucella nucleic acid assay only takes about 30 min, but also realizes the rapid detection of Brucella nucleic acid on-site screening, especially on the remote family pasture (Figure 1).
Materials and Methods
Materials
Brucella strain is B. melitensis from Kailu county of Tongliao city, Inner Mongolia autonomous region, China (GenBank No. CIT21: CP025819, CP025820). The positive quality control plasmid from T-Vector pMD19 inserted target sequence fragments were synthesized in our laboratory and extracted with TIANprep Mini Plasmid Kit purchased from Tiangen (Beijing, China). Brucella infection Pretreatment Kit (Zhai et al., 2017). The TwistAmp Basic kit was purchased from TwistDx Ltd (Hertfordshire, AL, U.K.). Cas12a protein, lateral flow strip purchased from Bio-Lifesci (Guangzhou, China). RNase inhibitor was purchased from TaKaRa Bio Inc. (Dalian, China). Yersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensisplasmids were stored in our laboratory.