3. Discussion
Infections caused by Brucella  have emerged as a considerable threat worldwide, particularly in the vast amount of livestock. It is essential to eradicate and control livestock infection with Brucella  from the source. Meanwhile, earlier detection and timely culling are equally important. Consequently, the screening used for livestock must be accurate, sensitive, specific, simple, and fast. At present, the methods of brucellosis diagnosis mainly include agglutination test, Real-Time PCR, ELISA, semi-quantitative PCR, colloidal gold test strip, and polarized light technology. No single diagnostic method can meet the required sensitivity and specificity criteria. Some methods have low specificity and sensitivity, such as the agglutination and colloidal gold test strip. Some require special equipment, complex procedures, and professional personnel; Therefore, they can only be carried out in professional laboratories, unsuitable for on-site testing herders at home, such as the Real-Time PCR. RBT is simple, rapid, and highly sensitive, which is the primary method currently used for screening brucellosis in livestock groups, and it is also the designated test for brucellosis in cattle, sheep, and pigs in international trade (Godfroid, Nielsen, & Saegerman, 2010). However, the interpretation of the results is subjective, and there are cross-antigens among Brucell a, Yersinia enterocoliticaO:9, Escherichia coli O157, Salmonella enterica serovarUrbana O:30, and Francisella tularensis , and cross-agglutination reaction with brucellosis-specific antibodies, prone to false positives. Therefore, RBT cannot be used as an objective and direct diagnostic evidence of brucellosis and cannot assay Brucella infection during the window period. That is, the antibodies were not produced at the initial infection stage. RPA is carried out under isothermal conditions, realizing nucleic acid detection independent of professional instruments and personnel. Although RPA is simple to operate and has high amplification efficiency, non-specific amplification is also inevitable.
As reported, a single RPA cannot assay low levels of targets (Gootenberg et al., 2018). These studies have confirmed Cas12a accessory splicing ssDNA activity for nucleic acid detection. The ternary complex consists of Brucella  DNA, Cas12a, and crRNA. Cas12a possesses a RuvC domain that can exert activity to cleave the ssDNA labeled with fluorescent signal arbitrarily. As for the Real-Time PCR and RPA detection, the probe corresponds to the template one by one. Theoretically, under identical circumstances, we can hypothesize that the fluorescent signals produced by the CRISPR-Cas12a reaction are higher than those of Real-Time PCR and RPA detection. However, the studies have been ambiguous regarding how much higher they are, so we need to conduct additional experiments. By detecting Brucella DNA with the fluorescence signal, we can determine whether livestock have ever been infected. The proposed method has high sensitivity and strong specificity. The test strip is portable and convenient for field assay. When designing the probe, one end of ssDNA was labeled with biotin, and the other was labeled with 6-FAM. Nucleic acid detection ofBrucella infection can be achieved without relying on large-scale instruments through lateral flow chromatography detection (Figure 1). This study developed a new, rapid, sensitive, and specific nucleic acid detection package (CRISPR/CAST package), which can be used in grassroots veterinary stations and farms at home. It is vitally significant for the earlier diagnosis of Brucella  infection, comprehensive prevention and control, and eliminating the threat of infected animals to environmental biosecurity.
The CRISPR/CAST package combines RPA, CRISPR-Cas12a, and nucleic acid detection test strips. The assay can complete within 30 min under isothermal conditions, with a sensitivity of 10 copies/μl (Figures 2B, 2C). In addition, the CRISPR/CAST package can detect Brucellawithout antigenic cross-reacting to Yersinia enterocolitica O:9,Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis (Figure 4). The high specificity is due to the specific primers designed in the RPA reaction, followed by the specific binding of crRNA to the target sequence. This dual-specific base-complementary binding allows detection even if non-specific amplification occurs in the RPA reaction; The second-step crRNA cannot complementarily pair with the target sequence, which leads to the cleavage reaction of Cas12a can not generate so that no fluorescent signal can be collected and illuminated. Cas9 needs to form a complex with two small RNA (sgRNA and tracrRNA), both of which are required for cleavage activity; However, Cas12a requires only one crRNA to form a complex (Yao et al., 2018), which is more highly efficient, flexible in binding to the target sequence and decrease the off-target probability. Logistically, Cas12a presents a more minimalistic system than Cas9 (Paul & Montoya, 2020).
CRISPR/CAST Package has unique advantages in nucleic acid detection. On the one hand, the assay, which takes a shorter time of around 30 min, is rapid. On the other hand, the package is portable; that is to say, the package requires no large instrument, professional laboratory, or professional and technical personnel, and the package attaches to strong specificity of Brucella nucleic acid assay with no cross-reacting to other organisms. Not only are the specific primers used during isothermal amplification, but the complex formed by crRNA and Cas proteins can be accurately located in the target sequence (Li, Li, Wang, & Liu, 2019). Such dual localization ensures the high specificity of the CRISPR/CAST package (Figure 4). The lower detection limit for the CRISPR/CAST package sensitivity experiment was 10 copies/μl (Figure 3B, 3C), while the one of RPA was 1000 copies/μl (Figure 3A). The strip facilitated naked-eye observation to determine the result (Figure 3C, 4A).
Thus, the CRISPR/CAST package is superior to the conventional serological methods and PCR for detecting Brucella infection. Field serum samples of 398 sheep and 100 cattle were tested by CRISPR/CAST package, of which 31 sheep and 8 cattle were BrucellaDNA positive. The detection rate was consistent with the qPCR and higher than that of the RBT (19 sheep, 5 cattle were serum positive). CRISPR/CAST package as a patron saint for livestock health enables nucleic acid detection technology to enter pastoral households successfully. The package can realize the earlier screening of infected animals, timely culling, and cleaning up the infected environment.
Meanwhile, it can control the incidence effectively, reduce the spread of Brucella infection and suppress large-scale infection, improving the quality of meat and dairy products. Furthermore, it ensures the safety of individual farmers’ transactions, and it can protect the livestock herd’s safety and reduce the probability of abortion in pregnant animals in the livestock. In other words, reduce farmers’ losses. Improving the quality of meat and dairy products can also promote the import and export trade and protect human health. CRISPR/CAST package with high sensitivity, strong specificity, and portability facilitates farmers and herders who live in remote areas to complete the test whenever and wherever. CRISPR/CAST package provides a tool for field screening, earlier and rapid detection of Brucellainfection, and new ideas for establishing rapid nucleic acid assays for other pathogens. We believe that the ”Patron Saint” will prevent livestock free from Brucella infection and safeguard the herders’ benefits and health eternally. The CRISPR-Cas system is leading to a new technological revolution. This new diagnostic tool will rewrite future diagnostic technologies, especially in developing countries with relatively poor sanitation and a high incidence of animal diseases.