Scientific Papers

CRISPR-Cas12a test strip (CRISPR/CAST) package: In-situ detection of Brucella from infected livestock | BMC Veterinary Research

Infections caused by Brucella have emerged as a considerable threat worldwide, particularly in vast amounts of livestock. It is essential to eradicate and control Brucella infections in livestock from the source. In addition, 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 diagnosing brucellosis mainly include agglutination tests, real-time PCR, ELISA, semiquantitative PCR, colloidal gold test strips, and polarized light technology. No single diagnostic method can meet the required sensitivity and specificity criteria. Some methods exhibit low specificity and sensitivity, such as agglutination and colloidal gold test strips. Some require special equipment, complex procedures, and professional personnel. Therefore, these methods, such as real-time PCR, can only be carried out in professional laboratories and are unsuitable for on-site testing by herders. RBT is simple, rapid, and highly sensitive and is the primary method currently used for screening brucellosis in livestock groups; in addition, RBT is the designated test for brucellosis in cattle, sheep, and pigs in international trade [20]. However, the results are subjective, and there are cross-antigens with other bacteria, such as Brucella, Yersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis, and cross-agglutination reactions with brucellosis-specific antibodies; thus, the method is prone to false-positive results. Therefore, RBT cannot be used as objective and direct diagnostic tool for brucellosis and cannot assay Brucella infection during the window period. That is, the antibodies are 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 exhibits high amplification efficiency, nonspecific amplification is also inevitable.

As reported, a single RPA cannot assay low levels of targets [13]. 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 arbitrarily cleave ssDNA labeled with a fluorescent signal. For real-time PCR and RPA detection, the probe corresponds to the template individually. 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, how much higher these signals are remains ambiguous, so we need to conduct additional experiments. By detecting Brucella DNA with the fluorescence signal, we can determine whether livestock have been infected. The proposed method exhibits high sensitivity and strong specificity. The test strip is portable and convenient for performing field assays. When designing the probe, one end of ssDNA was labeled with biotin, and the other end was labeled with 6-FAM. Through lateral flow chromatography detection, the nucleic acids present during Brucella infections can be identified without relying on large-scale instruments (Fig. 1). This study developed a new, rapid, sensitive, and specific nucleic acid detection package (CRISPR/CAST package) that can be used in grassroots veterinary stations and farms. The method is vitally significant for the earlier diagnosis of Brucella infection, comprehensive prevention and control, and elimination of the threat of infected animals to environmental biosecurity.

The CRISPR/CAST package combines RPA, CRISPR‒Cas12a, and nucleic acid detection test strips. The assay was completed within 30 min under isothermal conditions, with a sensitivity of 10 copies/μl (Fig. 3B, C). In addition, the CRISPR/CAST package can detect Brucella without involving antigenic cross-reactions to Yersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis (Fig. 4). The high specificity is due to the specific primers designed in the RPA reaction, which is followed by the specific binding of crRNA to the target sequence. Through this dual-specific base-complementary binding, detection is achieved even if nonspecific amplification occurs in the RPA reaction. The second-step crRNA cannot complementarily pair with the target sequence; as a result, the cleavage reaction of Cas12a does not occur and no fluorescent signal can be collected and illuminated. Cas9 must form a complex with two small RNAs (sgRNA and tracrRNA), both of which are necessary for cleavage activity; however, Cas12a only needs one crRNA to form a complex [21], which increases the efficiency, is flexible in binding to the target sequence and decreases the off-target probability. Logistically, Cas12a presents a more minimalistic system than that of Cas9 [22].

The CRISPR/CAST package exhibits unique advantages in nucleic acid detection. On the one hand, the assay, which is completed in a shorter time (approximately 30 min), is rapid. On the other hand, the package is portable; that is, no large instrument, professional laboratory, or professional and technical personnel are needed; in addition, the package utilizes the strong specificity of the Brucella nucleic acid assay and does not involve cross-reactions to other organisms. The specific primers are used during isothermal amplification, and the complex formed by crRNA and Cas12a proteins can be accurately located in the target sequence [23]. This dual localization ensures the high specificity of the CRISPR/CAST package (Fig. 4). The lower detection limit for the CRISPR/CAST package sensitivity experiment was 10 copies/μl (Fig. 3B, C), while that of RPA was 1000 copies/μl (Fig. 3A). Due to the strip, the results are observable by the naked-eye (Figs. 3C, 4A).

Thus, the CRISPR/CAST package is superior to conventional serological methods and PCR for detecting Brucella infection. Field serum samples of 398 sheep and 100 cattle were tested by the CRISPR/CAST package, of which 31 sheep and 8 cattle were Brucella DNA positive. The detection rate was consistent with the qPCR and higher than that of the RBT (19 sheep, 5 cattle were serum positive). Due to the CRISPR/CAST package, which significantly promotes livestock health, nucleic acid detection technology can be successfully utilized by pastoral households. With the package, infected animals can be screened earlier, culling can be performed in a timely manner and the infected environment can be cleaned.

Additionally, the method 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 livestock. Thus, farmers’ losses are reduced. Improving the quality of meat and dairy products can also promote the import and export trade and protect human health. The CRISPR/CAST package, which exhibits high sensitivity, strong specificity, and portability, helps farmers and herders who live in remote areas complete the test at their convenience. The CRISPR/CAST package provides a tool for screening in the field, detects Brucella infection in an early and rapid manner, and inspires new ideas for establishing rapid nucleic acid assays for other pathogens. We believe that this phenomenal technology will prevent livestock from incurring Brucella infection and safeguard the herders’ benefits and health indefinitely. 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.

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