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.