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.