Brucellosis is a worldwide major zoonotic disease caused by members of the genus Brucella, with more than half a million new cases reported annually (Hinić et al., 2008; Moeini-Zanjani et al., 2020). It can manifest as undulating fever with arthralgia, sometimes associated with chronic and severe complications (e.g., orchitis, spondylitis, and arthritis) and remains as a common cause of pyrexia of unknown origin (PUO) (Zhong et al., 2013; Daugaliyeva et al., 2018; Patra et al., 2019). Meanwhile, the symptoms of infection in animals include abortion, infertility, and decreased production. Brucella spp. are Gram-negative, facultative, and intracellular pathogens with 10 different species-specific host preferences (e.g., Brucella melitensis, Brucella abortus, Brucella suis, Brucella ovis, Brucella canis, Brucella neotomae, etc.) (Zhong et al., 2013; Daugaliyeva et al., 2018). Especially, Brucella abortus (B. abortus), an important pathogen of the genus Brucella, is a major causative agent of abortion and infertility in case of cattle population (namely, bovine brucellosis is an important infectious disease in the cattle population) (Ali et al., 2014; Islam et al., 2019; Khurana et al., 2021). Many countries have adopted different strategies to control this disease from their cattle herd. Thus, the ability to early, rapidly, and specially differentiate B. abortus is essential to control the disease.

Currently, isolation and identification of the Brucella spp. from blood culture are universally regarded as the standard diagnostic methods for laboratory examination (Patra et al., 2019). However, bacteriological isolation and identification methods (e.g., Rose Bengal plate test, serum agglutination test, and phage lysis test) are time-consuming and low sensitivity, and contain a risk of infection for laboratory personnel, so it is difficult to meet the requirements for early strategies (Li et al., 2019a; Khurana et al., 2021). Thus, rapid, safe, and sensitive identification techniques are required for the detection of specific-species of Brucella spp. Presently, several rapid detection methods based on immunological tests, including enzyme-linked immunosorbent assay (ELISA) and fluorescence polarization immunoassay (FPIA) have been successfully applied for the serological diagnosis of brucellosis (Xu et al., 2020; Dong et al., 2021). Although the sensitivity of the immunological techniques is between 65 and 95%, the low sensitivity of the acute phase, the low specificity of the antibodies, and the need for expensive reagents are major limitations of the methods (Özdemir et al., 2011; Karthik et al., 2014).

In recent years, various molecular detection techniques for the identification of Brucella spp. have been developed. Among them, polymerase chain reaction (PCR) and PCR-based assay (i.e., multiplex PCR and real-time PCR) are used as the conventional molecular detection methods in clinical examinations of brucellosis (Hinić et al., 2008; Surucuoglu et al., 2009; Kang et al., 2011). Despite these methods own outstanding analytical capabilities, the shortcomings (special apparatus’ requirements, poor availability, and long detection procedure) restrict their application in the point-of-care and field laboratories (Li et al., 2019a; Yang et al., 2021). To address the shortcomings of PCR-based techniques, loop-mediated isothermal amplification (LAMP), which includes 4 core primers (F3, B3, FIP, and BIP) and/or two loop primers (LF and LB) on target sequences, was developed in 2000 by Notomi et al. (2000). LAMP technique, as a fast, reliable, simple, and sensitive isothermal detection method, has been performed to detect various pathogens (containing bacteria, viruses, fungi, and emerging/re-emerging infectious agents) (Wang et al., 2017; Kashir and Yaqinuddin, 2020; Yang et al., 2021). In previous reports, the LAMP technique has been used to detect Brucella ssp. in diagnosis assay. Unfortunately, conventional validation methods for LAMP amplicons, including hydroxy naphthol blue (HNB), SYBR Green, and agarose gel electrophoresis, are difficult to accurately distinguish specific amplification from non-specific amplification, which can easily lead to misinterpretation of the results (Li et al., 2019a; Yang et al., 2021).

To overcome these defects, a target-specific, simple, and visual nanoparticle-based lateral flow immunoassay biosensor (LFIA) was successfully designed and applied to verify LAMP products (Li et al., 2019a; Yang et al., 2021). The detection mechanism of LFIA is that LAMP reaction amplicons, which are composed of 6-carboxyfluorescein (6-FAM) and biotin, combined with dye streptavidin-coated polymer nanoparticles (SA-PNPs) to form a complex (6-FAM-biotin-SA-PNPs). Then, after the complex is captured by the rabbit anti-fluorescein antibody (anti-FITC) on the LFIA, the detection for pathogen nucleic acid is realized by the visual test line (Gong et al., 2019; Li et al., 2020). Although LAMP- and MCDA-LFIA (multiple cross displacement amplification) methods targeting Bscp31, mcr-1, IS6110, and mtp40 gene for detection of Brucella spp. (genus level) and/or other target pathogens have been established, these methods cannot accurately detect and/or identify the Brucella abortus strains (species level) (Li et al., 2019a; Li et al., 2019b; Gong et al., 2019; Yang et al., 2021). Previously, conventional PCR and LAMP assays targeting BruAb2_0168 gene were developed and applied to identify the B. abortus in accurate diagnosis tests of brucellosis (Hinić et al., 2008; Kang et al., 2015).

In this report, a novel LFIA detector linked to LAMP technique (LAMP-LFIA) was developed and used for the visual, simple, sensitive, and specific identification of B. abortus (B. abortus-LAMP-LFIA) by highly specific region on the BruAb2_0168 gene. These improvements overcome the detection complexity of traditional methods and realize the accurate identification of specific-species of Brucella spp. (namely Brucella abortus). In B. abortus-LAMP-LFIA system, a unique-region of BruAb2_0168 gene was amplified in the reaction mixture, and results were indicated using LFIA. The optimal reaction conditions and feasibility of the B. abortus-LAMP-LFIA assay were confirmed by using DNA from pure cultures and whole blood samples.

Currently, although the isolation and identification (namely culture-biotechnical methods) of the Brucella spp. were commonly accepted as the gold standard method, these deficiencies (e.g., time-consuming, complicated operation steps and the risk of infection to laboratory personnel) cannot meet the requirements for early strategies (Schwarz et al., 2017; Li et al., 2019a). However, conventional PCR techniques (single PCR, multiplex PCR, and real-time PCR) and immunological tests (ELISA and FPIA) can realize more rapid detection of Brucella spp. than the culture-biotechnical methods, but the demands for PCR thermal cyclers and/or specific antibodies hinder their development in basic laboratories (Surucuoglu et al., 2009; Kang et al., 2011; Xu et al., 2020; Dong et al., 2021). Thus, it is extremely necessary to develop newly diagnostic methods that can meet the above-mentioned requirements.

LAMP, as a low-cost, fast, simple, and efficient nucleic acid amplification technique, seems to be more satisfactory. Presently, LAMP- and LAMP-based assay have been applied in the detection of various pathogens (containing viruses, bacteria, and fungi) (Sharma et al., 2016; Gomes et al., 2020). However, conventional validation tools for LAMP amplicons, including visual indicators, agarose gel electrophoresis, and real-time turbidimeter, are difficult to achieve simple, rapid, and accurate detection of target pathogens (Li et al., 2019a; Yang et al., 2021). A label-based polymer nanoparticle biosensor was developed and applied to specifically verify LAMP amplification products by labeling special primer (Ba-FIP primer was labeled 6-FAM) (Li et al., 2019b; Yang et al., 2021). The test results can be verified visually (approximately 2 min) by observing the color of the TL line (red and/or colorless) on the LFIA strips. Although LAMP- and MCDA-LFIA methods targeting Bscp31 gene for detection of Brucella spp. (genus level) have been established in previous publications, these two assays cannot accurately identify the B. abortus strains (species level) (Li et al., 2019b; Li et al., 2019a). Meanwhile, conventional LAMP techniques targeting the BruAb2_0168 gene have achieved rapid detection of B. abortus and demonstrated reliable specificity and sensitivity, the shortcomings of frequently used confirmation methods for LAMP amplicons are still unavoidable in a previous work (Karthik et al., 2014). Thus, the nanoparticle biosensor combined with the LAMP technique targeting BruAb2_0168 gene (B. abortus-LAMP-LFIA) has been successfully established and performed to achieve rapid and accurate detection of B. abortus in the current report.

A set of unique LAMP primers specifically recognized 8 regions on the BruAb2_0168 sequence, thus showing high selectivity for the diagnosis of B. abortus strains (Figure 2). In order to confirm the optimal reaction conditions of LAMP-LFIA to obtain more efficient amplification, different reaction temperatures (60–67°C, 1°C interval) and times (10–60 min, 10-min intervals) were carried out in our study. When the reaction condition was at 65°C for 50 min, B. abortus-LAMP-LFIA assay showed relatively stable amplification (Figure 4 and Table 3). Meanwhile, the specificity of B. abortus-LAMP-LFIA assay was 100%, which can not only detect all representative B. abortus strains (i.e., reference strains, vaccine strain, and isolates), but also exclude other Brucella species (including B. melitensis, B. suis, and B. canis) and non-Brucella strains (Figure 6).

In addition, B. abortus-LAMP-LFIA showed excellent detection sensitivity and could be detected when the genomic DNA concentration of B. abortus 544 was 100 fg per microliter (Figure 5). The sensitivity of the novel nanoparticle-based biosensors combined with LAMP assay was tenfold higher than that of PCR assay (Alamian et al., 2019; Moeini-Zanjani et al., 2020). In follow-up studies, after appropriately extending the amplification time (70–80 min), we found that B. abortus-LAMP-LFIA assay can detect the genomic DNA concentration of B. abortus 544 at about 80 fg per reaction (data not shown). In addition to using LFIAs to verify LAMP products, we also added MG amplification indicator to the reaction mixture in the experiment, and the results of amplification can be preliminarily judged with the naked eye. As expected, compared with the verification results of LFIAs, positive and negative amplifications do not seem to be clearly distinguished by visual indicators (e.g., the color change between 100 fg and 10 fg reaction tubes). However, the LAMP reaction is prone to aerosol contamination, so the whole experiment was performed in different laboratories (e.g., sample preparation, premixed reaction mixture, LAMP amplification, and LAMP amplicons verification).

In this study, in order to evaluate the practicality of B. abortus-LAMP-LFIA in practical examination, a total of 86 whole blood samples collected from suspected bovine brucellosis were tested using culture-biotechnical, conventional B. abortus-PCR, and B. abortus-LAMP-LFIA methods. The accurate diagnosis efficiency of B. abortus-LAMP-LFIA was consistent with the culture-biotechnical methods (Table 4). In addition, ROC analysis (AUC 1.000, the data not shown) based on the sensitivity and specificity of B. abortus-LAMP-LFIA was further carried out in our study using the R package (ver. 4. 0. 5). Interestingly, we found that two samples, which were tested as positive by B. abortus-LAMP-LFIA and culture-biotechnical, were detected as negative by B. abortus-PCR assay. The reasons for the different results are as follows: (i) the concentration of template DNA in the whole blood sample is low and cannot reach the minimum detection limit of B. abortus-PCR methods (PCR assay LoDs are usually 1–100 pg per microliter for genomic DNA) (Moeini-Zanjani et al., 2020). (ii) This phenomenon may be caused by false-positive amplification of B. abortus-LAMP-LFIA, and the non-specific amplification of conventional LAMP assay was occasionally reported in a previous publication; however, the possibility of false-positives for B. abortus-LAMP-LFIA is extremely low, due to these novel strategies were performed (including the specific labeling of primers, the design and application of LFIA biosensors, and the confirmation of conventional culture-biotechnical methods) (Li et al., 2019a; Yang et al., 2021). Meanwhile, 86 blood samples were also examined by the Brucella spp.-LAMP-LFIA assay established in our previous experiments (Li et al., 2019a), and the positive results were consistent with B. abortus-LAMP-LFIA (data not shown). These data indicate that although the B. abortus-LAMP-LFIA method is as sensitive as the culture-biotechnical methods for the detection of practical whole blood samples in the current experiment, B. abortus-LAMP-LFIA is simpler, faster, safer, and more applicable than the culture-biotechnical methods.

Moreover, compared to other verification tools for LAMP amplicons (including color indicator, real-time turbidity, and agarose gel electrophoresis), nanoparticle-based LFIA is more convenient (amplification products and running buffer are added to the sample pad), visual (the results are determined by directly observing the color changes of CL and TL line), and highly specific (the Ba-FIP primer is specifically labeled). Meanwhile, the total cost of a single B. abortus-LAMP-LFIA reaction is about 5 USD, including LAMP isothermal reagents (approximately 1.5 USD), LFIA biosensor (approximately 2.5 USD), DNA extraction reagents (approximately 0.2 USD), MG indicator reagents (approximately 0.3 USD), and other reagents and/or materials (approximately 0.5 USD).

In this report, a simple, visual, and reliable nanoparticle-based LFIA, which can eliminate the use of special equipment and simplify the detection procedure, was newly designed and applied in the current report. The B. abortus-LAMP-LFIA assay (B. abortus-LAMP combined with a nanoparticle-based LFIA) targeting the BruAb2_0168 gene was successfully established and performed, and the technique showed excellent sensitivity and specificity for the detection of bacterial strains (including reference strains, vaccine strains, and isolates) and whole blood samples. Hence, the B. abortus-LAMP-LFIA test developed was a simple, rapid, sensitive, and reliable detection technique, which can be used as a screening and/or diagnostic tool for B. abortus in the field and basic laboratories.