Rapid Detection of Toxoplasma gondii: Prevalence Investigation of T. gondii Infection Among Stray Cats and Dogs in Zhejiang Province Based on a New Loop-Mediated Isothermal Amplication-Lateral-Flow-Dipstick (LAMP-LFD) Device

Background: Toxoplasma gondii (T. gondii) is worldwide spread caused Toxoplasmosis threatening warm-blooded animal and human health, especially for immunodecient population and pregnant women. Simple and applicable diagnostic methods are urgently needed for the prevention of toxoplasmosis. The molecular diagnosis of T. gondii infection generally requires high technical skills, sophisticated equipment and a controlled lab environment. Methods: In this study, we developed a loop-mediated isothermal amplication-lateral-ow-dipstick (LAMP-LFD) assay that specically targets the 529 bp for the detection of T. gondii infection in a new kind of portable device, which is universal, fast, user-friendliness, experimental sensitivity and low risk of aerosol contamination. Results: The detection limit of the LAMP-LFD assay is 1 fg of T. gondii DNA and no cross-reaction with other parasitic pathogens including Leishmania donovani, Plasmodium vivax, Cryptosporidium parvum, etc. In total, 318 stray cat and dog blood samples were collected from Deqing, Wenzhou, Yiwu, Lishui and Zhoushan cities in Zhejiang province, Eastern China. The current infection prevalence of T. gondii was 4.76% and 4.69% in stray cats and dogs respectively, detected by LAMP-LFD device. Conclusions: In conclusion, the established LAMP-LFD was an ecient and avoidable aerosol-contaminated device that can detect 1 fg genomic DNA of T. gondii, and suitable for T. gondii detection in the basic medical institution and even in eld areas.


Introduction
Toxoplasma gondii(T. gondii) is an obligate intracellular protozoan parasite that is globally distributed and can infect a wide variety of warm-blooded animals including humans and causing Toxoplasmosis [1,2]. T. gondii infections are distributed worldwide, with an estimated one-third of the global population being seropositive for T. gondii but signi cant geographical variations in infection rates [3]. A recent analysis showed that the antibody positivity rate for T. gondii in the general population of China was 8.20% and 8.60% for pregnant women [4,5]. T. gondii can invade all nucleated host cells and obtain the vital nutrients needed to replicate itself [6]. It is now widely believed that the foodborne transmission of T. gondii for human occurs by the ingestion of undercooked meat containing tissue cysts, consumption of water or food contaminated with toxoplasmosis oocyst, etc [7,8], meanwhile, some scholars propose that arthropods such as ticks play a role in the spread of T. gondii [9,10]. In general, primary infection of T. gondii in immunocompetent individuals is usually subclinical such as some symptoms of malaise, fever, myalgias, and isolated cervical or occipital lymphadenopathy, and then the cysts are formed in the brain, skeletal muscle, heart, or other organs [11]. As an important opportunistic pathogen in immunocompromised patients, a global meta-analysis suggests that T. gondii infection rate is higher in patients with AIDS, malignancies, organ transplant, upon this severe immunosuppression, the T. gondii tachyzoites derived from activated encysted bradyzoites replicate rapidly and result in more severe clinical presentation of encephalitis, pneumonia, retinochoroiditis, other disseminated systemic diseases and even death [12,13]. Furthermore, pregnant women infected with T. gondii before or just during pregnancy can result in miscarriage, stillbirth, fetal abnormalities, neural and neurocognitive de cits, even congenital transmission to the fetus if the T. gondii cross the placenta [14]. Now, the treatment of toxoplasmosis in the active stage relies on the combination drugs of pyrimethamine and sulfadiazine(pyr-sulf), but the spiramycin for acute maternal infections is more suitable and pyr-sulf for established fetal infection [15]. Effective, rapid and accurate diagnosis is therefore essential and desirable for initiating appropriate treatment and achieving a good prognosis [16].
The pathogenetic examination of T. gondii infection mainly includes direct microscopy, trophozoite isolation culture and cyst examination, but it is di cult to con rm the diagnosis, and it is mostly used in animal infection diagnosis or strain isolation, as well as human pathological tissue examination under special circumstances. Currently, nucleic acid, serum circulating antigen, and speci c antibody assays of T. gondii are more commonly used in clinical settings [17]. In particular, the serum IgM/IgG antibody test is widely used as a primary screening method for toxoplasmosis infection. However, this method may fail to detect speci c anti-T. gondii antibody during the active phase of infection, because these antibodies can only be produced after several weeks of parasitemia [18]. In recent years, PCR, real-time PCR and nested-PCR strategies based on polymerase chain reaction have become essential tools for the molecular diagnosis of T. gondii. These PCR ampli cation techniques showed good sensitivity and speci city in the molecular detection of T. gondii, and the real-time PCR with probe hybridization has been reported to be the most sensitive assay [19]. Nevertheless, factors such as the need for sophisticated instruments and well-trained personnel limit the widespread clinical application of these techniques.
The loop-mediated isothermal ampli cation(LAMP) technique has attracted much attention since its discovery because of its simpler ampli cation conditions and high-e ciency ampli cation. The technique is based on strand displacement activity Bst DNA polymerase with and two pairs of specially designed primers, at a constant temperature of around 60-65 °C, and a set of ampli cation products consisting of stem-loop structures containing repetitive target sequences forms [20]. The LAMP product can be judged by the turbidity of the resulting magnesium pyrophosphate by visual inspection, or by the addition of a uorescent dye to the reaction system to make the product visible under UV light [21]. The advantages of LAMP technology over traditional PCR put it at the forefront of research in the search for new diagnostic tools for parasitic diseases [18]. In toxoplasmosis diagnosis, a series of LAMP-based assays targeting B1 gene or 529 bp repeat sequences, internal transcriptional spacer sequences (ITS 1), and 18S rDNA sequences have been established. Besides, the detection of LAMP products was further optimized by combining probe hybridization [22], ELISA [23], and lateral ow dipstick (LFD) [24], that improves the sensitivity and speci city of LAMP assay. Among these, LAMP-LFD is one of the optimal detection methods and has been validated in the detection of parasites and microbes, such as Mycoplasma ovipneumoniae [25], Toxoplasma gondii [26], Babesia bovis and Babesia bigemina [27], canine parvovirus [28], and African trypanosome [29]. LAMP-LFD is based on the principle that biotinprimers biotinylate the ampli cation product and uorecein isothiocyanate (FITC)-labeled probes dehybridize so that the product is simultaneously double-labeled and then captured by anti-FITC antibodies on the lateral ow dipstick. Conventional LAMP-LFD method requires the reaction tubes to be opened to add the reaction product to the LDF after the LAMP ampli cation completed. Yet, the e cient ampli cation mechanism in LAMP experiment makes it highly sensitive to aerosol contamination from previous LAMP reactions and serves as a template for repeated ampli cation, leading to inaccurate falsepositives results [30,31]. In order to address this problem, we have designed a simpli ed, portable, closed LAMP-LFD format. In the present study, we targeted the 529-repeated element of T. gondii, and developed a new sensitive and simple assay based on LAMP-LFD in a hermetic device that for T. gondii detection. This device performs well in a simply regular laboratory water bath and can simply read-out. The performance of the LAMP-LFD device is identi ed by the T. gondii tachyzoite genomic DNA and stray cats and dogs blood samples collected from Zhejiang province, China.

Ethics
This study was carried out in strict conformed to the recommendations in the Guide for the Care and

Strains and samples
The Toxoplasma gondii tachyzoites (RH strain) preserved by our laboratory were used in this study. T. gondii tachyzoites were cultured in vitro under standard procedures by serial passages in Vero cell and prepared as described previously [32]. The stray dogs and cats blood sample were provided by animal protection base of Zhejiang Small Animal Protection Association. A total of 318 blood samples were collected from Zhoushan, Deqing, Lishui, Yiwu, Wenzhou respectively, Zhejiang province. For nucleic acid extraction, each blood sample was anticoagulated. All the blood samples were collected by experienced staff from animal hospital and stored at 4℃ until been used.

DNA extraction and serum separation
As positive control, genomic DNA was extracted from Toxoplasma gondii RH strains according to the introduction of Animal Genomic DNA Quick Extraction Kit (Beyotime, Shanghai, China). The anticoagulant blood sample was extracted DNA by magnetic beads adsorption using KBM Blood Genomic DNA Extraction Kit (KBM, Hangzhou, China). The concentration of genomic DNA extracted was evaluated on NanoDrop spectrometry (Thermo Fisher Scienti c, MA,USA), then stored at -20℃ until use..

Design of primers and probe
According to reported of molecular tests on T. gondii, it's proved that B1 gene (GenBank AF179871) and 529-bp repeated element (GenBank AF146527) are the potential optimal targets. B1 gene, which is used in the molecular detection extensively of T.gondii previously, has 35 copies in the T.gondii genome [33]. 529-bp repeated element, as the newly discovered target gene, has up to 300 copies therefore been more sensitively and speci cally for detection [34]. Therefore, we target tne 529-bp repeated element for T. gondii detection in our study. The success of LAMP ampli cation depends on ideal primers target the gene. For the LAMP assay targeting the 529-bp repeated element of T.gondii, we design a set of speci c oligonucleotide primers on an online LAMP primer designing software Primer Explorer V3 (http://primerexplorer.jp/e/;). The forward inner primer FIP was labeled with biotin in the 5′ end and the probe labeled with uorescein isothiocyanate (FITC) was designed between primers B1c and B2 for molecular hybridization detecting FITC-biotinylated LAMP product ( Figure 1). The primer sequences used in our experiment are listed in Table 1. Table.1.
The speci c primers of PCR and LAMP used in this study.

LAMP-Lateral-Flow-Dipstick (LAMP-LFD)
To detect the products of LAMP, a universal rapid detection equipment LAMP-Lateral-Flow-Dipstick (LAMP-LFD) was designed by combining the LAMP reaction and lateral ow dipstick (Figure 2.A). The integrated equipment has connected micro-amplify reaction tube for LAMP reaction and another tube containing nucleic acid dilution buffer with the lateral-ow-dipstick module for LAMP product capture, and the LFD detection module consists of a plastic grooved pedestal and a hermetic plastic cover that contains a visualization window and two connectors. The lateral-ow-dipstick, which set on the plastic grooved pedestal, is composed of a sample pad, application pad, test line, control line, and waterabsorbing pad, that the application pad, test line, and control line covered with gold-streptavidin(SA) conjugates, immobilized anti-FITC mouse monoclonal antibody and biotin respectively. Before starting the assay, the user only needs to connect the micro-amplify reaction tube with the reserved connector after the LAMP reaction mixture adding the sample to be detected (Figure 2 The speci city of LAMP-LFD Same LAMP protocol and procedure mentioned above are executed to verifying the speci city of LAMP. The template was replaced by the genomic DNA extracted from Leishmania donovani, Plasmodium vivax, Cryptosporidium parvum, Entamoeba histolytica, Trypanosoma evansi. The ampli ed product was identi ed by 1.5% gel electrophoresis. LAMP-LFD was performed as a contrast.

Evaluation of sensitivity of LAMP-LFD
The sensitivity of LAMP-LFD was evaluated against 10-fold serial dilutions of positive control template(genomic DNA of T .gondii) ranging from 1 ng to 0.01 fg, nuclease-free water were used as negative controls. Meanwhile, as comparative study, PCR was carried out under the protocol described before. Outer forward primer (F3), outer backward primer (B3) of LAMP were used as a pair of upstream and downstream primers in PCR assay. Clinical application for detection of T. gondii After the establishment of the universal rapid detection LAMP-LFD device for T. gondii, it was applied to the test of blood samples of stray animals (dogs, cats). One of 318 blood samples of dogs and cats collected from Zhejiang Province, China were extracted the genomic DNA. LAMP-LFD and PCR target 529 gene to detect T. gondii nucleic acids in blood samples.

Result
The speci city of LAMP-LFD method.
Genomic DNA samples of Leishmania donovani, Plasmodium vivax, Cryptosporidium parvum, Entamoeba histolytica, Trypanosoma evansi and T. gondii (RH) were used to test the speci city of LAMP and LAMP-LFD methods. Ampli cation signals were detected with the templates of T. gondii while the rest DNA templates showed no signals (Fig. 3A). LAMP-LFD was carried out using the templates above, positive bands at test lines were found when ampli ed with DNA samples of T. gondii and the rest remained negative (Fig. 3B), indicating that the established T. gondii LAMP-LFD detection method had good speci city.
Sensitivity of LAMP-LFD and PCR.
Next, we performed the sensitivity analysis of LAMP-LFD using different concentrations of T. gondii genomic DNA as a template. The template concentration diluted by 10-fold from 1 ng to 0.01 fg was tested by real-time LAMP and LAMP-LFD. The signal curve of real-time LAMP shows that the detection limit of LAMP in 1 hour is 1 fg genomic DNA of T. gondii (Fig. 4A), same is LAMP-LFD (Fig. 4D). For conventional PCR assay, the minimum detectable concentration is found to be 100 fg (Fig. 4C).
Positive rate of T. gondii in stray dogs and cats.
A total of 294 dogs and cats blood samples collected from ve cities of Zhejiang province were detected by the established LAMP-LFD equipment, as well as the conventional PCR assay. As shown in Figure, positive detection rates were determined 4.72% (15/318) by LAMP-LFD (Fig. 6) and 0.63% (2/318) by conventional PCR assay.

Discussion
Considering the global burden of toxoplasmosis, the unbearable consequences and the lack of effective anti-toxoplasmosis drugs, the development of early, rapid and cost-effective diagnosis methods suitable for economically deprived areas and eld testing is essential for early screening, prevention, control, and treatment of toxoplasmosis. As LAMP is a molecular ampli cation technique characterized by high sensitivity and speci city that can amplify several copies of nucleic acid to 10 9 times. Thus, LAMP is gradually becoming an alternative to PCR methods in the molecular diagnosis of multiple pathogens. The key players in LAMP are the Bst DNA polymerase with strand displacement activity and a set of four primers recognizing six distinct sequences of the target fragment [20]. The application of LAMP in toxoplasmosis diagnosis has been widely reported. Burg et al. rst proposed the B1 gene as a target gene for molecular diagnosis of toxoplasmosis, with 35 copies [33]; then the 529-bp fragment became another preferred target due to it's up to 300 copies [34]. Therefore, we designed a set of optimal primers target 529 element in our study. The optimal system and reaction temperature for the LAMP reaction have been optimized in our previous work and therefore are not mentioned in this experiment [32]. Usually, the LAMP products can be identi ed by electrophoresis, turbidity measurement of magnesium pyrophosphate, uorescent dye method, etc. Subsequently, to avoid the tediousness of electrophoretic, a faster and simpler method of chromatographic lateral ow dipstick (LFD) format was applied to reveal LAMP products [27,35,36]. LAMP-LFD is more speci c for products detection attribute to it adopts molecular probe hybridization technique, biotin and uorescein labeling of LAMP products in combination with double-sandwiching.
In this experiment, we applied the LAMP and LFD mothed for products detection. The biotin-labeled internal primers and FITC-labeled probes were used to make the ends of the stem-loop structure of the LAMP product biotin-and FITC-labeled, respectively, and the product was captured and visualized as it owed through the anti-FITC antibody region of the lateral ow dipstick. This is more speci c and sensitive than the magnesium pyrophosphate turbidity measurement and the uorescent dye method.
This LAMP-LFD method can detect down to 1 fg in 1 hour with template of genomic DNA of the Toxoplasma gondii RH strain and no across reaction with Leishmania donovani, Plasmodium vivax, Cryptosporidium parvum, Entamoeba histolytica, Trypanosoma evansi. Zhibing Lin et al. reported their detection limit was 10 fg with target 529 element by LAMP method [37]. Marco Lalle et al. propose that can detect T. gondii oocysts down to 25 oocysts/50 g in ready-to-eat baby lettuce with the LAMP-LFD assay [26]. Besides, Shirzad Fallahi et al. reported the detection limit of 529-LAMP was 1 fg T. gondii DNA [38]. It proves that our LAMP-LDF method achieves equal detection potency with better speci city because only biotin-and FITC-amplicons resulting in a band detectable by the LFD strips.
Although infrequently reported, contamination by aerosol residues is an unavoidable obstacle in the eld application for molecular diagnosis. LAMP ampli cation technology can amplify a large number of amplicons in a short time, thus greatly increasing the risk of aerosol contamination. Besides, the LAMP-LFD technique typically required opening the LAMP reaction tube for subsequent LFD testing, which greatly introduced the risk of aerosol contamination leading to false-positives result. Sterile pipetting technology and LAMP partition can be performed in the laboratory to reduce the risk of contamination [32]. Haihong Xu, Ming Hong et al. attempted to load the DNA uorescent dye into a tin foil, microcrystalline wax-dye capsule and preloaded it into a LAMP reaction tube, which was centrifuged to mix the dye with the product and develop the color after the LAMP reaction was completed. [39,40]. However, it has not yet been reported how to avoid contamination in LFD testing. In our study, we rst report an integral hermetic LAMP-LFD device application for T. gondii detection. The operator only needs to prepare the LAMP reaction system in PCR tube according to the procedure, then connect the reaction tube to the device interface, and complete the LAMP ampli cation in a 65 ℃ water bath. The LFD assay is performed by turning the device upside down to mix the LAMP ampli cation product with the diluent, then laying the device at to allow the mixture to ow onto the strip, and results can be visual read-out in the viewing window after 5 minutes. The whole testing process is carried out in the device, which greatly ensures the airtightness of the experiment and reduces the possibility of aerosol contamination. This device requires only a water bath for the LAMP reaction and is simple, cost-effective and suitable for use in minimally equipped laboratories and even eld settings.
In this study, we detected 318 blood samples of stray cats and dogs collected from ve cities in Zhejiang province for Toxoplasma gondii with LAMP-LFD. In view of the large variation in the number of Toxoplasma gondii in the animal blood on different stages of infection, in order to improve the nucleic acid extraction yield and detection rate, we divided each blood sample into three parts to extract nucleic acid and tested it with LAMP-LFD device. If anyone of them is positive, it means that the sample is positive. The total positive rate of T. gondii for stray cats and dogs are 4.76% and 4.69%, respectively, in our study. It is quite different from the seropositivity rate of Toxoplasma gondii in stray animals in the previous report. Mona K. Hegazy et al. compared the detection rates of Toxoplasma gondii in the blood of mice at different stages after infection with Toxoplasma gondii ME49 strain. On the seventh day after infection in mice, the LAMP method detected 18 positives out of the 20 examined samples. However, on day 56 post-infection, PCR and LAMP failed to detect positive from 20 mice blood samples. LAMP and routine PCR targeting the 529 bp RE gene failed to detect toxoplasmosis in the blood of mice in the chronic phase of the disease DNA, which can be explained by a progressive decrease in parasitemia levels as infection continues [18]. The application of LAMP for toxoplasma detection is therefore limited to the early stages of infection and is di cult to detect in peripheral blood at the quiescent bradyzoite stage. This explains the low detection rate of Toxoplasma gondii in the blood samples of our stray dogs and cats in Zhejiang province, which may be due to that the stray animals are already at an advanced stage of infection instead of parasitemia stage. Among them, the positive detection rate of toxoplasmosis in cat blood samples was higher in Wenzhou. According to our eld observations, these stray cats are in a more messy and unrestrained state and therefore more susceptible to toxoplasmosis infection. As the economy grew and the standard of living improved, some people started adopting stray animals from animal protection base as pets for companionship. However, pets such as dogs and cats as intermediate and nal hosts of T. gondii increase the likelihood of human infection with T. gondii that supported by our prevalence rate investigation.

Conclusion
In conclusion, we developed a novel Toxoplasma gondii detection assay, based on closed-device design test format that the LFD assay can be performed directly after the LAMP reaction completed. Our current results show that this LAMP-LFD device can be a reliable and portable diagnostic tool of T. gondii. The device has good airtightness, exhibits excellent sensitivity and speci city in sample testing and suitable for under-equipped laboratories and primary health care that will facilitate clinical diagnosis and epidemiological investigations for T. gondii. Authors' contributions QK and SL conceived of and designed the study. HD, JD and DL participated in the preparation of the lateral-ow-dipstick strips. QT and CX carried out the nucleic acid extraction of the sample. YX carried out the LAMP-LFD experiments and drafted the manuscript. XZ and BZ analyzed the data. HL revised the manuscript. All authors read and approved the nal manuscript.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.  Figure 1 The nucleotide sequence of 529 showing a set of primers and the probe. The sequence with color red, purple, brown, blue, green, orange represent primer F3, F2, F1c, B1c, B2, B3 respectively. The forward inner primer (FIP = F1c-F2) was labeled with biotin in the in the 5′ end and the primer amplify from 5'end to 3'end. The yellow module shows the sequence of FITC-probe between primer B1c and B2.    Positive rate of Toxoplasma gondii in stray cats and dogs in ve cities of Zhejiang Province. Cats and Dogs represent positivity rates for cats and dogs, respectively. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.