Loop-mediated isothermal amplification (LAMP): Early detection of Toxoplasma gondii infection in mice
© Kong et al; licensee BioMed Central Ltd. 2012
Received: 30 March 2011
Accepted: 3 January 2012
Published: 3 January 2012
Toxoplasmosis is a widespread zoonotic parasitic disease that occurs in both animals and humans. Traditional molecular assays are often difficult to perform, especially for the early diagnosis of Toxoplasma gondii infections. Here, we established a novel loop-mediated isothermal amplification targeting the 529 bp repeat element (529 bp-LAMP) to detect T. gondii DNA in blood samples of experimental mice infected with tachyzoites of the RH strain.
The assay was performed with Bst DNA polymerase at 65°C for 1 h. The detection limit of the 529 bp- LAMP assay was as low as 0.6 fg of T. gondii DNA. The sensitivity of this assay was 100 and 1000 fold higher than that of the LAMP targeting B1 gene (B1-LAMP) and nested PCR targeting 529 bp repeat element (529 bp-nested PCR), respectively. The specificity of the 529 bp- LAMP assay was determined using the DNA samples of Trypanosoma evansi, Plasmodium falciparum, Paragonimus westermani, Schistosoma japonicum, Fasciola hepatica and Angiostrongylus cantonensis. No cross-reactivity with the DNA of any parasites was found. The assay was able to detect T. gondii DNA in all mouse blood samples at one day post infection (dpi).
We report the following findings: (i) The detection limit of the 529 bp- LAMP assay is 0.6 fg of T. gondii DNA; (ii) The assay does not involve any cross-reactivity with the DNA of other parasites; (iii) This is the first report on the application of the LAMP assay for early diagnosis of toxoplasmosis in blood samples from experimentally infected mice. Due to its simplicity, sensitivity and cost-effectiveness for common use, we suggest that this assay should be used as an early diagnostic tool for health control of toxoplasmosis.
Approximately one third of the global human population is infected with T. gondii, including populations in Europe, South America, Africa and several Asian countries [1–3]. This parasite can cause congenital toxoplasmosis in a developing fetus and is dangerous for patients with acquired toxoplasmosis and compromised immune systems, such as patients with acquired immune deficiency syndrome (AIDS) or patients undergoing organ transplantation [4, 5]. Congenital transmission of this parasite is found in a large variety of wild animal species and livestock, such as sheep, goats, pigs, and cattle [6, 7]. Ingestion of infected pork is considered to be the main source of T. gondii infection in humans in the United States . T. gondii is also recognized as a major cause of abortion in farm livestock such as sheep, goats, pigs, and other domestic animals [5, 9].
The diagnosis of T. gondii infection or toxoplasmosis can be established by isolation of the parasite, histological examination, serological tests, or polymerase chain reaction (PCR). Biological diagnosis classically relies upon serological examination and direct detection of the parasite by inoculation of laboratory animals. A serological assay is considered the most challenging procedure because specific antibodies may not be present in the early stages of infection, especially in immune deficient or pregnant patients . Nested PCR, real-time PCR and LAMP assays have been used to detect T. gondii DNA in animal materials , water , soil , and clinical specimens . In the past decade, the use of PCR has resulted in a significant improvement in both the prenatal diagnosis of congenital toxoplasmosis and the detection of acute disease in immunocompromised patients. Among these PCR techniques, nested PCR, followed by hybridization, has been reported to be the most sensitive assay for detection . Real-Time PCR to detect toxoplasmosis not only can quantify T. gondii in biological samples but also has superior sensitivity over nested PCR assays [16, 17]. Despite these advances, diagnosis of T. gondii infection remains unsatisfactory because PCR-toxoplasma assays have not yet attained a sufficient level of sensitivity, and are limited due to expensive equipment and long reaction time periods.
LAMP is one of the nucleic acid amplifications tests used in various fields, including infection diagnosis to identify organisms. This assay uses a DNA polymerase called Bst polymerase, which has displacement activity and a set of four specially designed primers that recognize a total of six distinct sequences of the target DNA . It has been used to perform highly specific and sensitive amplifications of DNA to detect pathogens including viruses, bacteria, protozoa, and fungi. Recently, this technique has proven to be very useful in the diagnosis of parasitic infections, such as malaria, trypanosomiasis, dirofilariasis, and babesiosis [19–22]. Rapid detection of T. gondii in water samples by LAMP was first described in a study by Sotiriadou et al. . Thereafter, the LAMP assay was developed and evaluated for the detection of T. gondii infection from the lymph nodes of pigs , various organs harvested from mice , and blood samples from patients .
In the present study, we developed a LAMP assay targeting the 529 bp repeat element for the detection of T. gondii. Furthermore, we evaluated the detection sensitivity of T. gondii LAMP in comparison with conventional nested PCR. This is the first report in which the LAMP assay has been used for early diagnosis of active toxoplasmosis in mouse blood samples.
Development and optimization of the 529 bp-LAMP assay
LAMP sensitivity and specificity
T. gondii tachyzoites (RH strain) were propagated in vitro under standard procedures by serial passages in HeLa cell monolayer in Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 2% fetal bovine serum at 37°C under 5% CO2 (Figure 2A). Tachyzoites were collected by scraping the cell monolayer and washing with cold phosphate-buffered saline (PBS). The final pellet was resuspended in cold PBS and passed three times through a 30-gauge needle syringe. Clarification and all subsequent centrifugations were performed at 4°C. The parasites were then diluted with 1.0 ml PBS and centrifuged to equilibrium in 13 ml non-linear 10% - 50% Percoll density gradients at 2,500 × g for 20 min. Purified parasites were diluted with PBS and pelleted by sedimentation at 2,500 × g for 20 min to remove the Percoll. The purity of parasites was confirmed microscopically to ensure that the tachyzoites had normal morphology and to exclude the possible inclusions of other cellular organelles and debris (Figure 2B). Tachyzoite genomic DNA extracted with DNeasy Blood & Tissue Kit (QIAGEN, Maryland, USA) was used in concentrations ranging from 6 ng to 0.06 fg. Three replicate assays showed high reproducibility of the LAMP and nested PCR. The DNA extraction procedure did not affect the sensitivity of the LAMP and PCR.
The sensitivity of the 529 bp- LAMP assay was determined and compared to the results of the 529 bp-nested PCR and the B1-LAMP. The LAMP products in positive reaction tubes were visually detectable on addition of SYBR Green I to the reaction tube. On gel electrophoresis, the amplified products showed ladder-like patterns. The detection limit of the 529 bp- LAMP assay was 0.6 fg of T. gondii DNA (Figure 2E). In contrast, the detection limit of B1-LAMP was 60 fg of the DNA template (Figure 2D) and the limit of the 529 bp-nested PCR was 600 fg of the DNA template (Figure 2C). Therefore, it appears that the sensitivity of the 529 bp- LAMP assay was 100 fold higher than that of the B1-LAMP assay and was 1000 fold higher than that of the 529 bp- nested PCR. These results demonstrate that the primers based on the 529 bp repeat element are more suitable than those based on the B1 gene. This supported by the finding that the sensitivity of the quantitative LightCycler PCR assay targeting the 529 bp repeat element of T. gondii is about 10 to 100-fold higher than that targeting the B1 gene . The sensitivity of LAMP assay is higher than that of the conventional PCR for detection of protozoan parasites such as Babesia spp., Theileria spp., Trypanosoma spp., which has been reported previously [30–32].
Application of LAMP to blood samples from infected mice
Six-week old BALB/c mice (Purchased from the Zhejiang Provincial Experimental Animal Center, China) were infected by intraperitoneal injection of 20 virulent tachyzoites of the T. gondii RH strain (Stored in our laboratory). Blood samples were collected at 1, 3, and 5 dpi from the vena orbitalis posterior plexus blood. The DNA was extracted from blood samples of infected mice by using DNeasy Blood & Tissue Kit (QIAGEN, Maryland, USA) according to the manufacturer's instructions. The use of animals was approved by the Institutional Animal Care and Use Committee (IACUC) of Zhejiang Academy of Medical sciences. The purified DNA from 50 μl blood samples was dissolved in 30 μl of double-distilled water and 1 μl of the resulting supernatant was used as the template for the subsequent detection. The DNA extracted from the blood samples of the uninfected mice was used as negative control. In order to exclude the presence of inhibitors in blood samples, we selected a pair of PCR primer
R: 5'-GGACATCTAAGGGCATCACA-3') to amplify a mouse 18s rRNA fragment (GenBank Accession No. NR_003278.2), with an amplified zone of the target is 489 bp . We also chose mouse 18s rRNA as an internal control target. The primers for internal control are as follows,
The 529 bp- LAMP assay was also applied to various organ samples including brain, heart, liver, spleen, kidney and lung from experimentally infected mouse. The assay showed positive results for heart samples at 1 dpi, for kidney samples at 3 dpi and for all organ samples except the brain at 5 dpi (Data not shown). Kaneko et al. also reported that the specificity and sensitivity of LAMP detection does not seem to be impaired by sample type, including plasma, serum, PBS, saline, urine, aqueous humor and vitreous substances. The tolerance of LAMP for biological substances was very high [34, 35]. Another advantage of the LAMP assay is that the requirements for LAMP are relatively simple, and that it does not require high technical skills or sophisticated equipments. However, there is a high risk of aerosol contamination due to the large amount of LAMP products. To reduce the risk of contamination, the LAMP partition in the laboratory, such as solution preparing partition, sample treatment partition, and gel electrophoresis detection partition, should be carried out in separate areas. Gloves should be changed regularly and sterile pipetting techniques should be applied during the entire LAMP experiment.
Here, we report a LAMP assay that specifically targets the 529 bp repeat element for the detection of T. gondii in mouse blood samples. We were able to demonstrate the successful amplification of T. gondii DNA at 1 dpi within 1 h at 65°C using the LAMP assay. On the basis of these results, the LAMP assay can be considered one of the most accurate molecular assays because it is a specific, sensitive, and rapid diagnostic tool for the early detection of Toxoplasma in blood samples.
Acknowledgements and Funding
This work was supported by the National Important S&T Project (2008ZX10004-011), Zhejiang Science and Technology Project (2007F30016, 2009F20036), the Projects of Zhejiang Health Department (XKQ-009-003, XKQ-010-001), and Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents. We acknowledge Wang Yue at Zhejiang Academy of Medical Sciences for kindly providing the DNA of Plasmondium falciparum in the LAMP specificity experiment.
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