Introduction
Toxoplasmosis is a world-wide parasitic zoonosis caused by the protozoan Toxoplasma gondii, which can infect almost all warm-blooded animals and humans (Liu et al., 2012). About one-third of human beings are chronically infected by this pathogen (Moncada and Montoya, 2012). In immunocompetent individuals, infection with T. gondii usually does not cause obvious symptoms, but primary and reactivated infections in immunocompromised individuals can result in lethal damage, such as toxoplasmosis pneumonitis, myocarditis and encephalitis (Eza and Lucas, 2006, Saadatnia and Golkar, 2012). Infection during pregnancy can lead to severe lesions to the fetus, abortion, fetal death or long-term disabling sequelae (Montoya and Liesenfeld, 2004).
T. gondii oocysts are shed into the environment by felids worldwide. The sporulated oocysts that have capability to infect definitive and intermediate hosts are very resistant to environmental conditions (Tenter et al., 2000, Yan et al., 2016), which makes the oocysts a major source of infection for humans and animals. It would be desirable to develop a useful nucleic acid detection method to monitor T. gondii in the environment. So far, various molecular methods have been developed to detect T. gondii DNA (Liu et al., 2015). Traditionally, polymerase chain reaction (PCR) is considered to be the gold standard method to detect T. gondii DNA with considerable sensitivity and specificity (Jalal et al., 2004). However, due to the laborious operation, long reaction time, and expensive cost of the instrument, PCR-based assays cannot be widely used in clinical settings (Su et al., 2010, Wiengcharoen et al., 2004).
Recombinase polymerase amplification (RPA) is a recently developed nucleic acid amplification technology (Piepenburg et al., 2006). Instead of heat denaturation of the template strand as in PCR, the RPA amplification reaction can be carried out at a constant low temperature (Rosser et al., 2015, Wu et al., 2016). The amplification product can be visualized by a lateral flow (LF) strip by adding a specific probe into the RPA reaction solution. LF-RPA is a complex assay that has a higher efficiency and is more suitable to be used in field detection than conventional PCR (Wu et al., 2016).
In this study, we established a LF-RPA assay targeting the B1 gene for the detection of T. gondii DNA (B1-LF-RPA). Furthermore, we evaluated the detection sensitivity of B1-LF-RPA compared with that of conventional nested PCR. This is the first report showing that the LF-RPA assay can be used for detection of T. gondii in the environment.
Section snippets
Sample collection and DNA extraction
Thirty-five soil samples and 15 water samples were collected from parks, residential areas, schools and gutterways in Lanzhou city, Gansu Province, China, during August 2016. The soil samples were taken from the surface layer (top 3 cm) of the ground where cats often appear. Approximately 10 g of each soil sample was transferred into a 15-ml centrifuge tube by a stainless-steel spoon. The soil sample was evenly mixed with 50 ml ddH2O and then filtered by a 60-mesh sieve. The turbid liquid was
Validity and specificity of LF-RPA
The validity of the B1-LF-RPA was assessed using a negative control and the genomic DNA of ten T. gondii isolates (Table 1). The results showed that all 10 of the T. gondii isolates can be detected by the B1-LF-RPA (Fig. 1). The B1-LF-RPA assay was tested for specificity using a negative control, DNA of T. gondii oocysts and six other closely related protozoan parasites. No cross-reaction was observed for C. parvum, G. lamblia, E. cylindrica, E. bieneusi, N. caninum and H. hammondi and the
Discussion
The transmission of T. gondii may result from three causes: ingestion of tissue cysts in undercooked meat, ingestion of oocysts from the environment, and mother-to-child transmission (Tenter et al., 2000, Yan et al., 2016). Oocysts in the environment are shed by domestic cats or wild felines in feces. One cat may excrete more than 100 million oocysts into the environment (Jackson and Hutchison, 1989, Dubey, 1996, Omata et al., 1990). The sporulated oocysts are infective and remain viable for
Conclusions
The B1-LF-RPA assay developed herein provides a more practical technique for detection of T. gondii DNA in the environment. This assay has the significant advantages of being highly sensitive, highly specific, cost-effective and time-saving, which makes B1-LF-RPA a promising method for monitoring T. gondii in the environment and a potential tool for the diagnosis of human and animal toxoplasmosis.
Acknowledgments
Project support was provided by the National Key Research and Development Program of China(2017YFD0501304), the Special Fund for Agro-scientific Research in the Public Interest (Grant No.201303037), the National Key Project of Scientific and Technical Supporting Program (Grant No.2012BAD12B07), and the Science and Technology Support Program of Gansu Province (Grant No.1504NKCA054-6).
TwistAmp RPA Kits & Later Flow Strip 문의 https://www.sciencedirect.com/science/article/abs/pii/S0304401717302947
Abstract
Toxoplasma gondii infects all warm-blooded vertebrates, resulting in a great threat to human health and significant economic loss to the livestock industry. Ingestion of infectious oocysts of T. gondii from the environment is the major source of transmission. Detection of T. gondii oocysts by existing methods is laborious, time-consuming and expensive. The objective of the present study was to develop a recombinase polymerase amplification (RPA) method combined with a lateral flow (LF) strip for detection of T. gondii oocysts in the soil and water. The DNA of T. gondii oocysts was amplified by a pair of specific primers based on the T. gondii B1 gene over 15 min at a constant temperature ranging from 30 °C to 45 °C using RPA. The amplification product was visualized by the lateral flow (LF) strip within 5 min using the specific probe added to the RPA reaction system. The sensitivity of the established assay was 10 times higher than that of nested PCR with a lower detection limit of 0.1 oocyst per reaction, and there was no cross-reactivity with other closely related protozoan species. Fifty environmental samples were further assessed for the detection validity of the LF-RPA assay (B1-LF-RPA) and compared with nested PCR based on the B1 gene sequence. The B1-LF-RPA and nested PCR both showed that 5 out of the 50 environmental samples were positive. The B1-LF-RPA method was also proven to be sufficiently tolerant of existing inhibitors in the environment. In addition, the advantages of simple operation, speediness and cost-effectiveness make B1-LF-RPA a promising molecular detection tool for T. gondii.
Introduction
Toxoplasmosis is a world-wide parasitic zoonosis caused by the protozoan Toxoplasma gondii, which can infect almost all warm-blooded animals and humans (Liu et al., 2012). About one-third of human beings are chronically infected by this pathogen (Moncada and Montoya, 2012). In immunocompetent individuals, infection with T. gondii usually does not cause obvious symptoms, but primary and reactivated infections in immunocompromised individuals can result in lethal damage, such as toxoplasmosis pneumonitis, myocarditis and encephalitis (Eza and Lucas, 2006, Saadatnia and Golkar, 2012). Infection during pregnancy can lead to severe lesions to the fetus, abortion, fetal death or long-term disabling sequelae (Montoya and Liesenfeld, 2004).
T. gondii oocysts are shed into the environment by felids worldwide. The sporulated oocysts that have capability to infect definitive and intermediate hosts are very resistant to environmental conditions (Tenter et al., 2000, Yan et al., 2016), which makes the oocysts a major source of infection for humans and animals. It would be desirable to develop a useful nucleic acid detection method to monitor T. gondii in the environment. So far, various molecular methods have been developed to detect T. gondii DNA (Liu et al., 2015). Traditionally, polymerase chain reaction (PCR) is considered to be the gold standard method to detect T. gondii DNA with considerable sensitivity and specificity (Jalal et al., 2004). However, due to the laborious operation, long reaction time, and expensive cost of the instrument, PCR-based assays cannot be widely used in clinical settings (Su et al., 2010, Wiengcharoen et al., 2004).
Recombinase polymerase amplification (RPA) is a recently developed nucleic acid amplification technology (Piepenburg et al., 2006). Instead of heat denaturation of the template strand as in PCR, the RPA amplification reaction can be carried out at a constant low temperature (Rosser et al., 2015, Wu et al., 2016). The amplification product can be visualized by a lateral flow (LF) strip by adding a specific probe into the RPA reaction solution. LF-RPA is a complex assay that has a higher efficiency and is more suitable to be used in field detection than conventional PCR (Wu et al., 2016).
In this study, we established a LF-RPA assay targeting the B1 gene for the detection of T. gondii DNA (B1-LF-RPA). Furthermore, we evaluated the detection sensitivity of B1-LF-RPA compared with that of conventional nested PCR. This is the first report showing that the LF-RPA assay can be used for detection of T. gondii in the environment.
Section snippets
Sample collection and DNA extraction
Thirty-five soil samples and 15 water samples were collected from parks, residential areas, schools and gutterways in Lanzhou city, Gansu Province, China, during August 2016. The soil samples were taken from the surface layer (top 3 cm) of the ground where cats often appear. Approximately 10 g of each soil sample was transferred into a 15-ml centrifuge tube by a stainless-steel spoon. The soil sample was evenly mixed with 50 ml ddH2O and then filtered by a 60-mesh sieve. The turbid liquid was
Validity and specificity of LF-RPA
The validity of the B1-LF-RPA was assessed using a negative control and the genomic DNA of ten T. gondii isolates (Table 1). The results showed that all 10 of the T. gondii isolates can be detected by the B1-LF-RPA (Fig. 1). The B1-LF-RPA assay was tested for specificity using a negative control, DNA of T. gondii oocysts and six other closely related protozoan parasites. No cross-reaction was observed for C. parvum, G. lamblia, E. cylindrica, E. bieneusi, N. caninum and H. hammondi and the
Discussion
The transmission of T. gondii may result from three causes: ingestion of tissue cysts in undercooked meat, ingestion of oocysts from the environment, and mother-to-child transmission (Tenter et al., 2000, Yan et al., 2016). Oocysts in the environment are shed by domestic cats or wild felines in feces. One cat may excrete more than 100 million oocysts into the environment (Jackson and Hutchison, 1989, Dubey, 1996, Omata et al., 1990). The sporulated oocysts are infective and remain viable for
Conclusions
The B1-LF-RPA assay developed herein provides a more practical technique for detection of T. gondii DNA in the environment. This assay has the significant advantages of being highly sensitive, highly specific, cost-effective and time-saving, which makes B1-LF-RPA a promising method for monitoring T. gondii in the environment and a potential tool for the diagnosis of human and animal toxoplasmosis.
Acknowledgments
Project support was provided by the National Key Research and Development Program of China(2017YFD0501304), the Special Fund for Agro-scientific Research in the Public Interest (Grant No.201303037), the National Key Project of Scientific and Technical Supporting Program (Grant No.2012BAD12B07), and the Science and Technology Support Program of Gansu Province (Grant No.1504NKCA054-6).