A novel loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) device for rapid detection of Toxoplasma gondii in the blood of stray cats and dogs

Abstract

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis and threatens warm-blooded animal and human health worldwide. Simple and applicable diagnostic methods are urgently needed to guide development of effective approaches for prevention of toxoplasmosis. Most molecular diagnostic tools for T. gondii infection require high technical skills, sophisticated equipment, and a controlled lab environment. In this study, we developed a loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) assay that specifically targets the 529 bp for detecting T. gondii infection. This novel portable device is universal, fast, user-friendly, and guarantees experimental sensitivity as well as low risk of aerosol contamination. Our LAMP-LFD assay has a detection limit of 1 fg of T. gondii DNA, and shows no cross-reaction with other parasitic pathogens, including Cryptosporidium parvum, Leishmania donovani, and Plasmodium vivax. We validated the developed assay by detecting T. gondii in DNA extracted from blood samples collected from 318 stray cats and dogs sampled from Deqing, Wenzhou, Yiwu, Lishui and Zhoushan cities across Zhejiang province, Eastern China. The LAMP-LFD device detected T. gondii DNA in 4.76 and 4.69% of stray cats and dogs, respectively. In conclusion, the developed LAMP-LFD assay is efficient, minimizes aerosol contamination, and is therefore suitable for detecting T. gondii across basic medical institutions and field settings.

Microfluidic chip and isothermal amplification technologies for the detection of pathogenic nucleic acid

“…At 60–65°C, a strand-displacing Bst DNA polymerase catalyzes the primer extension along the template and the strand displacement reaction for 1 h, ultimately giving rise to the target repeat fragments of different lengths. The amplified products can be detected using ELISA [ 33 ], gel electrophoresis, real-time turbidimetry, lateral flow test strips [ 34 , 35 ], and fluorescent probe method [ 36 , 37 ]. LAMP has become a research hotspot for the development of POCT in the last 20 years because of its advantages such as high sensitivity (two to five orders of magnitude higher than the traditional PCR assays), low instrument requirements, and judgment of the test results based on the visual observation of white turbidity or generated green fluorescence.…”

A novel rapid visual detection assay for Toxoplasma gondii combining recombinase-aided amplification and lateral flow dipstick coupled with CRISPR-Cas13a fluorescence (RAA-Cas13a-LFD)

“…In China, antibody-positive rates for T. gondii were described to be 8.20% in the general population and 8.60% in pregnant women [ 11 ]. Characteristic symptoms do not always manifest in individuals with normal immunity, following infection with T. gondii , whereas premature delivery, abortion, teratosis, or stillbirth may occur in pregnant women regardless of apparent or recessive infection with this protozoan parasite, and even worse, newborns may develop ocular and neurological lesions [ 4 , 5 , 32 ]. For people with weakened immunity or immunodeficiency, such as patients with HIV/AIDS, organ transplant recipients, and tumor patients, infection by T. gondii can be associated with toxoplasmic encephalitis or even death [ 18 , 37 ].…”

“…Direct pathogenic diagnosis of T. gondii involves microscopic detection of tachyzoites or smear of tissue cysts. However, pathologic examination of tissues or strain isolation is primarily applied to diagnose animal infection, and less commonly human toxoplasmosis [ 32 ]. Nucleic acid and specific antibody assays of T. gondii are commonly used in clinical settings [ 9 ], particularly serum IgM/IgG antibody tests, such as enzyme-linked immunosorbent assay (ELISA), have been widely accepted as a primary screening technology for diagnosis of toxoplasmosis.…”

“…To date, some new molecular biology technologies operating without thermocycling but at constant incubation temperature have been described, which can greatly reduce the reaction time and complexity of nucleic acid amplifying procedures. For instance, loop-mediated isothermal amplification (LAMP) performed in only a heating block, can achieve constant temperature, which is superior to an expensive thermocycler [ 32 ]. Unfortunately, the LAMP technique is prone to aerosol contamination that potentially results in false-positive findings [ 32 ].…”

“…For instance, loop-mediated isothermal amplification (LAMP) performed in only a heating block, can achieve constant temperature, which is superior to an expensive thermocycler [ 32 ]. Unfortunately, the LAMP technique is prone to aerosol contamination that potentially results in false-positive findings [ 32 ]. Recombinase-aided amplification (RAA) assay is a newly developed technology for nucleic acid amplification, and involves only three major proteins, i.e., recombinase UvsX, single strand DNA binding protein (SSB), and DNA polymerase at a constant temperature [ 35 ].…”