Detection of Trichinella spiralis DNA in mouse faeces during the early stage of infection

Gołąb, Elżbieta; Rozej, Wioletta; Wnukowska, Natalia; Rabczenko, Daniel; Masny, Aleksander

doi:10.1016/j.mimet.2009.05.019

Cited by 16 publications

(6 citation statements)

References 14 publications

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“…Several real-time PCR assays targeting different genes for specific detection of T. spiralis DNA have been described. Previous developed specific assays for T. spiralis detection were applied to the study of wild life samples (Atterby et al 2009;Cuttel et al 2012) or to the study of experimental models (Golab et al 2009;Li et al 2010). Guenter et al developed a real-time PCR methodology for T. spiralis, Trichinella britovi and Trichinella psedospiralis DNA detection in domestic pigs infected which showed a sensitivity of 0.1 LPG (Guenther et al 2008); but there is not background about specific T. spiralis real-time PCR performed on muscle samples derived to the diagnostic laboratory.…”

Section: Introductionmentioning

confidence: 99%

Development of A Real‐Time PCR Assay for the Detection ofTrichinella Spiralisin Muscle Tissue of Swine and Derivatives

Quintana¹,

Recavarren²,

Scialfa³

et al. 2015

Journal of Food Safety

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Trichinellosis is an emergent zoonosis in several regions of the world and it is considered a public health problem. Trichinellosis represent one of the most important zoonotic diseases in Argentina. The purpose of this study was to develop a real-time PCR assay with internal control to detect Trichinella spiralis DNA in samples of swine muscles and its derivatives. PCR amplification of DNA from muscle samples was performed by real-time PCR with an internal porcine DNA amplification control. The developed PCR assay specifically detects T. spiralis showing no amplification with other Trichinella genotypes and showed an estimated sensitivity of 0.024 larvae per gram in swine muscle samples. From the 21 samples analyzed, five samples negative by enzymatic digestion were positive by real-time PCR, which demonstrates that this technique is capable of detecting T. spiralis in cases when enzymatic digestion cannot. In this work, a new molecular assay with internal control for T. spiralis detection was successfully developed. PRACTICAL APPLICATIONSTrichinellosis is considered a public health problem in Argentina and also represents an economic problem in porcine animal production and food safety. Due to the predominantly zoonotic importance of infection, the main efforts have focused on the control of Trichinella or the elimination of Trichinella from the food chain. In our country, most human infections are caused by Trichinella spiralis, which is transmitted mainly by the ingestion of raw or undercooked infected swine meat or its derivative products. New controls in order to improve food safety for consumers should be developed. This new real-time PCR assay with internal control shows a substantial increase in diagnostic sensitivity in comparison with muscle artificial digestion and the possibility to avoid false negative results. This specific PCR for T. spiralis may be useful for detection of infection at early stages in humans and food animals.

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Section: Introductionmentioning

confidence: 99%

Development of A Real‐Time PCR Assay for the Detection ofTrichinella Spiralisin Muscle Tissue of Swine and Derivatives

Quintana¹,

Recavarren²,

Scialfa³

et al. 2015

Journal of Food Safety

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show abstract

“…To overcome the limitations of conventional and serological methods, several molecular techniques, i.e., conventional PCR (cPCR) (Dick et al 1992, Wu et al 1998, Golab et al 2009, Wang et al 2012, random amplified polymorphic DNA-PCR (Bandi et al 1995), PCR-based single-strand conformation polymorphism (Gasser et al 1998), PCR-restriction fragment length polymorphism (Wu et al 1999(Wu et al , 2007, and multiplex PCR (Zarlenga et al 1999, Borsuk et al 2003, have been developed to detect and diagnose Trichinella infection. Presently, real-time PCR is becoming more widely used for routine diagnostic purposes because it is accurate, sensitive, and fast, allowing the rapid quantitative analysis of a specific DNA in a biological sample.…”

Section: Introductionmentioning

confidence: 99%

Early Detection ofTrichinella spiralisin Muscle of Infected Mice by Real-Time Fluorescence Resonance Energy Transfer PCR

Tantrawatpan

Intapan²,

Thanchomnang³

et al. 2013

Vector-Borne and Zoonotic Diseases

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Real-time fluorescence resonance energy transfer (FRET) PCR and melting curve analysis using newly developed fluorophore-labeled hybridization probes were applied for the detection of Trichinella spiralis DNA in muscle of mice following oral inoculation with 300 T. spiralis larvae. The developed assay could detect and differentiate T. spiralis, Trichinella papuae, and Trichinella pseudospiralis DNAs by the different melting temperatures (Tm). The assay had a detection limit of 5 × 10(2) positive control plasmid copies, which was equivalent to 1 ng of T. spiralis DNA spiked into 250 mg of muscle sample. No fluorescence signal was detected when the technique was applied to the DNA of 27 parasites other than Trichinella spp. The assay could detect T. spiralis DNA in muscle at 7, 14, and 21 days postinoculation. The range, mean ± standard deviation, and median of the Tm values of all positive muscle tissue samples were 60.4-60.8, 60.6 ± 0.2, and 60.5, respectively. This assay provides an effective tool for the specific, sensitive, and high-throughput detection of T. spiralis DNA in muscle during the early stage of infection. In addition, the technique can be useful for epidemiologic surveillance in naturally infected wildlife.

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“…To develop the faecal DNA extraction protocol a QIAamp® DNA stool mini kit was used on faeces from mice infected with T . muris nematodes to see if an eDNA signal could be detected, using nematode species specific primers from the literature [ 33 , 34 ]. When the manufacturer’s protocol was followed there was no successful amplification from faecal extracted DNA.…”

Section: Resultsmentioning

confidence: 99%

A novel copro-diagnostic molecular method for qualitative detection and identification of parasitic nematodes in amphibians and reptiles

et al. 2017

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Anthropogenic disturbance via resource acquisition, habitat fragmentation and climate change, amongst other factors, has led to catastrophic global biodiversity losses and species extinctions at an accelerating rate. Amphibians are currently one of the worst affected classes with at least a third of species categorised as being threatened with extinction. At the same time, they are also critically important for many habitats and provide man with a powerful proxy for ecosystem health by acting as a bioindicator group. Whilst the causes of synchronised amphibian losses are varied recent research has begun to highlight a growing role that macroparasites are playing in amphibian declines. However, diagnosing parasite infection in the field can be problematic, principally relying on collection and euthanasia of hosts, followed by necropsy and morphological identification of parasites in situ. The current study developed a non-invasive PCR-based methodology for sensitive detection and identification of parasitic nematode DNA released in the faeces of infected amphibians as egg or tissue fragments (environmental DNA). A DNA extraction protocol optimised for liberation of DNA from resilient parasite eggs was developed alongside the design of a novel, nematode universal, degenerate primer pair, thus avoiding the difficulties of using species specific primers in situations where common parasite species are unknown. Used in conjunction this protocol and primer pair was tested on a wide range of faecal samples from captive and wild amphibians. The primers and protocol were validated and detected infections, including a Railletnema nematode infection in poison dart frogs from ZSL London Zoo and Mantella cowani frogs in the wild. Furthermore, we demonstrate the efficacy of our PCR-based protocol for detecting nematode infection in other hosts, such as the presence of pinworm (Aspiculuris) in two tortoise species and whipworm (Trichuris muris) in mice. Our environmental DNA approach mitigates problems associated with microscopic identification and can be applied to detect nematode parasitoses in wild and captive hosts for infection surveillance and maintenance of healthy populations.

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Detection of Trichinella spiralis DNA in mouse faeces during the early stage of infection

Cited by 16 publications

References 14 publications

Development of A Real‐Time PCR Assay for the Detection ofTrichinella Spiralisin Muscle Tissue of Swine and Derivatives

Development of A Real‐Time PCR Assay for the Detection ofTrichinella Spiralisin Muscle Tissue of Swine and Derivatives

Early Detection ofTrichinella spiralisin Muscle of Infected Mice by Real-Time Fluorescence Resonance Energy Transfer PCR

A novel copro-diagnostic molecular method for qualitative detection and identification of parasitic nematodes in amphibians and reptiles

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