Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Cuttell, Leigh; Corley, Sean W.; Gray, Christian; Vanderlinde, Paul; Jackson, Louise A.; Traub, Rebecca J.

doi:10.1016/j.vetpar.2012.03.054

Cited by 28 publications

(38 citation statements)

References 24 publications

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“…Molecular detection methods such as PCR are alternative assays that offer both high sensitivity and specificity (Cuttell et al 2012). In this work, specific primers for detection of T. spiralis DNA by real-time PCR were designed.…”

Section: Discussionmentioning

confidence: 99%

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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: Discussionmentioning

confidence: 99%

“…All previously described real-time PCR techniques for T. spiralis DNA detection lacked an internal amplification control to detect PCR inhibition (Guenther et al 2008;Atterby et al 2009;Cuttell et al 2012). Inclusion of internal amplification controls is mandatory when describing diagnostic tests based on PCR (Hoofar et al 2003).…”

Section: Discussionmentioning

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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“…Diaphragm muscle was prepared and tested by the real-time PCR method described in Cuttell et al (2012b). This test generically targets the small subunit of the ribosomal RNA (SSU-rRNA) gene of Trichinella parasites and the analytical sensitivity is 0.1 larvae/g when 10 g of muscle is tested.…”

Section: Molecular Methodsmentioning

confidence: 99%

Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa)

Cuttell

Morales

Cookson³

et al. 2014

Veterinary Parasitology

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 35A c c e p t e d M a n u s c r i p t Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were 35 examined using both an 'in-house' and a commercially available indirect-ELISA that used other parasitic infections, more work using well-defined cohorts of positive and negative samples is 53 required. Even if the specificity of the ELISAs is proven to be low, their ability to correctly classify 54 the small number of true positive sera in this study indicates utility in screening wild boar 55 populations for reactive sera which can be followed up with additional testing.56 57

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“…In addition, the various species or strains of various medically pathogenic microorganisms can be differentiated by melting curve analysis (Lyon and Wittwer 2009). Recently, SYBR Green detection-based (Guenther et al 2008, Cuttell et al 2012 and Taqman probe-based (Atterby et al 2009) real-time PCR approaches have been reported as a diagnostic tool for the detection of T. spiralis DNA in muscle tissue. Moreover, high-resolution melting (HRM) assay in a single tube real-time PCR reaction was used to detection of inter-and intraspecies polymorphisms of four Trichinella species-T. pseudospiralis, T. spiralis, T. britovi, and T. nativa (Masny et al 2012).…”

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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Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Cited by 28 publications

References 24 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

Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa)

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

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