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Comparison of three molecular detection methods for detection of Trichinella in infected pigs
Zhibing Lin1, Jie Cao, Houshuang Zhang
1Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
Abstract:
Different molecular detection methods require diverse molecular platforms, but there is no uniform standard for people to reference in the detection of Trichinella. In this study, real-time PCR, loop-mediated isothermal amplification (LAMP), and conventional PCR were developed for the detection of Trichinella by targeting mitochondrial large subunit ribosomal DNA (mt-lsrDNA). We compared the performance of the three newly developed assays. The results revealed that the detection limits of the real-time PCR, LAMP, and conventional PCR assays were 10 and 100 fg/μL and 1 pg/μL of Trichinella spiralis genomic DNA, respectively. The assays were used in the detection of Trichinella in the field. A total of 192 samples were obtained from pigs: 75 samples from free range farming and 117 from intensive feeding factory. The infection rate was 8/192 (4.2 %), 7/192 (3.6 %), and 1/192 (1.0 %) through the real-time PCR, LAMP, and conventional PCR assays, respectively. These data indicate that Taqman real-time PCR was a rapid, specific, and sensitive tool as a preferred option for investigation of valuable samples, but that LAMP assay was closed tube, highly sensitive, cost-effective, rapid, easy-to-perform, and was the optimal choice for detection of Trichinella in the field. The results of a model of experimental infection in mice indicated that spleen can be used as sampling site for the detection of early T. spiralis infection. However, the diaphragm and myocardium were the most suitable sampling sites for the detection of T. spiralis.
Insights
This study developed and compared real-time PCR, loop-mediated isothermal amplification (LAMP), and conventional PCR for detecting Trichinella. Real-time PCR is best for valuable samples, while LAMP is optimal for field detection due to its speed and ease of use.
Area of Science:
- Veterinary Parasitology
- Molecular Diagnostics
- Food Safety
Background:
- Standardized molecular detection methods for Trichinella are lacking.
- Diverse molecular platforms exist, leading to variability in detection.
- Accurate detection is crucial for public health and food safety.
Purpose of the Study:
- To develop and compare real-time PCR, loop-mediated isothermal amplification (LAMP), and conventional PCR assays for Trichinella detection.
- To evaluate the performance and applicability of these assays in field conditions.
- To identify optimal sampling sites for early detection of Trichinella spiralis infection.
Main Methods:
- Development of real-time PCR, LAMP, and conventional PCR assays targeting mitochondrial large subunit ribosomal DNA (mt-lsrDNA).
- Comparison of assay sensitivity, specificity, and detection limits using varying concentrations of Trichinella spiralis genomic DNA.
- Application of assays to detect Trichinella in 192 field samples from pigs and in experimentally infected mice.
Main Results:
- Detection limits: Real-time PCR (10 fg/μL), LAMP (100 fg/μL), conventional PCR (1 pg/μL).
- Field detection rates in pigs: Real-time PCR (4.2%), LAMP (3.6%), conventional PCR (1.0%).
- Spleen for early infection detection in mice; diaphragm and myocardium for later stages.
Conclusions:
- Taqman real-time PCR is a sensitive and specific tool for valuable samples.
- LAMP assay is the optimal choice for field detection of Trichinella due to its closed-tube, sensitive, cost-effective, rapid, and easy-to-perform nature.
- Diaphragm and myocardium are suitable sampling sites for detecting Trichinella spiralis in experimentally infected mice.
