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| Establishment and Application of Multiplex Fluorescent Real-time PCR for Detecting Six Kinds of Animal Derived Materials |
| FU Li-wen, ZHANG Yu, YI Han, LI Xue, ZHU Nai-shuo |
| Lab of Molecular Immunology, State Key Lab of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200438, China |
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Abstract Objective:To establish a highly efficient method for simutaneously detecting pig, bovine, sheep, chicken, duck and goose derived materials in animal products by use of the multiplex fluorescent real-time quantitative PCR(qPCR). Methods:The species-specific primers and probes for detection of beta-actin (actb) genes of pig, bovine, chicken, duck and goose, and the prolactin receptor gene of sheep in multiplex fluorescent real-time quantitative PCR assay were designed. Total DNA as templates were extracted respectively from the six kinds of animal materials. The specificity and sensitivity of these primer pairs and probes were separately confirmed using simplex real-time qPCR analysis. Six pairs of primers and six fluorescent probes were mixed up with the above 6 kinds of animal templates. And the most effective reaction system and condition for multiplex(hexa-plex) qPCR method were obtained experimentlly after lots of tests and optimization. The specificity, sensitivity and accuracy of this assay were evaluated. Results:The limits of detection (LOD) of the assay were 0.049ng, 0.048ng, 0.085ng, 0.13ng, 0.162ng, 0.074ng DNA for pig, bovine, sheep, chicken, duck and goose, respectively (50μl qPCR reaction system), and the amplification results of other animals were all negative. A total of 200 specimens tested by the multiplex qPCR method one more showed its accuracy, precision and high efficiency. Conclusions:The developed multiplex fluorescent real-time quantitative PCR method was a rapid, specific, sensitive and efficacious detection assay for pig, bovine, sheep, chicken, duck and goose derived materials in animal products, and was applicable to identifications in feed stuff, meat, milk, pelt and grease, etc.
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Received: 13 March 2017
Published: 25 September 2017
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