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| Establishment and Application of a TaqMan Real-time Fluorescent Quantitative PCR Assay for Detecting Transferrin Receptor of Carnivore |
| ZHANG Chuan-Mei*, CUI Yan-Lei*, WEN Li-Sen, YU Yong-Le, SHAN Hu**, YANG Hai-Yan** |
| College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China |
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Abstract Transferrin receptor (TfR) is a type Ⅱ transmembrane dimer glycoprotein located on the cell membrane, which is the receptor for the invasion of dog (Canis lupus familiaris), cat (Felinae) and other Carnivorous parvovirus into host cells. This study aimed to establish a TaqMan real-time fluorescent quantitative PCR assay for detecting the expression of TfR in carnivore, and to detect and analyze the expression of TfR gene in different tissues of carnivores and passage cells. A pair of primers and a specific TaqMan probe were designed and synthesized by referencing to the gene sequence of the TfR extracellular region protein published in GenBank. The reaction system and conditions of qPCR were optimized, a positive plasmid was constructed, a standard curve was established, and the sensitivity and repeatability test were carried out. The expression of TfR gene in different tissues of carnivores and passage cells were detected and analyzed by the established method. The results showed that the assay had a good linear relationship, sensitivity and stability with a correlation coefficient of 0.998 7, the detection limit was 67 copies/μL, the coefficient of variation of intra-assay and inter-assay evaluation test was less than 2.0%. This study provides a qPCR method for detecting the expression of TfR gene in carnivore with good sensitivity and stability, and it can be used for detecting the expression of TfR gene in different tissues and passage cells of carnivore.
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Received: 11 January 2022
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Corresponding Authors:
**shanhu67@163.com; hy386@126.com
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| About author:: * These authors contributed equally to this work |
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