猪δ冠状病毒TaqMan荧光定量RT-PCR检测方法的建立与初步应用

doi:10.11843/j.issn.0366-6964.2019.06.016

Abstract

Abstract: This experiment was conducted to develop a TaqMan based real-time fluorescent RT-PCR for the detection of PDCoV. Based on the M gene sequence of PDCoV in GenBank, a pair of primers was designed, and M gene of PDCoV was amplified and cloned into pMD18-T vector to get the recombinant plasmid. Then another pair of primers and a TaqMan probe was designed, and a TaqMan based real-time fluorescent RT-PCR assay was developed by optimizing the conditions and systems of reaction. Sensitivity, specificity and reproducibility assay were determined. The results showed that this assay could detect the PDCoV between 1.0×10¹-1.0×10⁹ copies·μL^-1 with a good linear relation, and the sensitivity limit was 1.0×10¹ copies·μL^-1. There were no cross-reaction with porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), pseudorabies virus (PRV) and other porcine viruses. The CV (Coefficient of Variation) of intra-assay and inter-assay were no more than 1%. The TaqMan based real-time RT-PCR assay was then used to detect 100 diarrhea clinical samples of porcine that collected from 2017 to 2018, and the PDCoV positive ratio was 23%, and the coincidence rate with SYBR Green Ⅰ real-time PCR assay was 100%. Piglets were inoculated with PDCoV, and fecal samples were collected and detected by TaqMan real-time RT-PCR and common RT-PCR assay, the results showed that the sensitivity of the former was much better than the latter. This assay provides a high sensitivity, specificity and reproducibility method for detection of PDCoV.

CLC Number:

S852.659.6

ZHENG Lanlan, ZHU Jingjing, WANG Pan, SHU Yan, LIANG Qingqing, LI Bingxiao, WANG Chaoqun, WEI Zhanyong. Development and Application of a TaqMan based Real-time Fluorescent RT-PCR for Specific Detection of Porcine Deltacoronavirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1261-1267.

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References

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Development and Application of a TaqMan based Real-time Fluorescent RT-PCR for Specific Detection of Porcine Deltacoronavirus

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