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Establishment and Application of Fluorescence Quantitative RT-PCR for Detection of the Velogenic Newcastle Disease Virus

LUO Yaoyao1,2 WANG Jingjing 1 LV Yan 1 ZHAO Yunling1 ZHENG Dongxia1 WEI Runyu1,3 YU Songmei1 ZUO Yuanyuan1 ZHANG Lecui2 SHAN Hu2 LIU Hualei1,2 WANG Zhiliang1

(1.China Animal Health and Epidemiology Center, Qingdao, China 266032)
(2.College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China 266109)
(3.Yangzhou University, Yangzhou, China 225009)
【Novelty】Based on the domestic epidemic strains, a fluorescence quantitative RT-PCR method was established to detecting velogenic NDVs. The results showed that the method was highly sensitive, specific, reproducible and accurate in diagnostic accuracy. It was proved that the area under ROC curve of 1,974 clinical samples was 0.9861. The sensitivity and specificity of diagnosis were 94.82 % and 98.3 % respectively. It was suitable for the active monitoring and epidemiological investigation of NDV on large-scale pathogens. It has a significant promotion prospect.

【Abstract】The primers and probe of a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were designed according to the characteristics of fusion protein cleavage sites of velogenic Newcastle disease viruses (NDVs). The analytical sensitivity of qRT-PCR was evaluated by standard curves using cRNA standards in vitro as positive controls. Analytical specificity was identified by detection of the common avian viruses and lentogenic NDV. Diagnostic sensitivity and specificity were validated based on receiver-operating characteristic (ROC) curves through detection of clinical samples. The limit of detection limit of this assay reached ≥ 2 copies and no cross-reaction was found. The coefficient of variation (CV) for intra-assay and inter-assay repeatability was < 1% and 1.5% respectively. The area under the ROC curve of 1974 clinical samples was 0.986 1. According to the Youden Index, the Cutoff value was 35. The sensitivity and specificity of this diagnosis was 94.82% and 98.3% respectively. The Kappa coefficient for isolation of the virus was 0.919. Our study demonstrated that a one-step qRT-PCR was highly sensitive, specific, reproducible and accurate, thereby providing a fast and practical method to detect the NDV in clinical samples.

【Keywords】 Newcastle disease virus (NDV); Virulent virus; Fluorescence quantitative RT-PCR; ROC curve;

【DOI】

【Funds】 National Key Research and Development Plan (2016YFD0500800) Public Welfare Industry (Agriculture) Research Project (201303033)

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This Article

ISSN:1000-8721

CN: 11-1865/R

Vol 34, No. 04, Pages 541-549

June 2018

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Article Outline

Novelty

Abstract

  • Materials and methods
  • Results
  • 7 Diagnostic performance evaluation
  • 8 Comparison test of different kits
  • Discussion
  • References