禽腺病毒血清4型纳米PCR检测试剂盒的研制及应用

doi:10.16431/j.cnki.1671-7236.2020.07.026

Abstract

Abstract: This experiment was aimed to establish an efficient,fast and sensitive kit for fowl aviadenovirus serotype 4 (FAdV-4),the primers and probe were designed from penton gene of FAdV-4,and FITC was labeled in the downstream primer,Biotin was labeled in the probe.The nano PCR-LFD method for detection of FAdV-4 was established by using nano PCR technology and LFD technology,and the reaction and hybridization conditions were optimized.At the same time,the specificity,sensitivity,intra- and inter-batch repeatability,stability,shelf-life evaluation and clinical samples of the kit were tested.The specificity test showed that the kit could detect FAdV-4 specifically,while for other major viral and bacterial pathogens of poultry,such as infectious laryngotracheitis virus (ILTV),infectious bronchitis virus (IBV),infectious bursal disease virus (IBDV),chicken Marek's disease virus (MDV),egg drop syndrome virus (EDSV),duck plague virus (DPV),Newcastle disease virus (NDV),avian influenza virus H9 subtype (AIV (H9)),avian reovirus (ARV),FAdV standard strain (FAdV-1,FAdV-2,FAdV-8a,FAdV-8b and FAdV-11),E.coli,Staphylococcus aureus (SA),Salmonella Pullorum (SP) and Mycoplasma gallisepticum (MG) were all negative.The sensitivity detections suggested that the kit was so sensitive that it could detect the 56.0 copies/μL level and 10 folds higher than conventional PCR.The results of intra- and inter-repeatability test showed that it had good consistency and stability.The stability test results showed that the kit could withstand at least 20 times of repeated freezing and thawing,and still had a good detection effect.The shelf life test showed that the kit could be stored for at least 3 months at 4 ℃ and 12 months at -20 ℃.The kit realized the visualization of the detection results of FAdV-4,simplified the operation process and improved the detection efficiency.The positive rate of FAdV-4 was 17.95% (21/117).It could be used in the clinical diagnosis and molecular epidemiological investigation of FAdV-4,which provided an important technical support for the early detection of FAdV-4 infection in poultry farms and the formulation of comprehensive prevention and control measures.

Key words: nano PCR; detection; FAdV-4

CLC Number:

S852.65

WANG Lili, ZHENG Li, TIAN Xiangxue, LI Xiuli, REN Weike, LU Chao, ZHANG Li, CHI Jingjing, YAN Minghua. Development and Application of Nanoparticle-assisted PCR Detection Kit for Fowl Aviadenovirus Serotype 4[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2207-2214.

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Development and Application of Nanoparticle-assisted PCR Detection Kit for Fowl Aviadenovirus Serotype 4

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