一种同时检测传染性喉气管炎病毒的多重PCR方法及…… （原文此处不完整）

A Multiplex PCR Method for Simultaneous Detection of Infectious Laryngotracheitis Virus and .

作者信息

Nguyen Van-Giap, Cao Thi-Bich-Phuong, Le Van-Truong, Truong Ha-Thai, Chu Thi-Thanh-Huong, Dang Huu-Anh, Nguyen Thi-Hoa, Le Thi-Luyen, Huynh Thi-My-Le

机构信息

Department of Veterinary Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, Vietnam National University of Agriculture (VNUA), Hanoi 100000, Vietnam.

Key Laboratory for Veterinary Biotechnology, Vietnam National University of Agriculture (VNUA), Hanoi 100000, Vietnam.

出版信息

Vet Sci. 2023 Apr 3;10(4):272. doi: 10.3390/vetsci10040272.

DOI:10.3390/vetsci10040272

PMID:37104427

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10144794/

Abstract

To date, many fluorescence- and gel-based multiplex polymerase chain reaction (PCR) assays have been developed for the simultaneous detection of multiple infectious agents of respiratory disease in poultry. However, PCR assays are not available for other important emerging respiratory bacteria, such as (ORT). We aimed to fill this gap by establishing a new duplex PCR method for the simultaneous detection of infectious laryngotracheitis virus (ILTV) and ORT. Multiplex primer design software was used to select the compatible multiplex primer pairs. It was determined that an annealing temperature of 65 °C and an initial concentration of 2.5 pmol/µL for each primer set were the most suitable conditions for multiplex PCR. The assay was confirmed to be specific, as it only detected the target pathogens, even in the presence of six non-target agents. The limit of detection was up to 10 copies/µL of template DNA for both ILTV and ORT. In the screening of 304 field samples, 23, 88, and 44 were positive for both ILTV and ORT, solely for ILTV, and solely ORT, respectively.

摘要

迄今为止，已经开发了许多基于荧光和凝胶的多重聚合酶链反应（PCR）检测方法，用于同时检测家禽中多种呼吸道疾病感染因子。然而，对于其他重要的新兴呼吸道细菌，如禽鼻炎（ORT），尚无PCR检测方法。我们旨在通过建立一种新的双重PCR方法来填补这一空白，用于同时检测传染性喉气管炎病毒（ILTV）和ORT。使用多重引物设计软件选择兼容的多重引物对。确定每个引物组的退火温度为65°C，初始浓度为2.5 pmol/µL是多重PCR最合适的条件。该检测方法被证实具有特异性，因为即使存在六种非目标病原体，它也只能检测到目标病原体。ILTV和ORT的检测限均高达10拷贝/µL模板DNA。在对304份现场样本的筛查中，ILTV和ORT均呈阳性的有23份，仅ILTV呈阳性的有88份，仅ORT呈阳性的有44份。