建立一种巢式 PCR 方法用于检测的感染。

Development of a nested PCR assay for specific detection of infecting .

机构信息

Aquaculture Department, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.

Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, Shenyang Agricultural University, Shenyang, China.

出版信息

Front Cell Infect Microbiol. 2022 Jul 22;12:930585. doi: 10.3389/fcimb.2022.930585. eCollection 2022.

DOI:10.3389/fcimb.2022.930585

PMID:35937694

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9352885/

Abstract

In recent years, the "milky disease" caused by has seriously affected the culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect , by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 10 copies/μL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 10 copies/μL) and internal transcribed spacer (ITS; 6.74 × 10 copies/μL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of infection. This will help to elucidate the transmission route of and to design effective management and control measures for disease.

摘要

近年来，由引起的“牛奶病”严重影响了文化产业。发现并阻断传播途径成为控制该病的关键。目前针对的聚合酶链反应（PCR）检测技术使用核糖体 DNA（rDNA）序列，但灵敏度和特异性低导致频繁出现假阳性。我们开发了一种针对菌丝调控细胞壁蛋白（HYR）基因的高度特异和敏感的嵌套 PCR 方法来检测。该嵌套 HYR-PCR 产生了一个单一清晰的条带，与其他病原体无交叉反应，在特异性和灵敏度方面优于 rDNA-PCR。嵌套 HYR-PCR 的灵敏度（6.10×10 拷贝/μL）大于大亚基核糖体 RNA 基因（LSU rRNA；6.03×10 拷贝/μL）和内部转录间隔区（ITS；6.74×10 拷贝/μL）PCR。嵌套 HYR-PCR 的阳性率（71.1%）也高于 LSU rRNA（16.7%）和 ITS rDNA（24.4%）。总之，我们开发了一种新的嵌套 HYR-PCR 方法，用于的特异性和敏感检测。这将有助于阐明的传播途径，并为病的有效管理和控制措施提供依据。