A TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of porcine enteric coronaviruses

被引：86

作者：

Huang, Xin ^{[1
,2
]}

Chen, Jianing ^{[2
]}

Yao, Gang ^{[1
]}

Guo, Qingyong ^{[1
]}

Wang, Jinquan ^{[1
]}

Liu, Guangliang ^{[1
,2
]}

机构：

[1] Xinjiang Agr Univ, Coll Vet Med, 311 East Noda Rd, Urumqi 830052, Xinjiang, Peoples R China

[2] Chinese Acad Agr Sci, State Key Lab Vet Etiol Biol, Lanzhou Vet Res Inst, 1 XuJiaPing, Lanzhou 730046, Gansu, Peoples R China

来源：

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY | 2019年 / 103卷 / 12期

基金：

国家重点研发计划; 中国国家自然科学基金;

关键词：

TaqMan probe; Multiplex real-time RT-qPCR; Diagnosis; Swine enteric coronaviruses; EPIDEMIC DIARRHEA VIRUS; SWINE; DELTACORONAVIRUS; DISCOVERY; PCR; ALPHACORONAVIRUS; CHINA; PIGS;

D O I：

10.1007/s00253-019-09835-7

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Swine enteric coronaviruses are a group of most significant pathogens causing diarrhea in piglets with similar clinical symptoms and pathological changes. To develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, specific primers and probes were designed based on the highly conserved regions of transmissible gastroenteritis virus (TGEV) N, porcine epidemic diarrhea virus (PEDV) M, porcine deltacoronavirus (PDCoV) M, and porcine enteric alphacoronavirus (PEAV) N genes respectively. A TaqMan-probe-based multiplex real-time RT-qPCR assay was developed and optimized to simultaneously detect these swine enteric coronaviruses. The results showed that the limit of detection can reach as low as 10 copies in singular real-time RT-qPCR assays and 100 copies in multiplex real-time RT-qPCR assay, with all correlation coefficients (R-2) at above 0.99, and the amplification efficiency at between 90 and 120%. This multiplex real-time RT-qPCR assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. The multiplex real-time RT-qPCR assay was then employed to detect the swine enteric coronavirus from 354 field diarrheal samples. The results manifested that TGEV and PDCoV were the main pathogens in these samples, accompanied by co-infections. This well-established multiplex real-time RT-qPCR assay provided a rapid, efficient, specific, and sensitive tool for detection of swine enteric coronaviruses.

引用

页码：4943 / 4952

页数：10