Yu Xuewu, Shi Lin, Lv Xiaoping, Yao Wei, Cao Minghui, Yu Hanxun, Wang Xiurong, Zheng Shimin
College of Veterinary Medicine, Northeast Agricultural University, No.59, Mucai street, Xiangfan District, Harbin, 150030, China.
Animal Epidemic Diseases Control and Prevention Center of Liaoning Province, Shenyang, China.
Virol J. 2015 May 14;12:76. doi: 10.1186/s12985-015-0297-1.
Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease characterized by severe enteritis, vomiting and watery diarrhea in swine. Recently, the outbreak of the epidemic disease has been a serious problem in swine industry. The objective of this study is to develop a rapid, sensitive, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of porcine epidemic diarrhea virus (PEDV) in less equipped laboratories.
The optimal reaction condition of the current real-time RT-LAMP for PEDV was 62 °C for 45 min. It was capable of detecting PEDV from clinical samples and differentiating PEDV from several related porcine viruses, while it did not require additional expensive equipment. The minimum detection limit of the real-time RT-LAMP assay was 0.07PFU per reaction for PEDV RNA, making this assay approximately 100-fold more sensitive than that of one-step RT-PCR. By screening a panel of clinical specimens, the results showed that this method presented a similar sensitivity with real-time RT-PCR and was somewhat sensitive than one-step RT-PCR in detection of clinical samples.
In this study, we have developed a new real-time RT-LAMP method, which is rapid, sensitive and efficient to detect PEDV.This method holds great promises not only in laboratory detection and discrimination of PEDV but also in large scale field and clinical studies.
猪流行性腹泻（PED）是一种急性且高度传染性的肠道疾病，其特征为猪出现严重肠炎、呕吐和水样腹泻。近来，这种疫病的爆发已成为养猪业的一个严重问题。本研究的目的是开发一种快速、灵敏的实时逆转录环介导等温扩增（RT-LAMP）方法，用于在设备较少的实验室中检测猪流行性腹泻病毒（PEDV）。
当前用于PEDV的实时RT-LAMP的最佳反应条件是62℃反应45分钟。它能够从临床样本中检测PEDV，并将PEDV与几种相关的猪病毒区分开来，同时不需要额外的昂贵设备。实时RT-LAMP检测的最低检测限为每个反应0.07PFU的PEDV RNA，这使得该检测方法比一步法RT-PCR的灵敏度高约100倍。通过对一组临床标本进行筛查，结果表明该方法与实时RT-PCR具有相似的灵敏度，并且在检测临床样本时比一步法RT-PCR稍灵敏。
在本研究中，我们开发了一种新的实时RT-LAMP方法，该方法快速、灵敏且高效地检测PEDV。这种方法不仅在实验室检测和鉴别PEDV方面，而且在大规模的现场和临床研究中都具有很大的前景。
