Chinese Academy of Inspection and Quarantine, Institute of Animal Inspection and Quarantine, Beijing 100176, China.
J AOAC Int. 2022 Sep 6;105(5):1437-1446. doi: 10.1093/jaoacint/qsac039.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread over the world since its emergence. Although the dominant route of SARS-CoV-2 infection is respiratory, a number of studies revealed infection risk from contaminated surfaces and products, including porcine-derived food and other products. The SARS-CoV-2 outbreak has been severely threatening public health, and disrupting porcine products trade and the pig industry. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was responsible for large-scale, fatal disease in piglets, emerged in 2017 and has caused enormous economic losses in the pig industry. Currently, reverse transcription real-time PCR (RT-rPCR) is the gold standard method for SARS-CoV-2 diagnosis and is most commonly used for SADS-CoV detection. However, inaccurate detection of the SARS-CoV-2 infection obtained by RT-rPCR is increasingly reported, especially in specimens with low viral load.
This study aimed to develop an accurate reverse transcription droplet digital PCR (RT-ddPCR) assay for the detection of SARS-CoV-2 and SADS-CoV simultaneously.
Two pairs of primers and one double-quenched probe targeting the RNA-dependent RNA polymerase (RDRP) region of the open reading frame 1ab (ORF1ab) gene of SARS-CoV-2 and the corresponding ORF1ab region of SADS-CoV were designed to develop the RT-ddPCR assay. The sensitivity, specificity, repeatability, and reproducibility were tested using complementary RNAs (cRNAs) and clinical specimens.
The detection limits of RT-ddPCR were 1.48 ± 0.18 and 1.38 ± 0.17 copies in a 20 μL reaction for SARS-CoV-2 and SADS-CoV cRNAs, respectively (n = 8), showing approximately 4- and 10-fold greater sensitivity than the RT-rPCR assay. This assay also exhibited good specificity, repeatability, and reproducibility.
The established RT-ddPCR assay was shown to be a highly effective, accurate, and reliable method for the sensitive detection of SARS-CoV-2 and SADS-CoV.
This RT-ddPCR assay could be used to detect both SARS-CoV-2 and SADS-CoV in a sample with one double-quenched probe, and is also the first reported RT-ddPCR assay for SADS-CoV detection.
自出现以来，严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）已在全球迅速传播。虽然 SARS-CoV-2 的主要感染途径是呼吸道，但许多研究表明，受污染的表面和产品（包括源自猪的食品和其他产品）存在感染风险。SARS-CoV-2 的爆发严重威胁着公共卫生，并扰乱了猪产品贸易和养猪业。猪急性腹泻综合征冠状病毒（SADS-CoV）于 2017 年导致仔猪大规模致命疾病，已给养猪业造成巨大经济损失。目前，逆转录实时 PCR（RT-rPCR）是 SARS-CoV-2 诊断的金标准方法，也是最常用于检测 SADS-CoV 的方法。然而，越来越多的报道表明，RT-rPCR 检测到的 SARS-CoV-2 感染并不准确，尤其是在病毒载量较低的标本中。
本研究旨在开发一种用于同时检测 SARS-CoV-2 和 SADS-CoV 的准确逆转录液滴数字 PCR（RT-ddPCR）检测方法。
设计了针对 SARS-CoV-2 的开放阅读框 1ab（ORF1ab）基因的 RNA 依赖性 RNA 聚合酶（RDRP）区和 SADS-CoV 的相应 ORF1ab 区的两对引物和一个双淬灭探针，以开发 RT-ddPCR 检测方法。使用互补 RNA（cRNA）和临床标本测试了该方法的灵敏度、特异性、重复性和重现性。
在 20 μL 反应中，RT-ddPCR 对 SARS-CoV-2 和 SADS-CoV cRNA 的检测限分别为 1.48±0.18 和 1.38±0.17 拷贝（n=8），比 RT-rPCR 检测方法的灵敏度高约 4 倍和 10 倍。该检测方法还表现出良好的特异性、重复性和重现性。
建立的 RT-ddPCR 检测方法是一种高效、准确、可靠的方法，可用于敏感检测 SARS-CoV-2 和 SADS-CoV。
该 RT-ddPCR 检测方法可在一个双淬灭探针中同时检测 SARS-CoV-2 和 SADS-CoV，也是首次报道的 SADS-CoV RT-ddPCR 检测方法。
