Laboratorio de Virología, Facultad de Ciencias Veterinarias (FCV), Universidad Nacional de La Plata (UNLP), 60 & 118, La Plata, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), (C1425FQB), Godoy Cruz 2290, Ciudad Autónoma de Buenos Aires, Argentina.
Laboratorio de Virología, Facultad de Ciencias Veterinarias (FCV), Universidad Nacional de La Plata (UNLP), 60 & 118, La Plata, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), (C1425FQB), Godoy Cruz 2290, Ciudad Autónoma de Buenos Aires, Argentina.
Prev Vet Med. 2024 Oct;231:106303. doi: 10.1016/j.prevetmed.2024.106303. Epub 2024 Aug 2.
SARS-CoV-2 emerged from an animal source and was then transmitted to humans, causing the COVID-19 pandemic. Since a wide range of animals are susceptible to SARS-CoV-2 infection, the zoonotic potential of SARS-CoV-2 increases with every new animal infected. The molecular gold standard assay for SARS-CoV-2 detection is real-time RT-PCR, where the Ct obtained is proportional to the amount of nucleic acid and can be a semi-quantitative measure of the viral load. However, since the use of real-time RT-PCR assays in animal samples is low due to the high costs, the use of validated nested PCR assays will help to monitor large-scale animal samplings, by reducing the costs of detection. In the present study, 140 samples from dogs and cats (15 SARS-CoV-2-positive samples with Ct values from 27 to 33, and 125 negative samples), previously analyzed by real-time RT-PCR, were analyzed by nested PCR. To increase the number of positive samples to determine the sensitivity of the assay, 40 human samples obtained during COVID-19 diagnosis in 2020 were included. The specificity of the primers was analyzed against samples positive to canine coronavirus (CCV) and feline infectious peritonitis virus (FIPV). To calculate the limit of detection (LoD) of the nested PCR, the viral load was estimated extrapolating the Ct value obtained by real-time RT-PCR. The Ct values obtained were considered as semi-quantitative and were able to distinguish between high, moderate and low viral loads. The Kappa value or "agreement" between assays and reliability of the nested PCR were also determined. Eleven of the animal samples analyzed by nested PCR targeting the N gene were detected as positive, while 129 were detected as negative to the virus, with Ct values ranging between17 and 31.5. All the samples from humans analyzed by nested PCR were positive. These results indicate that the assay has a sensitivity of near 95 % and a specificity of 100 %. No unspecific reactions analyzed by nested PCR were observed with the samples positive to CCV and FIPV. The samples detected as positive to SARS-CoV-2 by nested PCR were those that presented a Ct between17 and 31.5. The LoD of the nested PCR was estimated close to 50 copies/µL of viral load, corresponding with a Ct of 31.5. The Kappa value between assays was excellent (k = 0.829). The results obtained demonstrate that nested PCR is useful to detect SARS-CoV-2 low viral loads at a lower cost than with real-time RT-PCR.
SARS-CoV-2 源自动物源，然后传播给人类，引发了 COVID-19 大流行。由于广泛的动物易感染 SARS-CoV-2，因此每感染一种新动物，SARS-CoV-2 的人畜共患潜力就会增加。SARS-CoV-2 检测的分子金标准检测方法是实时 RT-PCR，其中获得的 Ct 值与核酸量成正比，并且可以作为病毒载量的半定量测量。然而，由于实时 RT-PCR 检测方法在动物样本中的使用成本较高，因此使用经过验证的巢式 PCR 检测方法将有助于通过降低检测成本来监测大规模的动物采样。在本研究中，对先前通过实时 RT-PCR 分析的 140 份狗和猫样本（15 份 SARS-CoV-2 阳性样本，Ct 值为 27 至 33，125 份阴性样本）进行了巢式 PCR 分析。为了增加阳性样本的数量以确定检测的灵敏度，还纳入了 2020 年 COVID-19 诊断期间获得的 40 份人类样本。针对犬冠状病毒 (CCV) 和猫传染性腹膜炎病毒 (FIPV) 阳性样本分析了引物的特异性。为了估算巢式 PCR 的检测限 (LoD)，通过实时 RT-PCR 获得的 Ct 值来估计病毒载量。获得的 Ct 值被认为是半定量的，能够区分高、中、低病毒载量。还确定了巢式 PCR 的 Kappa 值（“一致性”）或与检测的相关性以及可靠性。针对 N 基因的巢式 PCR 分析检测到 11 份动物样本为阳性，而 129 份样本为病毒阴性，Ct 值范围为 17 至 31.5。对巢式 PCR 分析的所有人类样本均为阳性。这些结果表明该检测方法的灵敏度接近 95%，特异性为 100%。用 CCV 和 FIPV 阳性样本进行巢式 PCR 分析未观察到非特异性反应。巢式 PCR 检测为阳性的 SARS-CoV-2 样本的 Ct 值在 17 至 31.5 之间。巢式 PCR 的 LoD 估计接近 50 拷贝/μL 的病毒载量，对应 Ct 值为 31.5。检测方法之间的 Kappa 值为极好（k = 0.829）。研究结果表明，巢式 PCR 比实时 RT-PCR 更经济，可用于检测 SARS-CoV-2 的低病毒载量。
