Kerachian Mohammad Amin, Amel Jamehdar Saeid, Azghandi Marjan, Keyvanlou Nasrin, Mozaffari-Jovin Sina, Javadmanesh Ali, Amini Mahnaz
Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Iran J Basic Med Sci. 2022 Jun;25(6):762-766. doi: 10.22038/IJBMS.2022.65260.14379.
Early, specific, and sensitive detection methods of COVID-19 are essential for force stopping its worldwide infection. Although CT images of the lung and/or viral RNA extraction followed by real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) are widely used; they have some limitations. Here, we developed a highly sensitive magnetic bead-based viral RNA extraction assay followed by rRT-PCR.
Case group included oropharyngeal/nasopharyngeal and blood samples from 30 patients diagnosed positive by PCR test for COVID-19 and control group included 30 same samples from COVID-19 negative PCR test individuals. RNA was extracted, using viral RNA extraction kit as well as using our hand-made capture bead-based technique. A one-step cDNA synthesis and Real Time PCR was conducted. A two-step comparison of the different viral RNA extraction methods for oropharyngeal/nasopharyngeal and blood samples was performed. Student t-test was applied with a 0.05 considered statistically significant.
In the case group, all 30 mucosal samples extracted either with viral RNA extraction kit or with beads-based assay were COVID-19 positive although in the latter category, Cqs were much lower. Although 43% of plasma samples extracted by bead-based method were found to be positive but no plasma samples extracted with column-based kit were detected positive by Real Time PCR.
Bead-based RNA extraction method can reduce RNA loss by its single-tube performance and enhance the test sensitivity. It is also more sensitive to lower viral loads as shown in the detection of blood samples and the lower Cqs of mucosal samples.
新冠病毒病（COVID-19）的早期、特异性和灵敏检测方法对于有效阻止其全球传播至关重要。尽管肺部CT图像和/或提取病毒RNA后进行实时逆转录聚合酶链反应（rRT-PCR）被广泛应用，但它们存在一些局限性。在此，我们开发了一种基于磁珠的高灵敏病毒RNA提取方法，随后进行rRT-PCR。
病例组包括30例经PCR检测确诊为COVID-19阳性患者的口咽/鼻咽和血液样本，对照组包括30例经PCR检测为COVID-19阴性个体的相同样本。使用病毒RNA提取试剂盒以及我们自制的基于捕获磁珠的技术提取RNA。进行一步法cDNA合成和实时PCR。对不同病毒RNA提取方法用于口咽/鼻咽和血液样本进行了两步比较。采用学生t检验，P<0.05认为具有统计学意义。
在病例组中，用病毒RNA提取试剂盒或基于磁珠的方法提取的所有30份黏膜样本均为COVID-19阳性，不过在后一种方法中，定量循环数（Cq）要低得多。尽管基于磁珠方法提取的血浆样本中有43%被检测为阳性，但基于柱式试剂盒提取的血浆样本经实时PCR检测均未呈阳性。
基于磁珠的RNA提取方法通过单管操作可减少RNA损失并提高检测灵敏度。如在血液样本检测及黏膜样本较低的Cq值中所示，其对较低病毒载量也更灵敏。
