Department of Laboratory Medicine, Affiliated Hospital of Academy of Military Medical Sciences, Beijing, P.R. China.
J Med Virol. 2018 Mar;90(3):464-468. doi: 10.1002/jmv.24974. Epub 2017 Nov 7.
crAssphage is a novel and by far the most abundant bacteriophage in human gut. This bacteriophage might modulate gut microbiota balance so as to be involved in some diseases like obesity, diabetes, metabolic disorders, hypertension, and cancer. Therefore, a rapid and reliable detection and quantification method for crAssphage is essential for studying its molecular epidemiology and pathogenicity in human diseases. The primers-probes set for the quantitative real-time PCR assay was designed based on the DNA polymerase gene (ORF00018) of crAssphage. The sensitivity and specificity, as well as comparison testing with the conventional PCR and sequencing were evaluated. The assay could specifically detect crAssphage, and no cross-reactions with other gut microbes were observed. The detection limit was 15.6 copies/μL of clinical samples (46.8 copies/reaction). When using clinical samples, the assay showed higher ability to detect samples with low viral DNA copies and had an agreement of 93.33% when compared with the conventional PCR amplification and sequencing. The established real-time PCR assay is a sensitive, specific, and repeatable method for quantitatively detecting crAssphage, and thus is a very useful tool for investigating the molecular epidemiology, dynamics, and pathogenicity of crAssphage in human diseases.
粪球菌是一种新型的噬菌体，也是迄今为止在人类肠道中最丰富的噬菌体。这种噬菌体可能会调节肠道微生物群落的平衡，从而参与一些疾病，如肥胖症、糖尿病、代谢紊乱、高血压和癌症。因此，快速可靠的 crAssphage 检测和定量方法对于研究其在人类疾病中的分子流行病学和致病性至关重要。用于定量实时 PCR 检测的引物探针集是基于 crAssphage 的 DNA 聚合酶基因（ORF00018）设计的。评估了该检测方法的灵敏度、特异性以及与常规 PCR 和测序的比较测试。该检测方法能够特异性地检测 crAssphage，并且没有与其他肠道微生物发生交叉反应。检测限为临床样本的 15.6 拷贝/μL（46.8 拷贝/反应）。当使用临床样本时，该检测方法显示出更高的能力来检测低病毒 DNA 拷贝的样本，并且与常规 PCR 扩增和测序相比具有 93.33%的一致性。该建立的实时 PCR 检测方法是一种敏感、特异、可重复的定量检测 crAssphage 的方法，因此是研究 crAssphage 在人类疾病中的分子流行病学、动态和致病性的非常有用的工具。
