采用数字 PCR 技术检测粪便样本中的巨细胞病毒（CMV）用于 CMV 性胃肠炎的非侵入性诊断。
Detection of cytomegalovirus (CMV) by digital PCR in stool samples for the non-invasive diagnosis of CMV gastroenteritis.
机构信息
Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Wuhan, 430030, Hubei, China.
出版信息
Virol J. 2022 Nov 11;19(1):183. doi: 10.1186/s12985-022-01913-z.
BACKGROUND
CMV gastroenteritis is common in patients receiving allogeneic hematopoietic stem cell transplantation and it is difficult to distinguish from acute graft-versus-host disease (aGvHD), which has very similar symptoms but needs quite different treatment. CMV gastroenteritis is caused by local infection or reactivation of CMV in the gastrointestinal tract while aGvHD is due to immune rejection. The gold standard of diagnosis of CMV gastroenteritis and aGvHD is gastrointestinal biopsy under endoscopy, which is invasive and can potentially lead to severe side effects. Stool samples testing with quantitative polymerase chain reaction (qPCR) may be an alternative, while the application in trace level measurements and precision are not all satisfactory enough in reported research.
METHODS
In this study, we designed a novel method that extracted the cell free DNA (cfDNA) from the fecal supernatant to perform digital PCR (dPCR) for the detection of CMV, analyzed the performance and compared it with the total DNA extracted by the current procedure.
RESULTS
Twenty-two paired stool samples using two DNA extraction methods proved that the cfDNA extraction method had markedly higher DNA concentrations and control gene copy number, suggesting that cfDNA may be more informative and more useful for the detection of CMV DNA segment. The dPCR approach in detecting CMV DNA segment also exhibit good linearity (R = 0.997) and higher sensitivity (limit of detection at 50% was 3.534 copies/μL). Eighty-two stool samples from 44 immunocompromised patients were analyzed, CMV-positive rate was 28%, indicating that more than one-quarter of the gastrointestinal symptoms within these patients may be caused by CMV infection or reactivation.
CONCLUSION
The combined results suggest that detection of CMV by dPCR in cfDNA of stool supernatant is a powerful method to identify CMV gastroenteritis and helps in clinical treatment decision making.
背景
巨细胞病毒（CMV）胃肠炎在接受异基因造血干细胞移植的患者中很常见，难以与急性移植物抗宿主病（aGvHD）相区分，两者具有非常相似的症状，但需要截然不同的治疗方法。CMV 胃肠炎是由胃肠道局部 CMV 感染或再激活引起的，而 aGvHD 则是由于免疫排斥引起的。CMV 胃肠炎和 aGvHD 的诊断金标准是在内镜下进行胃肠道活检，这是一种有创性检查，可能会导致严重的副作用。定量聚合酶链反应（qPCR）检测粪便样本可能是一种替代方法，但在报告的研究中，其在痕量测量和精密度方面的应用并不完全令人满意。
方法
在本研究中，我们设计了一种从粪便上清液中提取无细胞游离 DNA（cfDNA）进行数字 PCR（dPCR）检测 CMV 的新方法，分析了其性能并将其与现行方法提取的总 DNA 进行了比较。
结果
使用两种 DNA 提取方法对 22 对粪便样本进行了检测，结果表明 cfDNA 提取方法的 DNA 浓度和对照基因拷贝数明显更高，表明 cfDNA 可能更具信息量，更有助于检测 CMV DNA 片段。dPCR 方法检测 CMV DNA 片段也表现出良好的线性（R=0.997）和更高的灵敏度（检测限为 50%时为 3.534 拷贝/μL）。对 44 例免疫功能低下患者的 82 份粪便样本进行了分析，CMV 阳性率为 28%，表明这些患者中超过四分之一的胃肠道症状可能是由 CMV 感染或再激活引起的。
结论
综合结果表明，采用 dPCR 检测粪便上清液中的 cfDNA 是一种识别 CMV 胃肠炎的有力方法，有助于临床治疗决策。
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