Digital CRISPR/Cas13a-RPA based assay for rapid and sensitive detection of JC virus in renal transplant patients
Digital CRISPR/Cas13a-RPA based assay for rapid and sensitive detection of JC virus in renal transplant patients
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DOI：
10.1016/j.microc.2023.109655
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发表时间：
2024-01
影响因子：
4.8
通讯作者：
Yu Liu;Jing-ying Xu;Xiaoying Chen;Shuang Yang;Jiajun Li;Hongbo Zhou;Qian Liu;Wei Liu;Min Li;Hua Wang
中科院分区：
文献类型：
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作者：
Yu Liu;Jing-ying Xu;Xiaoying Chen;Shuang Yang;Jiajun Li;Hongbo Zhou;Qian Liu;Wei Liu;Min Li;Hua Wang
JC病毒（JCV）是多瘤病毒家族的成员，是一种环状双链DNA（dsDNA）病毒，可导致机会性感染。肾移植患者的JCV感染可导致多瘤病毒相关性肾病。因此，早期检测和定量尿液或血液中的JC病毒将有效提高肾移植患者的存活率，降低患者的死亡率。目前，PCR已作为JC病毒检测的常规方法应用于临床。但PCR不能实现病毒核酸的绝对定量检测。使得其不适合于具有特定要求的某些临床应用。在此，我们提出了一种JC病毒核酸检测平台，命名为MdCaR，其将微流控液滴装置与CRISPR-Cas 13 a和重组酶聚合酶扩增相耦合。MdCaR可通过数字滴定量检测JC病毒。检测限达到1拷贝/mL，检测时间仅需40 min。分析了12例肾移植患者和20例对照受试者的标本。MdCaR在检测这些样本时表现良好，特别是用于检测qPCR鉴定为阴性的低病毒载量样本。总之，MdCaR可被视为一种有前途的、快速的JCV检测策略，并广泛应用于JCV感染的诊断。
JC viruses (JCVs), members of the Polyomaviruses family, are a type of circular double-stranded DNA (dsDNA) viruses and can lead to opportunistic infections. Infection of JCVs in kidney transplant patients can lead to polyomavirus-associated nephropathy. Thus, early detection and quantification of JC virus in urine or blood will effectively improve the survival rate of kidney transplant patients and reduce the mortality of patients. At present, PCR has been used as a routine method for JC virus detection in clinical practice. However, PCR cannot achieve absolute quantitative detection of viral nucleic acid. rendering it unsuitable for certain clinical applications with specific requirements. Herein, we proposed a JC virus nucleic acid testing platform, named MdCaR, which couples amicrofluidicdroplet device with CRISPR-Cas13a andrecombinase polymerase amplification. MdCaR can quantitatively detect JC virus through digital droplet. The detection limit achieves 1 copy/mL, and the assay takes only 40 min. Twelve renal transplant patients and 20 control subjects’ specimens were analyzed. MdCaR conveyed a good performance in testing these samples, especially for detecting the low viral load samples identified as negative by qPCR. Taken altogether, MdCaR could be regarded as a promising, rapid JCV detection strategy and widely applied for diagnosis for JCV infection.