Development of rapid and high sensitivity detection methods for replication-competent viruses contaminated in viral vector products for gene therapy

可复制病毒的污染是影响基因治疗临床研究中病毒载体产品安全性和质量的重要问题之一。为了更灵敏、更快速地检测载体产物中污染的复制活性腺病毒（RCA）和复制活性逆转录病毒（RCR），我们建立了一种结合感染性实验和实时定量PCR （RT-）的混合检测方法，用于检测RCA。在该方法中，将RCA样本感染允许细胞，并通过实时荧光定量PCR对扩增的RCA进行定量。基于玻璃珠的DNA提取方法适用于快速提取rca感染细胞的DNA。通过感染性PCR， 1 pfu的RCA在3天内可被加入到10^9个腺病毒载体颗粒中。感染后第9天，常规细胞病变效应可检测到RCA的10 ^4 pfu。结果表明，感染型PCR技术可快速、灵敏地检测腺病毒载体产物中的RCA。建立感染性RT-PCR检测RCR。该方法将RCR样本感染允许细胞，扩增后的RCR通过实时RT-PCR进行定量。采用聚乙烯亚胺（PEI）偶联磁珠对培养上清液进行RCR富集。通过传染性RT-PCR，在3天内检测到1个感染单位（iu）的RCR加入到10^6 cfu/ml的逆转录病毒载体中。与传统的S+L试验相比，病毒检测的灵敏度提高了10倍以上。因此，感染性RT-PCR可以快速、灵敏地检测逆转录病毒载体产物中的RCR，且直接从RCR感染的细胞中提取RCR RNA可以更快地检测到RCR。利用pei磁珠和磁场对RCR进行感染，可以提高感染效率和检测灵敏度。少

Contamination by replication-competent viruses is one of the most important issues for safety and quality of virus vector products for gene therapy clinical research. In order to detect replication-competent adenovirus(RCA) and replication-competent retrovirus(RCR) contaminated in vector products more sensitively and rapidly, we have developed a novel detection method, a hybrid method that combines the infectivity assay and real-time quantitative(RT-) PCRInfectivity PCR was established for the detection of RCA. In this method, permissive cells were infected with RCA samples, and amplified RCA were quantified by real-time PCR. The glass-beads-based DNA extraction method was suitable for extracting DNA rapidly from RCA-infected cells. By infectivity PCR, 1 pfu of RCA spiked into 10^9 particles of adenovirus vectors could be detected within 3 days. In contrast, 1O^4 pfu of RCA could be detected by conventional cytopathic effect after 9 days of infection. These results showed that infectiv … More ity PCR was useful for the rapid and sensitive detection of RCA in adenovirus vector products.Infectivity RT-PCR was established for the detection of RCR. In this method, permissive cells were infected with RCR samples, and amplified RCR were quantified by real-time RT-PCR. Polyethyleneimine(PEI)-conjugated magnetic beads were used for concentration of RCR in the culture supernatants. By infectivity RT-PCR, 1 infectious unit(iu) of RCR spiked into 10^6 cfu/ml of retroviral vectors could be detected within 3 days. The sensitivity for viral detection was increased more than 10-fold compared with the conventional S+L- assay. Therefore, infectivity RT-PCR was useful for the rapid and sensitive detection of RCR in retrovirus vector products In addition, RCR could be detected more rapidly when RCR RNA were directly extracted from RCR-infected cells. Moreover, the infection efficiency and the detection sensitivity could be improved when PEI-magnetic beads and magnetic field were used for the infection of RCR. Less