Bertram S, Hufert F T, Neumann-Haefelin D, von Laer D
Abteilung Virologie, Inst. für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Germany.
Biotechniques. 1995 Oct;19(4):616-20.
A highly effective single-cell PCR method using a fluorescence-activated cell sorting (FACS)-based automated cell deposition unit (ACDU) that sorts single cells directly into PCR tubes was developed. To evaluate the sensitivity of this method, single ACH-2 cells (containing one HIV-1 genome per cell) were sorted, and 220 out of 228 samples (96.5%) were HIV DNA-positive by PCR. Furthermore, the number of samples accidentally containing more than one cell was determined by sorting single cells from a mixture of human cytomegalovirus (HCMV)-infected fibroblasts and ACH-2 cells. Multiplex nested PCR (nPCR) was then performed, detecting HCMV and HIV DNA simultaneously. From 66 sorted cells, 2 (3%) were double-positive for HIV and HCMV, 31 (47%) for HCMV alone, 30 (45.5%) for HIV alone and 3 (4.5%) were PCR-negative. The ACDU was then programmed to sort defined numbers of cells into PCR tubes. This is similar to classic dilution assays in that it allows the determination of the percentage of cells that was positive for a specific DNA. The accuracy of multiple cell deposition by the ACDU was evaluated by determining the percentage of HIV-positive cells in defined mixtures of ACH-2 and uninfected H9 cells. Infection rates determined by the ACDU correlated well with the rates expected from the given dilutions.
开发了一种高效的单细胞PCR方法，该方法使用基于荧光激活细胞分选（FACS）的自动细胞沉积单元（ACDU），可将单个细胞直接分选到PCR管中。为了评估该方法的灵敏度，对单个ACH-2细胞（每个细胞含有一个HIV-1基因组）进行了分选，228个样本中有220个（96.5%）通过PCR检测为HIV DNA阳性。此外，通过从人巨细胞病毒（HCMV）感染的成纤维细胞和ACH-2细胞的混合物中分选单个细胞来确定意外含有多个细胞的样本数量。然后进行多重巢式PCR（nPCR），同时检测HCMV和HIV DNA。在66个分选的细胞中，2个（3%）对HIV和HCMV呈双阳性，31个（47%）仅对HCMV呈阳性，30个（45.5%）仅对HIV呈阳性，3个（4.5%）PCR检测为阴性。然后对ACDU进行编程，将确定数量的细胞分选到PCR管中。这类似于经典的稀释试验，因为它可以确定特定DNA呈阳性的细胞百分比。通过确定ACH-2和未感染的H9细胞的特定混合物中HIV阳性细胞的百分比来评估ACDU进行多个细胞沉积的准确性。ACDU确定的感染率与给定稀释度预期的感染率相关性良好。
