聚合酶链反应芯片同时定性定量鉴定多个靶点
Simultaneous Qualitative And Quantitative Identification Of Multiple Targets By Polymerase Chain Reaction On Microarray
摘要：
提出了一种基于微阵列的多重定性和定量PCR分析方法。采用固定化正向引物和单一通用反向引物的寡核苷酸凝胶微阵列，研究了几种DNA靶点的PCR反应特性。研究了一阶段多重芯片上PCR用于同时扩增单纯疱疹病毒1型和2型、巨细胞病毒DNA和作为内部对照的噬菌体λ DNA。此外，在两阶段版本的检测中对增加数量的靶标（添加沙眼衣原体、人型支原体和解脲脲原体DNA）进行联合分析：第一阶段是使用靶标特异性引物的管内PCR，而反向引物包含5 '-接头区域;第二阶段是使用固定化靶标特异性正向引物和接头作为溶液中的常见反向引物的芯片上扩增。利用溶液中常见的含聚T的引物，进一步证明了一步反应用于人cDNA分析的可能应用。SYBR green I和Cy-5标记的dUTP用于特异性PCR产物的实时和终点检测。一阶段和两阶段反应的效率被证明是强烈依赖于镁和引物浓度。用噬菌体λ DNA的10倍系列稀释液研究了两种版本中的定量PCR。对于两种版本的检测试剂盒，该方法能够检测每个反应的6个DNA拷贝。一级反应的定量区间涵盖了8个浓度级。凝胶垫大小对微阵列PCR有效性的显着影响进行了讨论。
英文摘要：
An approach for multiplex qualitative and quantitative microarray-based PCR analysis has been proposed. The characteristics of PCR executed on a gel-based oligonucleotide microarray with immobilized forward primers and a single common reverse primer in solution were investigated for several DNA targets. One-stage multiplex on-chip PCR was studied for simultaneous amplification of herpes simplex viruses types 1 and 2, cytomegalovirus DNA, and bacteriophage lambda DNA as an internal control. Additionally the joint analysis of increased number of targets (with addition of Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum DNA) was done in two-stage version of assay: first stage was in-tube PCR with target-specific primers, while the reverse ones contained 5'-adapter region; the second stage was on-chip amplification with immobilized target-specific forward primers and adapter as common reverse primer in solution. The possible application of one-stage reaction for human cDNA analysis was additionally demonstrated with utilization of a common poly-T-containing primer in solution. SYBR green I; and Cy-5 labeled dUTP were used for real-time and end-point detection of specific PCR products. The efficiencies of both one-stage and two-stage reactions was shown to be strongly dependent on magnesium and primers concentrations. Quantitative PCR in the both versions was studied with 10-fold serial dilutions of phage lambda DNA. The method enabled detection of 6 DNA copies per reaction for both versions of assay. The quantitative interval for one-stage reaction covered eight orders of concentration. The revealed significant effect of gel pad size on microarray PCR effectiveness has been discussed.
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