Multicolor Combinatorial Probe Coding for Real-Time PCR

The target volume of multiplex real-time PCR assays is limited by the number of fluorescent dyes available and the number of fluorescence acquisition channels present in the PCR instrument. We hereby explored a probe labeling strategy that significantly increased the target volume of real-time PCR detection in one reaction. The labeling paradigm, termed “Multicolor Combinatorial Probe Coding” (MCPC), uses a limited number (n) of differently colored fluorophores in various combinations to label each probe, enabling one of 2n-1 genetic targets to be detected in one reaction. The proof-of-principle of MCPC was validated by identification of one of each possible 15 human papillomavirus types, which is the maximum target number theoretically detectable by MCPC with a 4-color channel instrument, in one reaction. MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction. This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR. MCPC was further explored in detecting combined genotypes of five β-globin gene mutations where multiple targets were co-amplified. MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

多重实时聚合酶链反应（PCR）检测的可同时检测靶标数量，受限于可用荧光染料的种类数量以及PCR仪所配备的荧光采集通道数目。本研究探索了一种探针标记策略，可显著提升单反应体系中实时PCR检测的可同时检测靶标数量。该标记范式被命名为多色组合探针编码（Multicolor Combinatorial Probe Coding, MCPC），其通过有限数量（n）的不同颜色荧光基团的多种组合对每条探针进行标记，从而可在单反应体系中实现2ⁿ−1种遗传靶标中的任意一种的检测。通过在单反应体系中检测15种人乳头瘤病毒分型中的任意一种，验证了MCPC的原理可行性——这也是搭载4色通道的PCR仪通过MCPC理论上可实现的最大靶标检测数量。随后，本研究通过同源标签辅助非二聚体（Homo-Tag Assisted Non-Dimer, HAND）系统，将MCPC从单引物对体系升级至多引物对体系，使单反应体系可容纳多组引物对。通过在单反应体系中加入10组引物对，实现对10种食源性致病菌候选株中的任意一种的鉴定，该改进得到了验证，其检测限与单重PCR相当。研究进一步将MCPC应用于5种β-珠蛋白基因突变的联合基因型检测，实现多靶标的共扩增。MCPC策略可拓展实时PCR检测的应用边界，覆盖现有标记策略无法实现的应用场景。