Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel

Abstract

Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number ( cn ) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals.

An Overview of Laboratory Diagnosis of Central Nervous System Viral Infections

“…112 Same goes to intercalating dye-based multiplex that require primers for each virus and having different melting temperatures. 113 Many laboratories apply singleplex real-time PCR for detection of RNA and DNA viruses. Diagnosis of cytomegalovirus (CMV) using qPCR in saliva sample had resulted with 100% sensitivity and 99.9% specificity as compared to rapid culture technique.…”

“…96,120 Thus, it is critical during designing the probes and primers which must be specifically able to detect specific virus only, otherwise interactions between them will affect the efficiency of multiplex PCR assay. 113 A research made to assess performance of duplex RT-qPCR for detection of DENV and ZIKV showed a great analytical sensitivity as the limit of detection was similar when tested using singleplex assay. Plus, it also produced high specificity as no signals were measured from Flaviviridae family such as WNV, JEV, HCV, YFV and CHIKV.…”

“…122 Moreover, when the number of viruses involved is higher, it will increase the number of detectable colours and making the system complex and high cost. 113 Furthermore, issue related to multiplex is associated with competition of primers to bind with target sequence, also competition between multiple targets towards shared reagents. 120 Thus, development of multiplex assay requires optimisation of reactions such as by adjusting the concentration of reagents, primers and annealing temperature set up so that each target sequence can be amplified with a similar manner without amplifying the nonspecific product.…”

An image-to-answer algorithm for fully automated digital PCR image processing

“…A compartmentalized sample forms a binary system of thousands or millions of subsamples, each containing a single copy or no copy of the target DNA. 5 This technique allows absolute quantification of selected target DNA sequences and, most importantly, performs multiplex PCR with minimal interaction between different targets, 6 distinguishing rare DNA targets from abundant ones. 7 The compartmentalization of the original sample is achieved either by filling predefined partitions forming a chip-based dPCR (cdPCR) 8 or by forming individual droplets as microreactors of a droplet-based dPCR (ddPCR).…”

Similar color analysis based on deep learning (SCAD) for multiplex digital PCR via a single fluorescent channel

“…The existing qPCR has achieved multiplex detection in a single fluorescent channel by collecting and analyzing the signals of the whole amplification procedure, e.g., amplification curves and melting curves, to classify different targets. [11][12][13][14][15] For dPCR, the multiplex detection in a single fluorescent channel can also apply a similar principle. 16 However, such a strategy needs to take images or measure fluorescence intensities after each amplification cycle to obtain a real-time signal, which requires sophisticated readout equipment and a complex analysis algorithm, limiting its widespread application and translation to commercial instruments.…”