Multiplex Polymerase Chain Reaction (MPCR)
Multiplex Polymerase Chain Reaction (MPCR) refers to a technology that amplifies multiple targets simultaneously in a single PCR reaction and then employs specific detection methods to analyze the amplification products, thereby enabling the diagnosis of several targets at once. MPCR is the subject of in-depth research due to its high efficiency, high throughput, and low cost.

At present, MPCR technology has been widely applied in fields such as scientific research and disease diagnosis. This text provides an overview of the development and application of MPCR technology from the perspectives of MPCR amplification and detection. It discusses the advantages and current issues of MPCR technology and suggests that segmenting the reaction system into small droplets or integrating it with Capillary Convective PCR (CCPCR) technology holds the promise of improving the amplification efficiency on solid-phase carriers, thus developing an MPCR technology with high amplification efficiency, good consistency, system stability, and high detection multiplicity.

I. Principles of Multiplex PCR Technology

Multiplex PCR technology involves adding multiple pairs of primers into a single PCR reaction system to simultaneously amplify several target sequences from the same or different DNAs. The different lengths of these target sequences allow the amplification products to be effectively distinguished by electrophoretic analysis. Multiplex PCR was established by Chamberlain et al. in 1988. Although the principle is the same as conventional PCR, the reaction system and conditions must be optimized to suit the different primer pairs and their target sequences.

II. Multiplex Fluorescence Quantitative PCR Technology

Multiplex Fluorescence Quantitative PCR builds upon conventional fluorescence quantitative PCR by utilizing combinations of several different fluorescent groups, alongside the instrument’s ability to detect fluorescence across multiple channels, to achieve real-time quantitative detection of multiple targets.

TaqMan hydrolysis probes are the most commonly used probes in multiplex fluorescence PCR systems. In these probes, one end is labeled with a fluorescent reporter group while the other end is labeled with a quencher group. By labeling different sequence ends with varying fluorescent groups and their corresponding quencher groups, different TaqMan hydrolysis probes can be created. When these probes and their corresponding amplification primers are added into the same reaction system, simultaneous detection of multiple targets becomes feasible.

III. Multiplex PCR Technology Based on Fluorescent Dyes

Non-specific fluorescent dyes, when intercalated into the minor groove of double-stranded DNA and excited by specific wavelengths of light, emit fluorescence at particular wavelengths.

Based on this, the melting curve analysis method was developed. This method takes advantage of the characteristic that different DNA sequences have different melting temperatures (Tm). After the PCR reaction is completed, the temperature is gradually increased so that the double-stranded DNA gradually denatures into single strands. When the temperature corresponding to a particular Tm value for a double-stranded DNA is reached, the fluorescence intensity sharply decreases. This principle can be utilized to analyze double-stranded products of varying lengths or double-stranded products of the same length but with different GC contents.

Using multiplex real-time PCR melting curve analysis enables virus identification.

Specific primer pairs are designed for different virus types to amplify the template and generate distinct amplicons. These amplicons have different melting temperatures (Tm), allowing identification based on the position of the peaks in the melting curve.

High-Resolution Melting (HRM) analysis, which evolved from conventional melting curve analysis, is a new technique that uses saturated fluorescent dyes and high-resolution instruments to detect differences in the melting temperatures of single nucleotides, producing distinct melting curve patterns for genetic analysis. This method offers extremely high sensitivity and can detect single-base differences. Currently, it is mainly applied in areas such as single nucleotide polymorphism (SNP) analysis, methylation analysis, gene mutation detection, and genotyping.

IV. Applications of Multiplex PCR Technology

Multiplex PCR (MPCR) technology has significant application value across various fields, including genetic research, clinical diagnostics, and forensic science. Below are several representative examples:

MPCR can be used to detect pathogens and illegal additives in food products.

Q1600 Series Real-time Fluorescence Quantitative PCR Instrument

Q3200 Series Real-time Fluorescence Quantitative PCR Instrument