Breakthrough! Highly multiplex fluorescence PCR technology by Chinese team reshapes the technology development road!

Real-time PCR

 

Real-time PCR is the most widely used nucleic acid detection technology. The detection mode uses closed-tube detection, which greatly reduces the chance of amplification product contamination compared to open-tube detection, saves time and manpower, facilitates automation, and supports quantitative detection using the threshold cycle number (Cq value).

However, a major limitation of RT- PCR is the limited number of target genes that can be detected in a single reaction. Currently, based on the number of detection channels of mainstream fluorescent PCR instruments, it is difficult to detect more than 4-6 target genes in a single reaction, limiting the application of this technique to complex diseases involving multiple targets.

The addition of a melting curve analysis step after the PCR reaction can increase the number of detectable target genes to some extent, however, the increase in fluorescent probe type is accompanied by an increase in fluorescence background and detection cost, especially, any nucleic acid variation in the probe binding region may cause a melting point shift and lead to misclassification of results, making the method still limited in terms of the number of targets.

 

New Fluorescent PCR Technology -”MeltArray”

 

In a study published online in the Proceedings of the National Academy of Sciences (PNAS) on February 24, 2022, Qingge Li’s team at the College of Life Sciences, Xiamen University, reported a new fluorescent PCR technique called “MeltArray”, which improves the single-tube detection capability of fluorescent PCR by more than an order of magnitude and demonstrates the powerful and flexible detection capability of the technique through several clinical application scenarios.

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“MeltArray” cleverly utilizes the 5′-flap endonuclease activity of TaqDNA polymerase, which cleaves the “media probe” located downstream of the primer to generate the “media primer” in the PCR reaction. The “mediator primer” then binds to the molecular beacon reporter probe and, under the action of Taqase polymerization activity, extends along the molecular beacon to create a fluorescent double chain with a specific melting point, which ultimately gives the target specifically recognized by the mediator probe a “two-dimensional” marker including fluorescence type and melting point value. ” label.

Since each molecular beacon allows multiple vector primers to form a series of fluorescent duplexes with different melting points, the total number of target genes that can be detected by a single MeltArray multiplex PCR reaction is equal to the number of molecular beacons in the reaction multiplied by the number of vector primers it holds. The authors show in the paper that a single fluorescent channel can detect 12 targets, and an instrument with 6 fluorescent channels can detect up to 72 targets! This is the maximum number of target genes that can be detected by a single closed-tube PCR one-step method to date.

  “Multi-berth” reporter probes

 

The key to implementing this technology is to obtain a reporter probe with “multiple-berth” to accommodate many mediator primers. The researchers first compared the advantages and disadvantages of using linear probes and molecular beacons as separate reporter probes, with molecular beacons winning out due to their clear context. Next, the authors examined the “multi-berth” capability of molecular beacons. Carefully starting with a two-plex PCR, they first demonstrated that a molecular beacon could allow two “mediator primers” to be moored, and then with a four-plex PCR, they demonstrated that a molecular beacon can allow four “mediator primers” to be moored in a stack, thus The hypothesis that a molecular beacon can accommodate any number of mediator primers was proposed. Based on the melting point resolution of current fluorescent PCR instruments, it was verified that a single fluorescent channel could detect 12 target genes using two molecular beacons. Finally, an array structure of “mediator” libraries and their corresponding molecular beacon reporter probes was constructed to standardize the detection.

To reduce the interference of primer dimerization that may arise from the coexistence of multiple primer pairs in multiplex PCR, the researchers also introduced the concept of “PCR suppression”, thus presenting the complete technical principle of MeltArray (Figure 1).

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Fig 1 Technical principle of MeltArray

Multi- detection capability of “MeltArray”

 

To verify the Multi- detection capability of MeltArray, the authors first designed a 20-plex PCR MeltArray assay system covering 18 sequence-tagged loci and 2 reference genes (SRY and ZFX/Y genes) in the human Y chromosome azoosperm factor region (AZFa, AZFb, AZFc and AZFd). In normal male samples, all 20 loci are present, i.e. all have melting peaks, while patient males show the absence of 1 or more melting peaks – this experiment is a typical example of simultaneous appearance of multiple targets (Figure 2).

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Fig 2 MeltArray technology applied to the detection of 20-fold human Y chromosome microdeletion

The showed that this MeltArray system achieved simultaneous detection of 20 targets using only three fluorescence channels. To assess the effect of template volume on MeltArray detection capacity, the authors examined genomic DNA from 100 ng to 100 pg of healthy males and females, and all templates were stably detected in this range. Melt peak heights decreased as the amount of DNA template was gradually reduced, but the melting point values of all 20 peaks remained unchanged. Finally, the authors confirmed the accuracy of the MeltArray system by double-blind testing of 757 samples.

PCR MeltArray detection system

 

Encouraged by the above results, the authors set out to challenge a larger number of targets.  They designed a 62-plex PCR MeltArray assay system using six fluorescent channels (Figure 3) to identify 61 O antigen synthesis genes and one E. coli housekeeping gene yccT. When we look at the number of detections per channel at this point, the number of targets detected by the six fluorescent channels, in descending order of fluorescence wavelength, are 11, 9, 12, 12 , 8 and 10! Repeated experimental results for each channel are shown using a 3-fold standard deviation, and the melting point values corresponding to each target are stable and clearly distinguishable. Finally, the authors identified 167 E. coli cultures serotyped using this system, and except for 4 strains outside the coverage area, the assay results were in full agreement with the whole genome sequencing results, and 11 more strains were identified than the traditional antiserum method, and additionally corrected the 1 strain it misidentified.

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Fig3 MeltArray technology for serotyping 61 E. coli O

Quantitative detection by MeltArray

 

Both of the above examples are qualitative assays, can MeltArray be additionally quantified? To answer this question, the team designed a 24-plex PCR assay system for respiratory pathogens (Figure 4), in which they set 19 of the non-deterministic bacteria/viruses and one internal control gene in melt analysis mode, i.e., qualitative detection, and the other four bacteria in real-time PCR detection mode – i.e., TaqMan probe mode, i.e. quantitative detection.

It is important to note that chose the denaturing phase for real-time fluorescence detection. They believe that the real-time detection is for the fluorescence signal accumulated after probe digestion, and the detection results should be consistent regardless of the denaturation or extension phase. During the denaturation phase, the fluorescence generated by any reporter probe would converge without affecting the real-time detection results.

This assumption of the authors was proved. The system successfully achieved quantitative detection of four target genes along with qualitative detection of 19 target genes. the analytical sensitivity of the MeltArray assay system for 24-plex PCR reached 10copies/μl. The reliability of the assay results was confirmed by validation on 67 alveolar lavage fluid samples from patients with pneumonia or respiratory

tract infection.

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Fig 4 Real-time PCR and melting curve analysis applied simultaneously for qualitative and quantitative detection of respiratory pathogens

 

Mutation analysis by MeltArray

 

To examine whether MeltArray can be used for mutation analysis, the team designed a mutation identification system called “microsequencing”, in which nine types of mutations occurring at codon 12 and codon 13 of the KRAS gene are identified in a single reaction tube (Figure 5).

Here, the authors took advantage of the specificity of probe hybridization and used 10 mediator probes to label each of the nine mutation types and the wild type in two dimensions. The system was shown to have excellent mutation detection specificity, tolerating up to 500 ng of wild-type genomic DNA and detecting mutant allele frequencies of 5%-10%, superior to Sanger sequencing-which has a minimum mutant allele frequency between 10%-20% and a detection limit as low as 30 copies per reaction.

Finally, they used the system to detect KRAS mutation types in 167 colorectal cancer tissue samples, obtaining a mutation frequency distribution consistent with the Cosmic database and also demonstrating that MeltArray has more sensitive mutation detection capabilities than Sanger sequencing.

图片7Fig 5 MeltArray technology for microsequencing of KRAS mutations

 MeltArray drives new developments in fluorescent PCR technology

The above four application scenarios of MeltArray actually correspond to the three major application areas of molecular diagnostics, i.e., genetic diseases, infectious diseases (which involve pathogen identification and qualitative and quantitative detection of pathogens respectively) and molecular diagnosis of tumors, showing the typical characteristics of MeltArray as a universal nucleic acid detection technology. Currently, the detection of similar target numbers requires “open tube detection” technologies, such as microarrays, mass spectrometry, microspheres, electrophoresis, etc. However, these technologies generally have expensive equipment, complex operation, long turnaround time and other shortcomings, and the localization rate is low, in the domestic and international market is not hot, long-term slow progress. In contrast, MeltArray uses widely available and localized fluorescence PCR instruments, which have many advantages such as cost-effective, easy operation, high automation and short turnaround time, etc. Combined with the powerful and flexible technical advantages shown in this paper, It not only successfully promotes the development of fluorescence PCR technology, but also perfectly fills the technical gap that has long existed between low-order PCR and high-throughput assays!

—— Forwarded from IVDworker

 


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