Targeted sequencing: Opening the "bronze gate" of ultra-multiplex PCR

Since the beginning of this year, targeted next-generation sequencing (tNGS) has become quite popular in clinical practice. It combines multiplex PCR amplification with sequencing technology, allowing the detection of dozens to hundreds of known pathogenic microorganisms and their virulence and/or drug-resistant genes in tested samples. Compared with metagenomic sequencing (mNGS), tNGS has the advantages of a clear pathogen spectrum and low sequencing costs for the detection of low-concentration pathogenic microorganisms, particularly their virulence and/or drug-resistant genes. Therefore, it has received widespread attention in clinical testing.

In June of this year, the National Institute for Viral Disease Control and Prevention of China CDC issued a notice on carrying out a joint research project on the quality evaluation of the tNGS multiple nucleic acid detection technology for pathogenic targets, in preparation for a pre-study of the quality evaluation of tNGS multiple nucleic acid detection technology for pathogenic targets.

The ultimate technique of ultra-multiplex PCR

From a technical perspective, although targeted sequencing is called sequencing, it actually belongs to the category of ultra-multiplex PCR. Sequencers are part of the result analysis and detection function in the entire technical path. To achieve detection of ultra-multiplex targets, there are mainly the following paths:

1. PCR amplification + sequencer or chip After the laboratory obtains the sample, full nucleic acid extraction is performed, and then multiple probes and primers are used to amplify the target genes in the nucleic acid. Then, the amplified nucleic acid is subjected to final result analysis through gene sequencers or chip hybridization equipment. Since the amplification is targeted at specific target points, the pathogen can be recognized well during sequencing or chip detection, avoiding the dilemma of human gene interference in mNGS, hence the name targeted sequencing.

1. Ultra-multiplex PCR technology This is a branch derived from fluorescence PCR technology and is currently a very popular field. It can identify ultra-multiplex target points through multi-color fluorescence combined with melting curve analysis. In this field, Xiamen ZhiShan is currently a leading enterprise. Another method is to separate reaction regions through pores and achieve ultra-multiplex target detection. For example, Tengfei Gene, which was recently approved, uses this method.

Commonly used targeted sequencing detection products on the market

1. Bingyuan Diagnostics - GenSeizerTM ultra-multiplex PCR technology The GenSeizerTM ultra-multiplex PCR technology independently developed by Bingyuan Diagnostics has been used by many domestic and foreign institutions for the development of research or clinical diagnostic products. It can complete more than 10,000 PCR reactions in a single tube in 3.5 hours. According to information on its official website, it currently offers four products that can detect 100, 200, 400, and one Pro product.

2. Genecast Medical - ultra-multiplex rapid PCR technology The targeted high-throughput sequencing (tNGS) technology platform jointly developed by Genecast Medical and Jin Qi Rui uses the KM MiniSeqDx-CN system. It combines the advantages of PCR and NGS, uses ultra-multiplex PCR forward enrichment to target pathogens, and eliminates host nucleic acid interference, effectively improving detection sensitivity. The platform sequencing time is 5 hours, which is relatively fast. Genecast relies on its own chain of testing institutions. Once the product is launched, it has been widely used in various places. Currently, the main products are used in the diagnosis of respiratory pathogens, hepatitis B virus, and tuberculosis.

Overall, the mainstream approach for implementing ultra-multiplex target detection through multiple PCR amplification and gene sequencing is due to the maturity of both technologies. The difficulties lie in the probe primer design and sequencing data analysis in the tNGS application. Compared to the multi-color PCR and melting curve technology platform, although it has the advantages of lower detection costs, easier operation, and shorter detection time, it will encounter significant challenges in the process of gradual implementation based on the theoretical feasibility, such as competitive inhibition, signal collection, and identification.

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Compared with traditional gene sequencing, targeted sequencing can significantly improve the detection of RNA viruses, fungi, and intracellular bacteria by designing specific primers, with less interference from human genes and lower detection costs. However, the disadvantage is that the operation is still complicated, and it cannot identify new pathogens. Therefore, it is relatively limited in responding to some virus tracing and difficult sample exclusion, but this does not affect its clinical application.

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According to research data from the IVD industry website, the fees for targeted gene testing products on the market are mostly concentrated between 120-250 USD. This pricing actually seizes the market positioning of multi-respiratory tract detection. For upper-level targeted sequencing products, it will cut off a portion of the market for metagenomics and divert some patients who do not need metagenomics to small panel targeted sequencing. For lower-level targeted sequencing, it will cut off a portion of the multi-respiratory tract detection market and bring in respiratory tract projects with fees above 120USD.

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On the other hand, with the pilot of Shanghai LDT, the LDT model will be the only way for hospital laboratories to solve the problem of unlicensed products in the future. A good project will ultimately be brought back to the laboratory, so how to position future products and how to use the time difference is particularly important in this trend.