Source: IVD Practitioner Network Author: Suo Yan
Molecular diagnostics of genetic nucleic acids is driven by the needs of modern life sciences, utilizing various advanced manufacturing and instrumentation technologies to develop detection instruments. It combines various testing techniques, analytical techniques, and scientific principles from other foundational disciplines to qualitatively and quantitatively observe, compare, analyze, and judge biological entities, trachea, tissues, cells, and genes.
This paragraph is the most comprehensive description and definition of the molecular diagnostics industry. Molecular diagnostics in China began in the 1960s and 1970s, but at that time, due to technological and equipment limitations, it was not widely recognized. Later, with the emergence and widespread application of PCR technology, molecular diagnostic technology began to gradually penetrate the clinical application market from the field of infectious diseases, such as hepatitis, sexually transmitted diseases, and SARS.
Currently, molecular diagnostic technology and products are widely used in clinical settings. Today, I will introduce the main instruments and innovative products related to the molecular diagnostics industry one by one.
Nucleic Acid Extraction Instruments
Nucleic acids are the genetic material of organisms and the cornerstone of molecular diagnostics. Traditional nucleic acid extraction methods generally use inorganic or organic materials to lyse cells, releasing nucleic acids, and then employ various methods to separate them, achieving the goal of nucleic acid extraction, such as thermal lysis, alkaline lysis, ultrasonic lysis, etc.
With the use of materials such as nano-magnetic beads and silica membranes, nucleic acid extraction has entered the era of automation. The silica membrane-based column centrifugation method has become less commonly used due to its cumbersome operation, while magnetic bead-based nucleic acid extraction methods have become mainstream.
From a product design perspective, nucleic acid extraction instruments can be divided into upward suction and downward suction. The former refers to extraction completed by transferring magnetic beads, while the latter refers to extraction completed by transferring liquids.
From a functional perspective, they can be divided into semi-automatic nucleic acid extraction instruments and fully automatic nucleic acid extraction workstations. The former uses pre-loaded plates for extraction, requiring manual sample addition and nucleic acid transfer, with throughput typically in multiples of 12 samples, such as 12, 24, 36, 48, and 96. Popular products include the GeneRotex fully automatic rotary nucleic acid extractor, NP968-S nucleic acid extractor, EX3600, etc. After the COVID-19 pandemic, almost every PCR laboratory is equipped with nucleic acid extraction instruments, and many new products have emerged with improved design and functionality.
Fully Automatic Nucleic Acid Extraction Workstations differ from semi-automatic nucleic acid extraction instruments primarily in that the fully automatic workstations can complete all operations from sample to PCR preparation (sample addition, extraction, system configuration). In the past, manual lid opening was required; now, with the launch of various cup systems, lid opening is performed by machines, making it more intelligent. Popular brands include Tianlong Technology’s 9600E, Shengxiang’s Natch CS, and Zhijiang’s Autrax, while imported products include Roche’s 4800, PE, and Holotest brands.
PCR Instruments (Systems)
After nucleic acid extraction, we enter the nucleic acid testing phase, where the main instrument designed for this phase is the PCR instrument. PCR instruments can be classified into three types based on their functions: qualitative PCR instruments, fluorescent quantitative PCR instruments, and digital PCR instruments.
Qualitative PCR Instruments are the earliest products, primarily functioning to complete nucleic acid amplification based on a set temperature gradient. Their emergence replaced the awkward situation of needing a gradient water bath for early PCR experiments. In simple terms, this is a “temperature control system,” so the market price is relatively low. Examples include Genesy-96T, iCycling 96 gradient PCR instrument, Life Touch gene amplification instrument, etc.
Fluorescent Quantitative PCR Instruments have upgraded components compared to qualitative PCR instruments, adding an optical system. Their amplification system and PCR principles differ, incorporating fluorescent dyes or probes to monitor the entire PCR process in real time through signal changes. By integrating amplification and detection functions, closed-tube reactions significantly reduce contamination risks. Well-known domestic brands include Shanghai Hongshi, Hangzhou Bori, Da’an Gene, Tianlong Technology, Yaruai, and Anjiese, while mainstream foreign brands include ABI, Bio-Rad, Roche, and Thermo Fisher.
Here, I will also share a method to differentiate between qualitative and quantitative PCR instruments in the laboratory: check if it connects to a computer. Generally, qualitative instruments have screens built into the machine, while quantitative ones require a computer connection; this is for reference only.
Digital PCR Instruments are emerging technology products in the PCR instrument field, characterized by absolute quantification and ultra-high sensitivity. They utilize microfluidics and micro-nano manufacturing technology to disperse solutions into individual micro-reaction units, paired with software algorithms to achieve true absolute quantification.
The foreign market mainly features Bio-Rad and Thermo Fisher’s digital PCR as mainstream, while the domestic digital PCR industry is developing rapidly, with companies such as Hangzhou Linghang, Xinyi Biotechnology, Zhenzhun Biotechnology, Shunde Yongnuo, and Xiaohai Turtle continuously launching products.
Many people have previously asked me, do you think digital PCR instruments will replace fluorescent PCR instruments? Personally, I believe it is unlikely. Cost is one aspect, and on the other hand, true clinical applications do not necessarily require such precise detection technology. With the precise advantages of digital PCR technology, its future applications will focus on monitoring transplant rejection free DNA, liquid biopsies, NIPT, and other areas. It’s like using a kitchen knife to kill a chicken and an electric knife to kill a cow; there’s no need to use an electric knife to kill a chicken.
Gene Sequencing Instruments
Gene sequencing is one of the hottest fields in molecular diagnostics, aiming to obtain as many nucleic acid sequences as possible from the tested substances to analyze their potential value. Currently, gene sequencing technology has developed to the fourth generation. The first-generation sequencing technology is the classic Sanger sequencing (dideoxy nucleotide termination sequencing), and commercial products still available include Haier’s respiratory 13 multiplex detection, which is a product combining Sanger sequencing with capillary electrophoresis;
Second-generation sequencing technology is currently the most widely used, characterized by high throughput, with major players in the market being Illumina and BGI, along with many service companies based on further research and development;
Third-generation sequencing technology, also known as single-molecule sequencing technology, addresses the library preparation and PCR amplification steps required before sequencing in the first and second generations. If we look at molecular diagnostics as a whole process, sequencing before the third generation was performed after PCR instrument processing, but the third-generation sequencing technology places sequencing parallel to fluorescent quantitative PCR, significantly lowering the threshold for sequencing technology and making it more universal.
Fourth-generation sequencing technology is, strictly speaking, an upgraded version of third-generation technology, which can be referred to as 3.5 generation technology. However, the leaps it brings are also significant, mainly reflected in controlling the costs of detection equipment through nanopore detection technology, allowing signal capture without relying on high-speed cameras or high-resolution CCD cameras.
Gene chip technology, also known as nucleic acid chips or microarray chips, is currently one of the most widely used types of biochip technology. It involves fixing designed nucleic acid fragments in an orderly manner on a solid-phase support, followed by hybridization with products after PCR amplification, and interpreting results through signal detection.
Compared to fluorescent PCR technology, gene chip technology addresses the issue of target detection throughput, as a single chip can integrate many detection targets. This gives the technology a significant advantage in gene mutation and bacterial resistance detection. The main players now include BGI, Kappa, Yanan, and Bohui Innovation.
Previously, the clinical application of gene chips was primarily conducted in the PCR area, requiring PCR amplification before hybridization. However, automation products from companies like BGI and Bohui Innovation have provided greater application scenarios for gene chips, with automation becoming the main avenue for gene chip technology in the future.
Molecular POCT Instruments
Molecular POCT instruments belong to one of the currently popular subfields, with industry expectations for them to move out of laboratories and into grassroots and clinical departments. However, this path is not easy.If interested, you can look up historical articles from the IVD Practitioner Network for more relevant information.
Current popular technical directions for molecular POCT include microfluidics, micro-droplets, chips, nucleic acid rapid testing, etc. Major brands already on the market include Yousida, BGI Crystal Core, Aoran, Baikangxin, and Wanfu Beite, with successful domestic products such as Cepheid’s GeneXpert system and Merieux’s Flimarray system.
Nucleic Acid Mass Spectrometry Instruments
After the rise of molecular diagnostics, many sub-technologies and fields have also become popular, with nucleic acid mass spectrometry being one of them. The principle of mass spectrometry is to ionize sample molecules and apply force in an electric field to separate them, subsequently obtaining mass spectrometry graphs.
Nucleic acid mass spectrometry primarily employs ESI and MALDI ionization techniques, both of which are soft ionization methods. Currently available nucleic acid mass spectrometry instruments include Antu’s Autof ms1000, Clin-TOF II clinical time-of-flight mass spectrometry system, and Zhongyuan Huiji mass spectrometer EXS3000, among others.
This is aoften-overlooked subfield of molecular diagnostics, as the products are mainly used in pathology departments, which are separated from the main battlefield of testing departments, resulting in lower attention and fewer market players. Compared to PCR and NGS, FISH technology has unique advantages in chromosome number, structural abnormalities, gene deletions, rearrangements, and gene fusions.
FISH testing is also the gold standard for detecting the Her-2 gene in breast cancer and the ALK gene break in lung cancer. The applicable instruments mainly include fluorescence microscopes.
Reference Materials: 2018 Volume “Blue Book on the Development of China’s In Vitro Diagnostic Industry,” some representative personal views of the author Suo Yan, welcome criticism and correction! Please indicate the source when reprinting!
