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Main Techniques of Molecular Diagnosis in China

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(I) domestic distinctive technologies

one-step PCR detection technology

"One-step PCR detection technology" is a domestic technology with independent intellectual property rights and is unique in the world. It uses rapid nucleic acid release technology to eliminate heating, centrifugation, pipetting, tube change and other operations, and sample processing time Compared with similar products, it is greatly shortened. Sample processing and PCR amplification can be completed in a PCR reaction tube, and high-sensitivity detection can be achieved with only a small sample, which not only has low detection cost, but also greatly reduces the requirements for operators, and is easy to popularize the application of molecular diagnosis. It is conducive to automated large-scale sample processing, and is suitable for large-scale diseases such as HBV and HPV screening. At the same time, one-step technology can provide the simplest and fastest nucleic acid detection scheme for the instant detection platform (POCT) of molecular diagnosis, greatly simplify the detection device and process, and ensure the mobility and timeliness of the instant detection platform for molecular diagnosis. Real-time detection of critical patients and prevention and control of major epidemics have unique advantages.

Magnetic Bead PCR Detection Technology with Magnetic Bead Amplification

Compared with the traditional boiling method, column formulation and other nucleic acid extraction technologies, the internationally leading fifth-generation magnetic bead method nucleic acid extraction technology independently developed in China, which enriches the nucleic acid molecules in the sample through super-paramisane magnetic beads. The pathogen is lysed at room temperature without heating, boiling and centrifugation. The magnetic beads do not need to elute nucleic acid. The magnetic beads can be used for PCR amplification with magnetic beads to simplify the operation steps, high sensitivity, wide linear range, good repeatability, strong anti-interference ability, can realize automatic PCR quantitative detection. In terms of hepatitis B detection, the sensitivity can reach the internationally leading 5-10 IU/mL, which meets the needs of clinical early diagnosis, virological response, drug resistance monitoring, efficacy observation and prognosis evaluation of viral hepatitis infection.

Integrated Automation and Microfluidic Technology

in vitro diagnosis are robotic arm and microfluidic technology. These two types of instruments have their own characteristics and advantages and are active in different application scenarios. In vitro diagnostic instruments based on microfluidic technology are often fully automated and integrated, and the operation is relatively simple. However, the cost of individual sample detection may be higher than that of a mechanical arm type instrument. Domestic products that integrate fully automated robotic arm and microfluidic chip technology for molecular diagnosis have been developed. The precise pipetting operation of the robotic arm and the detection chip prepared by microfluidic technology can realize fully automated nucleic acid extraction, The whole process of amplification and hybridization detection realizes the sample-to-answer detection mode, and at the same time realizes multiple detection of samples. Based on this model, detection of multiple types of pathogens, such as HPV typing, can be completed.

Super-ARMs PCR technology

mutation amplification blocking system (amplification refractory mutation system,ARMS) is one of the most important and widely used technologies for individualized molecular detection of tumors in the world. Its advantages in clinical application have been widely recognized by experts in the industry. "Super-ARMS" independently developed by Xiamen Ed is an upgraded version of ARMS technology, which retains the characteristics of ARMS technology, such as simplicity, rapidity, good specificity, and easy popularization, while further improving the detection sensitivity (sensitivity up to 0.2%), used for clinical detection of EGFR gene mutation status in blood ctDNA of patients with advanced non-small cell lung cancer (NSCLC), and screening suitable for patients receiving first-to third-generation EGFR targeted drug therapy.

(II) Automated Nucleic Acid Extraction Technology

nucleic acid extraction instrument is an instrument that uses matching nucleic acid extraction reagents to automatically complete the extraction of sample nucleic acids. The target users are usually molecular diagnosis and research laboratories interested in standardization, quality improvement and automation, involving nucleic acid extraction experiments. Small, medium and large laboratories. Roche was the first to put the full-automatic nucleic acid extraction instrument into the market. In recent years, manufacturers developing nucleic acid extraction instruments have sprung up. Domestic manufacturers mainly include Daan, Shengxiang, Tianlong, Zhijiang and Ampley, while foreign manufacturers include Roche, Abbott, Cage, Perkin Elmer, etc. In 2017, various nucleic acid extraction instruments on the market have been greatly applied and promoted.

According to the function of the nucleic acid extraction instrument, the nucleic acid extraction instrument is divided into two categories: one is a large-scale fully automated nucleic acid extraction instrument, generally called an automatic liquid workstation, and all sample reagents are added to the instrument for automatic processing; the other is A small automatic nucleic acid extraction instrument, also known as a semi-automatic nucleic acid extraction instrument, uses packaged supporting reagents to manually add samples, and then place them in the instrument to automatically complete the extraction and purification process. The automatic liquid workstation is a device with relatively comprehensive functions. The liquid separation and liquid absorption are automatically completed. Some can even integrate the functions of amplification and detection to achieve the results of sample entry and exit. The sample size extracted at one time is very large, and the flux of general extraction ranges from 96 to several hundred specimens. The small automatic instrument achieves the purpose of automatic nucleic acid extraction through the special design of the operating structure. The equipment and operating cost are low, the operation is simple and convenient, and 24-48 samples are extracted at a time. At present, there are more than ten types of clinical laboratory medical test samples, such as serum, plasma, secretions, exfoliated cells, throat swabs, anal swabs, urine, feces, sputum, whole blood, tissues, paraffin sections, etc. Different analysis systems and reagents have inconsistent nucleic acid extraction operations, resulting in cumbersome clinical operations, prone to operating errors, and affecting the accuracy of clinical reports. Some domestic manufacturers have taken the lead in developing a unified extraction reagent, which can extract multiple sample types at the same time on its automatic liquid workstation, simplifying the operation of nucleic acid extraction, and opening up broad prospects for downstream applications.

(III) digital PCR

the end of the 20th century, Vogelstein and others proposed the concept of digital PCR(digital PCR,dPCR), that is, by dividing a sample into a large number of equal parts and then distributing it to different independent reactions, each independent reaction unit contains at least one copy of the target molecule (Nucleic acid template), PCR amplification of the target template molecule in each independent reaction, and then statistical analysis of the fluorescence signal of each reaction unit.

The current digital PCR mainly uses microfluidic chip or droplet method to disperse the nucleic acid solution into the microreactor or droplet reaction unit of the chip, and the number of nucleic acid templates for each reaction is less than or equal to 1. After PCR, reactions containing nucleic acid molecular templates will produce fluorescent signals, and reactions without templates will have no fluorescent signals. According to the relative ratio and the volume of the reaction unit, the nucleic acid concentration of the original solution can be calculated. At present, digital PCR is mainly used in scientific research fields such as rare mutation detection of cancer markers, detection of pathogenic microorganisms, gene expression analysis, and copy number variation analysis. It can also be seamlessly connected with high-throughput sequencing to verify sequencing results.

On April 21, 2017, the State Food and Drug Administration approved the chip-based digital PCR analysis system of Nanjing Covies Company to enter the list of new batch of innovative medical device special approval applications ("Green Approval Channel"). This product is expected to be the first to be certified by the State Food and Drug Administration and become the first clinical-grade testing product based on digital PCR in China.

(IV) Domestic Sequencing Technology

2017, domestic manufacturers introduced several domestic sequencers such as GenoCare, MGISEQ-200 and MGISEQ-2000. In January 2017, the State Food and Drug Administration approved the change in the adaptation range of the DA8600 high-throughput gene sequencer, from the original "only used for fetal chromosome 21 trisomy, 18 trisomy, and 13 aneuploidy detection" was expanded to "used for fetal chromosome 21 trisomy, 18 trisomy, and 13 trisomy aneuploidy detection, human gene locus detection". In March 2017, the State Food and Drug Administration approved the registration of medical device products for gene sequencer NextSeq 550AR and fetal chromosome aneuploidy (T21, T18, T13) detection kits (reversible end termination sequencing). In December 2017, the BGISEQ-50 gene sequencer obtained the national medical device registration certificate.

In November 2017, the team of Professor Huang Yanyi of Peking University developed a new technology called ECC(Error-Correction Code) sequencing, which was published in Nature Biotechnology. This technology is a high-accuracy fluorescent DNA sequencing method based on information theory to correct errors, which will further greatly improve the accuracy of high-throughput sequencers. Through the test of laboratory prototype, the sequencing reading length can reach 250bp, of which the accuracy rate of the first 200 bases can reach 100%. This new sequencer creatively combines "fluorescence generation" and "error correction coding" technology. The sequencing results will have the remarkable characteristics of more accurate, more efficient and more economical, and will bring fresh blood to the manufacture and market application of domestic high-end medical equipment.

In 2017, the universal sequencer and its supporting reagents, NIFTY detection kit and nucleic acid extraction kit of the domestic BGISEQ-500 platform were all certified by CE Mark of EU medical devices. This means that the high-throughput sequencers and their products made by China Smart meet the regulatory requirements of the European in vitro diagnostic device-related directives and are expected to be more promoted.


According to the space occupied by normal microbiota, human micro-ecosystems are divided into the following categories, namely, human oral micro-ecosystems, gastrointestinal micro-ecosystems, urinary tract micro-ecosystems, reproductive tract micro-ecosystems, skin micro-ecosystems, and respiratory tract micro-ecosystems. Among them, the research on the correlation between intestinal microecology and diseases has become a hot research topic in 2017. It has been reported that intestinal bacterial diversity has a high correlation with Crohn's disease, diabetes, cardiovascular and cerebrovascular diseases, colorectal cancer and other diseases. For example, the content of Fusobacterium nucleatum can be specifically detected in the intestines of colorectal cancer patients. At the same time, there are also research reports showing that the composition of intestinal bacterial flora is related to the treatment of cancer and the effect of chemotherapy, which provides a new idea for the individualized treatment of cancer. However, the clinical research of intestinal microecology is still in the scientific research stage, and more data support is still needed to clarify the significance of clinical diagnosis.

On June 20, 2017, Nature Medicine published an article on the discovery of an intestinal microorganism that inhibits obesity-Bacteroides polymorpha. The team of Academician Ning Guang and Professor Wang Weiqing of Ruijin Hospital of Shanghai Jiaotong University took Han young people in China as the research object. The study found that the content of glutamic acid in obese people is very high, which is significantly different from that of slim people, and the content of glutamic acid in obese people is inversely proportional to the number of Bacteroides polymorpha. Through animal experiments on mice transplanted with Bacteroides polymorpha and analysis of the intestinal microbes of 23 volunteers who underwent sleeve gastrectomy, it is further verified that it is feasible to treat obesity through intestinal microbes.

(VI) Non-invasive Prenatal Single Gene Disease Detection Technology

With the development and promotion of high-throughput sequencing technology, non-invasive prenatal gene detection technology (NIPT) has been widely used in clinical practice, providing an accurate and convenient solution for the screening of down syndrome and other chromosomal mutations. However, NIPT technology only screens for chromosome-level mutation detection, and does not involve another major category of factors that cause birth defects --- single-gene genetic diseases. There are many types of single-gene diseases, and the comprehensive incidence is high, which causes serious damage to human health (Death, disability or teratogenicity), and lack of effective diagnosis and treatment methods, once it occurs, it will cause a heavy burden on society and families.

In order to solve the technical defects in the detection of non-invasive prenatal monogenic diseases, Professor Lu Yuming of the Chinese University of Hong Kong and the Genomics of 10X, a new sequencing company in the United States, jointly applied 10X Genomics Linked Read technology to type the genomes of both parents and distinguish the effective single nucleotide polymorphism (SNP) near the mutation site of the single gene disease, and then sequence the mother's peripheral blood DNA to infer whether the fetus has related genetic mutations. This technology can carry out non-invasive prenatal detection of monogenic diseases, and can use one method to complete the detection of a variety of monogenic diseases. It is a major breakthrough in the application of non-invasive prenatal genetic detection technology.

(VII) Infection

infectious diseases. Traditional serology, culture and other methods are time-consuming and laborious in identifying the pathogens of infectious diseases, and it is difficult to provide reliable diagnostic basis for clinic in time. As a result, many patients with infectious diseases fail to receive timely and effective treatment, resulting in poor prognosis. Domestic companies use high-throughput metagenomic sequencing technology to provide genetic testing for patients infected with pathogens. Through professional microbial database comparison and intelligent algorithm analysis, they can obtain suspected pathogenic microbial species information, which can provide rapid and accurate diagnosis basis for patients with difficult and critical infections. Promote the rational use of antibiotics. This test can assist the clinical rapid identification and identification of the patient's infectious pathogens, especially the unique advantages of unknown or uncultured diseases. At the same time of qualitative and quantitative infection of pathogens, the detection of pathogen genome variation and the study of pathogen variation over time are helpful for epidemic origin tracking and epidemic monitoring, and new pathogens can be found. It is a powerful means for hospital infection control and community infection outbreak investigation.

Source: Blue Book on the Development of China's In Vitro Diagnostic Industry

Created on:2020-06-29 13:49