At present, molecular diagnostic technology mainly includes nucleic acid molecular hybridization technology, nucleic acid amplification technology, gene chip technology, gene sequencing technology, has been widely used in clinical departments.

Nucleic acid molecular hybridization technology refers to the specific formation of two single strands of nucleic acid from different sources by the principle of base complementary pairing under certain conditions. They include Southern blot hybridization, Northern blot hybridization, in-situ hybridization and fluorescence in situ hybridization (FISH). FISH technology is used to form hybridization between fluorescent-labeled probe and target fragment, and obtain relevant data of DNA to be tested through fluorescence detection system. It has the characteristics of strong specificity and fast detection speed, and is widely used in prenatal diagnosis [1]. In 1993, the American Society of Medical Genetics published relevant instructions on the application of FISH in prenatal diagnosis [2], and this technique has become an expert consensus as a supplement to karyotype analysis in the field of prenatal diagnosis.

Figure 1 Principle of nucleic acid molecular hybridization technology (Source: Network)

In the 1980s, Mullis developed polymerase chain reaction (PCR), a technique for in vitro amplification of target nucleic acid fragments. Nucleic acid amplification technology is widely used in clinic, and a variety of PCR technologies have been developed so far, including quantitative real-time PCR(qPCR) and digital PCR(dPCR). Because the operation process of qPCR is carried out in a closed system, the contamination probability is reduced, and quantitative detection can be carried out by monitoring fluorescence signals. Therefore, the clinical application of QPCR is the most widespread, and it has become the leading technology in PCR [3]. dPCR is also known as single-molecule PCR. Its principle is to place a single nucleic acid molecule in an independent reaction unit for PCR amplification, then detect the fluorescence at the end of the reaction chamber and carry out molecular counting statistics to achieve quantitative analysis. Due to the characteristics of high sensitivity and accuracy, it is not easy to be interfered by PCR reaction inhibitors, and absolute quantification in real sense can be achieved without standard substances, which has become a research hotspot [4].

Gene chip technology is to fix a large number of nucleic acid probes with known sequences on the substrate surface, then hybridize them with target nucleotides, and obtain the sequence information of the sample to be tested through the detection of the probes. According to the types of probes, gene chips can be divided into two categories, namely cDNA microarray chips and oligonucleotide microarray chips [5]. The probe of the former is the corresponding cDNA of the specific mRNA in the cell, while the probe of the latter is the oligonucleotide fragment synthesized in situ. Gene chip technology is widely used in genotyping, gene expression and single nucleotide polymorphism detection due to its high automation, simple operation and high throughput.

Gene sequencing technology started from the DNA dideoxy end termination sequencing method invented by Sanger in 1977. Because of its high accuracy, it is considered as the "gold standard" of gene sequencing. However, its clinical application is limited by its high cost and low throughput.

With the continuous development of molecular diagnostic technology, it has become a new clinical means and ideas for effective disease diagnosis. Especially in the fields of infectious diseases, tumors and genetic diseases, it not only effectively makes up for the deficiency of traditional methods, but also provides important evidence for disease prevention, diagnosis, treatment monitoring and prognosis assessment.

The occurrence of tumor is the result of the joint action of genes and environment. The individualized and precision medicine of malignant tumor is of great significance. Molecular diagnostic technology not only contributes to the prevention and early diagnosis of cancer, but also plays a role in the molecular classification, prognosis prediction and treatment program screening of cancer.

FISH technology is used to detect changes in the number and structure of chromosomes, and the status of chromosomes is closely related to the development of tumors. The detection of specific chromosomes by FISH technology can predict the prognosis of cancer. Mian et al. [8] conducted long-term follow-up of 81 patients with uroepithelial carcinoma (UC), and used multicolor fluorescence in situ hybridization to detect 9p21(p16) sites and chromosomes 3, 7 and 17 of the patients, so as to screen those at high risk of bladder cancer recurrence and predict the development of the disease.

In nucleic acid amplification technology, fluorescence quantitative PCR has been widely used in tumor therapy for its unique advantages. Squamous cell carcinoma of the head and neck (HNSCC) is one of the most common cancers, and several studies have shown that human papillomavirus (HPV) infection is one of the pathogenic factors. Polanska et al. [9] collected tissue samples from 74 primary HNSCC patients and analyzed 15 HNSCC-related molecular markers by real-time quantitative polymerase chain reaction (qRT-PCR). Meanwhile, PCR was applied to detect HPV. Studies have confirmed differences in the expression of common biomarkers MT2A, MMP9, FLT1, VEGFA and POU5F in HPV-negative and HPV-positive HNSCC patients, which has important significance for predicting disease development and survival in HNSCC patients.

At the same time, HTS technology is also widely used in liquid tumor biopsies due to its high sensitivity. Lung cancer is the cancer with the highest incidence among Chinese men, among which the most common type is non-small cell lung cancer (NSCLC). In the targeted treatment of NSCLC, gene mutation detection is essential. Dono et al. collected blood samples from 42 patients and used HTS to detect mutations in exon T790M of EGFR-country 20 in plasma ct DNA to assess whether patients who had developed resistance to first - and second-generation EGFR-TKI therapy could continue to receive oxitinib, the third-generation EGFR-TKI drug. This study found that due to the extremely high sensitivity and detection rate of HTS, liquid biopsy could more effectively replace tissue detection and improve patient compliance [10].

Statistics from the China Report on the Prevention of Birth Defects show that about 1 million babies are born with birth defects every year in China, and heredity is one of the factors causing birth defects. Genetic diseases are diseases caused by changes in genetic material or control of pathogenic genes, which are generally divided into chromosome syndrome, single gene inherited diseases and polygene inherited diseases. In recent years, molecular diagnostic technology has played an important role in genetic disease diagnosis, newborn screening and other fields.

As a new technology, gene chip is gradually replacing karyotype analysis in prenatal diagnosis. aCGH technique can rapidly detect chromosome CNV and the source of variation at the whole genome level. Chong et al. applied aCGH technology for prenatal diagnosis of more than 1000 fetuses with abnormal ultrasound examination. Due to the high resolution of aCGH chip technology, its combined application with ultrasound examination will improve the accuracy of detection [11].

High-throughput sequencing technology can sequence hundreds of thousands to millions of DNA molecules at a time, and is one of the most popular technologies at present. For the detection of chromosome aneuploidy disease, maternal cfDNA can be sequentially sequed by HTS, and the differences of different chromosomes in peripheral blood can be analyzed using biological information tools, so as to accurately identify the changes in fetal chromosomes. Bianchi et al. applied HTS to conduct large-scale parallel sequencing and detected trisomy 21 and 18 in blood samples of 1,914 pregnant women from 21 centers in the United States. Compared with traditional screening methods, the false positive rate of HTS technology decreased significantly and the accuracy rate was high [12].

Infectious diseases are diseases caused by human infection with bacteria, viruses, fungi and other pathogenic microorganisms. According to the WHO, 17 million people die each year from infectious diseases. Traditional pathogen diagnosis is time-consuming and less accurate, while molecular diagnosis is faster and more accurate, enabling early treatment and virus control, which is the main development trend of pathogen detection.

Molecular diagnosis has become an important subject to solve many scientific and medical problems. It has been widely used in clinical practice to promote disease prevention, diagnosis, prognosis prediction and the formulation of individualized medical programs. With the aging of the Chinese population, the incidence of cancer and chronic diseases will continue to rise, and the national health awareness is also gradually improving. Molecular diagnostics will be automated to reduce labor consumption and cost, and automated interpretation of test results will also reduce manual errors. At the same time, the breakthrough of single molecule technology also makes the detection of high sensitivity become the future development trend of molecular diagnostic technology, improve the detection efficiency. In addition, non-invasive detection and minimally invasive detection will improve patient compliance, which is conducive to the further promotion and application of molecular diagnostic technology in clinical practice. In the next few years, molecular diagnostic technology will be more widely used in physical examination, early diagnosis, prevention and treatment of major diseases.