Molecular Methods – strategie used in diagnosis of human Infectious diseases


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Infectious diseases are a significant burden on public health and economic stability of societies all over the world. They have been among the leading causes of death and disability and presented growing challenges to health security and human progress for centuries. Infectious diseases are generally caused by microorganisms. The routes of them entry into host is mostly by the mouth, eyes, genital openings, nose, and the skin. Damage to tissues mainly results from the growth and metabolic processes of infectious agents intracellular or within body fluids, with the production and release of toxins or enzymes that interfere with the normal functions of organs and/or systems [1]. Advances in basic science research and development of molecular technology and diagnostics have enhanced understanding of disease etiology, pathogenesis, and molecular epidemiology, which provide basis for appropriate detection, prevention, and control measures as well as rational design of vaccine [2]. The diagnosis of infectious diseases is particularly critical for the prevention and control of the epidemic. Here we introduce the insights and detection methods of infectious disease, aiming to provide some helps for clinical diagnosis as well as epidemic prevention and control of infectious diseases.

Molecular Methods - strategie used in diagnosis of human Infectious diseases

The development of molecular methods for the direct identification of a specific viral genome from the clinical sample is one of the greatest achievements of the 21st century. Clearly nucleic acid amplification techniques including Reverse Transcription-Polymerase Chain Reaction (RT-PCR), nucleic acid sequence-based amplification (NASBA) and Lawrence Livermore Microbial Detection Array (LMDA) are proven technology leaders for rapid detection and molecular identification for most known human viruses [9].

RT-PCR assays for virus detection provides faster results than end-point assays and in many cases have sensitivities equal to or better than culture [10]. The novel coronavirus, 2019-nCoV, was detected through real-time RT-PCR with primers against two segments of its RNA genome [11]. The particular primer sets and specific guideline for detection of COVID-19 through RT-PCR were made available by the Center for Disease Control (CDC) USA, according to CDC [12]. However, high mutation rates may lead to extensive changes in viral nucleic acid sequences making dedicated PCR primer use irrelevant, therefore there is high demand for the development of rapid and universal virus identification and detection technologies. In contrast, although NASBA assay is considered sensitive; it has not been widely used because of the difficulties in the preparation of NASBA master mix in-house and the high cost of commercial kits. A new molecular biology-based microbial detection method for rapid identification of multiple virus types in the same sample has been developed by a research group at Lawrence Livermore National Laboratory. Lawrence Livermore Microbial Detection Array (LLMDA) detects viruses using probes against genomic DNA sequence within 24 hours [13,14]. In addition, the oligonucleotide probes were selected to enable detection of novel, divergent species with homology to sequenced organisms [14].

Figure 1. Principle of RT-PCR.


Infectious diseases are a real public health threat, outbreaks can have serious social, political, and economic effects. A complex number of factors relating to human behavior and activities, pathogen evolution, poverty, and changes in the environment as well as dynamic human interactions with animals have been found to contribute to infectious disease emergence and transmission. Aggressive research is warranted to unravel important characteristics of pathogens necessary for diagnostics, therapeutics, and vaccine development. Here we describe some strategies for the diagnosis of human infectious diseases, hoping to be helpful for clinical diagnosis and epidemic prevention and control of infectious diseases. To date, multiple diagnostic techniques have been developed. Various diagnostic tools show both significances and limitations. Conventional approaches to quantify infective viral particles are labor-intensive, time-consuming, and often associated with poor reproducibility. Immunological tests generally provide quick results, however, is quite expensive due to the requirement of antigen-specific antibody. While RT-PCR may be able to provide results within a matter of hours, it is laborious, requires a skilled operator, and is sensitive to contamination. TEM-based quantification, although highly accurate in determining the shape and the total number of viral particles, often considered time-consuming, extremely expensive and impractical for high sample numbers. Moreover, TEM sample preparation is tedious, and the technique requires sophisticated instrument and a skilled operator. To alleviate these limitations, there is still a need to develop new cost-effective analytical methods that can allow users to quickly and easily determine virus concentrations and reduce constrictions coupled with current assays. Nevertheless, any such emerging methods must be carefully evaluated in terms of their efficiency, precision and linear range. The evaluation of each diagnostic technique and approval from the FDA are necessary before practical application.

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