Infectious diseases remain a leading cause of morbidity and mortality worldwide, significantly impacting public health and socioeconomic stability. Estimates indicate that infectious diseases account for at least 25% of the approximately 60 million annual deaths globally. While vaccines and antimicrobial/antiviral drugs are crucial in combating infections, their effectiveness can be compromised by the emergence of new pathogens and drug-resistant strains. The development of new vaccines and medicines is a lengthy process, highlighting the need for rapid and accurate diagnostic tools. Traditional diagnostic techniques, such as microbial culture, hemagglutination inhibition tests, and ELISAs, suffer from limitations including low detection rates, long turnaround times, and limited automation. These limitations necessitate the development of superior diagnostic methods for efficient disease management and public health response. Molecular diagnostic techniques offer significant advantages over traditional methods due to their enhanced sensitivity and specificity, faster detection times, and greater automation. These advancements facilitate early and rapid detection of infectious disease pathogens, improving treatment efficiency and reducing disease spread.

The review cites numerous studies comparing the performance of molecular diagnostic techniques to traditional methods. Several studies demonstrated the superior sensitivity and speed of qPCR compared to culture-based methods for detecting pathogens such as pneumococci, Porphyromonas gingivalis, and Helicobacter pylori. The use of digital PCR (dPCR) is highlighted for its ability to detect low levels of pathogens and rare mutations, making it suitable for monitoring viral load and drug resistance. High-resolution melting (HRM) analysis is presented as a rapid and cost-effective method for species identification and genotyping. The application of isothermal amplification techniques like LAMP and RPA are discussed for their suitability in resource-limited settings due to their simplicity and speed. Gene chip technology's ability to detect multiple pathogens simultaneously is mentioned, although its limitation in detecting novel pathogens is noted. Finally, high-throughput sequencing (NGS and TGS) is presented as the most accurate method for pathogen identification and is crucial for epidemiological investigations and tracking emerging pathogens. The reviewed literature underscores the need for and potential of improved molecular techniques for various pathogens.

This is a review article, not an original research study. The methodology involved a comprehensive literature search and analysis of published studies on various molecular diagnostic techniques used for detecting infectious disease pathogens. The authors systematically reviewed and summarized the principles, advantages, limitations, costs, and applications of PCR (including qPCR, dPCR, and HRM), isothermal amplification (LAMP, RPA, NASBA), gene chip technology (solid-phase and liquid-phase), and high-throughput sequencing (NGS and TGS). The information was synthesized to provide a comparative analysis of these techniques. Specific examples of the application of each technology in diagnosing various infectious diseases were provided, alongside their respective limitations and cost considerations. The review also discussed emerging technologies like biosensors, FISH, and mass spectrometry, briefly outlining their potential for infectious disease diagnostics.

The review highlights the strengths and weaknesses of various molecular diagnostic techniques. qPCR is presented as a mature, low-cost technology well-suited for routine pathogen detection in well-equipped laboratories. dPCR excels in absolute quantification and is ideal for samples with low pathogen loads or rare mutations. HRM offers a rapid and cost-effective approach to genotyping and mutation detection. Isothermal amplification methods (LAMP, RPA, NASBA) offer simpler, faster, and more portable alternatives, especially beneficial in resource-limited settings. Gene chip technology allows for simultaneous detection of multiple pathogens, facilitating the diagnosis of mixed infections. However, it's unable to detect novel pathogens. High-throughput sequencing (NGS and TGS) provides the highest accuracy for pathogen identification and is crucial for epidemiological studies. NGS offers high throughput but longer processing times and higher costs, while TGS provides ultra-long reads but has a higher error rate. Emerging technologies such as biosensors, FISH, and mass spectrometry are also mentioned as promising avenues for future advancements. The review points out that many molecular techniques require nucleic acid extraction, a complex and time-consuming step. Furthermore, several techniques rely on expensive equipment, potentially limiting their use in resource-poor regions. Therefore, the development of ready-to-use reagents, room temperature-stable reaction mixtures, and portable, automated instruments is crucial for expanding the accessibility and applicability of molecular diagnostics.

This review effectively addresses the need for advanced molecular diagnostic techniques in infectious disease management. The comparative analysis of various methods highlights their strengths and limitations, providing valuable insights for researchers and healthcare professionals. The emphasis on the need for cost-effective and portable technologies for resource-limited settings is particularly significant. The review's discussion of emerging technologies suggests promising future directions in infectious disease diagnostics. The challenges in nucleic acid extraction and the reliance on expensive equipment necessitate further research into streamlined extraction methods and the development of more accessible diagnostic tools. The findings of this review contribute to the ongoing efforts to improve global infectious disease surveillance and control.

Molecular diagnostic techniques provide significant advancements over traditional methods for diagnosing infectious diseases. The choice of technology depends on factors like cost, availability of equipment, and the specific pathogen being detected. Future research should focus on improving nucleic acid extraction procedures, developing room temperature-stable reagents, and creating more portable and automated instruments to expand the accessibility and applicability of molecular diagnostics globally.

As a review article, this study is limited by the scope of the literature included. The authors' selection of studies may not represent the entirety of relevant research. Furthermore, the rapid evolution of molecular diagnostic technologies means that some findings may quickly become outdated.