مقالات پذیرفته شده در هشتمین کنگره بین المللی زیست پزشکی
Rapid and Accurate Detection of Brucellosis: A Comparative Study of Molecular Techniques
Rapid and Accurate Detection of Brucellosis: A Comparative Study of Molecular Techniques
Fatemeh Karimiyan,1Zahra Gholizadeh farshi,2,*
1. School of Medicine, Fasa University of Medical Sciences, Fasa, Iran 2. Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
Introduction: Brucellosis, a zoonotic infection triggered by Brucella bacteria, is a significant zoonotic illness that poses a substantial global public health challenge, affecting millions of lives yearly. This illness is primarily transmitted through close contact with diseased animals or the consumption of tainted animal products.
It is highly prevalent in regions with extensive livestock farming, such as the Middle East and Iran. The disease affects over half a million people annually worldwide, leading to significant morbidity. In the Middle East and Iran, the incidence is exceptionally high due to consuming unpasteurized dairy products and close human-animal interactions. Brucellosis presents various symptoms, including fever, malaise, and severe complications like neuro brucellosis, which can lead to chronic meningitis and other neurological issues. The socio-economic burden of brucellosis is substantial, affecting livestock productivity and human health. Hence, the urgency of timely detection and efficient treatment to avert long-term complications and curb the disease's spread cannot be overstated . This study not only evaluates and compares three advanced molecular diagnostic techniques—droplet digital PCR (ddPCR), one-tube nested PCR (OTN-PCR), and loop-mediated isothermal amplification PCR (LAMP-PCR)—for the detection of human brucellosis but also presents a promising future for the field of brucellosis detection.
Methods: Droplet Digital PCR (ddPCR) is an advanced molecular technique used to quantify DNA or RNA with high precision. The process starts by partitioning a sample into thousands of tiny droplets, each acting as an individual PCR reaction chamber. The target DNA or RNA is amplified within these droplets using standard PCR reagents. After amplification, the droplets are passed through a detector that counts the number of positive (fluorescent) and negative droplets. Compared to conventional PCR (RT-qPCR), which offers relative quantification based on fluorescence during amplification cycles, ddPCR provides higher precision and accuracy by eliminating the need for standard curves. It also boasts greater sensitivity, can detect low-abundance targets, and is less affected by sample inhibitors, resulting in more consistent results. While ddPCR is more expensive and time-consuming due to specialized equipment and additional processing steps, its significant advantages make it ideal for applications requiring precise and sensitive nucleic acid quantification.
One-Tube Nested Quantitative Real-Time PCR (qPCR) is an advanced molecular diagnostic technique designed to enhance the sensitivity and specificity of DNA detection compared to conventional PCR. This method integrates a nested PCR approach within a single tube, utilizing two sets of primers and two probes that amplify target DNA sequences sequentially. One-tube nested qPCR minimizes handling, unlike standard PCR, which can be less sensitive and prone to contamination. It reduces contamination risks by maintaining a closed-tube system throughout the process. This method significantly improves detection rates, especially for low-abundance DNA targets, with sensitivity and specificity reaching 98.6% and 100%, respectively. This advancement in molecular diagnostics minimizes the likelihood of false negatives, thereby improving the reliability and precision of clinical diagnostics.
Results: LAMP (Loop-mediated Isothermal Amplification) is an innovative nucleic acid amplification technique that operates at a constant temperature of 63°C, eliminating the need for a thermal cycler required in conventional PCR. Unlike PCR, which requires multiple temperature cycles, LAMP uses four to six primers to recognize distinct regions of the target DNA, enabling continuous amplification through a strand-displacement reaction. The primary advantages of LAMP include high sensitivity, rapid results, often within 35 minutes, and the capability to detect low levels of DNA. Additionally, it allows for visual result inspection, making it ideal for low-resource settings. However, it can be prone to non-specific amplification if not carefully optimized, and the primer design process is complex. Despite these drawbacks, LAMP's efficiency and simplicity make it a powerful tool for diagnostic applications, especially in environments lacking sophisticated laboratory infrastructure.
Conclusion: In conclusion, this study's findings highlight the advantages and limitations of each method. With its high sensitivity, rapid results, and operational simplicity, LAMP-PCR is the most suitable method for detecting human brucellosis quickly and precisely. Its potential in low-resource settings underscores its practicality and instills hope for improved healthcare in these areas, reassuring the audience.