Discuss the molecular methods for the identification of microorganisms. (10)
Rapid detection and identification of microorganisms is a challenging and important aspect in a wide range of fields, from medical to industrial, affecting human lives. Unfortunately, classical methods of microorganism identification are based on time-consuming and labor-intensive approaches. Screening techniques require the rapid and cheap grouping of bacterial isolates; however, modern bioanalytics demand comprehensive bacterial studies at a molecular level. Modern approaches for the rapid identification of bacteria use molecular techniques, such as 16S ribosomal RNA gene sequencing based on polymerase chain reaction or electromigration, especially capillary zone electrophoresis and capillary isoelectric focusing.
However, there are still several challenges with the analysis of microbial complexes using electromigration technology, such as uncontrolled
aggregation and/or adhesion to the capillary surface. Thus, an approach using capillary electrophoresis of microbial aggregates with UV
and matrix-assisted laser desorption ionization time-of-flight MS detection is presented.
Microorganisms are well known for both positive and negative properties. They are used on a wide scale in the food industry, biotechnology, and modern genetic engineering. However, some microorganisms can cause food
spoilage or serious disease. Because of these two negative effects, the unequivocal and rapid identification of microorganisms in real samples represents a very important area of focus (1, 2). This is particularly important in medical diagnostics. Microbial infections can lead to dangerous diseases,
such as sepsis (3), diabetic foot infections (4), or meningitis (5). Rapid and unambiguous identification of the pathogen causing the infection is a necessary factor for the implementation of an appropriate therapy to save patient lives (6, 7).
Conventional techniques currently used for the identification of microorganisms are based on the culture of microorganisms and the determination of the phenotypic characteristics thereof. However, these methods are labor-intensive, time-consuming (taking up to 3 days), and often inadequate for the differentiation of phenotypically similar species (8–12). These limits are especially important from a medical diagnostics point of view
because the rapid and proper identification of pathogens is an essential factor in the implementation of the appropriate therapy (10, 11). In addition, screening identification of microorganisms is also a key issue in areas such as the pharmaceutical industry, food QC, and environmental research (1, 11, 13). Therefore, much emphasis has been placed on the development of alternative methods for the rapid and unambiguous identification of
microorganisms directly from tested material (8–11). Nowadays, in the taxonomy of microorganisms, molecular biology methods—such as 16S ribosomal RNA (rRNA) gene sequencing (8, 14), polymerase chain reaction (PCR; 15), and other related PCR-based methods (16–18)—are very popular.
These methods are characterized by high sensitivity and reproducibility. 16S rRNA gene sequencing is considered the most accurate method and deemed the gold standard for the identification of microorganisms at the species level (19). Unfortunately, due to the high cost of this analysis, in routine diagnostics, it is impossible to use this method. Moreover, such studies are usually performed by external institutions, resulting in prolonged wait times for results (14, 20). One of the strategies to reduce the time required for the
identification of microorganisms in routine diagnostics is the use of semiautomatic and automatic systems based on biochemical methods. Such methods allow the attainment of results in maximum of 24 h (21–23). However, this time frame is still too long, and the obtained results are not always satisfactory (24). An alternative could be MS methods, such as
matrix-assisted laser desorption ionization time-of-flight mode (MALDI-TOF MS) or electromigration techniques (8). These methods allow the performance of quick and clearanalysesofmicroorganisms directly from infected liquid samples while maintaining unit costs low (25–30). The most recent approach involves the use of multivariate techniques, such as PCR electrospray ionization (ESI) MS (31, 32), PCR-microchip capillary electrophoresis (ME; 33), PCR-capillary electrophoresis (PCR-CE; 34), capillary isoelectric focusing (CIEF)-MALDI-TOF MS (34), or micropreparative
solution CIEF (sCIEF)-HPLC (35). Such connections allow the attainment of conclusive results in a very short amount of time and have great potential for further research