BACKGROUND

Accurate identification of microorganisms detected in test samples is a crucial and necessary requirement. Classical methods are now rarely used because for bacteria, identification based on biochemical reactions via automated equipment such as VITEX®2 or gas chromatography via Maldi-tof is highly reliable. However, these solutions and equipments still have limitations in identifying rare bacterial agents; and especially fungal agents.

AIMS OF THE STUDY

This study evaluates the effectiveness of solutions implemented in accurately identifying bacterial and fungal agents isolated from test samples based on the detection and analysis of genetic structural differences between these agents. Furthermore, it also presents solutions for detecting agents that cannot be cultured, such as viruses and atypical bacteria, as well as the possibility of direct detection from test samples.

MATERIALS AND METHODS

The solutions with materials and methods currently applied by us for detection and identification of the bacteria, virus and fungi include: (1) Multiplex real-time PCR (MPL-rPCR) with real-time PCR devices and MPL-rPCR master mixes; (2) Sanger sequencing with the Sanger sequencer ABI3500 and related reagents; (3) Next-generation sequencing with the GeneMind next-generation sequencer and related reagents.

RESULTS

The effective methods we implemented were: (1) Sanger sequencing, targeting highly conserved gene regions such as the 16S-rRNA gene for bacteria^[1], the ITS region for fungi, and specific coding regions (e.g., E or RdRp) for RNA viruses. (2) In cases requiring direct detection of bacterial, fungal, viral, and parasitic agents from test samples, we used MPL-rPCR (Multiplex real-time PCR) solution; this solution is currently used for rapid diagnosis of pathogenic microorganisms from various specimens taken from patients, however, it only detects agents for which we use specific primers and probes and cannot detect agents outside of the specific primers and probes used. (3) Currently, thanks to the new generation sequencing system through short-sequence sequencing, we have applied it to sequence the entire genome of isolated bacterial strains, including identification and detection of antibiotic resistance genes, and from there we have established software to analyze the collected data so that scientists can apply it^[2,3]. (4) Metagenomic solution based on new generation sequencing technology for the presence of 16S-rRNA genes is also being used by us in detecting bacterial agents and their distribution in test samples, thereby assessing the abnormality of the microbiome in test samples. (5) In the near future, we will also try to deploy new generation sequencing technology in sequencing the entire genome of pathogenic viruses that we have detected such as RSV, Influenza A virus, SARS- CoV2…