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[Improvement of a method for detecting of strains of the plague microbe using polymerase chain reaction]
Abstract:
Three pairs of oligonucleotide primers, complementary to nucleotide sequences of Yersinia pestis plasmids (pPst, 9.5 kb; pCad, 70 kb; pFra, 95 kb), were used in polymerase chain reaction for high-sensitive specific detection of plague pathogen. Primer pairs P1,P2,C1,C2, and F1,F2 were used to amplify fragments of pla gene (plasmid pPst), yop1 gene (plasmid pCad of plague microbe), and caf1 gene (plasmid pFra), respectively. The method developed enables specific detection of strains from all natural loci in Russia and contiguous states as well as strains of oceanic origin. Sensitivity of method is 50-100 CFU/ml. Primer sequences enable to amplify gene fragments, located in three own plasmides of plague microbe, in the same reaction mixture. The method offers identification of plague microbe and determination of its virulence and epidemic significance.
Insights
This study developed a highly sensitive polymerase chain reaction (PCR) method for detecting Yersinia pestis, the plague pathogen. The technique accurately identifies plague strains from diverse origins, aiding in virulence assessment.
Area of Science:
- Microbiology
- Molecular Biology
- Epidemiology
Background:
- Yersinia pestis is the causative agent of plague, a serious infectious disease.
- Accurate and sensitive detection methods are crucial for disease control and surveillance.
- Plague pathogen virulence is often associated with specific plasmid-borne genes.
Purpose of the Study:
- To develop a highly sensitive and specific polymerase chain reaction (PCR) method for detecting Yersinia pestis.
- To target key virulence genes located on different Yersinia pestis plasmids.
- To enable simultaneous detection and assessment of plague pathogen significance.
Main Methods:
- Oligonucleotide primers were designed to target nucleotide sequences on Yersinia pestis plasmids pPst, pCad, and pFra.
- Polymerase chain reaction (PCR) was employed to amplify specific gene fragments (pla, yop1, caf1).
- A multiplex PCR approach was developed to detect multiple targets in a single reaction mixture.
Main Results:
- The developed PCR method demonstrated high sensitivity, detecting as few as 50-100 CFU/ml.
- Specific detection of Yersinia pestis strains from various geographical origins, including Russia and oceanic strains, was achieved.
- The method successfully amplified gene fragments from the three key plasmids (pPst, pCad, pFra) simultaneously.
Conclusions:
- The established PCR method provides a sensitive and specific tool for identifying Yersinia pestis.
- This technique facilitates the determination of the plague microbe's virulence and epidemic potential.
- The multiplex PCR approach offers an efficient means for plague pathogen surveillance and diagnostics.