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Detection of Bacillus piliformis by specific amplification of ribosomal sequences
Abstract:
In an effort to explore a sensitive species-specific detection system using the polymerase chain reaction (PCR) for B. piliformis, we sequenced 16S ribosomal DNA (rDNA) of the organism (MSK strain) isolated from the mouse and compared it with known rDNA sequences of the RJ strain isolated from the rat. Sequence homology between the MSK strain and the RJ strain was over 97%, but homology between the MSK strain and other bacterial species was less (70-83%). The results indicated that the sequences included B. piliformis species-specific regions. On the basis of the sequences, we designed a PCR primer set which amplifies B. piliformis rDNA specifically. The PCR with the primer set detected not only these two strains but also an HN strain of hamster origin, although it did not detect other organisms. Therefore, this primer set was considered to be specific for B. piliformis species. More than one organism (RJ strain) could be detected by the PCR method. Nine Jcl:Wistar rats were infected perorally with 2x10(4) RJ strain organisms, and three rats each were sacrificed on days 1, 3 and 5 postinoculation (p.i.) to investigate the presence of the organism in the liver, heart, cecum, spleen and mesenteric lymph nodes by PCR and the immunofluorescence test. On days 1 and 3 p.i., B. piliformis was not detected in any tissues of the six rats, but B. piliformis was detected in two of the three rats sacrificed on day 5 p.i. The presence of the pathogen was seen in both liver and heart (1/3), or in the cecum (1/3) by both methods.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Researchers developed a specific polymerase chain reaction (PCR) test to detect B. piliformis, a pathogen. This new PCR method accurately identified B. piliformis in infected rat tissues, offering a sensitive diagnostic tool.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- B. piliformis is an important pathogen causing significant economic losses in animal husbandry.
- Accurate and sensitive detection methods are crucial for controlling B. piliformis infections.
Purpose of the Study:
- To develop a sensitive and species-specific detection system for B. piliformis using polymerase chain reaction (PCR).
- To evaluate the efficacy of the developed PCR method in detecting B. piliformis in experimentally infected rats.
Main Methods:
- Sequencing of 16S ribosomal DNA (rDNA) from B. piliformis strains (MSK, RJ, HN).
- Design and validation of a species-specific PCR primer set based on rDNA sequences.
- Experimental infection of Wistar rats with B. piliformis (RJ strain) and detection of the pathogen in various tissues using PCR and immunofluorescence tests.
Main Results:
- The designed PCR primer set specifically amplified B. piliformis rDNA with high homology (>97%) among strains and low homology (<83%) with other bacteria.
- The PCR method successfully detected B. piliformis in experimentally infected rats on day 5 post-inoculation, with the pathogen found in liver, heart, and cecum.
- Both PCR and immunofluorescence tests confirmed the presence of B. piliformis in infected tissues.
Conclusions:
- A novel, species-specific PCR primer set was successfully developed for the sensitive detection of B. piliformis.
- The PCR method demonstrated high specificity and sensitivity for B. piliformis detection in animal tissues.
- This PCR assay represents a valuable tool for the diagnosis and control of B. piliformis infections.