Abstract
Background:The Sanger sequencing technique has been questioned and challenged by advanced high-throughput sequencing approaches. Sanger sequencing seems to be an obsolete technology. However, there are still research problems that could be answered using the Sanger sequencing technology. Fastidious obligate anaerobic bacteria are mostly associated with abscesses in animals. These bacteria are difficult to isolate from abscesses and are frequently excluded due to the bias of conventional bacterial culturing.Aim:This study demonstrated the usefulness of a broad-range polymerase chain reaction (PCR) with Sanger sequencing to identify the majority population of bacteria in abscesses from exotic pet animals.Materials and Methods:This study performed a pilot investigation of abscesses from 20 clinical cases (17 rabbits, 2 hedgehogs, and 1 sugar glider) using standard culture methods for both aerobes and anaerobes and broad-range nested PCR targeting the 16S rRNA gene followed by the Sanger sequencing technique.Results:The standard culture and PCR techniques detected bacteria in 9 and 17 of 20 samples, respectively. From the 17 sequencings of the 16S rRNA, 10 PCR products were found to be closely related with obligate anaerobes including Bacteroides spp., Fusobacterium spp., Prevotella spp. Phylogenetic analysis using the rpoB gene revealed that the species for the Bacteroides was thetaiotaomicron and for the Fusobacterium was varium and nucleatum. However, the amplification of the rpoB gene for the Prevotella spp. was unsuccessful. Correlations between the standard culture and PCR techniques were found in 9 (6 positive and 3 negative samples) of 20 samples. Eleven samples were discordant between the standard culture and PCR techniques which were composed of eight samples negative by culture but positive by PCR and three samples had different bacteria by the culture and PCR techniques.Conclusion:According to this study, broad-range PCR combined with Sanger sequencing might be useful for the detection of dominant anaerobic bacteria in abscesses that were overlooked based on conventional bacterial culture.
Highlights
Broad-range polymerase chain reaction (PCR) detections have long been used for the studies of microorganism diversity within complex environments
Aim: This study demonstrated the usefulness of a broad-range polymerase chain reaction (PCR) with Sanger sequencing to identify the majority population of bacteria in abscesses from exotic pet animals
According to this study, broad-range PCR combined with Sanger sequencing might be useful for the detection of dominant anaerobic bacteria in abscesses that were overlooked based on conventional bacterial culture
Summary
Broad-range polymerase chain reaction (PCR) detections have long been used for the studies of microorganism diversity within complex environments. These PCR and Sanger sequencing techniques have discovered large numbers of novel bacterial species and their ecosystems. Including soil, water, and plant as the main habitats of these novel species [1] These broad-range PCRs targeting the 16S rRNA gene have altered routine diagnosis in clinical microbiology as conventional bacteriological techniques have failed to identify some groups of organisms known as culture-negative bacteria [2]. Huge numbers of novel pathogens and high microbial diversity have been revealed in human specimens using these broad-range PCRs followed by Sanger or high-throughput sequencing technologies that have opened a new era of the study of Veterinary World, EISSN: 2231-0916. These bacteria are difficult to isolate from abscesses and are frequently excluded due to the bias of conventional bacterial culturing
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